BIOLOGICAL OVERVIEW

The BMP receptor Wishful thinking (Wit) is required for synapse stabilization. In the absence of BMP signaling, synapse disassembly and retraction ensue. Remarkably, downstream Smad-mediated signaling cannot fully account for the stabilizing activity of the BMP receptor. LIM Kinase1 (LIMK1)-dependent signaling has been identified as a second, parallel pathway that confers the added synapse-stabilizing activity of the BMP receptor. LIMK1 binds a region of the Wit receptor that is necessary for synaptic stability but is dispensable for Smad-mediated synaptic growth. A genetic analysis demonstrates that LIMK1 is necessary, presynaptically, for synapse stabilization, but is not necessary for normal synaptic growth or function. Furthermore, presynaptic expression of LIMK1 in a wit or mad mutant significantly rescues synaptic stability, growth, and function. LIMK1 localizes near synaptic microtubules and functions independently of ADF/cofilin, highlighting a novel requirement for LIMK1 during synapse stabilization rather than actin-dependent axon outgrowth (Eaton, 2005).

Throughout the vertebrate nervous system, precisely wired neural circuitry is established through an initial overproduction of synaptic connections followed by the selective loss of a subset of these synapses. The molecular mechanisms that specify which synapses will be retained and which will be lost are unknown. There is additional complexity because synapse disassembly occurs coincidently with synapse growth in most systems. For example, live imaging studies have shown that single motoneurons can simultaneously disassemble synapses at some target muscles while increasing the size of synaptic connections at other muscle targets. Similar observations have been made in live imaging studies of dendritic remodeling of vertebrate central neurons. Thus, a molecular understanding of synapse development and neural circuit formation will require investigation of both synaptic growth and stabilization and an understanding of how these two processes are coordinately controlled (Eaton, 2005).

It is well established that cell growth, synapse formation, and synaptic competition are influenced by the action of trophic signaling molecules. Increased expression or activity of the neurotrophins can promote synapse formation and enhance synapse dynamics. Conversely, impaired neurotrophin signaling can lead to decreased synapse number through effects on synaptic growth and stability in both the central and peripheral nervous systems. The current challenge is to dissect the signaling systems downstream of these trophic receptors to determine how receptor activation could influence the seemingly disparate processes of synaptic growth, synaptic stability, and synapse disassembly. To investigate this, a trophic signaling system recently identified at the Drosophila NMJ has been investigated (Eaton, 2005).

The canonical bone morphogenic protein (BMP) signaling system has been implicated in diverse cellular and developmental processes ranging from cell growth to tissue patterning. At the Drosophila NMJ, a BMP signaling system has been identified that controls synaptic growth via canonical Smad-mediated signaling to the cell body. It has been demonstrated that mutations in the BMP ligand glass bottom boat (gbb), the type I and type II BMP receptors thick veins (tkv) and wishful thinking (wit), and the Smad homologs mad and medea all significantly impair synaptic growth and function. These data define a retrograde trophic signaling system that functions through transcriptional mechanisms in the cell soma to control motoneuron synaptic growth. BMP signaling at the Drosophila NMJ is not only required for normal synaptic growth, but also for synaptic stabilization. In the absence of BMP signaling, significant increases are observed in synapse retraction and disassembly. Signaling downstream of the BMP receptors can be genetically separated into two pathways: Smad-dependent synaptic growth and LIM Kinase1-dependent synaptic stability (Eaton, 2005).

LIM Kinase1 (LIMK1) is a cytoplasmic serine/threonine kinase that was originally isolated in screens for novel kinases expressed in the nervous system (Bernard, 1994; Cheng, 1995; Mizuno, 1994; Proschel, 1995). Findings in LIMK1 knockout mice reveal defects in dendritic spine morphology and activity-dependent plasticity, although neither synaptic growth nor synaptic stability has been specifically analyzed (Endo, 2003). In the Drosophila central nervous system, LIMK1 has been implicated in the mechanisms of axonal outgrowth during metamorphosis, acting through ADF/cofilin (see Drosophila Twinstar) to modulate the actin cytoskeleton, a mechanism also documented in other tissues (Arber, 1998; Ng, 2004; Ohashi, 2000; Eaton, 2005 and references therein).

Genetic analyses define a new function for LIMK1 during synaptic stability in comparison with its function in axonal outgrowth. Synaptic LIMK1 is closely associated with the synaptic microtubule cytoskeleton. In addition, genetic manipulation of ADF/cofilin activity does not affect synaptic stability at the NMJ. These data highlight differences in LIMK1 function during the rapid, dynamic process of axon outgrowth compared to the slower, more prolonged mechanisms that govern synapse stabilization at the NMJ. Together, these data define genetically separable signaling pathways downstream of the BMP receptor that could allow a single trophic signaling event to coordinately control synaptic growth and synaptic stabilization (Eaton, 2005).

