Despite the complexity of DM1 pathogenesis, it is now well
established that non-coding CUG repeat transcripts play a toxic
gain-of-function role. Abnormal DMPK transcripts form
secondary structures, which aggregate into foci within muscle
nuclei and which sequester RNA-binding proteins such as
Muscleblind-like 1 (MBNL1). Also, by a still undetermined
mechanism activating protein kinase C, CUG-binding protein 1
(CUGBP1) is stabilized by phosphorylation. MBNL1 and CUGBP1 are
both splicing factors, but play antagonistic roles. Thus, in DM1
patients, several transcripts are mis-spliced due to an inverse
ratio of MBNL1/CUGBP1. Among mis-spliced transcripts, insulin
receptor (IR), chloride ion channel-1 (ClC-1), Bin1 and troponin T
(cTNT) are involved in insulin resistance, myotonia, muscle
weakness and reduced myocardial function observed in patients.
However, recent reports indicate that other molecular aberrations
such as altered maturation of miRNAs or CUG repeat-dependent
transcription factors leaching can also contribute to the
pathogenesis of DM1. To characterize molecular defects underlying
this pathology, several animal models have been generated. The
first to be developed were mice models, but it was found that Drosophila
also represents an accurate model system to study DM1.
Accordingly, fruit flies expressing CUG repeats in adult muscles
develop DM1 symptoms and can be used to screen modifiers of
transcript toxicity. Recently, applying a Drosophila
model has revealed the role of the anti-sense DMPK transcript in
DM1 pathogenesis. It has also been shown that Muscleblind (Mbl),
the Drosophila MBNL1 ortholog, as in humans, is involved
in muscle phenotypes observed in DM1 flies
(Picchio, 2013 and references therein).
Myotonic dystrophy type 1 (DM1, OMIM no. 160900) is a
neuromuscular disorder linked to a major misregulation of
alternative splicing and is considered to be the first described
spliceopathy. DM1 is caused by the expansion of a CTG
trinucleotide repeat tract located in the 3′ untranslated
region (UTR) of the dystrophia myotonica-protein kinase (DMPK)
gene. The main pathogenic effect in DM1 is a deleterious
gain-of-function of the mutant expanded CUG-containing mRNA
(CUG-RNA), which triggers the biochemical and clinical features of
DM1. The current model of disease progression derives from the
strong interaction of expanded CUG-RNA with splicing regulators
such as the muscleblind-like proteins (MBNL1–MBNL3) and
CUG-BP Elav-like family member 1 (CELF1), key proteins involved in
DM1 pathophysiology. Importantly, MBNL1 is sequestered by the
expanded CUG-RNA in anomalous ribonuclear aggregates (foci), which
causes the deregulation of alternative splicing in a large group
of pre-mRNAs. The muscleblind gene (mbl) is not
only conserved in the Drosophila genome, but it also
plays a role in alternative splicing in this organism, suggesting
the conservation of key disease pathways in Drosophila.
This has been confirmed by the successful reproduction of
tissue-specific DM1 hallmarks such as nuclear foci formation,
muscleblind sequestration, missplicing, muscle atrophy and reduced
lifespan in flies expressing a disease-associated CTG repeat tract
[UAS-i(CTG)480 flies] (García-Alcover, 2014 and references therein).
Oculopharyngeal muscular dystrophy (OPMD) is another triplet
expansion disease which results from short expansions of a GCN
repeat in the gene encoding poly(A) binding protein nuclear 1
(PABPN1). OPMD is an autosomal dominant muscular dystrophy, which
has a late onset and is characterized by progressive weakness and
degeneration of specific muscles. Triplet expansion in PABPN1
leads to extension of a polyalanine tract from 10 alanines in the
normal protein to a maximum of 17 alanines at the N-terminus of
the protein. Nuclear aggregates in muscle fibres are a
pathological hallmark of OPMD. These aggregates contain mutant
insoluble PABPN1, ubiquitin, subunits of the proteasome, as well
as poly(A) RNA. Polyalanine expansions in PABPN1 are thought to
induce misfolding and formation of aggregates, which are targeted
to the ubiquitin-proteasome degradation pathway. However, it is
still unknown whether these nuclear aggregates have a pathological
function, a protective role, or are a consequence of a cellular
defence mechanism (Chartier, 2015 and references therein).
For modeling purposes, the study took advantage of the competence
of D. melanogaster both to allow a stable integration of
human DNA in its genome and to mirror disease phenotypes. The
‘humanized’ spliceosensor flies accurately reproduce
the alternative splicing deregulation described in DM1 patients in
the presence of the disease mutation. Simultaneously, the study
used the latest technology applied to small organisms to achieve
in vivo HTS capabilities. For this, the standards for valid
screening parameters (positive Z-factor and a z-score≥3) were
determined on the use of the spliceosensor flies, thus allowing
the assessment of chemical entities in a large-scale format.
Importantly, some of the confirmed hits identified by positive
modulation of the DM1 spliceosensor system were confirmed as
molecules with positive roles in relevant independent DM1 assays,
and for this reason they are currently the subject of further
evaluation in additional DM1 systems (García-Alcover,
2014).
At this point, the evaluation of treatments that simultaneously
combine use of compounds with different anti-DM1 features or
chemical properties is an experimental strategy in need of
exploration in order to improve the potential anti-DM1 response.
Confirmatory data from this study suggest that the method
presented is able to identify high-quality hits from screened
compounds, one of the advantages anticipated from the use of whole
animals in screening (García-Alcover, 2014).
The reliable use of a sensitive reporter-based system in an in
vivo situation establishes a novel and interesting option for
pharmacological evaluation in addition to previously established Drosophila-specific
phenotypic outputs, such as behavioral assays, or assays of
lethality or eye roughness, which are commonly limited to low- and
medium-throughput assays, are difficult to miniaturize or automate
and show higher heterogeneity in their final measurements.
Furthermore, the use of the spliceosensor flies offers the
possibility of establishing distinct types of mechanistic outputs
from the compounds identified. The screening described in this
study involves a splicing-phenotype-based approach, as induced by
the expression of CUG repeats, without connecting the hit
evaluation to a specific mechanism of action. That said, a
screening approach that is closer to a target-based approach is
also possible by using the spliceosensor flies alone and looking
for direct modulators of the splicing event. Given that this
approach entails a HTS format, the in vivo screening capabilities
exhibited are very promising. Most pharmacological screens
described in Drosophila are on the order of 500 to 1000
molecules tested per month (~15 to 30 daily). The approach
presented in this study is around a 10-fold increase in the in
vivo throughput because it can test up to 240 compounds daily
(García-Alcover, 2014).
A limitation on the development of in vivo screening methods is
the unfeasibility of traditional brute-force traditional methods
that usually involve mass 384-, 1536- or 3456-well plate formats.
In contrast, Drosophila-based screening methods offer
the ability to test compound activity directly in a living animal
with the simultaneous evaluation of toxicity and drug-like
properties. Moreover, and of significance, the use of flies allows
for an accurate control of the expression system, as demonstrated
in this study by targeting the DM1 transgenes only in somatic
muscles, a key tissue in DM1 progression (García-Alcover,
2014).
Success in the identification of novel valid compounds for the
potential development of a DM1 treatment suggests that the method
developed in this study could be adapted to any particular type of
alternative splicing deregulation (exon skipping, intron retention
and exon extension, among others) linked to human disease, such as
in myotonic dystrophy type 2 (DM2), progeria, Alzheimer’s
disease or cancer, where missplicing events are already
well-described and for which key disease aspects are conserved in
D. melanogaster. The versatile use of reporter-based
platforms in whole organisms, where it is at the moment still very
limited to cell culture, should serve to rapidly expand the kind
of in vivo HTS screens for which D. melanogaster can be
widely used (García-Alcover, 2014).
It is unclear how loss of the single TRIM32 protein in muscle tissue initiates or promotes LGMD2H pathology. Interpretation of mouse TRIM32 models created to mimic LGMD2H pathology has proven complex, as both neurogenic and myopathic abnormalities are present in TRIM32KO or TRIM32 D489N knock-in mice. Moreover, loss of TRIM32 in satellite cells limits muscle regeneration, thereby promoting further tissue deterioration. This stud took advantage of the lack of satellite cells in Drosophila larval muscles to separate the muscle-intrinsic function of TRIM32 from its role in mammalian muscle regeneration. It was confirmed that a decrease in neuronal TRIM32 does not contribute to muscle pathology, while the expression of myopathic mutations in larval and adult muscle leads to pathogenic defects with reduced TRIM32 protein levels. Most importantly, this study has substantiated a role for costameric proteins in disease progression (Biwa, 2020).
TRIM32 is a multidomain protein that consists of RING domains and NHL repeats that are structurally and functionally conserved among different species. At least 12 different molecules, consisting of either RNA or protein, have been shown to interact with NHL-containing proteins. Moreover, mutations and/or deletions in the NHL region of TRIM family members are linked to diverse human diseases. In addition to LGMD2H caused by mutations in TRIM32, axonal neuropathy and gliomas result from mutations in TRIM2 and TRIM3, respectively. However, understanding of how these repeats function in maintaining adequate protein-protein interactions, especially in muscle cytostructure, is limited (Biwa, 2020).
Despite the moderate amino acid identity between Drosophila and human TRIM32, this study has demonstrated that structural homology of the entire NHL region is remarkably close and serves as an ideal model to investigate the downstream consequences of these myopathic mutations. Previous modeling of the R394H and D487N mutations used the crystal structure of the related NHL-containing protein Drosophila Brat as a template. The curren domain mutation analysis, based upon the structure solved for TRIM32, reveals alterations that impact not only local protein structure. For example, the R394H mutation located in NHL domain 1 causes backbone perturbations and residue energy changes in NHL domains 3 and 4, thus providing a molecular understanding for the destabilization of the R394H mutant relative to WT NHL by DSC. These results provide evidence that mutations exert debilitating effects on NHL structure and reduce protein levels, further contributing to disease progression (Biwa, 2020).
Similar to LGMD2H patients, the expression of disease-causing alleles (R394H, D487N and 520fs) in Drosophila larval muscle causes muscle degeneration with reduced TRIM32 protein levels and locomotor defects. It has been reported that RNAi-mediated knockdown of TRIM32 disrupts adult myofiber architecture at day 7 and day 11 after tn RNAi induction posteclosion. In contrast, the current genetic assays show that expression of the R394H and 520fs mutations in IFMs cause severe damage to the myofibers by day 1, which continue to degenerate with age. Expression of the D487N mutation also triggered muscle deterioration, but delayed compared with other mutations. It is also clear that TRIM32 protein levels correlate with protein destabilization and muscle degeneration. TRIM32 did not localize in the IFMs of R394H and 520fs mutants, but remained relatively well localized in D487N mutant myofibers until day 3. In all destabilizing mutations, variable accumulation of TRIM32 puncta was observed. The presence of puncta, rather than the complete absence of protein, may indicate that TRIM32 no longer retains its correct sarcomeric association or possibly represents aggregated TRIM32 protein in response to the damage caused by the myopathic mutations. It is believed that the differences in muscle degeneration and TRIM32 expression in different alleles partially explains the variable onset and phenotype severity observed in LGMD2H patients (Biwa, 2020).
Do components of the dystrophin-glycoprotein components (DGC) and integrin adhesion system play a critical role in LGMD2H disease progression? Mutations in the DGC and integrin adhesion complex are associated with muscular dystrophies and cardiomyopathies. Deficiency of δ-sarcoglycan in mammalian skeletal muscle results in the absence of α-, β-, and γ-sarcoglycan, suggesting that sarcoglycan is a core component of sarcoglycan complex assembly. Similarly, integrin adhesion molecules at the costamere are indispensable for the development and maintenance of sarcomeric architecture. Work performed in mice and flies showed that integrins play an important role in Z-disk formation. Previous work revealed that TRIM32 is essential for costamere integrity, whereby βPS integrin, talin, spectrin, vinculin, and Scgδ accumulate abnormally along the sarcolemma (LaBeau-DiMenna, 2012). The current results further support that loss of costamere stability may be involved in disease pathogenesis, as the expression of TRIM32 mutations also phenocopies the mislocalization of βPS integrin and Scgδ. Similar data were obtained upon expression of a catalytically inactive version of TRIM32 in murine myoblasts, extending in mammals the novel role of TRIM32 in promoting the degradation of Drosophila costamere components. Importantly, although TM protein is mislocalized upon loss of TRIM32, it retains its normal localization when pathogenic alleles are expressed in muscle tissue. These results suggest that TRIM32 can regulate the levels of sarcomeric proteins such as TM, but this accumulation is not sufficient to cause muscle degeneration as observed with costamere proteins (Biwa, 2020).
