InteractiveFly: GeneBrief

Ubiquilin: Biological Overview | References

BIOLOGICAL OVERVIEW

dDsk2 (Ubqln2/Ubiquilin) is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. This study reports that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1 (monoubiquitylated H2B), a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications (Kessler, 2015).

This study reports that the extraproteasomal ubiquitin receptor dDsk2 interacts with the dHP1c complex, localizes at promoters and is required for transcription. Binding sites of the dHP1c complex are marked by H2Bub1 and the results suggest that, through the UBA domain, dDsk2 binds H2Bub1. However, reducing H2Bub1 levels affects binding of the complex only weakly, indicating that the interaction with H2Bub1 has only a minor contribution to recruitment. Actually, dDsk2 contains a single ubiquitin-binding site of low affinity (Kd~400 μM), which is in contrast to most ubiquitin receptors that contain several ubiquitin-binding sites that act synergistically to provide high-affinity binding. Similarly, the interaction of dDsk2 with H2Bub1 is of low specificity since selective recognition of ubiquitylated substrates is largely based on the recognition of the linkage type, length and anchoring site of a polyubiquitin chain, and, consequently, requires the presence of several ubiquitin-binding sites. In fact, the UBA domain of dDsk2 recognizes a monoubiquitylation in PTEN with a similar affinity as in H2B. On the other hand, binding of the dHP1c complex likely involves the recognition of specific DNA sequences since it depends on the zinc-finger proteins WOC and ROW. Noteworthy, dHP1c complex-binding sites are significantly enriched in a specific DNA sequence motif. However, although the interaction with H2Bub1 is weak, binding of the complex unexpectedly depends on dDsk2. Besides, dHP1c is dispensable for WOC and ROW binding, as well as for dDsk2 binding). These effects do not appear to be the consequence of changes in gene expression levels since dDsk2 mRNA levels do not significantly change on WOC, ROW or dHP1c depletion. Furthermore, dDsk2 depletion upregulates ROW and weakly downregulates dHP1c mRNA levels. Finally, it was reported that dHP1c interacts with RNA pol II, suggesting that binding of the dHP1c complex might depend on RNA pol II. However, arguing against this possibility, it was observed that binding of the complex at promoters is resistant to treatment with Actinomycin D. Altogether, these results suggest that WOC, ROW and dDsk2 constitute the actual binding module of the complex, being fully interdependent for binding to chromatin and required for binding of dHP1c. In this regard, the slight reduction of ROW and dHP1c protein levels observed on dDsk2 depletion is most likely due to their inability to bind chromatin, as described previously for dHP1c in ROW and WOC knockdowns, as well as for other chromatin-associated proteins when their binding to chromatin is impaired (Kessler, 2015).

These results suggest that the main function of dDsk2 in the dHP1c complex is to prevent H2Bub1 deubiquitylation by dUbp8/Nonstop, as H2Bub1 levels are strongly reduced on dDsk2 depletion and recovered after dUbp8/Nonstop co-depletion. Protection against deubiquitylation has also been reported for Rad23 and appears to be a common feature of many ubiquitin receptors. Simultaneous dDsk2 and dUbp8/Nonstop depletion also restores expression of target genes, whereas it has no effect on recruitment of the complex at promoters. Furthermore, overexpression of the ΔUBA–dDsk2 construct, which misses the UBA domain that mediates interaction with H2Bub1 in vitro, reduces H2Bub1 levels at promoters and downregulates expression of target genes. Altogether, these results strongly suggest that the contribution of dDsk2 to transcription regulation is mainly based on this protective function (Kessler, 2015).

dHP1c complex target genes show features associated with strong RNA pol II pausing. The results support a contribution of dDsk2 to pausing since its depletion reduces RNA pol II occupancy preferentially at TSS and strongly decreases NELF-E levels. dDsk2 depletion downregulates expression and, in good agreement, total RNA pol II occupancy across target genes is reduced. Interestingly, the majority of NELF target genes are also downregulated on NELF depletion in S2 cells51, and NELF potentiates gene expression in the Drosophila embryo. Actually, ~80% of dHP1c complex target genes that change expression in NELF knockdown conditions are downregulated. Furthermore, NELF-E depletion shows a similar reduction of total RNA pol II occupancy across target genes. Altogether, these observations suggest that disrupting RNA pol II pausing does not generally increase productive transcription, but results in reduced total RNA pol II occupancy and decreased expression. It is possible that premature pause release interferes with RNA pol II activation into elongation, resulting in abortive transcription that, in turn, could affect RNA pol II recruitment and/or re-initiation. Notice, however, that dDsk2 depletion does not affect the extent of histone acetylation detected at target promoters, suggesting that they retain the transcriptional active chromatin state (Kessler, 2015).