Thus BMP-receptor signaling at the Drosophila NMJ controls both synaptic growth and synaptic stabilization. The data support a model in which signaling from the BMP receptor can coordinately activate two genetically separable developmental programs: (1) cell-wide regulation of neuronal growth via nuclear Smad signaling and (2) LIMK1-dependent synaptic stabilization. This organization of synaptic signaling systems involved in synaptic growth versus synaptic stabilization could have important consequences for neural development. Since a single trophic receptor can increase both cell-wide growth and synaptic stabilization, the efficiency of trophic signaling could be increased due to the coupling of new synapse growth with synaptic stabilization and retention. Another scenario is also possible that could help to explain how synaptic growth and elimination could occur simultaneously at the terminals of a single motoneuron. If LIMK1 functions locally at the synapse, then cell-wide growth signaling could be uncoupled from the local control of synaptic stability. Thus, the loss of trophic signaling at a single muscle target could lead to target-specific synapse destabilization while the same trophic signaling system at other muscle targets could simultaneously promote synaptic growth throughout the motoneuron via cell-wide growth signaling. While there is no direct evidence that LIMK1 functions locally at the synapse, the synaptic activation of kinase signaling downstream of the BMP receptor could support such a model (Eaton, 2005).

The data support a model in which LIMK1 functions downstream of the Wit receptor and in parallel to Smad-mediated nuclear signaling in order to achieve wild-type synaptic stability. Several lines of evidence indicate that LIMK1 functions downstream of the Wit receptor. LIMK1 binds to a region of the Wit receptor that is necessary for synaptic stabilization, but which is dispensable for Smad-mediated synaptic growth. LIMK1 mutations specifically disrupt synaptic stabilization, and both LIMK1 and Wit are found to be are necessary within the presynaptic neuron for synapse stability. A strong transheterozygotic genetic interaction has been demonstrated between mutations in LIMK1 and wit, resulting in a specific loss of synaptic stability. Further, neuronal overexpression of a dominant negative LIMK1 in the wit mutant background does not result in an additive increase in the number of synapse retractions. From these data, it is concluded that LIMK1 functions downstream of Wit to control synaptic stability. Finally, it is shown that the stabilizing activity of LIMK1 does not require the presence of the Smad signaling system, since expression of LIMK1 in either the wit or the mad mutant background is able to restore synaptic stability to the NMJ. While these data are consistent with LIMK1 functioning either downstream or in parallel to Smad-mediated signaling, the conclusion that LIMK1 functions in parallel to Smad is favored because LIMK1 binds a region of the Wit receptor that is dispensable for Smad-mediated synaptic growth (Eaton, 2005).

The data demonstrate that signaling via the Wit receptor stabilizes the synapse via both Smad-dependent and LIMK1-dependent signaling. Mutations in mad, medea, and expression of the inhibitory Smad dad (DAD-GOF) all cause an increase in synapse retractions as well as a decrease in bouton number. The data indicate that Smad-mediated signaling accounts for approximately 50% of the stabilizing activity of the Wit receptor, and LIM Kinase functions in parallel to mediate the other 50% of the Wit receptor’s stabilizing activity. It is hypothesized that the stabilizing functions of Smad signaling and LIMK1 signaling are quite different. It is suspected that Smad-dependent synaptic stabilization is directly coupled to the growth-promoting function of the nuclear Smad signaling system. It is hypothesized that Smad-dependent growth regulation involves transcriptional programs that produce the necessary raw material for synaptic growth. In the absence of Smad signaling, these raw materials may become limiting, not only for new growth but also for the maintenance of the existing synapse, since synaptic proteins will need to be turned over and replaced at some rate. According to this logic, the synapse retractions caused by mutations in the Smad signaling system are related to synaptic atrophy. In contrast, LIMK1 is necessary for synaptic stability, but is not required for normal synaptic growth. Thus, it is suspected that synapse retractions observed in LIMK1 mutants are caused by modulation of the synaptic stabilization machinery resident at the synapse (Eaton, 2005).

Smad-independent signaling of TGF-β, including the BMP receptors, has been observed in a number of systems and includes the activation of other diverse signaling cascades such as MAPK, PI3K, Rho-like GTPases, and LIMK1. However, only LIMK1 has been associated biochemically with the region of the BMP type II receptor that is specifically required for synaptic stabilization in the Wit receptor (Foletta, 2003; Lee-Hoeflich, 2004). In vitro kinase assays demonstrate that the interaction of LIMK1 with the BMPRII tail leads to changes in LIMK1 kinase activity (Foletta, 2003; Lee-Hoeflich, 2004). Although these studies reached different conclusions about the effects of receptor binding on LIMK1 activity, both studies support a model in which binding of BMP to the receptor complex leads to an increase in LIMK1 activity. It is proposed that ligand binding to the Wit receptor activates LIMK1 to stabilize the NMJ (Eaton, 2005).