Several studies have shown that upregulation of utrophin or integrin α7 partially compensates for the lack of dystrophin in mdx mice or in human DMD patients. Although the integrin adhesion complex (assayed by βPS integrin) and the DGC complex (assayed by Scgδ) are also up-regulated upon loss of TRIM32, this buildup of costameric proteins at the sarcolemma compromises the attachment between the membrane and myofibrils, leading to muscle degeneration. Some or all of these costamere proteins may be substrates for TRIM32 E3 ligase activity. The abnormal accumulation of βPS integrin and Scgδ along the sarcolemma may result from the inability to be ubiquitinated and turned over by the proteasome and/or the inability to maintain its normal localization due to protein damage during muscle contraction (Biwa, 2020).
There are conflicting data about whether LGMD2H mutations in the NHL domain of TRIM32 inhibit multimer formation or alter ubiquitination activity. It is speculated that disease-causing mutations elevate protein levels of at least a subset of costamere proteins, either by altering protein interactions or by abolishing the catalytic activity of TRIM32. However, additional work is required to characterize the ubiquitination signature by TRIM32 and the resulting fate of substrate proteins. The structural and functional conservation of TRIM32, combined with the muscle-intrinsic Drosophila genetic model, will continue to provide novel insights into LGMD2H initiation and progression not currently available through study in other model organisms (Biwa, 2020).
Myotonic dystrophy is the most common form of adult-onset muscular dystrophy, originating in a CTG repeat expansion in the DMPK gene. The expanded CUG transcript sequesters MBNL1, a key regulator of alternative splicing, leading to the misregulation of numerous pre-mRNAs. This study reports an RNA-targeted agent as a possible lead compound for the treatment of myotonic dystrophy type 1 (DM1) that reveals both the promise and challenges for this type of small-molecule approach. The agent is a potent inhibitor of the MBNL1-rCUG complex with an inhibition constant (Ki ) of 25±8 nm, and is also relatively nontoxic to HeLa cells, able to dissolve nuclear foci, and correct the insulin receptor splicing defect in DM1 model cells. Moreover, treatment with this compound improves two separate disease phenotypes in a Drosophila model of DM1: adult external eye degeneration and larval crawling defect. However, the compound has a relatively low maximum tolerated dose in mice, and its cell uptake may be limited, providing insight into directions for future development (Luu, 2016).
Dialynas, G., Shrestha, O.K., Ponce,
J.M., Zwerger, M., Thiemann, D.A., Young, G.H., Moore, S.A., Yu,
L., Lammerding, J. and Wallrath, L.L. (2015). Myopathic
lamin mutations cause reductive stress and activate the
nrf2/keap-1 pathway. PLoS Genet 11: e1005231. PubMed ID: 25996830
Abstract
Mutations in the human LMNA gene cause muscular
dystrophy by mechanisms that are incompletely understood. The LMNA
gene encodes A-type lamins, intermediate filaments that form a
network underlying the inner nuclear membrane, providing
structural support for the nucleus and organizing the genome. To
better understand the pathogenesis caused by mutant lamins, this
study performed a structural and functional analysis on LMNA
missense mutations identified in muscular dystrophy patients.
These mutations perturb the tertiary structure of the conserved
A-type lamin Ig-fold domain. To identify the effects of these
structural perturbations on lamin function, these mutations were
modeled in Drosophila Lamin C and the mutant
lamins were expressed in muscle. It was found that the structural
perturbations have minimal dominant effects on nuclear stiffness,
suggesting that the muscle pathology is not accompanied by major
structural disruption of the peripheral nuclear lamina. However,
subtle alterations in the lamina network and subnuclear
reorganization of lamins remain possible. Affected muscles have
cytoplasmic aggregation of lamins and additional nuclear envelope
proteins. Transcription profiling revealed upregulation of many
Nrf2 target genes. Nrf2 is normally sequestered in the
cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from
Keap-1, translocates into the nucleus, and activates gene
expression. Unexpectedly, biochemical analyses revealed high
levels of reducing agents, indicative of reductive stress. The
accumulation of cytoplasmic lamin aggregates correlates with
elevated levels of the autophagy adaptor p62/SQSTM1,
which also binds Keap-1, abrogating Nrf2 cytoplasmic
sequestration, allowing Nrf2 nuclear translocation and target gene
activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2
were identified in muscle biopsies from the corresponding muscular
dystrophy patients, validating the disease relevance of the Drosophila
model. Thus, novel connections were made between mutant lamins and
the Nrf2 signaling pathway, suggesting new avenues of therapeutic
intervention that include regulation of protein folding and
metabolism, as well as maintenance of redox homoeostasis
(Dialynas, 2015).
Highlights
- Mutant lamins alter the tertiary structure of the Ig-fold
domain.
- Mutant lamins have minimal dominant effects on nuclear
stiffness.
- Mutant lamins alter muscle gene expression.
- Mutant lamins cause reductive stress.
- Mutant lamins activate the Nrf2/Keap-1 pathway.
Discussion
Structural studies of the lamin Ig-fold demonstrate that single
amino acid substitutions in the loop regions perturb the tertiary
structure, leaving the secondary structure of the folded domain
largely intact. These data are consistent with single-molecule
force spectroscopy showing that the lamin Ig-fold possessing an
R453W substitution requires less force to unfold than the wild
type Ig-fold domain. Structural data of this study are consistent
with in silico modeling in which amino acid substitutions in the
Ig-fold that cause muscular dystrophy were predicted to alter the
structure, more so than those that cause lipodystrophy or
progeria. NMR analysis of the mutant Ig-fold domains identifies
surfaces on opposite sides of the Ig-fold barrel that are critical
for muscle function. This finding predicts that substitution of
other amino acids that comprise these surfaces might result in
muscular dystrophy. Consistent with this prediction, amino acid
substitutions in eight of the 21 amino acids that make up these
surfaces cause muscular dystrophy (Dialynas, 2015).
It is interesting to note that the largest structural
perturbations were observed for the G449V and W514R mutants, which
correspond to the most severe patient phenotypes. The
corresponding amino acid substitutions in Drosophila
Lamin C cause the greatest percentage of lethality. The N456I
mutant shows the least structural perturbations in the Ig-fold
domain, though the relative severity of symptoms in this patient
was not ascertained. Consistent with the structural data, the
corresponding amino acid substitution in Drosophila
Lamin C gives the least percentage of lethality. Thus, these data
show an obvious correlation between the severity of the Ig-fold
structural perturbations and phenotypic severity (Dialynas, 2015).
The structural perturbations within the Ig-fold might generate
novel interaction surfaces that promote lamin aggregation. Both
nuclear and cytoplasmic aggregation of mutant lamins have been
reported, however, they are not commonly observed in human muscle
biopsy tissue or tissue from a laminopathy mouse model.
Cytoplasmic aggregation has been observed for a truncated form of
A-type lamin that causes Hutchinson-Gilford progeria syndrome.
Lamin aggregation is supported by X-ray crystallography studies of
a R482W substitition in the A-type lamin Ig-fold domain that
causes lipodystrophy. The R482W Ig-fold domain possesses unique
interaction surfaces not present in the wild type Ig-fold that
form a unique platform for tetramerization (Dialynas, 2015).
Cytoplasmic protein aggregation has been linked to reductive
stress. This study shows that cytoplasmic lamin aggregation
correlates with elevated levels of both GSH and NADPH, hallmarks
of reductive stress. Elevated levels of isocitrate dehydrogenase
enzyme activity contribute to the additional NADPH. In a similar
manner, dominant negative forms of alphaB-crystallin (CryAB)
result in cytoplasmic CryAB misfolding/aggregation and reductive
stress in the mouse heart, ultimately leading to dilated
cardiomyopathy. These findings suggest that reductive stress might
contribute to the dilated cardiomyopathy in cases of lamin
associated muscular dystrophy. Interestingly, mutations in the
human CRYAB gene cause disease phenotypes that are
strikingly similar to those observed for lamin associated muscular
dystrophy, including skeletal muscle weakness and dilated
cardiomyopathy in cases of lamin-associated muscular dystrophy. It
is worthwhile to note that CryAB functions as a chaperone to
prevent aggregation of intermediate filament proteins such as
desmin, suggesting a common link between intermediate filament
aggregation and reductive stress (Dialynas, 2015).
An imbalance in redox homeostasis can provide an environment that
promotes protein misfolding and aggregation. The redox state
influences aggregation of lamins; aggregation has been observed
under both oxidative and reductive conditions. In fact, the
formation of the novel tetramer generated by the R482W mutant
Ig-fold domain requires a reductive environment. Reductive stress
has also been observed in healthy individuals predisposed to
Alzheimer disease, a disease of protein aggregation. Alzheimer
disease is typically accompanied by oxidative stress, however,
lymphocytes from patients carrying an ApoE4 allele that
predisposes them to Alzheimer disease show reductive stress. It is
hypothesized that continual activation of antioxidant defense
systems, such as Nrf2/Keap-1 signaling, becomes exhausted over
time, particularly later in life, resulting in the inability to
properly defend against oxidative stress. This study analyzed the
redox status in Drosophila muscle 24–48 hours post
expression of the mutant lamins; their findings suggest reductive
stress at the onset of pathology that could resolve into oxidative
stress later in disease progression (Dialynas, 2015).
Typically lamins are thought to regulate gene expression from
inside the nucleus, by interacting with transcription factors and
organizing the genome. Data from this study support a novel model
in which genes are misregulated as a consequence of mutant lamin
aggregation in the cytoplasm. Cytoplasmic lamin aggregates have
been found in high molecular weight complexes in cases of liver
injury. Such complexes contain nuclear pore proteins, signaling
mediators, transcription factors and ribosomal proteins, which are
thought to disrupt the normal cellular physiology. Lamin
aggregation might also serve a cytoprotective function by
facilitating the coalescence of mutant lamin so that the
contractile apparatus can properly function. A similar mechanism
exists in Huntington’s disease, where sequestration of
mutant huntingtin in inclusion bodies correlates with better
neuron health (Dialynas, 2015).
Collectively, findings from this study continue to support this Drosophila
model of laminopathies, as many of the phenotypes discovered in Drosophila
have been validated in human muscle biopsies. It is now possible
to use this rapid genetic model to (1) determine if mutations in
other domains of lamin produce similar phenotypes and (2) if lamin
mutations have similar effects in other tissues, such as the
heart. Data suggest that cytoplasmic lamin aggregation contributes
to muscle pathology. Consistent with this idea, increased rates of
autophagy suppress phenotypes caused by mutant A-type lamin in
cultured cells and mouse models. Furthermore, electron microscopy
of skeletal muscle biopsies from patients with LMNA
mutations show large perinuclear autophagosomes, similar to the
localization of lamin aggregates and p62 foci in the Drosophila
muscle. Thus, the regulation of autophagy, a process that removes
both damaged organelles and proteins, might be central to the
development of therapies. The Drosophila model will
allow for genetic dissection of both the autophagy and reductive
stress pathways to identify the key factors responsible for the
muscle pathogenesis and its suppression (Dialynas, 2015).
Go to top
Chartier, A., Klein, P., Pierson, S.,
Barbezier, N., Gidaro, T., Casas, F., Carberry, S., Dowling, P.,
Maynadier, L., Bellec, M., Oloko, M., Jardel, C., Moritz, B.,
Dickson, G., Mouly, V., Ohlendieck, K., Butler-Browne, G.,
Trollet, C. and Simonelig, M. (2015). Mitochondrial
dysfunction reveals the role of mRNA poly(A) tail regulation in
oculopharyngeal muscular dystrophy pathogenesis. PLoS Genet 11:
e1005092. PubMed ID: 25816335
Abstract
Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder
characterized by progressive degeneration of specific muscles,
results from the extension of a polyalanine tract in poly(A)
binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in
nuclear polyadenylation and regulation of alternative poly(A) site
choice are established, the molecular mechanisms behind OPMD
remain undetermined. This study shows, using Drosophila
and mouse models, that OPMD pathogenesis depends on affected
poly(A) tail lengths of specific mRNAs. The study identifies a set
of mRNAs encoding mitochondrial proteins that are down-regulated
starting at the earliest stages of OPMD progression. The
down-regulation of these mRNAs correlates with their shortened
poly(A) tails and partial rescue of their levels when
deadenylation is genetically reduced improves muscle function.
Genetic analysis of candidate genes encoding RNA binding proteins
using the Drosophila OPMD model uncovers a potential
role of a number of them. The study focuses on the deadenylation
regulator Smaug and shows that it is expressed in adult muscles
and specifically binds to the down-regulated mRNAs. In addition,
the first step of the cleavage and polyadenylation reaction, mRNA
cleavage, is affected in muscles expressing alanine-expanded
PABPN1. The study proposes that impaired cleavage during nuclear
cleavage/polyadenylation is an early defect in OPMD. This defect
followed by active deadenylation of specific mRNAs, involving
Smaug and the CCR4-NOT deadenylation complex, leads to their
destabilization and mitochondrial dysfunction. These results
broaden the understanding of the role of mRNA regulation in
pathologies and might help to understand the molecular mechanisms
underlying neurodegenerative disorders that involve mitochondrial
dysfunction (Chartier, 2015).
