A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish

Background

Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro.

Results

We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required.

Conclusion

Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.     

Localization and Mobility of Synaptic Vesicles in Myosin VI Mutants of Drosophila

Background

At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila.

Results

In Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching.

Conclusion

This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton.     

Neurexin in Embryonic Drosophila Neuromuscular Junctions

Background

Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described.

Methodology/Principal Findings

We use a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). Our results surprisingly suggest that neurexin in embryos is present both pre and postsynaptically. Presynaptic neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, we find that loss of neurexin only affects receptors containing the subunit GluRIIA.

Conclusions/Significance

Our study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and our results provide new insights into the role of neurexin in synapse development.     

Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Background

Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ).

Methodology/Principal Findings

Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins.

Conclusions/Significance

In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.     

Neuronal function and dysfunction of Drosophila dTDP

Background

TDP-43 is an RNA- and DNA-binding protein well conserved in animals including the mammals, Drosophila, and C. elegans. In mammals, the multi-function TDP-43 encoded by the TARDBP gene is a signature protein of the ubiquitin-positive inclusions (UBIs) in the diseased neuronal/glial cells of a range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U).

Methodology/Principal Findings

We have studied the function and dysfunction of the Drosophila ortholog of the mammalian TARDBP gene, dTDP, by genetic, behavioral, molecular, and cytological analyses. It was found that depletion of dTDP expression caused locomotion defect accompanied with an increase of the number of boutons at the neuromuscular junctions (NMJ). These phenotypes could be rescued by overexpression of Drosophila dTDP in the motor neurons. In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ. Significantly, constitutive overexpression of dTDP in the mushroom bodies caused smaller axonal lobes as well as severe learning deficiency. On the other hand, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately. Overexpression of dTDP also led to the formation of cytosolic dTDP (+) aggregates.

Conclusion/Significance

These data together demonstrate the neuronal functions of dTDP, and by implication the mammalian TDP-43, in learning and locomotion. The effects of mis-expression of dTDP on Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses. The conservation of the regulatory pathways of functions and dysfunctions of Drosophila dTDP and mammalian TDP-43 also shows the feasibility of using the flies as a model system for studying the normal TDP-43 function and TDP-43 proteinopathies in the vertebrates including human.     

Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

Background

Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.

Principal Findings

We report here that Meltrin β, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor α mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin β–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin β–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin β and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin β and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin β and EphA4 strongly blocked vesicular internalization of ephrin-A5–EphA4 complexes without requiring the protease activity of Meltrin β, suggesting a regulatory role of Meltrin β in ephrin-A5-Eph signaling.

Conclusion

Meltrin β plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin β stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.     

Anterograde Activin Signaling Regulates Postsynaptic Membrane Potential and GluRIIA/B Abundance at the Drosophila Neuromuscular Junction

Members of the TGF-β superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. In this study, we analyzed whether the Activin branch of the TGF-β superfamily also contributes to NMJ development and function. We find that elimination of the Activin/TGF-β type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. Our results demonstrate that anterograde Activin/TGF-β signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling, our findings indicate that the two branches of the TGF-β superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function.     

Natural Spike Trains Trigger Short- and Long-Lasting Dynamics at Hippocampal Mossy Fiber Synapses in Rodents

Background

Synapses exhibit strikingly different forms of plasticity over a wide range of time scales, from milliseconds to hours. Studies on synaptic plasticity typically use constant-frequency stimulation to activate synapses, whereas in vivo activity of neurons is irregular.

Methodology/Principal Findings

Using extracellular and whole-cell electrophysiological recordings, we have here studied the synaptic responses at hippocampal mossy fiber synapses in vitro to stimulus patterns obtained from in vivo recordings of place cell firing of dentate gyrus granule cells in behaving rodents. We find that synaptic strength is strongly modulated on short- and long-lasting time scales during the presentation of the natural stimulus trains.

Conclusions/Significance

We conclude that dynamic short- and long-term synaptic plasticity at the hippocampal mossy fiber synapse plays a prominent role in normal synaptic function.     