It is remarkable that the overexpression of LIMK1 rescues all aspects of the wit receptor mutant phenotype, including synaptic growth, synaptic function, and animal viability. LIMK1 expression is also sufficient to restore synaptic growth and stability to the mad mutation. These data contrast with the genetic analysis demonstrating that LIMK1 is necessary for synaptic stability but is not required for normal synaptic growth and has only a minor role in functional synapse development. How canLIMK1 expression restore synaptic growth, function, and viability in the absence of the BMP receptor? Even if LIMK1 independently signals to the nucleus, it seems unlikely that LIMK1 activity would be sufficient to restore the entire transcriptional program normally mediated by nuclear Smad signaling. Instead, it is proposed that the overexpression of LIMK1 in the wit receptor mutant background hyperstabilizes the synapse, consistent with synaptic stabilization being the primary function of LIMK1. If UAS-LIMK1 hyperstabilizes the synapse, a second, parallel growth factor signaling system may thereby be allowed to assume the growth-promoting functions normally mediated by BMP signaling. This hypothesis invokes the existence of an unidentified second growth factor signaling cascade at the Drosophila NMJ. It seems likely that additional growth factor signaling exists since the synapse still grows to nearly 50% of its normal size in the wit mutant background. One candidate for a second synaptic growth signaling system is the activin signaling system. In the Drosophila central nervous system, ecdysone-regulated axonal remodeling during metamorphosis is regulated, in part, by signaling via the activin type I receptor Babo, the type II BMP receptor Put, and DSmad2. It should be noted, however, that the Put type II receptor lacks the C-terminal LIMK1 binding region found in the Wit receptor. Therefore, the Put receptor probably does not normally signal via direct interactions with LIMK1 during the remodeling of the CNS or during stabilization at the NMJ (Eaton, 2005).

In lower motor diseases, such as ALS, two events are thought to contribute significantly to disease progression: (1) the loss of nuclear trophic signaling due to impaired axonal transport, and (2) the loss of access to trophic signal due to synapse retraction. The ability of LIMK1 to stabilize the NMJ in the absence of trophic signaling might suggest a model in which activation of stability-promoting proteins, such as LIMK1, may counteract inappropriate synaptic disassembly during disease. It is interesting to note that LIMK1 has been shown to accumulate within the presynaptic nerve terminal during the late maturation of the mammalian NMJ, after the period of developmental plasticity observed during embryonic and early postnatal development (Wang, 2000). This observation supports an intriguing model in which the maturation of the mammalian NMJ from a highly plastic synapse early in development to a more stable adult synapse involves both the reduction of plasticity-associated proteins, such as CAP-23 and GAP-43, and the enrichment of stability-promoting proteins such as LIMK1. It remains to be determined whether synaptic loss in degenerative disease is directly related to the loss of stabilizing factors such as LIMK1 and, indeed, whether enhanced signaling from these factors could lead to restabilization of diseased synapses. However, the possibility that LIMK1 may function locally at the NMJ to promote synaptic stabilization, even in the absence of an essential source of trophic signaling, suggests a potent activity of LIMK1 in the nervous system that may have important therapeutic value as a future avenue for intervention in neuromuscular degenerative disease (Eaton, 2005).

GENE STRUCTURE

cDNA clone length - 4569 bp

Bases in 5' UTR - 240

Exons - 6

Bases in 3' UTR - 555

PROTEIN STRUCTURE

Amino Acids - 1235 aa (LIMK1-PA)

Structural Domains

LIMK1 contains two LIM domains and a PDZ domain in the N-terminal half and a kinase domain in the C-terminal half. The role was examined of the extra-catalytic region in the regulation of kinase activity of LIMK1. Limited proteolysis of LIMK1 results in the production of the 35-40-kDa kinase core fragments with 3.5 to 5.5-fold increased kinase activity. The LIMK1 mutants with deleted LIM domains (DeltaLIM) or conserved cysteines in the two LIM domains replaced with glycines (dmLIMK1) had 3-7-fold higher kinase activities in vitro, compared with the wild-type LIMK1. The C-terminal kinase fragment of LIMK1 binds to the LIM domain but not to the PDZ domain. Furthermore, the LIM fragment dose-dependently inhibits the kinase catalytic activity of the kinase core fragment of LIMK1. Taken together, these results suggest that the N-terminal LIM domain negatively regulates the kinase activity of LIMK1 by direct interaction with the C-terminal kinase domain. In addition, expression of the DeltaLIM mutant in cultured cells induces punctate accumulation of actin filaments, an event distinct from the pattern of actin organization induced by expression of the wild-type LIMK1, suggesting that the LIM domain plays a role in the function of LIMK1 in vivo (Nagata, 1999).