Highlights
- Genes encoding mitochondrial proteins are down-regulated in Drosophila
muscles expressing PABPN1-17ala.
- Defects in mitochondrial function play an important role in
OPMD physiopathology in the Drosophila model.
- Role of mRNA poly(A) tail length regulation in the defects
induced by PABPN1-17ala expression.
- Affected nuclear cleavage/polyadenylation in the Drosophila
model of OPMD.
- Smg regulates mRNAs down-regulated in muscles expressing
PABPN1-17ala.
- Rescue of PABPN1-17ala-induced phenotypes by reducing
deadenylation does not involve decreased PABPN1 aggregation.
- Early down-regulation of mRNAs encoding mitochondrial proteins
is conserved in the OPMD mouse model.
- Mitochondrial protein levels are decreased in OPMD patient
muscles.
Discussion
The molecular defects underlying OPMD pathology remain largely
undetermined, although recent advances have implicated apoptosis
and a general deregulation of the ubiquitin-proteasome system.
However, these are downstream events in the pathogenesis. This
study investigates the molecular mechanisms involved by analysing
early defects in the disease. It was shown that specific mRNAs
encoding proteins involved in mitochondrial activity are present
at lower levels in pre-symptomatic OPMD muscles; this reduced
expression results from the shortening of their poly(A) tails
which leads to their destabilization. Poly(A) tail length
regulation plays a key role in OPMD since muscle function is
improved when deadenylation is decreased using mutants. It was
further shown that nuclear cleavage/polyadenylation of pre-mRNAs
is inefficient in PABPN1-17ala-expressing muscles. This defect
occurs both on genes which are and on those which are not
down-regulated, indicating that it does not per se systematically
lead to reduced steady-state mRNA levels. The decreased levels of
specific mRNAs result from their active deadenylation, itself
dependent, at least in part, on the specific interaction of these
mRNAs with the Smg RNA binding protein and the recruitment by Smg
of the CCR4-NOT deadenylation complex. In addition, genetic data
revealing the potential involvement of other RNA binding proteins
suggest that OPMD pathogenesis is complex and probably involves
additional mechanisms of RNA regulation (Chartier, 2015).
The function of PABPN1 during cleavage/polyadenylation has been
documented. Nuclear polyadenylation occurs in two steps: first,
cleavage of the pre-mRNA at the poly(A) site, which is
co-transcriptional, and second, polyadenylation which potentially
occurs after dissociation of the RNA from the RNA polymerase II.
PABPN1 has been shown to be involved in the second step,
polyadenylation, for the control of poly(A) tail lengths. More
recent data have also implicated PABPN1 in the cleavage step for
the regulation of weak poly(A) sites. This study shows impaired
cleavage at poly(A) sites in the Pabp2 loss-of-function
mutant, revealing a more general role of PABP2/PABPN1 in this step
of the reaction. In the regulation of weak poly(A) sites, PABPN1
binds to non-canonical polyadenylation signals and prevents the
binding of CPSF (Cleavage and polyadenylation specificity factor)
required for cleavage. A general function of PABPN1 in cleavage
would require other interactions, for example with proteins
required for cleavage, such as the poly(A) polymerase known to
associate with PABPN1. Although a global shift to proximal poly(A)
sites has been reported in the mouse model of OPMD, this study
shows that the down-regulation and poly(A) tail shortening of
specific mRNAs participating in OPMD pathogenesis are independent
of alternative poly(A) site utilization (Chartier, 2015).
The molecular defects observed in PABPN1-17ala-expressing
muscles, reduction of mRNA poly(A) tail length and decreased
efficiency of cleavage at poly(A) sites are similar to those
observed in Pabp2 loss-of-function mutants. This
suggests that part of the defects in OPMD could result from
partial PABPN1 loss-of-function. However, the genetic
suppression of wing posture phenotypes by reducing the dosage of Pabp2
does not favour a simple loss-of-function model. It has been
proposed for polyglutamine diseases that the pathogenesis could
result from both the gain-of-function and the loss-of-function of
the same protein. The protein responsible for the disease would
exist in two different conformations with two different yet normal
functions. Extension of the polyglutamine tract would favor one
conformer resulting in increased amounts of this conformer and the
partial loss-of-function of the other conformer; the pathology
would result from both these effects. In this model, the mutant
protein would have the same function as the normal protein but
would have the ability to alter the balance between both protein
forms. Several properties of PABPN1 are consistent with this model
for OPMD. It has been shown previously that the normal function of
PABPN1 and more specifically its RNA binding activity is required
for OPMD-like defects in the Drosophila model. In
addition, PABPN1-17ala half-live has been reported to be longer
than that of PABPN1 in cell models, leading to higher accumulation
of PABPN1-17ala and protein aggregation. Thus, expansion of the
polyalanine tract results in protein "overexpression" which
contributes to the pathology. Given this data, overepression of
the normal protein might be expected to induce similar defects as
expression of the mutant protein, as it is the case in Drosophila
models for other disorders. Consistent with this, it has been
previously reported that PABPN1 expression in Drosophila
muscles induces wing posture defects, although at lower levels
than PABPN1-17ala expression. Finally, normal PABPN1 is also known
to form oligomers during nuclear polyadenylation and can form
nuclear aggregates that recruit ubiquitin and proteasomes under
specific physiological conditions (Chartier, 2015).
Because the presence of nuclear aggregates and muscle defects can
to some extent be uncoupled, it has been previously proposed that
nuclear aggregates are not always pathological. This is consistent
with results concerning polyglutamine diseases where aggregates
can have a protective role. It was found that the improvement of
muscle function when deadenylation is genetically reduced
correlates with an increased number of PABPN1 aggregates, again
strengthening the notion that the aggregates are not always
causative of muscle defects. In that case, muscle protection that
results from the reduction of molecular defects could allow the
formation of more PABPN1 aggregates. Thus, these aggregates might
not always be pathological, in particular during early stages of
the disease, although they might become so at later stages, when
their increased size could interfere with nuclear function
(Chartier, 2015).
A major conclusion from this study is that the specificity of the
defect in OPMD does not depend per se on PABPN1 defect in pre-mRNA
cleavage, but on Smg-dependent regulation occurring in the
cytoplasm. Because of the shift to proximal poly(A) sites that
correlates with mRNA up-regulation, described in the OPMD mouse
model, this study analyzed whether a similar mechanism could lead
to increased Smg levels in Drosophila muscles expressing
PABPN1-17ala and underlie increased deadenylation. However, the
same poly(A) site was used in normal and PABPN1-17ala-expressing
muscles, and a major deregulation of smg mRNA and
protein levels in PABPN1-17ala-expressing muscles was not found.
The study proposes that normal Smg-dependent deadenylation,
following inefficient pre-mRNA cleavage, could lead to the reduced
levels of specific mRNAs that were observed. In addition, other
processes such as mRNP remodelling could contribute to enhanced
mRNA decay in the course of the disease progression. Indeed, Smg
forms cytoplasmic foci which are distinct, but related to other
cytoplasmic RNA granules such as processing (P) bodies or stress
granules, in which mRNAs are degraded or translationally
repressed, and the regulation of which affects mRNA regulation. A
recent study also revealed the implication of Smg/SAMD4A in
Myotonic Dystrophy Type 1 (DM1). In that case, Smg mechanism of
action appeared to be different, since overexpression of Smg
decreased DM1 muscle defects by reducing unproductive
CUGBP1-eIF2α translational complexes (Chartier, 2015).
Mitochondrial dysfunction has been shown to play a major role in
most neurodegenerative diseases including Parkinson's,
Alzheimer's, Huntington's and other polyglutamine diseases. More
recent data have uncovered that aside mitochondrial function in
energy production, mitochondrial dynamics including trafficking
and quality control is instrumental in pathogenesis. Mitochondria
also have a key role in muscle function. Drosophila
mutants of pink1 and parkin, mutations of
which cause Parkinson's disease in humans, lead to mitochondrial
dysfunction and flight muscle degeneration. It was shown that
mitochondrial dysfunction is also an important component of OPMD:
Muscle function is improved when mitochondrial biogenesis and
activity are genetically increased; in addition, mitochondrial
proteins are down-regulated in OPMD muscle biopsies from patients.
The molecular defects leading to early mitochondrial dysfunction
in OPMD were identified: mRNAs encoding mitochondrial proteins are
down-regulated due to their Smg-dependent deadenylation.
Therefore, this study reveals Smg as a regulator of mRNAs involved
in mitochondrial function. This finding might have important
implications on the role of Smg in several neurodegenerative
diseases that involve mitochondrial dysfunction and/or RNA
toxicity (Chartier, 2015).
Go to top
Picchio, L., Plantie, E., Renaud, Y.,
Poovthumkadavil, P. and Jagla, K. (2013). Novel Drosophila
model of myotonic dystrophy type 1: phenotypic characterization
and genome-wide view of altered gene expression. Hum Mol Genet 22:
2795-2810. PubMed ID: 23525904
Abstract
Myotonic dystrophy type 1 (DM1) is a multisystemic RNA-dominant
disorder characterized by myotonia and muscle degeneration. In DM1
patients, the mutant DMPK transcripts containing
expanded CUG repeats form nuclear foci and sequester the
Muscleblind-like 1 splicing factor, resulting in mis-splicing of
its targets. However, several pathological defects observed in DM1
and their link with disease progression remain poorly understood.
In an attempt to fill this gap, this study generated inducible
transgenic Drosophila lines with increasing number of
CTG repeats. Targeting the expression of these repeats to the
larval muscles recapitulates in a repeat-size-dependent manner the
major DM1 symptoms such as muscle hypercontraction, splitting of
muscle fibers, reduced fiber size or myoblast fusion defects.
Comparative transcriptional profiling performed on the generated
DM1 lines and on the muscleblind (mbl)-RNAi
line revealed that nuclear accumulation of toxic CUG repeats can
affect gene expression independently of splicing or Mbl
sequestration. Also, in mblRNAi contexts, the largest
portion of deregulated genes corresponds to single-transcript
genes, revealing an unexpected impact of the indirect influence of
mbl on gene expression. Among the single-transcript Mbl
targets is Muscle protein 20 involved in myoblast fusion and
causing the reduced number of nuclei in muscles of mblRNAi
larvae. Finally, by combining in silico prediction of Mbl targetsbwith mblRNAi microarray data, the calcium pump dSERCA
was found to be a Mbl splice target and it was shown that the
membrane dSERCA isoform is sufficient to rescue a DM1-induced
hypercontraction phenotype in a Drosophila model
(Picchio, 2013).
Highlights
- Expanded CUG repeats accumulate in nuclear foci and sequester
Mbl in a new Drosophila larval model of DM1.
- Reduced motility and affected muscle morphology of DM1 larvae.
- Impaired muscle relaxation in a Drosophila larval
model of DM1.
- Transcriptional profiling of DM1 CTG-repeat lines and mblRNAi
line reveals splice-independent gene deregulation.
- Mp20, an indirect Mbl target involved in DM1-associated
myoblast fusion defects.
- In silico prediction of Mbl targets and microarray analyses
identify transmembrane isoform of the Drosophila sarco
endoplasmic reticulum calcium-ATPase (dSERCA) as required for
proper muscle contraction in DM1.
Discussion
Drosophila has already proved to be a powerful tool for
conducting genetic screening and global analyses on the effect of
CTG repeats in DM1. So far, an inducible line expressing 480
interrupted CTG repeats has been used at the adult stage and shows
age-dependent muscle degeneration. This study generated a set of
three inducible site-specific lines expressing 240, 600 or 960
interrupted CTG repeats. In some cases of DM1, the existence of
variant repeats interrupting the pure CTG expansions have been
observed. Interestingly, interruptions allow either repeat
stabilization or repeats contraction. If in one peculiar DM1
family CCG and CGG variants are associated with
Charcot-Marie-Tooth symptoms, the role of interruptions remains
unclear in other patients. The interrupted CTG repeats have
already been used to generate different animal models of DM1. One
could consider a possibility of additive toxic effect in all these
models. However, the CTCGA interruption motif commonly used for
these models is different from already described variants and its
toxicity as well as unstability have not been not reported so far
(Picchio, 2013).
This study used CTG size variation to compensate for age effect
in third instar larvae. The study assessed larval muscles instead
of adult muscle for three reasons: (i) segmentally repeated larval
musculature is organized in a stereotyped network of muscle fibers
and is easy to analyze at morphological and functional levels,
(ii) establishing and characterizing larval DM1 model appears
attractive for future genetic rescue approaches and molecular
screening applications and (iii) adult lethality of the Mef>mblRNAi
line prevents comparative analyses with DM1 lines in adult flies
(Picchio, 2013).
As observed in patients, it was found that expressing an
increasing number of CTG repeats in larval somatic muscles leadd
to the formation of nuclear foci and that these foci co-localize
with Drosophila MBNL1 ortholog, Mbl. As the number of
repeats positively influences the number of foci per nucleus and
worsen muscle phenotypes, the new Drosophila model of DM1
presented here could be of interest for simulating disease
progression and (or) severity (Picchio, 2013).