Notably, co-depletion of dUbp8/Nonstop, which rescues H2Bub1, also rescues the pausing defect caused by dDsk2 depletion and expression levels are restored, suggesting that dynamic regulation of H2Bub1 levels at promoters of dHP1c target genes plays a role in RNA pol II pausing. In this regard, work performed in yeast suggested that transcriptional activation involves sequential cycles of H2B ubiquitylation and deubiquitylation and that Ubp8 promotes Ctk1-dependent phosphorylation of Ser2 in the CTD33, a modification that is required for activation into the elongating Pol IIoser2 form. Nevertheless, H2Bub1 deubiquitylation at TSS does not appear to be sufficient by itself to induce pause release since dBre1 depletion, which also reduces H2Bub1 at TSS, has no significant effect in pausing. In this regard, it must be noted that dBre1 travels with the elongating RNA pol II along coding regions to induce H2Bub1, which stimulates Facilitates-Chromatin-Transcription (FACT) activity and, thus, facilitates elongation. On the other hand, dUbp8/Nonstop activity is mainly restricted to promoters since its depletion in dBre1-deficient cells has little effect in H2Bub1 levels at coding regions. On the contrary, dUbp8/Nonstop depletion in dDsk2-deficient cells strongly rescues H2Bub1 levels at coding regions. Interestingly, whereas dUbp8/Nonstop depletion restores expression of target genes in dDsk2-depleted cells, it has only a slight effect in dBre1-deficient cells. Altogether, these results suggest that dBre1 depletion impairs elongation and, thus, might prevent the release of paused RNA pol II by disturbing its actual engagement into elongation. Further work is required to better understand the mechanisms that regulate RNA pol II pausing, the actual contribution of dDsk2 and whether it involves H2Bub1 and/or additional factors also targeted by ubiquitylation (Kessler, 2015).

The dHP1c complex appears to have a particularly important contribution to nervous system development and function since target genes are enriched in related functions and knockdown conditions preferentially affect gene expression in the nervous system. Actually, WOC and ROW are highly expressed in the nervous system during embryo and larval development, and mutant larvae show brain defects. Furthermore, in humans, the WOC homologue DXS6673E/ZNF261 has been implicated in X-linked mental retardation. Interestingly, mutations in the human Dsk2 homologues (Ubqln-1/2) have been associated with Alzheimer's disease as well as other neurodegenerative diseases. Noteworthy, Ubqln-1/2 are detected in both the nucleus and the cytoplasm, and the development and progression of neurofibrillary tangles in Alzheimer's disease brains associate with an altered nuclear Ubqln-1 content. Whether the role of dDsk2 in transcription regulation is conserved in humans and contributes to disease remains to be determined (Kessler, 2015).

In summary, these results indicate that the ubiquitin receptor dDsk2 plays a nonproteolytic function in the regulation of H2Bub1 and RNA pol II pausing at promoters of dHP1c complex target genes. Ubiquitin receptors have been previously reported to play nonproteolytic functions in DNA repair and transcription elongation. Furthermore, in response to DNA damage, human Rad23B was found to interact with ubiquitylated p53, localize at chromatin and accumulate at the p21 promoter. In addition, in mouse embryonic stem cells, several components of the NER complex, including Rad23B, have been shown to act as an Oct4/Sox2 co-activator complex that associates with chromatin and is required for stem cell maintenance. Recruitment of NER factors to active promoters has also been reported in HeLa cells in the absence of DNA damage. However, in these cases, the precise function of the ubiquitin receptor has not been elucidated. In this regard, these results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications and transcription initiation. It must also be noted that ubiquitylation participates in the regulation of multiple genomic functions and that the number of proteins containing ubiquitin-binding domains is large, ~100 in humans. Therefore, a role of ubiquitin-binding proteins as epigenetic regulators of chromatin emerges as a distinct possibility (Kessler, 2015).

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. This study shows that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth (Wang, 2017).

A synapse is a specialized structure where signals are transmitted from a neuron to another neuron or other target cells such as muscles. Proper synapse formation is prerequisite to building functional synapses and constructing neuronal circuits. Synapse abnormalities are suggested to induce neurological and psychological disorders such as autism spectrum disorders and fragile X syndrome. Formation of postsynapses requires coordinated formation of several specialized structures. One prominent postsynaptic feature at neuromuscular junctions (NMJs) is the extensively folded muscular membranes. Specialized folding of postjunctional membranes is thought to increase the area exposed to the synaptic cleft and ensure the effectiveness of neuromuscular transmission. In addition to membrane specializations, the postsynaptic density (PSD) is also a common element whose size requires proper control. The PSD contains scaffolding proteins that recruit signaling protein complexes and neurotransmitter receptors, matching precisely the presynaptic active zones. Formations of postsynaptic membrane and PSD are tightly controlled and coordinated yet these processes remain elusive (Wang, 2017).

The Drosophila NMJ is a model to study synapse formation and activity-dependent synapse remodeling. Synaptic boutons are swollen structures of axonal terminals embedded in highly folded muscular membranes called the subsynaptic reticulum (SSR) and each bouton contains tens of neurotransmitter release sites paired with PSDs. During larval development, the SSR and the PSD concomitantly form and gradually increase their sizes. Two crucial factors, postsynaptic density protein-95/Discs large (Dlg) localized at the SSR and Drosophila p21-activating kinase (dPak) localized at the PSD, regulate SSR formation. At the PSD, two types of localized glutamate receptors (GluRs), IIA and IIB, appear in distinct GluR clusters. The abundance of GluRIIA at the PSD is regulated by PSD-localized dPak and the SSR-localized NF-κB complex, NF-κB/Dorsal (Dl), IκB/Cactus (Cact) and IRAK/Pelle (Pll) (Zhou, 2015 and references therein). Thus, the postsynaptic protein could localize at either SSR or PSD, and confer growth regulation on SSR, PSD or both (Wang, 2017).