Two isoforms of LIMK transcripts were identified, coding for proteins with 1235 and 1257 amino acids, possessing the structure composed of two LIM domains, a PDZ domain, a protein kinase domain, and an unusual long C-terminal extension. By scanning the Drosophila EST database, a 656-bp sequence of an EST clone was identifed showing a significant homology to the cDNA sequence corresponding to the PDZ region of human LIMK1. Sequence analysis of the 4.0-kb insert of the EST clone GH23615 revealed that it codes for DLIMK with a poly(A) sequence at the 39-terminal, but is incomplete in the 59-terminal. To isolate clones containing the 59-region of DLIMK cDNA, 59-RACE analysis was carried out, using as a template a Drosophila embryo cDNA library. Of the 12 clones isolated, 9 encoded the N-terminal region of DLIMK containing LIM and PDZ domains (termed the short form) with various lengths of 5'-untranslated region, while one clone contained a 66-bp extra sequence in the open reading frame corresponding to the PDZ region of the short form (termed the long form). Two other clones and the original GH23615 clone contained this extra 66-bp but 5'-terminal sequences were completely different from those of the short and long forms of DLIMK. Alignment of the assembled cDNA sequences of DLIMK with the Drosophila genome sequence revealed relationships between the DLIMK gene (CG1848) and its cDNA sequences and the exon/intron organization of the DLIMK gene. The gene spans approximately 6.7 kb. The cDNA for the short form of DLIMK, coding for a protein of 1235 amino acids, is derived from 5 exons, while the cDNA for the long form of DLIMK (coding for a protein of 1257 amino acids) is derived from 6 exons including a 66-bp extra exon (exon 29) in between exons 2 and 3. The GH23615 clone, which contains exon 2' and a part of an intron between exons 2 and 2' at the 5'-terminal, appears to be transcribed from a position in the intron between exons 2 and 2' to produce the N-terminally truncated form (1043 amino acids) of DLIMK, similar to the case of a testis-specific alternative transcript (LIMK2t) of mouse LIMK2 gene. The amino acid sequence of DLIMK-(short) deduced in this study is distinct from that in the genome database (AAF48176), which contains extra 20 amino acids by predicting a 60-bp intron between exons 4 and 5 as a coding region. Analysis of the Drosophila genome database revealed that the DLIMK gene (CG1848) is localized in the cytological position 11B10-13 on chromosome X (Ohashi, 2000).

Alignment of the deduced amino acid sequence of the short form of DLIMK with those of human LIMK1 and LIMK2 reveals that DLIMK(long) has extra amino acids DLFCNFFLWLTSMAEQCCGAPYR, in place of E at the position of 203 in the short form of DLIMK. DLIMK shares characteristic structural features of vertebrate LIMKs, consisting of two LIM domains, a PDZ domain and a protein kinase domain, but in contrast to vertebrate LIMKs, DLIMK uniquely contains a long C-terminal extension, with no significant similarity to any known sequence. DLIMK is related to LIMK1 and LIMK2 to a similar extent; amino acid identities of DLIMK to human LIMK1 (or LIMK2) in LIM, PDZ and protein kinase domains are 50% (46%), 26% (32%) and 53% (54%), respectively. These highly conserved regions are flanked by non-conserved 'linker' regions, thus indicating the important role of conserved LIM, PDZ and protein kinase domains for functions of LIMKs. In the subdomain VIB (catalytic loop) in the kinase domain, DLIMK contains a short sequence motif DLNSMN (residues 500-505), which does not match the consensus sequence for either serine/threonine kinases (DLKxxN) or tyrosine kinases (DLRxxN or DLxxRN). This is also the case of vertebrate LIMKs, which possess a sequence motif DLNSHN in this region. DLIMK contains a threonine residue (Thr-569), corresponding to Thr-508 in human LIMK1, within the activation loop in the kinase domain. Residues surrounding this threonine (RYTVV) are highly conserved between DLIMK and vertebrate LIMKs. Since Thr-508 in human LIMK1 is the site of phosphorylation by ROCK and PAK1 and responsible for its activation, DLIMK may be activated by Drosophila homologs of ROCK and PAK by phosphorylation of this threonine (Ohashi, 2000).

date revised: 18 February 2024

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