Global analysis of muscle pattern in this model reveals a
histopathological defect called ‘splitting fibers’
already observed in mbnl1 knockout mice as well as in
DM1 patients. Here, splitting occurs during larval stages
characterized by rapid muscle growth. As observed in dorsal
oblique fibers, it is initiated at muscle endings at the level of
interaction with tendon cells. This suggests that splitting
results from affected muscle attachment to tendon cells and (or)
abnormal sarcomeric organization that weaken the integrity of
myofibrils. This latter hypothesis is supported by decreased
expression of two sarcomere components (Mhc, up) in the DM1960
line. Surprisingly, the Mef>240CTG line which doesn
not exhibit visible foci within muscle nuclei displays altered
motility associated with muscle splitting but no fiber defects.
This observation suggests that splitting is sufficient to alter
motility (Picchio, 2013).
Also, SBM and VL3 fiber examination shows reduced muscle size. So
far, it has been shown in primary cell culture of myoblasts from
DM1 patients that the ability of DM1-derived myoblasts to fuse is
affected, consequently reducing myotube length. This study reports
that expressing non-coding CTG repeats affects in vivo myoblast
fusion. Interestingly, microarray data and RT–qPCR performed
at embryonic and larval stages on mutant lines have shown
decreased expression of Mp20 encoding an actin-binding protein
involved in Drosophila myoblast fusion. Mp20 appears as an
attractive candidate gene for myoblast fusion defects in DM1,
since by overexpressing Mp20 during myogenesis, the number of
nuclei per fiber was rescued in DM1 lines and in the mbl
attenuated line. Surprisingly, Mp20 does not appear to undergo
alternative splicing (single-transcript gene according to
Flybase), suggesting that its Mbl-dependent down-regulation could
occur through an indirect effect of Mbl. It is also noteworthy
that one human counterpart of Mp20, the Calponin 3
gene, has been found to be involved in myoblast fusion in vitro.
Thus, genes of the Mp20/Calponin family appear as
attractive candidates to be tested for their role in DM1 muscle
defects in humans (Picchio, 2013).
Finally, this study reports that mutant larvae and in particular
those from DM1960 and mbl attenuated lines display
altered motility with affected complex movements. Interestingly,
by measuring the contractility index and sarcomere size, it was
found that both the lines exhibit hypercontraction, a phenotype
related to myotonia. It has been previously shown that mbnl1
disruption in the mouse also leads to myotonia. In the Drosophila
DM1 model, the effect of CTG repeat size on the severity of
myotonia was observed, so that the DM1600 line exhibits
intermediate hypercontraction phenotypes when compared with DM1240
and DM1960. As not only hypercontraction but also affected
myoblast fusion account for a reduced muscle size in pathological
lines, this study assume that both parameters need to be repaired
to fully rescue muscle length (Picchio, 2013).
It was also found that the severity of several phenotypes is
positively correlated with the size of the CTG repeats. This was
followed by comparative transcriptional profiling on DM1600 and
DM1960 lines. First, during validation of selected candidate genes
from microarray analyses, repeat-size-dependent deregulation of
genes involved in carbohydrate and nitrogen metabolism was
observed. A more systematic classification of candidates
deregulated in a repeat-size-dependent manner and having human
orthologs was then performed based on the ratio of their
fold-change between the two conditions and on their function. Data
reveals that genes encoding transporter proteins are significantly
enriched among gene categories down-regulated in larvae carrying
high repeat numbers (Picchio, 2013).
Among these, repeat-size-dependent deregulation of smvt,
whose human orthologs (SLC5A3, SLC5A5, SLC5A8 and SLC5A12)
encode myo-inositol transporters, and CG17597/SCP-2,
involved in phosphatidylinositol transfer and signaling, was
validated. It is known that phosphatidylinositol is a derivative
of myo-inositol, suggesting that both transporters may work in the
same pathway. However, how the alterations of transporters
influence the accumulation of the inositol forms and how this is
connected to muscle defects remain to be investigated. It was also
found that two genes involved in the sarcomere structure Mhc
and up were both down-regulated specifically in the
DM1960 context. In DM1 patients, it has been shown that Mhc
ortholog MYH14 and up orthologs TNNT2
and TNNT3 are mis-spliced. Besides, another report
provides evidence that in a Drosophila mbl
null mutant, up transcripts are mis-spliced as well.
However, the link between the Mhc and up gene
deregulations and DM1 muscle phenotypes and their impact on DM1
pathogenesis have not yet been investigated. This study speculate
that down-regulation of Mhc and up might be
involved in splitting fiber phenotype observed in DM1 larvae
(Picchio, 2013).
Comparative genomic analyses shows that a high percentage of
genes with misregulated expression (∼70%) do not undergo
alternative splicing but are sorted out in the Mef>mblRNAi
context. As Mbl binds specifically to double-stranded RNA
structures, the study hypothesized that it may influence
transcript stability of this class of genes as already observed
with MBNL1 in C2C12 cells. Alternatively, Mbl might play an
indirect role on single-transcript genes via mis-splicing of
transcription factors that regulate their expression. In order to
gain insights into the second hypothesis, potential common
regulators of Mbl-deregulated single-transcript genes were
identified using the bioinformatics i-cisTarget approach.
Interestingly, several transcription factors known to act in
musclessuch as dMef2 and GATA factor Panier (Pnr) were found as
potential transcriptional regulators of candidate genes. More
importantly, the same transcription factors were found deregulated
in transcriptional profiling experiments under all pathological
conditions and most of them (including dMef2 and Pnr) were also
predicted in silico to be targets of Mbl. Thus, these data reveal
an important contribution of single-transcript gene deregulation
in the Drosophila DM1 model and point to an indirect
role of Mbl in the regulation of gene expression via mis-splicing
of key myogenic factors. As a matter of fact, this mechanism may
play a role in the regulation of Mp20 expression, one of
dMef2 targets. Interestingly, both qPCR and microarray
experiments showed that Mp20 expression is down-regulated in
pathological contexts leading to myoblast fusion defects.
Consequently, the study suggests an indirect role of Mbl in Mp20
expression through misregulation of dMef2 alternative
splicing (Picchio, 2013).
In DM1960 larvae, an Mbl-dependent muscle hypercontraction
phenotype related to myotonia was observed. By associating
microarray data with in silico prediction of Mbl direct targets,
the dSERCA gene was identified as a putative candidate
for Mbl-driven mis-splicing and hypercontraction phenotype. By
RT–qPCR, it was confirmed that the isoforms B-H of dSERCA
containing exons 8 or 11 encoding the transmembrane domain show
decreased expression in mbl attenuated and in DM1960
lines. This indicates that in the Mbl-deficient context, the exons
8 or 11 of dSERCA are spliced out, leading to the
production of dSERCA isoforms devoid of the transmembrane domain.
This switch in dSERCA isoforms is consistent with the
immunostaining of DM1 larval muscles, in which the
membrane-associated dSERCA protein is barely detectable at muscle
surface or even in sarcomere for the Mef>mblRNAi
line, whereas the level of free dSERCA in nuclei appears to be
enhanced in DM1 lines. It has been previously shown that in DM1
patient muscles, as a result of MBNL1 sequestration, SERCA1 exon
22 in the 3′ part of the transcript is excluded leading to
the formation of a neonatal isoform of SERCA1. This isoform is
expected to cause muscle degeneration but so far, no functional
analysis has been performed to confirm this hypothesis. However,
patients with Brody's disease, which is caused by different
mutations in the SERCA1a gene, manifest impairment of
skeletal muscle relaxation among other symptoms. In addition, it
has been shown that dSERCA plays a key role in muscle contraction
and heartbeat frequency and rythmicity in flies, suggesting that
it might be involved in muscle hypercontraction phenotypes and
myotonia in DM1 muscles (Picchio, 2013).
To date, the only gene functionally implicated in myotonia in DM1
is the CIC-1 encoding a muscle-specific chloride
channel. CIC-1 transcripts have been found to undergo MBNL1- and
CUGBP1-dependent splice modifications causing muscle delayed
relaxation and pathogenic muscle defects. However, analyses
performed on HSA(LR) myotonic mice reveals that ClC-1
channels account for muscle hyperexcitability in young but not in
old DM1 animals, suggesting alteration of conductance other than
chloride currents. Consequently, this study tested if the loss of
dSERCA function and in particular depletion in its isoforms
carrying the transmembrane domain could indeed affect muscle
contractility. By using a pharmacological tool, CPA, a highly
specific inhibitor of SERCA, which binds to the entry channel, it
was found that the contractility of CPA-treated larval muscles is
severely affected. Next, by performing rescue experiments by
overexpressing the transmembrane isoform of dSERCA in
DM1 lines with hypercontracted phenotypes, it was found that the
contractility index is significantly improved. Thus, these data
provide the first evidence in an animal model of DM1 that SERCA
mis-splicing is involved in muscle hypercontraction (Picchio,
2013).
Go to top
Pantoja, M., Fischer, K.A.,
Ieronimakis, N., Reyes, M. and Ruohola-Baker, H. (2013).
Genetic elevation of sphingosine 1-phosphate suppresses dystrophic
muscle phenotypes in Drosophila. Development 140:
136-146. PubMed ID: 23154413
Abstract
Duchenne muscular dystrophy is a lethal genetic disease
characterized by the loss of muscle integrity and function over
time. Using Drosophila, this study shows that dystrophic
muscle phenotypes can be significantly suppressed by a reduction
of wunen, a homolog of lipid phosphate phosphatase 3,
which in higher animals can dephosphorylate a range of
phospholipids. Suppression analyses include assessing the
localization of Projectin protein, a titin homolog, in sarcomeres
as well as muscle morphology and functional movement assays. The
study hypothesizes that wunen-based suppression is
through the elevation of the bioactive lipid Sphingosine
1-phosphate (S1P), which promotes cell proliferation and
differentiation in many tissues, including muscle. The role of S1P
in suppression is confirmed by genetically altering S1P levels via
reduction of S1P
lyase (Sply) and by upregulating the serine
palmitoyl-CoA transferase catalytic subunit gene lace,
the first gene in the de novo sphingolipid biosynthetic pathway
and these manipulations also reduce muscle degeneration.
Furthermore, it was shown that reduction of spinster
(which encodes a major facilitator family transporter, homologs of
which in higher animals have been shown to transport S1P) can also
suppress dystrophic muscle degeneration. Finally, administration
to adult flies of pharmacological agents reported to elevate S1P
signaling significantly suppresses dystrophic muscle phenotypes.
These data suggest that localized intracellular S1P elevation
promotes the suppression of muscle wasting in flies (Pantoja,
2013).
Highlights
- Establishment of a dystrophic myofibril phenotype.
- Suppression of dystrophic muscle phenotypes by genetically
reducing wunen.
- Suppression of dystrophic muscle phenotypes by genetically
increasing S1P levels.
- Suppression of the dystrophic myofibril phenotype by
genetically reducing spinster, a putative S1P
transporter.
- Suppression of dystrophic muscle function phenotypes as
assessed by fly movement assays.
- Suppression of dystrophic muscle phenotypes using
pharmacological agents that increase S1P levels or S1P
signaling.
Discussion
This study established an easy-to-score myofibril phenotype for
dystrophic flies that can even be scored in relatively young flies
in strong Dystrophin mutants. This assay complements the
histological sections used previously to score gross morphological
muscle degeneration. In using both assays, it was shown that a
reduction of the LPP3 homolog wunen prevents, to a
significant degree, Dystrophin-dependent muscle wasting. This is
likely to occur through the increase of S1P levels as other
avenues used to raise the level of S1P phenocopy this suppression.
Both reduction of Sply, encoding the Drosophila
S1P lyase, and overexpression of lace, encoding the
catalytic subunit of serine palmitoyl CoA transferase in the de
novo sphingolipid synthesis pathway, prevent muscle wasting. It
was also shown that muscle function as assayed by fly movement is
also improved in dystrophic animals when S1P is genetically
elevated. Furthermore, S1P-based suppression is not occurring
during development as feeding experiments utilizing adult flies
revealed that S1P elevation in these animals can suppress
dystrophic myofibril and activity phenotypes (Pantoja, 2013).
it was also found that minimal levels of S1P are necessary for
viability in Drosophila as global reduction of
Sphingosine Kinase 2 (SK2) results in lethality. Interestingly,
global reduction of Sphingosine Kinase 1 (SK1) is not lethal in
non-dystrophic flies yet is lethal in dystrophic flies, owing to
exacerbation of the phenotype. These data indicate that S1P levels
regulated by SK1 are crucial for Dystrophin mutant
survival. These data argue for a precise requirement of minimal
levels of S1P for muscle development and/or function. Future
analyses of the activity of both sphingosine kinases in different
tissues and cellular compartments might separate the roles of each
kinase in homeostasis and muscle (Pantoja, 2013).