Ubiquitination plays essential roles in various cellular processes including synaptic growth. Ubiquitin species are dynamically balanced among free and substrate-conjugated forms of mono-ubiquitin and ubiquitin chains. Ubiquitin homeostasis, i.e. the maintenance of diverse ubiquitin species in proper proportions and levels, is regulated in cellular growth and differentiation, a large superfamily of ubiquitin regulators, participate in the dynamic equilibrium of ubiquitin species. While some DUBs process newly synthesized ubiquitin precursors for ubiquitin supply, others recycle ubiquitin by cleaving ubiquitin chains from protein substrates prior to proteasomal degradation. USP5, the focus of this study, is dedicated to disassembly of free ubiquitin chains for recycling. Physiologically, heat shock stress in yeast causes a reduction of the mono-ubiquitin level. To compensate for ubiquitin depletion, the level of the DUB Doa4 is elevated, leading to an increase in the mono-ubiquitin level by cleaving free ubiquitin chains. The ataxia mice axJ, carrying mutations in the DUB USP14, displayed nerve swelling and abnormal neurotransmission at NMJs. The defects are caused by a reduction in the ubiquitin level as lower ubiquitin levels were detected in the mutant mice and introducing an ubiquitin transgene suppressed the axJ phenotypes. Thus, regulation of the ubiquitin level is a critical step in synapse development and for preventing neurological disorders (Wang, 2017).

Drosophila USP5/Leon is essential to maintain ubiquitin homeostasis during tissue formation and controls activation of apoptosis and the JNK pathway during eye development (Fan, 2014; Wang, 2014). This study characterized the role of Leon in postsynaptic growth after synapse formation. In leon mutants, while the presynapse maintains normal morphology, the postsynapse overelaborates, displaying expanded SSR, enlarged PSD and excess PSD-localized GluR clusters. Free ubiquitin chains and ubiquitinated substrates accumulate in leon postsynapses, revealing defects in ubiquitin homeostasis. Genetic analysis shows that accumulations of several postsynaptic proteins accounts for overelaborated postsynaptic structures. The ubiquitin receptor Ubiquilin (Ubqn) recognizes and transfers ubiquitinated substrates to the proteasome for degradation (Finley, 2009; Lipinszki, 2011). The Ubqn level is elevated in leon postsynapses and reducing the Ubqn level suppresses leon mutant phenotypes. Importantly, co-overexpression of free ubiquitin chains and Ubqn promotes expansion of these postsynaptic features. Thus, ubiquitin homeostasis such as disassembly of free ubiquitin chains, timely degradation of proteins, and normal function of the ubiquitin receptor Ubqn are compromised in leon mutants, leading to postsynaptic overgrowth (Wang, 2017).

Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly

The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. Previous work has characterized new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. The present study demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub(+)) TBPH pathology. Although it was found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, this study showed that an augmented soluble cytoplasmic Ub(+)-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. It was demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases (Jantrapirom, 2018b).

The protein turnover and disposal of misfolded proteins are fundamental processes that support normal cell functions. Protein synthesis, folding, conformational maintenance, and degradation require precise control in order to ensure normal cellular protein homeostasis. Post-mitotic cells, such as neurons, are markedly affected by aberrant protein turnover or the altered degradation of misfolded proteins, which are hallmarks of aging-associated proteinopathies, including neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's (PD) diseases (Jantrapirom, 2018b).

Previous studies reported that the cellular capacity to maintain proteostasis decreases with aging, rendering the organism susceptible to proteinopathies. However, mutations in genes that regulate protein homeostasis, such as valosin-containing protein (VCP) and p62/SQSTM1, which are involved in proteasomal and autophagosomal protein degradation; Tank-binding kinase 1 (TBK1) and optineurin (OPTN), which regulate autophagy; and VAPB, CHMP2B, and Alsin, which are critical for endosome trafficking and membrane remodeling , have also been associated with the early onset of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) with or without frontotemporal dementia (FTD). Furthermore, mutations in the ubiquitin (Ub) chaperone ubiquilin 2 (UBQLN2) have been identified as a cause of the X-linked forms of ALS/FTD. Collectively, these findings strongly suggest that the Ub pathology is a common pathway in several neurodegenerative diseases and aging (Jantrapirom, 2018b).

UBQLN2 belongs to a family of UBQLN proteins and plays a critical role in both proteasomal and autophagosomal protein degradation as a Ub receptor with the ability to recognize and bind polyubiquitinated substrates in order to target them for degradation. Different ALS-related UBQLN2 mutants show an aberrant trend towards accumulating in the cytosolic aggregates of degenerating neurons, which suggests the ability of these mutants to induce proteinopathy. Furthermore, recent studies demonstrated that some ALS-related UBQLN2 mutants exhibit defective Ub-binding abilities because they are unable to bind ubiquitinated substrates and fail to deliver them to proteasomes for degradation. A loss-of-function (LOF) and/or haploinsufficiency have been proposed as the predominant disease mechanisms for UBQLN2 mutations in ALS patients (Jantrapirom, 2018b).