Transmission,Development, and Plasticity of Synapses

Chemical synapses are sites of contact and information transfer between a neuron and its partner cell. Each synapse is a specialized junction, where the presynaptic cell assembles machinery for the release of neurotransmitter, and the postsynaptic cell assembles components to receive and integrate this signal. Synapses also exhibit plasticity, during which synaptic function and/or structure are modified in response to activity. With a robust panel of genetic, imaging, and electrophysiology approaches, and strong evolutionary conservation of molecular components, Drosophila has emerged as an essential model system for investigating the mechanisms underlying synaptic assembly, function, and plasticity. We will discuss techniques for studying synapses in Drosophila, with a focus on the larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Vesicle fusion, which underlies synaptic release of neurotransmitters, has been well characterized at this synapse. In addition, studies of synaptic assembly and organization of active zones and postsynaptic densities have revealed pathways that coordinate those events across the synaptic cleft. We will also review modes of synaptic growth and plasticity at the fly NMJ, and discuss how pre- and postsynaptic cells communicate to regulate plasticity in response to activity.     

Kismet Positively Regulates Glutamate Receptor Localization and Synaptic Transmission at the Drosophila Neuromuscular Junction

The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the Drosophila larvae. Here, we show that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. Our data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, our data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome.     

Discs-large (DLG) is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila

Background  

Drosophila discs-large (DLG) is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ) is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors.     

Role of Mental Retardation-Associated Dystrophin-Gene Product Dp71 in Excitatory Synapse Organization,Synaptic Plasticity and Behavioral Functions

Background

Duchenne muscular dystrophy (DMD) is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR), likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood.

Methodology/Principal Findings

Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory.

Conclusions/Significance

Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.     

Quantal glutamate release is essential for reliable neuronal encodings in cerebral networks

Background

The neurons and synapses work coordinately to program the brain codes of controlling cognition and behaviors. Spike patterns at the presynaptic neurons regulate synaptic transmission. The quantitative regulations of synapse dynamics in spike encoding at the postsynaptic neurons remain unclear.

Methodology/Principal Findings

With dual whole-cell recordings at synapse-paired cells in mouse cortical slices, we have investigated the regulation of synapse dynamics to neuronal spike encoding at cerebral circuits assembled by pyramidal neurons and GABAergic ones. Our studies at unitary synapses show that postsynaptic responses are constant over time, such as glutamate receptor-channel currents at GABAergic neurons and glutamate transport currents at astrocytes, indicating quantal glutamate release. In terms of its physiological impact, our results demonstrate that the signals integrated from quantal glutamatergic synapses drive spike encoding at GABAergic neurons reliably, which in turn precisely set spike encoding at pyramidal neurons through feedback inhibition.

Conclusion/Significance

Our studies provide the evidences for the quantal glutamate release to drive the spike encodings precisely in cortical circuits, which may be essential for programming the reliable codes in the brain to manage well-organized behaviors.     

ApoE receptor 2 regulates synapse and dendritic spine formation

Background

Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation—processes critical for learning and memory.

Methodology/Principal Findings

In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.

Conclusions/Significance

These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.     

Glutamatergic neurons induce expression of functional glutamatergic synapses in primary myotubes

Background

The functioning of the nervous system depends upon the specificity of its synaptic contacts. The mechanisms triggering the expression of the appropriate receptors on postsynaptic membrane and the role of the presynaptic partner in the differentiation of postsynaptic structures are little known.

Methods and Findings

To address these questions we cocultured murine primary muscle cells with several glutamatergic neurons, either cortical, cerebellar or hippocampal. Immunofluorescence and electrophysiology analyses revealed that functional excitatory synaptic contacts were formed between glutamatergic neurons and muscle cells. Moreover, immunoprecipitation and immunofluorescence experiments showed that typical anchoring proteins of central excitatory synapses coimmunoprecipitate and colocalize with rapsyn, the acetylcholine receptor anchoring protein at the neuromuscular junction.

Conclusions

These results support an important role of the presynaptic partner in the induction and differentiation of the postsynaptic structures.     

Brain deletion of insulin receptor substrate 2 disrupts hippocampal synaptic plasticity and metaplasticity

Objective

Diabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits and the corresponding neurophysiological structural and functional alterations are linked to both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. To elucidate the specific role of brain insulin signalling in neuronal functions that are relevant for cognitive processes we have investigated the behaviour of neurons and synaptic plasticity in the hippocampus of mice lacking the insulin receptor substrate protein 2 (IRS-2).