In mammals, there are five S1P receptors that share homology with
G protein-coupled receptors. Though flies do have G
protein-coupled receptors, they do not appear to have the S1P
receptors seen in vertebrates, suggesting that S1P
receptor-mediated signaling might have evolved later in higher
organisms. S1P lyase mutants increase intracellular S1P levels and
S1P is generated and has been shown to function inside the cells,
indicating that the suppression of muscle wasting in Drosophila
occurs intracellularly. With this in mind, this study hypothesized
that if spinster, like its mammalian homolog spns2,
is an S1P transporter, its reduction would prevent S1P from
leaving the cytoplasmic compartment and it would then behave like
reduced Sply and suppress muscle wasting. Data support
this hypothesis yet more work is required to connect this
transporter to S1P in Drosophila. Interestingly, Drosophila
spinster has been reported to interact with genes of the
cell death pathway and it is known that ceramide in the
sphingolipid pathway can induce cell death. Perhaps spinster
alters the cell death pathway by perturbing the equilibrium of
sphingolipids, particularly S1P, in different subcellular
compartments. Another report has revealed S1P epigenetic
regulation of gene expression through direct intracellular
interaction with histone deacetylases (HDACs). Through this
mechanism, perhaps increasing intracellular S1P levels alters gene
expression, which ultimately leads to elevated translation of
muscle proteins, such as Projectin, which then reduces muscle
wasting (Pantoja, 2013).
S1P has been shown to be necessary for the proliferation of
satellite cells in mammals and is required for differentiation of
myoblasts to myotubes. As there do not appear to be canonical
satellite cells in Drosophila, i.e. muscle precursor
cells located on the surface of muscle fibers, perhaps S1P-based
suppression of muscle wasting occurs as a result of the
requirement of S1P for proper differentiation. It has been
reported in Drosophila that sarcomeres are formed by an
assembly of latent protein complexes. It would be consistent with
this if S1P elevates muscle protein synthesis (in turn increasing
the level of latent protein complexes) so that after muscle
contraction-induced damage these complexes can assemble and
produce new myofibrils. Data from this study on small molecule
effectors of S1P signaling indicate that the above mentioned
possibilities for suppression occur in actively contracting adult
muscle. S1P-based suppression of muscle wasting can be dissected
further in Drosophila with the abundance of genetic
tools available. Furthermore, given the observations with THI, THI
oxime and FTY720, Drosophila may be used to screen small
molecules for their efficacy in suppressing muscle wasting
(Pantoja, 2013).
Go to top
Fernandez-Costa, J.M., Garcia-Lopez,
A., Zuñiga, S., Fernandez-Pedrosa, V., Felipo-Benavent,
A., Mata, M., Jaka, O., Aiastui, A., Hernandez-Torres, F.,
Aguado, B., Perez-Alonso, M., Vilchez, J.J., Lopez de Munain, A.
and Artero, R.D. (2013). Expanded CTG repeats trigger
miRNA alterations in Drosophila that are conserved in
myotonic dystrophy type 1 patients. Hum Mol Genet 22: 704-716.
PubMed ID: 23139243
Abstract
Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG
repeats in the 3′ untranslated region of the DMPK
gene. Several missplicing events and transcriptional alterations
have been described in DM1 patients. A large number of these
defects have been reproduced in animal models expressing CTG
repeats alone. Earlier studies have also reported miRNA
dysregulation in DM1 patients. This study uses a Drosophila
model to investigate miRNA transcriptome alterations in the
muscle, specifically triggered by CTG expansions. Twenty miRNAs
were found to be differentially expressed in CTG-expressing flies.
Of these, 19 are down-regulated, whereas 1 is up-regulated. This
trend was confirmed for those miRNAs conserved between Drosophila
and humans (miR-1,
miR-7
and miR-10)
in muscle biopsies from DM1 patients. Consistently, at least seven
target transcripts of these miRNAs are up-regulated in DM1
skeletal muscles. The mechanisms involved in dysregulation of miR-7
include a reduction of its primary precursor both in
CTG-expressing flies and in DM1 patients. Additionally, a
regulatory role for Muscleblind (Mbl) is also suggested for miR-1
and miR-7, as these miRNAs are down-regulated in flies
where Mbl has been silenced. Finally, the physiological
relevance of miRNA dysregulation is demonstrated for miR-10,
since over-expression of this miRNA in Drosophila
extends the lifespan of CTG-expressing flies. Taken together,
these results contribute to our understanding of the origin and
the role of miRNA alterations in DM1 (Fernandez-Costa, 2013).
Highlights
- Expression of expanded CTG repeats in Drosophila
causes reduction of defined miRNAs.
- CTG-induced down-regulation of miR-1, miR-7
and miR-10 is conserved between Drosophila
and DM1 patients.
- Expanded CTG repeats alter the levels of defined pri-miRNA
precursors.
- Muscleblind is necessary for the regulation of miR-1
and miR-7 in Drosophila.
- Over-expression of miR-10, but not miR-1
or miR-7, increases lifespan in CTG-expressing flies.
Discussion
Dysregulation of specific miRNAs in DM1 patients had been
previously described. This study assessed the contribution of CTG
expansions to miRNA defects in DM1, by analyzing changes in the
muscle miRNome of a Drosophila model of CTG toxicity
(2). Using SOLiD™ 3 sequencing, 20 miRNA alterations caused
by expression of CTG repeats were identified. Of these, 19 are
specifically down-regulated in the Drosophila model,
whereas only 1 is up-regulated. Therefore, the alterations on
miRNA regulation caused by CTG expression seem to trigger a
reduction, rather than an increase, of miRNA expression levels.
This effect is also observed in DM1 patients for all altered
miRNAs that are conserved between Drosophila and humans:
miR-1, an miRNA previously associated with DM1; and miR-7
and miR-10a, for which no previous link had been
described (Fernandez-Costa, 2013).
Importantly, the conservation of miR-1, miR-7
and miR-10 defects between the fly model and DM1
patients confirms that: (i) the miRNA down-regulation found in Drosophila
is specific, and not the consequence of a reduced contribution of
the muscle transcriptome to the total transcriptome; and (ii) the
dysregulation of these three miRNAs occurs in the presence of
CUG-repeat transcripts devoid of additional DMPK sequences.
Although it is possible that other coding or non-coding regions
within the DMPK gene contribute to miRNA defects in DM1,
this is the first demonstration that CTG expansions are directly
linked to alterations in miRNA regulation. Of note, the fly model
used in this work contains 480 CTG repeats interrupted every 20
units by the CTCGA sequence: i(CTG)480. The i(CUG)480 RNA is
predicted to form a double-stranded structure that closely
resembles the hairpin formed by 480 pure repeats, both of them
having similar folding energies. The existence of complex repeat
interruptions at the DM1 locus has been reported to attenuate the
severity of symptoms in patients. Although the CTCGA interruption
in the i(CTG)480 transgene does not resemble any of these variant
repeat alleles, it is possible that its presence might also modify
CTG-induced phenotypes in the flies. For example, the CUCGA
interruption would determine the length of any putative
repeat-associated non-ATG (RAN) translation products, should these
be generated in Drosophila, as i(CAG)480 transcripts
would produce polyS, polyA and polyQ peptides in consecutive
tracts of 20 amino acids linked by 1–2 amino acids. It
should be noted that RAN translation from pure CAG repeats
produces individual polyS, polyA and polyQ peptides. Bearing all
this in mind, the conservation of miR-1, miR-7
and miR-10 defects between the fly model and DM1
patients represents important evidence that dysregulation of at
least these three miRNAs occurs independently of the CUCGA repeat
interruption in the UAS-i(CTG)480 transgene
(Fernandez-Costa, 2013).
By studying the expression levels of the predicted target genes
of miR-1, miR-7 and miR-10 in
skeletal muscles from DM1 patients, a total of 42 targets were
found to be dysregulated, 41 of them being up-regulated and only 1
down-regulated. The up-regulation of these targets is consistent
with a reduced degradation by their respective miRNA regulators.
qRT–PCR analysis confirmed this general trend, and validated
at least seven of these alterations in DM1 patients, which had not
been previously described to be triggered by miRNA dysregulation.
Affected genes do not fall into related functional categories, but
instead involve multiple cellular processes. Moreover, miR-1,
miR-7 and miR-10 down-regulation could have an
even higher impact on gene expression, if it is taken into
consideration that these miRNAs might also affect the translation
of additional gene targets, without affecting the levels of their
messenger transcripts. Therefore, these results highlight the wide
number of cellular mechanisms potentially affected by CTG-mediated
disruption of miRNA regulation (Fernandez-Costa, 2013).
A number of miRNAs found altered in DM1 to date are encoded in
introns, thus suggesting a link between pre-mRNA splicing and
miRNA processing. Given that splicing alterations are a hallmark
of DM1, both defects could have a common origin. In this study,
two Drosophila miRNAs affected by CTG expression, miR-1003
and miR-1006, are miRtrons. The precursor intron of miR-1006
is completely spliced out both in control and in DM1 flies,
suggesting that miR-1006-reduced levels in
CTG-expressing flies do not originate from defects in the splicing
regulation of its host transcript, but would instead occur at a
more downstream level. For miR-1003, it was found that
its precursor intron is spliced out at higher levels in DM1 flies
than in control individuals. However, mature miR-1003
levels are reduced in DM1 flies. Increased levels of spliced-out
miR-1003 precursor could arise from a response mechanism triggered
by the cells to compensate for the reduced levels of mature miR-1003,
whereas the mature miRNA reduction itself would occur at a
downstream level. In this study, altered miRNAs that belong to the
same cluster (i.e. single-transcription units containing several
miRNAs regulated by an upstream promoter) were also found. In Drosophila,
the pri-miRNA levels of clusters miR-310–313 and miR-959–964
are reduced in CTG-expressing flies compared with controls.
Additionally, the levels of pri-miRNA for miR-7, but not for miR-1
or miR-10, are down-regulated in CTG-expressing flies and in
skeletal muscle of DM1 patients. Therefore, these results
demonstrate that pri-miRNA transcription/stability is involved in
at least part of the miRNA defects described in this work,
supporting the idea of different origins for miRNA dysregulation
in DM1 (Fernandez-Costa, 2013).
In the DM1 model flies, the CTG-mediated reduction of miR-1
seems to be dependent on Mbl, as over-expression of MblC in
CTG-expressing flies rescues miR-1 levels. Moreover, mbl
silencing in a wild-type background causes a strong reduction of miR-1.
These results are consistent with previous reports that describe a
direct implication of MBNL1 in the biogenesis of human miR-1.
It has been reported that MBNL1 binds to a UGC motif located
within the loop of the pre-miRNA, facilitating the Dicer
processing that generates the mature miR-1. According to
this model, MBNL1 sequestration by CUG repeats would lead to a
reduction of miR-1 levels in DM1, which has been
validated in cardiac muscle from DM1 patients (2.1-fold
reduction), and is consistent with results in flies and DM1 muscle
biopsies from this study. However, other reports have described a
different situation for miR-1: miR-1 from
biceps muscles of DM1 patients, a 1.9-fold up-regulation of this
miRNA, together with an increase in eight of its predicted
targets, has been found. This difference may be explained by the
different types of muscles analyzed and/or their use of controls
with suspected neuromuscular disorders. Intriguingly, another
study reported no changes in miR-1 levels in the vastus
lateralis muscle of DM1 patients. It is, therefore,
possible that miR-1 dysregulation is particularly
sensitive to cellular contexts, which could include factors such
as the number of CTG repeats or the age of the patients
(Fernandez-Costa, 2013).
It was found that mbl silencing also reduces miR-7
levels. However, this reduction is weaker than that observed for miR-1.
Moreover, over-expression of MblC does not rescue the effect of
CTG expansions on miR-7 levels. In the
transdifferentiation cell model, miR-7 levels are
reduced both before and after myogenesis, whereas miR-1
and miR-10 are only significantly affected after
differentiation. In addition, pri-miRNA down-regulation occurrs
for miR-7, but not for miR-1 or miR-10.
These observations further suggest that miR-7
alterations in DM1 occur via a different mechanism, although
further studies will be required to clarify the specific factors
involved in each case (Fernandez-Costa, 2013).
The different behavior of miR-1, miR-7 and miR-10
in the presence of CTG expansions might translate into different
consequences to the homeostasis of the cells. The pathological
relevance of miRNA dysregulation in DM1 is unclear, as alterations
previously described in miRNA levels could correspond either to a
response mechanism or to a pathogenic consequence. It was shown
that partial restoration of miR-10 levels by
over-expression of this miRNA in the Drosophila muscles
partially rescues the reduced lifespan phenotype of DM1 flies.
This demonstrates that miR-10 down-regulation
contributes to CTG-mediated toxicity. On the other hand, not all
miRNA alterations triggered by CTG expression seem to have a
phenotypic impact, as over-expression of miR-1 or miR-7
does not rescue the CTG-induced phenotype, and even reduce the
survival of flies. For miR-7, this effect could
originate from additive toxicity, as miR-7
over-expression alone affects the lifespan of flies. However, the
case of miR-1 is more intriguing, since this miRNA is
not toxic per se. Given that human MBNL1 has been described to
bind to miR-1 directly, it would be possible that the
CTG-specific detrimental effect observed for miR-1
over-expression results from a sequestration of Drosophila
Mbl by excess of miR-1 (Fernandez-Costa, 2013).