The 43-kDa TAR DNA-binding protein (TDP-43) is a component of cytosolic inclusions in patients with ALS, Ub-positive frontotemporal lobar degeneration (FTLD-U) and another related disorder. The reduction of TDP-43 in the nucleus and its accumulation as the Ub-positive (Ub+) hyperphosphorylated cytoplasmic inclusions in spinal motor neurons have since been recognized as a common pathological feature of approximately 97% of ALS cases. Therefore, the pathomechanisms of ALS and aging-related dementia are now considered to be directly or indirectly linked to the TDP-43 pathology. The emerging role of the Ub-related pathology in neurodegenerative diseases, such as evidence for TDP-43 UBQLN2-positive inclusions and TDP-43 aggregates in ALS and FTLD patients with or without UBQLN2 and TDP-43 mutations, has revealed a relationship between UBQLN and TDP-43. Moreover, studies on yeast and HeLa cells have shown that UBQLN2 is a polyubiquitin-TDP-43 co-chaperone that mediates the autophagosomal delivery and/or proteasome targeting of TDP-43 aggregates. However, the mechanisms by which UBQLN2 functions in TDP-43 proteinopathies currently remain unclear (Jantrapirom, 2018b).

As described above, VCP is a highly conserved mechanoenzyme that contributes to the maintenance of protein homeostasis and has specialized functions in distinct cell types. VCP plays a role in essential cellular processes, and dysfunctional VCP was shown to be involved in pathophysiological states such as cancer, neurodegenerative disorders, and premature aging. Drosophila models have recently been developed to show that VCP physically and genetically interacts with TDP-43, and disease-causing mutations in VCP also affect the sub-cellular localization of TDP-43, similar to the above described effect induced by UBQLN2 mutations (Jantrapirom, 2018b).

Drosophila melanogaster is proving to be a very useful model for deepening understanding of UBQLN-associated neurological diseases because its genome carries only one human UBQLN orthologue (dUbqn), which shows highly conserved functional domains that are similar to human UBQLN1 and UBQLN2. A Drosophila UBQLN knockdown pathology has been modeled by the depletion of dUbqn, which induced locomotive dysfunctions and cognitive impairments in combination with severe structural defects in neuromuscular junctions (NMJs) and mushroom bodies (MBs). Therefore, this model can be used to gain insights into neurotoxicity associated with the knockdown of UBQLN and ultimately the TDP-43-related pathology. Furthermore, this study investigated the role of Drosophila VCP (dVCP) in dUbqn knockdown neurotoxicity, with a focus on its involvement in aberrantly ubiquitinated and mislocated TBPH (Jantrapirom, 2018b).

Depletion of dUbqn in the fly recapitulates a TDP-43 pathology, since TBPH was found to be aberrantly located in the cytoplasm as Ub+ partitions. Of relevance to the field, this study showed that a decline in dUbqn functions allowed the formation of at least two different Ub+ TBPH partitions, with the soluble partition being neurotoxic and the insoluble not being neurotoxic or potentially being tolerated by neurons. Moreover, by down-regulating the expression of TBPH in a proteostasis-impaired background with the TBPH pathology, this study rescued the locomotive abilities of flies. Under these conditions, the depletion of TBPH exerted positive effects by reducing the amount of soluble Ub+ TBPH (Jantrapirom, 2018b).

This study has highlighted the ability of dVCP to restore ubiquitin-dependent proteostasis that is impaired by the depletion of dUbqn and, more importantly, its ability to protectively restore proper TBPH turnover in order to reduce neurotoxic soluble Ub+ TBPH partitions. The expression of dVCP rescued dUbqn knockdown toxicity without recovering the nuclear localization of TBPH. This is of interest and warrants future extensive characterization. The present results suggest that in a proteostasis-impaired background, cytoplasmic TBPH gain-of-function may be more critical than nuclear TBPH LOF (Jantrapirom, 2018b).

Although the knockdown of dUbqn widely alters the turnover of several proteins, the present study revealed that the TBPH pathology is a crucial source of toxicity associated with the knockdown of dUbqn. Future investigations may benefit from these results, potentially leading to the development of therapeutic treatments for dysfunctional proteostasis in aging-associated neurodegenerative diseases (Jantrapirom, 2018b).

A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. This study performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. The depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. The results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD (Jantrapirom, 2018a).

Protein homeostasis is finely regulated by an extensive network of components and plays a critical role in maintaining normal cellular processes, and its deficiency, such as with aging, results in abnormal protein functionality. Apart from loss-of-function effects, a critical consequence of impaired proteostasis is the accumulation of cytotoxic protein aggregates, which have also been implicated in neurodegenerative diseases (Jantrapirom, 2018a).

UBQLN is a key component in maintaining appropriate proteostasis because it is involved in the transportation of polyubiquitinated proteins to proteasomes and autophagosomes. UBQLN was recently linked to ALS/FTLD because of its engagement in TDP43 and/or FUS-containing aggregates. Moreover, UBQLN2 mutations have been detected in ALS patients. This evidence confirmed the critical involvement of proteostasis in a wide spectrum of neurodegenerative diseases and suggests an important role for UBQLN in these diseases. However, the mechanistic involvement of UBQLN in ALS and FTLD has not yet been elucidated in detail (Jantrapirom, 2018a).

Drosophila carries a single dUbqn gene that is the only human UBQLN orthologue. dUbqn shows high similarity to human UBQLN1 and UBQLN2, both of which have been implicated in ALS/FTLD. No Drosophila model of ALS/FTLD targeting of dUbqn has yet been established (Jantrapirom, 2018a).