Research Design and Methods

To study neuronal function and synaptic plasticity in the absence of confounding factors such as hyperglycaemia, we used a mouse model with a central nervous system- (CNS)-restricted deletion of IRS-2 (NesCreIrs2KO).

Results

We report a deficit in NMDA receptor-dependent synaptic plasticity in the hippocampus of NesCreIrs2KO mice, with a concomitant loss of metaplasticity, the modulation of synaptic plasticity by the previous activity of a synapse. These plasticity changes are associated with reduced basal phosphorylation of the NMDA receptor subunit NR1 and of downstream targets of the PI3K pathway, the protein kinases Akt and GSK-3β.

Conclusions

These findings reveal molecular and cellular mechanisms that might underlie cognitive deficits linked to specific defects of neuronal insulin signalling.     

NESH regulates dendritic spine morphology and synapse formation

Background

Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown.

Methodology/Principal Findings

NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines.

Conclusions/Significance

NESH is a novel F-actin binding protein that likely plays important roles in the regulation of dendritic spine morphogenesis and synapse formation.     

The intracellular threonine of amyloid precursor protein that is essential for docking of Pin1 is dispensable for developmental function

Background

Processing of Aβ-precursor protein (APP) plays an important role in Alzheimer''s Disease (AD) pathogenesis. Thr residue at amino acid 668 of the APP intracellular domain (AID) is highly conserved. When phosphorylated, this residue generates a binding site for Pin1. The interaction of APP with Pin1 has been involved in AD pathogenesis.

Methodology/Principal Findings

To dissect the functions of this sequence in vivo, we created an APP knock-in allele, in which Thr⁶⁶⁸ is replaced by an Ala (T⁶⁶⁸A). Doubly deficient APP/APP-like protein 2 (APLP2) mice present postnatal lethality and neuromuscular synapse defects. Previous work has shown that the APP intracellular domain is necessary for preventing early lethality and neuromuscular junctions (NMJ) defects. Crossing the T⁶⁶⁸A allele into the APLP2 knockout background showed that mutation of Thr⁶⁶⁸ does not cause a defective phenotype. Notably, the T⁶⁶⁸A mutant APP is able to bind Mint1.

Conclusions/Significance

Our results argue against an important role of the Thr⁶⁶⁸ residue in the essential function of APP in developmental regulation. Furthermore, they indicate that phosphorylation at this residue is not functionally involved in those APP-mediated functions that prevent (NMJ) defects and early lethality in APLP2 null mice.     

Heterogeneous nuclear ribonucleoprotein k interacts with Abi-1 at postsynaptic sites and modulates dendritic spine morphology

Background

Abelson-interacting protein 1 (Abi-1) plays an important role for dendritic branching and synapse formation in the central nervous system. It is localized at the postsynaptic density (PSD) and rapidly translocates to the nucleus upon synaptic stimulation. At PSDs Abi-1 is in a complex with several other proteins including WASP/WAVE or cortactin thereby regulating the actin cytoskeleton via the Arp 2/3 complex.

Principal Findings

We identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a 65 kDa ssDNA/RNA-binding-protein that is involved in multiple intracellular signaling cascades, as a binding partner of Abi-1 at postsynaptic sites. The interaction with the Abi-1 SH3 domain is mediated by the hnRNPK-interaction (KI) domain. We further show that during brain development, hnRNPK expression becomes more and more restricted to granule cells of the cerebellum and hippocampal neurons where it localizes in the cell nucleus as well as in the spine/dendritic compartment. The downregulation of hnRNPK in cultured hippocampal neurons by RNAi results in an enlarged dendritic tree and a significant increase in filopodia formation. This is accompanied by a decrease in the number of mature synapses. Both effects therefore mimic the neuronal morphology after downregulation of Abi-1 mRNA in neurons.

Conclusions

Our findings demonstrate a novel interplay between hnRNPK and Abi-1 in the nucleus and at synaptic sites and show obvious similarities regarding both protein knockdown phenotypes. This indicates that hnRNPK and Abi-1 act synergistic in a multiprotein complex that regulates the crucial balance between filopodia formation and synaptic maturation in neurons.