In summary, this study sheds light onto our understanding of the
molecular mechanisms behind gene expression dysregulation in DM1
and CTG toxicity, providing a direct link between miRNA
dysregulation and RNA toxicity in DM1, identifying a number of
mechanisms and predicted target genes that are affected by CTG
expansions and supporting the pathogenic potential of at least
part of them (Fernandez-Costa, 2013).
Go to top
Marrone, A.K., Edeleva, E.V.,
Kucherenko, M.M., Hsiao, N.H. and Shcherbata, H.R.
(2012). Dg-Dys-Syn1 signaling in Drosophila regulates
the microRNA profile. BMC Cell Biol 13: 26. PubMed ID: 23107381
Abstract
The Dystrophin Glycoprotein Complex (DGC) is at the center of
significant inheritable diseases, such as muscular dystrophies
that can be fatal and impair neuronal function in addition to
muscle degeneration. Recent evidence has shown that it can control
cellular homeostasis and work via Dystrophin
signaling to regulate microRNA gene expression which implies that
disease phenotypes hide an entourage of regulatory and homeostatic
anomalies. Uncovering these hidden processes could shed new light
on the importance of proper DGC function for an organism’s
overall welfare and bring forth new ideas for treatments. To
better understand a role for the DGC in these processes, this
study used the genetically advantageous Drosophila
muscular dystrophy model to conduct a whole animal microarray
screen. Since it has been shown earlier that dystrophic symptoms
can be caused by stress even in wild type animals and are enhanced
in mutants, this study screened stressed animals for microRNA
misregulation as well. Data support the hypothesis that there is a
Dystrophin and Dystroglycan dependent circuitry of processes
linking stress response, dystrophic conditions and cellular
signaling and that microRNAs play an important role in this
network. Verification of a subset of the results was conducted via
q-PCR and it was found that miR-956, miR-980
and miR-252 are regulated via a Dystroglycan-Dystrophin-Syntrophin
dependent pathway. These results support the hypothesis that there
is a Dystrophin and Dystroglycan dependent circuitry of processes
that includes regulation of microRNAs. Dystrophin signaling has
already been found to occur in mammalian musculature; however,
data from this study reveals that this regulation is
evolutionarily conserved and is also present in at least neuronal
tissues. Also, Dystroglycan-Dystrophin-Syntrophin signaling,
through control of multiple microRNAs, is involved in highly
managed regulation of gene expression required to adapt cellular
homeostasis that is compromised under stress and dystrophic
conditions (Marrone, 2012).
Highlights
- Stress and dystrophy alter the miRNA profile in Drosophila.
- miRNA expression profiles link stress, dystrophy and DGC
signaling.
- Verification of microarray data points to miRNA regulation by
stress.
- Verification of microarray data points to miRNA regulation by
the Dg-Dys-Syn1 pathway.
- Putative mRNA targets of Dg-Dys-Syn1 regulated miRNAs are
involved in general cellular processes, the nervous system, and
muscle development and maintenance.
- Dg-Dys-Syn1 regulated miRNAs are expressed in the nervous
system and musculature.
- miR-252 can regulate levels of DGC interacting
components and is required for proper muscle development.
- miR-980 regulates stress response in Drosophila.
Go to top
Uchino, R., Nonaka, Y.K., Horigome, T.,
Sugiyama, S. and Furukawa, K. (2013). Loss of Drosophila
A-type lamin C initially causes tendon abnormality
including disintegration of cytoskeleton and nuclear lamina in
muscular defects. Dev Biol 373: 216-227. PubMed ID: 22982669
Abstract
Lamins are the major components of nuclear envelope architecture,
being required for both the structural and informational roles of
the nuclei. Mutations of lamins cause a spectrum of diseases in
humans, including muscular dystrophy. This study reports that the
loss of the A-type lamin gene, lamin C in Drosophila
results in pupal metamorphic lethality caused by tendon defects,
matching the characteristics of human A-type lamin revealed by
Emery–Dreifuss muscular dystrophy (EDMD). In tendon cells
lacking lamin C activity, overall cell morphology is
affected and organization of the spectraplakin family cytoskeletal
protein Shortstop which is prominently expressed in tendon cells
gradually disintegrates, notably around the nucleus and in a
manner correlating well with the degradation of musculature.
Furthermore, lamin C null mutants are efficiently
rescued by restoring lamin C expression to
shortstop-expressing cells, which include tendon cells but exclude
skeletal muscle cells. Thus the critical function of A-type lamin
C proteins in Drosophila musculature is to
maintain proper function and morphology of tendon cells (Uchino,
2013).
Highlights
- Lethality of lamin C null alleles is associated with
muscle defects during pupal metamorphic stages.
- The primary defect of loss of lamin C is abnormality
of the tendon cells.
- Mutation of lamin C leads to aberrant tendon cell
morphology during larval development.
- Tendon cells lacking lamin C proteins exhibit changes in the
organization of cytoskeleton.
- The requirement for lamin C is in cells that express
shortstop.
Discussion
Drosophila lamin C alleles have been described
to show recessive pre-pupal lethality accompanied by aberrant
nuclear morphology in imaginal discs and muscle cells. However,
the direct cause of the lethality resulting from the loss of lamin
C function had not been determined. This study reports the
phenotypes of newly isolated and previously reported lamin C
alleles. Under modified culture conditions, these alleles were
found to develop to pharate adult stages and show head eversion
defects. Although head eversion is known to be dependent on
peristaltic contraction of the abdominal muscles and the abdominal
muscle patterns in lamin C mutants are obviously
abnormal, this defect is not rescued by lamin C
transgene expression in muscle cells. However it could be rescued
by expression in tendon cells, indicating that lamin C is critical
in the tendons rather than the muscles (Uchino, 2013).
In contrast to these results, a direct effect of lamin C on
muscle cells has been previously speculated, based on the
observation that the expression of N-terminal truncated lamin C in
muscle cells induces lethality accompanied by altered nuclear
structures including lamin C aggregation. Similar head domain-less
A-type lamins have been reported to aggregate in nucleoplasm in
vertebrates. These aberrant lamin structures have been shown to
disrupt the nuclear rim-distribution of endogenous lamins
including B-type lamins, by causing them to co-aggregate, and
result in the inhibition of replication and transcription. As
N-terminal truncated A-type lamins interfere with lamin-nuclear
function through an artificial dominant effect, the defects caused
by their expression in Drosophila muscle cells could
have been caused by their disruption of the normal localization of
lamin Dm0 (B-type lamin). As endogenous lamin Dm0 retains
localization at the nuclear rim in the lamin C null
mutants, the recessive phenotypes caused by lamin C loss described
in this study are probably different from, but not in conflict
with the dominant negative phenotypes reported to be induced by
head domain-less lamin C expression (Uchino, 2013).
The shot gene is essential in tendon cell
differentiation. Its expression is detected in the tendon cells
but not in the muscle cells from around stages 11 onward of
embryonic development. Mutations of shot are reported to
affect the EGF-receptor signaling pathway by inducing
mislocalization of Vein activator at the MTJ and result in the
disruption of somatic muscle pattern at stage 16 of embryonic
development. Shot belongs to the spectraplakin family of
cytoskeletal linker proteins and has also been shown to contribute
to the mechanical strength of tendon cells. Thus, Shot has both
structural and informational functions (Uchino, 2013).
It was found that the expression of a lamin C transgene
with a shot-promoter driven GAL4 transgene is restricted
to tendon cells in musculature, and its expression levels are
similar between LamCL58 and wild type animals. Further,
shot-GAL4 driver induced lamin C proteins are capable of
effectively rescuing lamin C null mutants to adults.
Although loss of lamin C induces disintegration of cytoskeletal
and nuclear lamina structures in the tendon cells, they are
relatively normal at the early larval stages in contrast to the
muscular abnormality which occurs in shot alleles at
embryonic stages. In lamin C null mutants,
disintegration of the cytoskeleton which includes Shot is
initiated before nuclear lamina deformation and progresses
gradually during larval growth. This alteration eventually causes
destruction of tendon cells and muscle detachment from epidermis
in a manner similar to shot mutant phenotypes. A-type lamin is
known to be major contributor to the mechanical properties of
nuclei as well as the cytoskeleton and cytoskeleton-based
processes, whereas B-type lamin contributes to nuclear integrity.
Thus, these findings with lamin C alleles are coincident
with these interpretations and strongly suggest that lamin C
null mutation directly induces mechanical instability of the
cytoskeleton and nuclear structures, which probably leads to
weakening of tendon cell strength and function (Uchino,
2013).
Interactions between the cytoskeleton and the nuclear lamina in
which A-type lamins are localized are generally mediated through
the double layer lipid nuclear membrane by LINC complexes formed
by Klarsicht/ANC-1/Syne homology (KASH) domain proteins directly
linked to Sad1/UNC-84 (SUN) domain proteins in the nuclear
envelope. In mammals, KASH domain proteins are divided into the
three major groups by association with actin filaments,
microtubules, and intermediate filaments (IF). In Drosophila,
the nesprin-ortholog MSP-300 associated with actin filaments and
Klarsicht associated with microtubules are the known KASH domain
proteins, and Klaroid and testis specific Spag4 are the known SUN
domain proteins. However, KASH domain deleted MSP-300
alleles and klarsicht, spag4 and klaroid
null alleles have been reported not to result in lethality.
Further, when the distribution of MSP-300 was studied in wild type
and LamCLC58 muscles, it was detected in the sarcomere
of both as previously described. In tendon cells its distribution
could not be evaluated because of its low expression. Klarsicht
and Klaroid were detected in the region of the nuclear envelope
(NE) in wild type, and their localizations were similar to the
distribution of lamin Dm0 at the NE in the tendon cells of LamCLC58
mutant larvae. Thus there is no indication that these known LINC
complex proteins play a central role in the lamin C-depleted
phenotypes of tendon cells (Uchino, 2013).
In humans, an IF-associating KASH domain protein Nesprin 3α
has also been reported in addition to actin and microtubule
associating KASH domain proteins, and its IF-binding is mediated
by plectin protein containing a plakin (or plectin) repeat domain
which can directly bind IF proteins. Although, interaction between
Lamin C and Spectraplakin-related Shot which also has a plakin
repeat domain has not been proven and an IF-associating type of
KASH domain protein has not yet been reported in Drosophila,
the mammalian spectraplakin; Bpag1/dystonin isoform has been
reported to also interact with Nesprin 3α. Furthermore, for
interaction of Shot with the nucleus, the actin–plakin and
plakin domains of Shot have been shown to be targeted to the
nucleus and to predominantly concentrate at the nuclear periphery
in tendon cells. Thus, it is possible that lamin C connects to
Shot mediated cytoskeletal complexes through a novel LINC complex
protein with characteristics similar to Nesprin 3α and
protects tendon cell and nuclei from forces produced by the
contractions of the muscles (Uchino, 2013).
In human EDMD, mutations of A-type lamins are characterized by
early onset contractures of the Achilles tendons and tendons of
the elbows and neck, whereas muscle weakness and wasting shows
slow progression compared to other types of muscular dystrophy. In
LMNA null model mice, the interdigitation between muscle
and tendon is reduced, and the muscle nuclei show abnormal
morphology and tend to cluster at the MTJ. Thus tendon defects are
strongly implicated in muscular dystrophy resulting from the loss
of A-type lamin in mammals, which could be analogous to what this
study reports in Drosophila (Uchino, 2013).
In vertebrates, tendons are morphologically diverse tissues and
their strength depends mainly on extracellular collagen fibrils.
However, well developed actin and myosin bundles are also observed
in cytoplasm and around the nucleus in rabbit tendon cells. In Drosophila,
the spectraplakin family protein Shot has a major role in
formation and maintenance of cytoskeletal organization in tendon
cells, but the mammalian spectraplakins, Bpag1/dystonin and MACF1,
which are Shot orthologs, have not yet been analyzed in tendon
cells. However, Bpag1 isoforms are known to be expressed in
musculature, and show nuclear envelope targeting, nesprin 3α
interaction, and functions in organizing the actin cytoskeleton
around the nucleus in myogenic and other culture cells. As mature
mammalian tendon cells are relatively large cells, with lengths up
to 300 μm, and their volume is almost entirely taken up by a
large nucleus. These cytoskeletal proteins might similarly protect
the mammalian tendon cell and nucleus against mechanical stress
from muscle contraction. The fact that the nuclear defects
observed in LMNA model mice are concentrated at the MTJ
structures suggests that the defects in the mouse and fly
musculatures by loss of A-type lamins may be initially caused by
similar mechanisms at sites of mechanical stress, namely tendon
cells (Uchino, 2013).