UBQLN is an UBL protein that binds ubiquitinated proteins and proteasome, thereby acting as a chaperone for protein degradation. Insufficient proteasome degradation of abnormal protein aggregates has been proposed to be a contributory factor to neuropathogenesis. Several studies have reported on the functional roles of UBQLN/dUbqn on neuronal functions such as RNAi silencing of dUbqn in CNS led to age-dependent neurodegeneration and shortened life span in flies, the overexpression of dUbqn in Drosophila eye-imaginal disc resulted in age-dependent retinal degeneration, mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have shown cognitive deficits, shortened life span and motor neuron degenerations. The present study confirmed a significant increase in ubiquitin chains and polyubiquitinated proteins as a consequence of the dUbqn knockdown, indicating that dUbqn is needed to maintain normal in the Drosophila brain. However, from these data it cannot be evaluated whether the effect of dUbqn knockdown on proteostasis is cell autonomous or not. Further analysis is necessary to clarify this point. The effects were examined of dUbqn depletion on locomotive abilities and learning/memory functions in the fly, which represent two distinctive features of ALS/FTLD. This study reports that neuron-specific dUbqn knockdown reduced locomotive abilities and induced a marked decline in cognitive activities and dUbqn loss-of-function could be responsible for these defects (Jantrapirom, 2018a).

Moreover, this study also demonstrated that the depletion of dUbqn caused morphological alterations in the neuronal structures designed for locomotion and learning/memory. For example, neuron-specific dUbqn knockdown resulted in an abnormal arrangement of abs, which are the primary neurons that form junctions with muscle-interacting secondary neurons. NMJs, which are required for appropriate synaptic transmission to muscles, also showed an aberrant morphology. MBs, which are the brain region required for learning and memory in the fly, also exhibited an altered morphology. Collectively, these results suggest that dUbqn is involved in neuronal dysfunction because of its role in the formation or maintenance of critical neuronal circuits; therefore, impaired proteostasis may alter the structures of fundamental synapses, thereby driving the distinctive deficits observed in ALS/FTLD (Jantrapirom, 2018a).

This study provides a new model to examine the role of impaired proteostasis in neurodegenerative diseases, which may expand knowledge on the critical functions of UBQLN in the nervous system. Due to the key role of UBQLN in the fate of a number of proteins, future studies are needed in order to examine whether the targets of UBQLN are associated with aberrant neurological functions. Therefore, the use of the novel Drosophila model described herein may provide interesting data and be applicable to the screening of promising ALS/FTLD therapies (Jantrapirom, 2018a).

Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS

Members of the conserved ubiquilin (UBQLN) family of ubiquitin (Ub) chaperones harbor an antipodal UBL (Ub-like)-UBA (Ub-associated) domain arrangement and participate in proteasome and autophagosome-mediated protein degradation. Mutations in a proline-rich-repeat region (PRR) of UBQLN2 cause amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD); however, neither the normal functions of the PRR nor impacts of ALS-associated mutations within it are well understood. This study shows that ALS mutations perturb UBQLN2 solubility and folding in a mutation-specific manner. Biochemical impacts of ALS mutations were additive, transferrable to UBQLN1, and resulted in enhanced Ub association. A Drosophila melanogaster model for UBQLN2-associated ALS revealed that both wild-type and ALS-mutant UBQLN2 alleles disrupted Ub homeostasis; however, UBQLN2^ALS mutants exhibited age-dependent aggregation and caused toxicity phenotypes beyond that seen for wild-type UBQLN2. Although UBQLN2 toxicity was not correlated with aggregation in the compound eye, aggregation-prone UBQLN2 mutants elicited climbing defects and NMJ abnormalities when expressed in neurons. An UBA domain mutation that abolished Ub binding also diminished UBQLN2 toxicity, implicating Ub binding in the underlying pathomechanism. It is proposed that ALS-associated mutations in UBQLN2 disrupt folding and that both aggregated species and soluble oligomers instigate neuron autonomous toxicity through interference with Ub homeostasis (Kim, 2018).

This study has characterized biochemical properties of wild-type and ALS-mutant UBQLN2 proteins and constructed Drosophila models to understand phenotypic impacts of disease-associated mutations in the UBQLN2 PRR. ALS mutations were shown to exert non-identical effects on UBQLN2 solubility and folding and cause mutation- and tissue-specific toxicities in Drosophila. These findings further implicate Ub binding as a central feature of UBQLN2 pathomechanisms (Kim, 2018).

The clustering of ALS mutations within or proximal to the functional-orphan PRR domain imply that such mutations interfere with cellular processes that are unique to UBQLN2 and/or initiate toxic folds that disrupt cellular regulation through GOF mechanisms. This study tested five clinical mutations (P497H, P497S, P506T, P509S, P525S) and found that only P497H consistently promoted insolubility beyond wild-type levels (Fig. 1). His substitutions at disease codons Pro-506 and Pro-509 did not elicit the same changes in solubility as the P497H mutation, indicating that both the nature of the substitution (i.e. Pro to His) and its position within the PRR critically determine the extent of insolubility. Although individual P506T, P509S, and P525S mutations had minor effects on solubility, their combination in UBQLN2^2P3X (P506T, P509S, and P525S) and UBQLN2^2P4X (P497H, P506T, P509S, and P525S) proteins led to severe reductions in solubility and patent neuronal aggregation. Although results using compound mutants must be interpreted cautiously, these findings suggest that ALS mutations elicit additive or synergistic effects on UBQLN2 folding (Kim, 2018).