In conclusion, this study hypothesizes that loss of A-type lamins
causes a reduction in spectraplakin mediated
cytoskeleton-integrity and cytoskeleton-based processes that
perturbs tendon cell morphology and function leading to symptoms
seen in human autosomal recessive EDMD. Further studies on the
involvement of tendon cell-cytoskeletal structures in the
progression of EDMD in both vertebrates and Drosophila
are therefore warranted (Uchino, 2013).
Go to top
Marrone, A.K., Kucherenko, M.M., Wiek,
R., Göpfert, M.C. and Shcherbata, H.R. (2011).
Hyperthermic seizures and aberrant cellular homeostasis in
Drosophila dystrophic muscles. Sci Rep 1: 47. PubMed ID: 22355566
Abstract
In humans, mutations in the Dystrophin Glycoprotein Complex (DGC)
cause muscular dystrophies (MDs) that are associated with muscle
loss, seizures and brain abnormalities leading to early death.
Using Drosophila as a model to study MD, this study
found that loss of Dystrophin (Dys) during development leads to
heat-sensitive abnormal muscle contractions that are repressed by
mutations in Dys's binding partner, Dystroglycan (Dg).
Hyperthermic seizures are independent from dystrophic muscle
degeneration and rely on neurotransmission, which suggests
involvement of the DGC in muscle-neuron communication.
Additionally, reduction of the Ca2+ regulator, Calmodulin
or Ca2+ channel blockage rescues the seizing phenotype, pointing
to Ca2+ mis-regulation in dystrophic muscles. Also, Dys and Dg
mutants have antagonistically abnormal cellular levels of ROS,
suggesting that the DGC has a function in regulation of muscle
cell homeostasis. These data show that muscles deficient for Dys
are predisposed to hypercontraction that may result from abnormal
neuromuscular junction signaling (Marrone, 2012).
Highlights
- Dystrophin mutants have elevated ROS and hyperthermic
seizures.
- Dystrophic seizures are dependent upon neuronal input that
require Dys during development.
- Loss of Dystroglycan, Coracle or Calmoduline represses the
seizure phenotype of Dystrophin mutants.
- Cora mutants have a mild seizure phenotype that does
not look like and overrides the dystrophic phenotype.
- Elevated Ca2+ from the sarcoplasmic reticulum (SR) leads to
hyperthermic seizures.
Discussion
Muscle seizures accompany multiple human neurological and muscular
disorders. Seizures can occur when a group of neurons becomes
hyperexcited and discharge action potentials irregularly without
suppression, which causes muscle fibers to contract
inappropriately. The uncontrolled action potential is dependent
upon K+ and Na+ channel permeability or Ca2+ mis-regulation.
Ryanodine receptors that are regulators of fast Ca2+ release from
the sarcoplasmic reticulum have been found to play a role in the
disease pathology of seizures. Additionally, inositol
1,4,5-triphosphate receptors (IP3Rs) which regulate slow Ca2+
release from the SR are necessary in the central nervous system,
causing epilepsy when mutated. Seizures can be alleviated by
manipulation of ion channels or by blocking neurotransmission
(Marrone, 2012).
It has been shown that Laminin binds directly to voltage gated
Ca2+ channels (CaV) at the presynapse in mice, specifically P/Q-
and N-type channels, and this binding induces vesicle clustering.
Laminin also binds the transmembrane protein Dg providing a direct
link between Dys and the presynaptic motoneuron in mammals.
Importantly, not only does the motoneuron send signals to the
muscle, but also retrograde signaling exists, where a signal
travels from the muscle back to the presynaptic neuron. The
current pathway by which synaptic retrograde signaling
communicates is not known, however Dys has been implicated
previously to play a role in the process. If it is assumed that
hyperthermic seizures reported in this study are related to the
role of Dys in retrograde signaling, then the CaV-Laminin-Dg
signaling cascade could explain why the P/Q- and N-type, but not
the L-type Ca2+ channel blocker affected Dys seizures.
Similarly, the reduction of Dg or Cora reduces the seizure
occurrence possibly by preventing propagation of signaling via
loss of communication with Laminin. Another pathway that plays a
role in retrograde signaling at the NMJ32 is TGF-β and
interestingly, mutants of the TGF-β pathway have a
hyperthermic seizure phenotype similar to Dys mutants.
However, a genetic interaction between the DGC and TGF-β
pathway components was not observed suggesting that they might act
in parallel (Marrone, 2012).
Dys and Dg have been reported to have opposing functions in
control of neurotransmitter release at the NMJ. Dg
mutants show a decrease and Dys loss of function and
heterozygous mutants an increase in release of neurotransmitter,
however both mutants do not show a change in response to altered
neurotransmitter levels. These opposing phenotypes are similar to
what is reported in this study, where Dys mutants have
seizures and Dg mutants do not. Additionally, Dys
mutants have an increase in synaptic vesicle docking sites
(T-bars) at larval NMJs, which could explain the developmental
requirement for Dys. Once the NMJ is established with a normal
number of active sites, animals would not be prone to seizures
(Marrone, 2012).
The study also shows that Dys and Dg mutants have altered
cellular homeostasis. In vertebrates, multiple metabolic disorders
have been implicated in seizure activity; for example,
mitochondrial encephalopathy, the most common neurometabolic
disorder, presents various symptoms including seizures and mice
that are partially deficient for mitochondrial superoxide
dismutase have an increased incidence of spontaneous seizures.
Additionally, mdx mice, a model for MD has sustained
oxidative stress in skeletal muscle. In Drosophila, it
has been shown that Dg mutant larvae have an altered
state of cellular homeostasis and are sensitive to ambient
temperature. A constant increase in mitochondrial oxidative
metabolism, caused by a Dg hypomorphic mutation, results
in a change in thermoregulatory behavior. In addition, it has been
reported that suboptimal temperatures and energetic stress
accelerate age-dependent muscular dystrophy in both, Dys
and Dg mutants. This study shows that Dys and
Dg mutants have antagonistically abnormal cellular levels
of ROS (Marrone, 2012).
ROS are derived from elemental oxygen (O2), and ROS cascades
begin with the superoxide anion radical. Sources for superoxide
anion radicals include xanthine oxidase, prostanoid metabolism,
catecholamine autooxidation, NAD(P)H oxidase activity and NO
synthase. These radicals are generated in normal muscle, and the
rate of generation is increased by muscle contraction. In Duchenne
MD the absence of dystrophin at the sarcolemma delocalizes and
downregulates neuronal nitric oxide synthase (nNOS), which in turn
leads to increase in inducible nitric oxide synthase (nNOS) that
generates excessive NO free radicals. This mechanism can explain
extremely high ROS levels in Dys mutants. This high
level of ROS in dystrophic Drosophila can be alleviated
by transheterozygous interaction with Dg and Cam,
which indicates a genetic interaction between Dys and
these two genes in control of cellular homeostasis (Marrone,
2012).
Thus study provides the first in vivo measurements on dystrophic
animals showing that they have hyperthermic seizures that are
dependent upon neurotransmission. Dystrophin is required during
development, since Dys downregulation in adulthood,
after muscles and NMJs are already established precludes
hyperthermic seizures. Data suggest that the DGC has a role in
signaling at the NMJ: reduction of Dg, a protein that binds Dys
and regulates localization of the NMJ specific proteins, prevents
dystrophic seizure occurrence. Seizures are associated with
abnormal Ca2+ release from the SR; introduction of a mutation of
Ca2+ mediator Calmodulin and supply of calcium channel
blockers reduce seizures. Taken together, these data show that the
DGC acts at the muscle side of the NMJ to regulate muscle cell
homeostasis in response to neuronal signaling and implies that Dys
is involved in muscle-neuron communication (Marrone, 2012).
Go to top
Yu, Z., Tengm X. and Bonini, N.M.
(2011). Triplet repeat-derived siRNAs enhance RNA-mediated
toxicity in a Drosophila model for myotonic dystrophy.
PLoS Genet 7: e1001340. PubMed ID: 21437269
Abstract
More than 20 human neurological and neurodegenerative diseases are
caused by simple DNA repeat expansions; among these, non-coding
CTG repeat expansions are the basis of myotonic dystrophy (DM1).
Earlier work, however, has also revealed that many human genes
have anti-sense transcripts, raising the possibility that human
trinucleotide expansion diseases may be comprised of pathogenic
activities due both to a sense expanded-repeat transcript and to
an anti-sense expanded-repeat transcript. This study established a
Drosophila model for DM1 and tested the role of
interactions between expanded CTG transcripts and expanded CAG
repeat transcripts. These studies reveal dramatically enhanced
toxicity in flies co-expressing CTG with CAG expanded repeats.
Expression of the two transcripts leads to novel pathogenesis with
the generation of dcr-2
and ago2-dependent
21-nt triplet repeat-derived siRNAs. These small RNAs target the
expression of CAG-containing genes, such as Ataxin-2
and TATA binding protein
(TBP), which bear long CAG repeats in both fly and
humans. These findings indicate that the generation of triplet
repeat-derived siRNAs may dramatically enhance toxicity in human
repeat expansion diseases in which anti-sense transcription occurs
(Yu, 2011).
Highlights
- Expression of expanded CUG RNA causes repeat-length dependent
toxicity.
- Enhancement of CTG-repeat toxicity by CAG-repeat transcripts.
- CTG/CAG transcripts are processed into small RNAs.
- Toxicity of co-expressed CTG/CAG transcripts is dependent on
Dcr2 and Ago2.
- Triplet repeat derived siRNAs compromise the expression of
genes containing short CAG stretches.
Discussion
Like many genes within the mammalian genome, the DM1
gene displays bi-directional transcription, generating an
anti-sense CAG repeat transcript in addition to the
disease-associated CTG transcript. These transcripts have been
shown to interact in human cells to generate small RNAs, with one
effect being local gene silencing; however additional ways in
which this may contribute to pathogenicity in disease is largely
unknown. In order to provide new insight into DM1, this study
generated a Drosophila model by expressing pure,
uninterrupted CTG repeat expansions; fly models for various
disorders have revealed critical insight into a number of human
disease situations. Interestingly, targeted expression of the long
CTG repeats in the fly eye causes a variable toxic effect. This
has also been observed in a fly model of SCA8, which carries an
uninterrupted CTG repeat expansion. In contrast, fly models
generated using interrupted CTG repeats have not been reported to
show variable phenotype. It is thus possible that phenotypic
variability may be a feature of pure repeat sequences, which is in
line with the fact that DM1 is among the most variable human
disorders. To define potential effects of bi-directional
transcription, the expanded CAG repeat transcripts were
co-expressed with the DM1 CTG repeats. This resulted in
dramatically enhanced toxicity concomitant with the generation of
triplet repeat-derived siRNAs. These results are in striking
contrast with previous findings that co-expression of CGG and CCG
expansions in flies leads to mitigated toxicity in a
ago2-dependent manner, suggesting that toxicity derived from
interactions between sense and anti-sense repeat transcripts may
be specific to CTG/CAG situations. Both CAG and CUG strands can be
processed into ~21 nt small RNAs when coexpressed and small RNAs
derived from both strands are methylated in a Hen1-dependent
manner. These results suggest that both CAG and CUG small RNAs can
be loaded into mature, holo-RISCs presumably due to the
symmetrical thermodynamic properties of the repeat small RNA
duplex. Although a direct cleavage of CAG containing transcripts
was detected, however cleavage of CUG containing transcripts
mediated by CAG small RNAs was not detected. Although underlying
reasons for this differential effect remain unclear, CUG and CAG
transcripts may have differential expression levels or translation
efficiencies, and/or CUG-containing and CAG-containing transcripts
may be associated with different RNA binding proteins of various
affinities, making CUG-transcripts less accessible to the RISC
complex than CAG-containing transcripts. A number of CUG-binding
proteins have been defined, such as MBNL1, CUGBP1 and PKR.
Interestingly, in-vitro gel retardation analysis indicated that
MBNL1 has a much lower affinity for CAG repeat RNA than CUG repeat
RNA. Moreover, expanded CAG transcripts, although co-localizing
with MBNL1 in ribonuclear foci similarly to expanded CUG
transcripts, do not appear to cause mis-regulation of alternative
splicing in cells, further highlighting differential properties of
these repeats in interacting with RNA binding proteins (Yu, 2011).
The toxicity caused by co-expression of expanded CAG and CTG was
associated with deleterious effects on transcripts of other CAG
containing genes within the genome; additional mechanisms that
contribute to toxicity may also exist. A large number of genes
contain CAG stretches in fly and human genomes. The enhanced
toxicity observed in flies expressing expanded CAG and CTG may
therefore be reflecting an additive effect of knockdown of
multiple CAG-containing genes, with each individual gene
contributing only partially to the overall outcome. Although
further reducing atx2 dosage did not enhance toxicity of
co-expressed CTG/CAG expansions, the compromised activities of
many target genes may be involved and further compromising any
single one has minimal impact. The toxic effects seen of the
CAG/CTG situation may also be complicated by the later-onset and
progressive nature of the toxicity. Further study will clarify the
contribution of this mechanism, and key targets among all possible
transcripts, to the overall phenotype of the disease. Moreover,
the deleterious effects caused by triplet repeat derived small
RNAs may be further exacerbated by the wide prevalence of CAG
stretches in the human transcriptome and the relative low
specificity of RNA interference when siRNAs and/or RNA targets
contain simple repeats like CAG. Such interactions may represent a
novel activity of endo-siRNAs that characterize disease situations
where bi-directional transcription spanning the repeat region
occurs (Yu, 2011).
Two CAG containing genes, atx2 and tbp, were
conformed to be targets of the triplet repeat-derived siRNAs.
Interestingly, CAG repeat expansions in ATXN2 (the human
Ataxin-2 gene) and TBP define two of the
CAG-repeat expansion diseases (SCA2 and SCA17, respectively). In
such diseases, the expanded polyglutamine domain is thought to
confer toxicity; however, increasing evidence suggests that the
loss-of-function of gene activity, and not just dominant
activities of the protein with an expanded polyglutamine region,
occur in disease. These findings raise the possibility that
bi-directional transcription of the repeat region in diseases like
DM1 may confer additional components of pathogenicity due to
deleterious interactions between the two overlapping
repeat-containing transcripts through the generation and activity
of triplet repeat-derived siRNAs (Yu, 2011).
Earlier studies indicate that bi-directionally transcribed RNAs,
and presumably resultant endogenous double-stranded RNAs, are
processed into ~21–23 nt small RNAs in human cells. This is
despite the fact that in most mammalian cells, long exogenous
double-stranded RNAs can elicit the interferon response. That
response presumably occurs in a threshold-dependent manner; cells
may also respond differentially to long exogenous double-stranded
RNAs versus endogenous double-stranded RNAs. Thus, these findings
suggest that the biogenesis pathway of small RNAs from endogenous
double-stranded RNAs is conserved in mammalian cells. Many loci
are bi-directionally transcribed throughout the mammalian genome,
and among these are a number of human trinucleotide disease genes,
including SCA8 and DM1. In SCA8, an anti-sense transcript is
proposed to encode a polyglutamine protein, which itself may have
deleterious actions. In DM1, the two transcripts interact to
produce small RNAs that can have local effects on gene silencing.
Data from this study raise another possibility that processing of
co-expressed transcripts containing CUG/CAG expansions into
triplet repeat-derived siRNAs in vivo, may contribute to toxicity
with widespread deleterious effects. These effects may include
downregulating the expression of other genes containing CAG
repeats. Among the genes that could be targets are the
polyglutamine disease genes themselves, one of which is TBP.
Expansion of the TBP polyglutamine repeat underlies SCA17;
intriguingly, general transcriptional compromise has been shown to
be a component of repeat expansion diseases. Another reason why
these diseases share transcriptional compromise may be that they
share bi-directional transcript interactions that compromise
common elements like TBP. This possibility underscores the idea of
shared therapeutic targets and mechanisms in repeat expansion
diseases (Yu, 2011).
It has been proposed that siCAG and siCUG may be used for therapy
of triplet repeat expansion diseases based on findings in cell
culture that these siRNAs seem to specifically target mutant
transcripts with expanded repeats. This study's data suggest
caution in designing such siRNA-based therapy, as in the intact
organismal situation, pathogenic activities may be noted. Although
previous findings suggest that expanded CUG alone can be processed
into small RNAs, this study's data suggest that both expanded CAG
and CTG are required for triplet repeat-derived siRNA generation
and toxicity in vivo. Thus, co-expressed CAG and CTG expansions
may contribute to DM1 pathogenesis through a fundamentally
different mechanism from that of CTG expansions alone. Targeting
human trinucleotide expansion diseases at the transcriptional
level may therefore be a promising therapeutic approach that would
minimize not only the effects of single expanded repeat
transcripts, but deleterious interactions between sense and
anti-sense repeat transcript domains (Yu, 2011).
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Goldstein, J.A., Kelly, S.M., LoPresti,
P.P., Heydemann, A., Earley, J.U., Ferguson, E.L., Wolf, M.J.
and McNally, E.M. (2011). SMAD signaling drives heart and
muscle dysfunction in a Drosophila model of muscular
dystrophy. Hum Mol Genet 20: 894-904. PubMed ID: 21138941
Abstract
Loss-of-function mutations in the genes encoding dystrophin and
the associated membrane proteins, the sarcoglycans, produce
muscular dystrophy and cardiomyopathy. The dystrophin complex
provides stability to the plasma membrane of striated muscle
during muscle contraction. Increased SMAD signaling due to
activation of the transforming
growth factor-β (TGFβ) pathway has been described
in muscular dystrophy; however, it is not known whether this
canonical TGFβ signaling is pathogenic in the muscle itself.
Drosophila deleted for the γ/δ-sarcoglycan
gene (Sgcd) develop progressive muscle and heart
dysfunction and serve as a model for the human disorder. This
study used dad-lacZ flies to demonstrate the signature
of TGFβ activation in response to exercise-induced injury in
Sgcd null flies, finding that those muscle nuclei
immediately adjacent to muscle injury demonstrate high-level
TGFβ signaling. To determine the pathogenic nature of this
signaling, it was found that partial reduction of the co-SMAD Medea,
homologous to SMAD4, or the r-SMAD, Smox,
corrects both heart and muscle dysfunction in Sgcd
mutants. Reduction in the r-SMAD, MAD, restores muscle function
but interestingly not heart function in Sgcd mutants,
consistent with a role for activin but not bone morphogenic
protein signaling in cardiac dysfunction. Mammalian sarcoglycan
null muscle was also found to exhibit exercise-induced SMAD
signaling. These data demonstrate that hyperactivation of SMAD
signaling occurs in response to repetitive injury in muscle and
heart. Reduction of this pathway is sufficient to restore cardiac
and muscle function and is therefore a target for therapeutic
reduction (Goldstein, 2011).
Highlights
- Exercise induces muscle disruption in Sgcd[840]
mutants.
- Exercise-induced muscle tears in the Sgcd[840]
mutant lead to SMAD signaling.
- TGFβ signaling is increased in Sgcd[840] mutant
muscle.
- Reducing TGFβ signaling rescues mobility in sarcoglycan
mutant flies.
- r-SMAD mutants rescue negative geotaxis in Sgcd[840]
mutants.
- Reducing TGFβ signaling rescues heart function in
sarcoglycan mutant flies.
- Exercise induces TGFβ signaling in Sgcg null
mice.
Discussion
TGFβ signaling is mediated through canonical and
non-canonical pathways. In mammalian muscle, pSMAD is detected in
nuclei, and reduction of nuclear pSMAD has been reported in mdx
mice treated with TGFβ-neutralizing antibodies. In dystrophic
muscle, both myonuclei and those nuclei in non-muscle cells
display enhanced nuclear pSMAD. This study shows that the
orthologous SMAD-type signaling is increased in a Drosophila
model of muscular dystrophy. The fly model was utilized for these
studies because dystrophic fly muscle is characterized by a
degenerative process. This is in contrast to dystrophic mammalian
muscle where there is degeneration alongside regeneration and
infiltration by non-muscle elements. Because in mammalian muscle
these non-muscle elements, particularly the fibroblasts, have much
greater pSMAD signaling, the simpler fly muscle allows examination
of the myogenic component of injury response. With this approach,
it was found that exercise enhances muscle disruption and the
degree of β-gal activity from the dad-lacZ
indicator allele in Sgcd[840] muscle. The myonuclei most
proximal to the region of disruption display the greatest amount
of β-gal activity, and this is consistent with a model of
local TGFβ activity inducing response nearest to the area of
injury. Injury alone, in the absence of disease, also elicits this
signaling response, if the injury was severe. Another study
profiled gene expression of mechanically injured Drosophila
thorax and found elevated transcript levels for the BMP-like
ligand gbb and the activin ligand daw at 1 and
6h after injury, suggesting that these ligands may be the
TGFβ orthologs (Goldstein, 2011).
In addition to fibroblast activation and proliferation, mammalian
muscular dystrophy is accompanied by an inflammatory response in
muscle, in part mediated by mononuclear cells derived from the
bone marrow. The equivalent in the fly is a hemocyte-like cell,
which was very occasionally observed in sarcoglycan mutant flies.
Hemocyte-derived cell lines are known to secrete TGFβ family
ligands. However, this behavior has not been investigated in Drosophila
adult muscle. In mammalian models of muscular dystrophy, immune
cells are prominent and a plausible source of TGFβ and other
cytokines. The use of the dad-lacZ indicator
demonstrates that muscle cells clearly possess the capacity to
respond to downstream signaling. In mammalian muscle, the finding
of myofiber nuclei positive for pSMAD2/3 supports a parallel
pathogenesis in mammalian muscle (Goldstein, 2011).
In mammalian muscle affected by muscular dystrophy, degeneration
occurs concomitantly with regeneration. In this model reducing
TGFβ signaling has been thought to primarily mediate its
effect by improving muscle regeneration, suggesting that TGFβ
signaling has a negative effect on satellite cell function.
Although the Sgcd[840] model exhibits many features of
mammalian muscular dystrophy, previous studies did not find
evidence of muscle regeneration in this model. The absence of
obvious regeneration in the fly model suggests that enhanced
TGFβ signaling is exerting its effect directly by hastening
muscle degeneration either by inhibiting membrane-mediated repair
mechanisms or other cellular adaptations. Data suggest that the
effects of inhibiting TGFβ signaling likely extend beyond the
regenerative response, mitigating degeneration and enhancing
repair. An additional possibility, raised by the improved climbing
performance of normal flies with heterozygous SMAD alleles, is
that reducing SMAD signaling developmentally increases muscle
function, perhaps by antagonizing the myostatin homolog,
myoglianin. Further understanding the genes regulated by nuclear
pSMADs in injured muscle may help to define the precise pathways
that are most critical for eliciting muscle dysfunction. Thus far,
results point to the r-SMADs and co-SMADs as targets for
therapeutic intervention (Goldstein, 2011).
It was found that exercising a mouse model of muscular dystrophy
increases the pSMAD signaling in those nuclei positioned centrally
within myofibers. Centrally positioned nuclei, as opposed to those
in the normal peripheral position, are indicative of recent
regeneration. However, increased pSMAD in central nuclei was
detected immediately after exercise without sufficient time for
myoblast fusion and regeneration. These findings indicate that all
myonuclei, including those in the central position, are affected
by pathogenic TGFβ signaling. Newly regenerated fibers are
particularly important to protect and so targeting this pathway
for therapy may offer additional protection (Goldstein, 2011).
It was found that heterozygous mutations of Medea, Smox
or MAD are dominant suppressors of the loss of walking
function in Sgcd[840] mutants. This finding implicates
broad downstream signaling, including both the activin and BMP
pathways. Curiously, the MAD[12] allele did not improve
heart tube function while the Medea[13] and Smox[G0348]
alleles did. MAD[12] is the r-SMAD for BMP, and
therefore the differential response of the heart tube versus
skeletal muscle implies that BMP is more important for muscle
versus heart tube function. The Drosophila heart tube is
a linear structure. During larval stages, the posterior region is
lined with cells expressing cardiac-specific markers. Expression
of the Hox gene AbdA is sufficient to determine the
cardiac fate while the anterior portion is the aorta. In the
adult, the more anterior segments, A1 through A4, assume cardiac
function and are lined with cardiomyocytes derived from the larval
cells. This developmental paradigm suggests a more intimate
relationship between cardiac and vascular structures where activin
but not BMP signaling may be essential. Although the SMAD family
is more complex in mammals, there still may a differential
response to SMAD reduction between heart and skeletal muscles, as
inhibition of SMAD signaling may differ between vascular cells and
skeletal muscle in mammals (Goldstein, 2011).
This study focused on canonical TGFβ signaling in the Sgcd[840]
model. Non-canonical TGFβ signaling includes JNK, p38, ERK
and Akt signaling. Notably, each of these pathways has been shown
to be important for muscle regeneration and function. P38 MAP
kinase and JNK2 signaling are induced by an exercise regimen in
the mdx mouse. Akt mediates the muscle proliferative and
differentiation functions of insulin-like growth factor-1. In
addition, SMADs are not exclusively activated by TGFβ
receptors, and the non-transcription factor roles of SMADs should
be considered when targeting these pathways for therapeutic
intervention (Goldstein, 2011).
Go to top
Reviews
Pantoja, M. and Ruohola-Baker, H.
(2013). Drosophila as a starting point for developing
therapeutics for the rare disease Duchenne Muscular Dystrophy.
Rare Dis 1: e24995. PubMed ID: 25002997
Plantié, E., Migocka-Patrzałek,
M., Daczewska, M. and Jagla, K. (2015). Model organisms
in the fight against muscular dystrophy: lessons from Drosophila
and Zebrafish. Molecules 20: 6237-6253. PubMed ID: 25859781
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