The increased immunoreactivity of UBQLN2^P497H, UBQLN2^2P3X, and UBQLN2^2P4X with antibodies directed against amino-terminal epitope tags and endogenous UBQLN2 peptides supports the notion that localized mutations in the PRR lead to global changes in UBQLN2 folding, which is further bolstered by the enhanced chymotryptic sensitivity of UBQLN2^2P3X and UBQLN2^2P4X in cell culture and fly heads. Interestingly, age-dependent increases in chymotryptic cleavage of UBQLN2 proteins expressed in fly heads was observed, and aging dependent increases in the aggregation of UBQLN2^WT and UBQLN2^P497H in fly neurons suggesting that UBQLN2 folding was sensitive to the aging cellular environment. It is speculated that ALS mutations disrupt intramolecular folding between the PRR and amino-terminal domains and/or inhibit functional intermolecular oligomerization, which is thought to be mediated by centrally located STI1 repeats. Structural studies of wild-type and ALS-mutant UBQLN2 proteins should further inform these possibilities (Kim, 2018).

The Drosophila findings revealed significantly stronger degenerative phenotypes in flies expressing UBQLN2^ALS alleles versus UBQLN2^WT and support the idea that Ub-binding figures prominently in UBQLN2-mediated toxicity. Both wild-type and ALS-mutant UBQLN2 proteins altered Ub homeostasis in an UBA domain-dependent manner, likely contributing to non-specific impacts on eye structure, survival, and climbing behavior observed in this and other studies. Nevertheless, enhanced toxicity of UBQLN2^ALS mutants was seen across a host of assays. Neuronally expressed UBQLN2^P497H reduced viability, conferred NMJ abnormalities, and diminished climbing to a greater extent than UBQLN2^WT; whereas eye-directed expression of UBQLN2^P497H and UBQLN2^P525S elicited severe bristle loss and hyperpigmented eye patches. Enhanced phenotypes in UBQLN2^ALS flies may enable identification of disease-specific modifier genes (Kim, 2018).

Somewhat unexpectedly, UBQLN2^P4X was less toxic than UBQLN2^WT, UBQLN2^P497H and UBQLN2P525S in the eye, and was homozygous viable when expressed in neurons at room temperature, indicating that cellular toxicity of UBQLN2 is not directly correlated to its aggregation potential. Nevertheless, Elav > UBQLN2^2P4X/UAS-UBQLN2^2P4X flies exhibited severe climbing defects and UBQLN2^2P4X conferred NMJ abnormalities that were comparable to those seen for UBQLN2^P497H. From the combined findings, it is proposed that soluble, partially misfolded UBQLN2 oligomers and/or microaggregates are the primary toxic species as has been proposed for neurodegeneration-associated mutants of SOD1 and Huntingtin. UBQLN2 macroaggregates, amplified by the artificial P4X mutation, also contribute to neurodegeneration, perhaps through pathways that are distinct from those initiated by soluble proteins. Critical pathways mediating each mode of UBQLN2 toxicity should be illuminated through genetic modifier screens using the suite of Drosophila UBQLN2^ALS models described in this study (Kim, 2018).

Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality in Drosophila melanogaster that can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit

Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference-mediated silencing of these receptors can be induced. Flies overexpressing Flag-p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag-p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag-Dsk2 and Flag-p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2-26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with DeltaRpn10 proteasomes and not with the wild-type one. In Drosophila, Dsk2 does not interact with Deltap54 proteasomes, but the interaction can be fully restored by complementing the Deltap54 deletion with Flag-p54 (Lipinszki, 2011).

Ubiquilin modifies TDP-43 toxicity in a Drosophila model of amyotrophic lateral sclerosis (ALS)

TDP-43 (43-kDa TAR DNA-binding protein) is a major constituent of ubiquitin-positive cytosolic aggregates present in neurons of patients with amyotrophic lateral sclerosis (ALS) and ubiquitin-positive fronto-temporal lobar degeneration (FTLD-U). Inherited mutations in TDP-43 have been linked to familial forms of ALS, indicating a key role for TDP-43 in disease pathogenesis. This study describes a Drosophila melanogaster model of TDP-43 proteinopathy. Expression of wild-type human TDP-43 protein in Drosophila motor neurons led to motor dysfunction and dramatic reduction of life span. Interestingly, coexpression of ubiquilin 1, a previously identified TDP-43-interacting protein with suspected functions in autophagy and proteasome targeting, reduced steady-state TDP-43 expression but enhanced the severity of TDP-43 phenotypes. Finally, ectopically expressed TDP-43 was largely localized to motor neuron nuclei, suggesting that expression of wild-type TDP-43 alone is detrimental even in the absence of cytosolic aggregation. These findings demonstrate that TDP-43 exerts cell-autonomous neurotoxicity in Drosophila and further imply that dose-dependent alterations of TDP-43 nuclear function may underlie motor neuron death in ALS (Hanson, 2010).

Role of X11 and ubiquilin as in vivo regulators of the amyloid precursor protein in Drosophila

The Amyloid Precursor Protein (APP) undergoes sequential proteolytic cleavages through the action of beta- and gamma-secretase, which result in the generation of toxic beta-amyloid (Abeta) peptides and a C-terminal fragment consisting of the intracellular domain of APP (AICD). Mutations leading to increased APP levels or alterations in APP cleavage cause familial Alzheimer's disease (AD). Thus, identification of factors that regulate APP steady state levels and/or APP cleavage by gamma-secretase is likely to provide insight into AD pathogenesis. Using transgenic flies that act as reporters for endogenous gamma-secretase activity and/or APP levels (GAMAREP), and for the APP intracellular domain (AICDREP), this study identified mutations in X11L and ubiquilin (ubqn) as genetic modifiers of APP. Human homologs of both X11L (X11/Mint) and Ubqn (UBQLN1) have been implicated in AD pathogenesis. In contrast to previous reports, this study showed that overexpression of X11L or human X11 does not alter gamma-secretase cleavage of APP or Notch, another gamma-secretase substrate. Instead, expression of either X11L or human X11 regulates APP at the level of the AICD, and this activity requires the phosphotyrosine binding (PTB) domain of X11. In contrast, Ubqn regulates the levels of APP: loss of ubqn function leads to a decrease in the steady state levels of APP, while increased ubqn expression results in an increase in APP levels. Ubqn physically binds to APP, an interaction that depends on its ubiquitin-associated (UBA) domain, suggesting that direct physical interactions may underlie Ubqn-dependent regulation of APP. Together, these studies identify X11L and Ubqn as in vivo regulators of APP. Since increased expression of X11 attenuates Abeta production and/or secretion in APP transgenic mice, but does not act on gamma-secretase directly, X11 may represent an attractive therapeutic target for AD (Gross, 2008).

ubiquilin antagonizes presenilin and promotes neurodegeneration in Drosophila

The majority of familial Alzheimer's disease (AD) cases are caused by mutations in presenilins, therefore, identifying regulators of presenilins is crucial for understanding AD pathogenesis. Ubiquilin 1 (UBQLN1) binds Presenilins in mammalian cells; however, the functional significance of this interaction in vivo remains unclear. Moreover, while genetic variants in UBQLN1 have recently been reported to associate with an increased risk for AD, whether these variants have altered function is unknown. This study shows that Drosophila Ubiquilin (Ubqn) binds to Drosophila Presenilin (Psn), and that loss of ubqn function suppresses phenotypes that arise from loss of psn function in vivo. In addition, overexpression of ubqn in the eye results in adult-onset, age-dependent retinal degeneration, which is at least partially apoptotic in nature. The degeneration associated with ubqn overexpression can also be suppressed by psn overexpression and enhanced by expression of a dominant negative version of Psn. Remarkably, expression of the human AD-associated variant of UBQLN1 leads to more severe degeneration than does comparable expression of the human wildtype UBQLN1. Together, these data identify Ubqn as a regulator of Psn, support an important role for UBQLN1 in AD pathogenesis, and suggest the possibility that expression of a human AD-associated variant can cause neurodegeneration independent of amyloid production (Ganguly, 2008).

Isolation and characterization of the Drosophila ubiquilin ortholog dUbqln: in vivo interaction with early-onset Alzheimer disease genes

UBQLN1 variants have been associated with increased risk for late-onset Alzheimer's disease (AD). This study produced transgenic Drosophila models that either silence (by RNAi) or overexpress the Drosophila ortholog of human UBQLN1, dUbqln. Silencing of dUbqln in the central nervous system led to age-dependent neurodegeneration and shortened lifespan. Silencing of dUbqln in the wing led to wing vein loss that could be partially rescued by mutant rhomboid (rho), a known component of epidermal growth factor receptor signaling pathway. Conversely, overexpression of dUbqln promoted ecotopic wing veins. Overexpression of dUbqln in the eye rescued a small, rough eye phenotype induced by overexpression of Drosophila presenilin (dPsn), and also rescuing dPsn-induced malformations in bristles. In contrast, RNAi silencing of dUbqln enhanced the retinal degenerative defect induced by overexpression of dPsn. Finally, co-overexpression of dUbqln and the human amyloid precursor protein (APP) in the eye significantly reduced the levels of full-length APP and its C-terminal fragment. Collectively, these data support in vivo functional interaction between UBQLN1 and the AD-associated genes, presenilin and APP, and provide further clues regarding the potential role of UBQLN1 in AD pathogenesis (Li, 2007).

Identification of Ubiquilin, a novel Presenilin interactor that increases Presenilin protein accumulation

Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. The properties are described of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, ubiquilin promotes presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life. Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies reveal prominent staining of neurons. Moreover, the anti-ubiquilin antibodies robustly stain neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively. These results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain (Mah, 2000).

It will be interesting to determine the precise mechanism by which ubiquilin induces increased presenilin protein synthesis. Ubiquilin could increase presenilin synthesis by simply increasing presenilin transcription, increasing presenilin translation, or facilitating correct polypeptide folding, maturation, and intracellular targeting of the polytopic transmembrane presenilin protein. The possibility that ubiquilin may act as a molecular chaperone is especially intriguing. Studies of the Xenopus ubiquilin homologue, XDRP1, have suggested that ubiquilin can act posttranscriptionally like a molecular chaperone and prevent degradation of in vitro translated cyclin A protein. Chap1 (ubiquilin 2) has been shown to bind Stch, an Hsp70-like protein. In turn, many heat-shock proteins have been shown to function as molecular chaperones, preventing protein aggregation and protein degradation. Recent evidence has linked ubiquilin proteins to the proteasome. Meanwhile, the 19S regulatory subunit of the 26S proteasome (the degradation complex for ubiquitin-tagged proteins) has been shown to possess protein-unfolding activity. Indirect evidence that ubiquilin may aid in presenilin protein folding or targeting comes from the observation that the presenilin construct PS2(DeltaLC), with deletions of both the loop and COOH-terminal ubiquilin-interaction sites, frequently accumulates into large cytoplasmic aggregates. In contrast, presenilin molecules containing ubiquilin interaction sites, rarely form large protein aggregates. Finally, the presenilins have themselves been linked to molecular chaperones of the ER, that are involved in the unfolded-protein response. Another mechanism by which ubiquilin might increase presenilin accumulation is to alter presenilin degradation rates, especially those of the ubiquitinated forms of presenilins. In fact, evidence has been found that overexpression of human ubiquilin proteins, hPLIC-1 (ubiquilin 1) and hPLIC-2 (ubiquilin 2), interfers with ubiquitin-dependent degradation of p53 and IkBalpha. Although no significant change in the turnover rate of the major 54-kD PS2 polypeptide species (corresponding to full-length PS2) has been found, the possibility that certain ubiquitinated forms of presenilins may have altered turnover rates cannot be excluded. It will be important in future studies to determine if ubiquilin is involved in ubiquitin-dependent degradation of presenilins (Mah, 2000 and references therein).

Search PubMed for articles about Drosophila Ubiquilin

Fan, X., Huang, Q., Ye, X., Lin, Y., Chen, Y., Lin, X. and Qu, J. (2014). Drosophila USP5 controls the activation of apoptosis and the Jun N-terminal kinase pathway during eye development. PLoS One 9(3): e92250. PubMed ID: 24643212

Finley, D. (2009). Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78: 477-513. PubMed ID: 19489727

Ganguly, A., Feldman, R. M. and Guo, M. (2008). ubiquilin antagonizes presenilin and promotes neurodegeneration in Drosophila. Hum Mol Genet 17(2): 293-302. PubMed ID: 17947293

Gross, G. G., Feldman, R. M., Ganguly, A., Wang, J., Yu, H. and Guo, M. (2008). Role of X11 and ubiquilin as in vivo regulators of the amyloid precursor protein in Drosophila. PLoS One 3(6): e2495. PubMed ID: 18575606

Hanson, K. A., Kim, S. H., Wassarman, D. A. and Tibbetts, R. S. (2010). Ubiquilin modifies TDP-43 toxicity in a Drosophila model of amyotrophic lateral sclerosis (ALS). J Biol Chem 285(15): 11068-11072. PubMed ID: 20154090

Jantrapirom, S., Piccolo, L. L., Yoshida, H. and Yamaguchi, M. (2018b). Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly. Biochim Biophys Acta. PubMed ID: 29936333

Jantrapirom, S., Lo Piccolo, L., Yoshida, H. and Yamaguchi, M. (2018a). A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities. Exp Cell Res 362(2): 461-471. PubMed ID: 29247619

Kessler, R., Tisserand, J., Font-Burgada, J., Reina, O., Coch, L., Attolini, C. S., Garcia-Bassets, I. and Azorin, F. (2015). dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes. Nat Commun 6: 7049. PubMed ID: 25916810

Kim, S. H., Stiles, S. G., Feichtmeier, J. M., Ramesh, N., Zhan, L., Scalf, M. A., Smith, L. M., Pandey, U. B. and Tibbetts, R. S. (2018). Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS. Hum Mol Genet 27(2):322-337. PubMed ID: 29161404

Li, A., Xie, Z., Dong, Y., McKay, K. M., McKee, M. L. and Tanzi, R. E. (2007). Isolation and characterization of the Drosophila ubiquilin ortholog dUbqln: in vivo interaction with early-onset Alzheimer disease genes. Hum Mol Genet 16(21): 2626-2639. PubMed ID: 17704509

Lipinszki, Z., Pal, M., Nagy, O., Deak, P., Hunyadi-Gulyas, E. and Udvardy, A. (2011). Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality in Drosophila melanogaster that can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit. FEBS J 278(24): 4833-4844. PubMed ID: 21973017

Mah, A. L., et al. (2000). Identification of Ubiquilin, a novel Presenilin interactor that increases Presenilin protein accumulation. J. of Cell Bio. 151: 847-862. 11076969

Wang, C. H., Chen, G. C. and Chien, C. T. (2014). The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development. Biochem Biophys Res Commun. PubMed ID: 25152394

Wang, C. H., Huang, Y. C., Chen, P. Y., Cheng, Y. J., Kao, H. H., Pi, H. and Chien, C. T. (2017). USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin. Elife 6. PubMed ID: 28489002

Zhou, B., Lindsay, S. A. and Wasserman, S. A. (2015). Alternative NF-κB isoforms in the Drosophila neuromuscular junction and brain. PLoS One 10: e0132793. PubMed ID: 26167685

date revised: 14 September 2018

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