Unlocking mechanosensitivity: integrins in neural adaptation.

Mechanosensitivity extends beyond sensory cells to encompass most neurons in the brain. Here, we explore recent research on the role of integrins, a diverse family of adhesion molecules, as crucial biomechanical sensors translating mechanical forces into biochemical and electrical signals in the brain. The varied biomechanical properties of neuronal integrins, including their force-dependent conformational states and ligand interactions, dictate their specific functions. We discuss new findings on how integrins regulate filopodia and dendritic spines, shedding light on their contributions to synaptic plasticity, and explore recent discoveries on how they engage with metabotropic receptors and ion channels, highlighting their direct participation in electromechanical transduction. Finally, to facilitate a deeper understanding of these developments, we present molecular and biophysical models of mechanotransduction.

Nanoscale organization of CaV2.1 splice isoforms at presynaptic terminals: implications for synaptic vesicle release and synaptic facilitation.

The distance between CaV2.1 voltage-gated Ca2+ channels and the Ca2+ sensor responsible for vesicle release at presynaptic terminals is critical for determining synaptic strength. Yet, the molecular mechanisms responsible for a loose coupling configuration of CaV2.1 in certain synapses or developmental periods and a tight one in others remain unknown. Here, we examine the nanoscale organization of two CaV2.1 splice isoforms (CaV2.1[EFa] and CaV2.1[EFb]) at presynaptic terminals by superresolution structured illumination microscopy. We find that CaV2.1[EFa] is more tightly co-localized with presynaptic markers than CaV2.1[EFb], suggesting that alternative splicing plays a crucial role in the synaptic organization of CaV2.1 channels.

Regulation of dendritic spine length in corticopontine layer V pyramidal neurons by autism risk gene ß3 integrin.

The relationship between autism spectrum disorder (ASD) and dendritic spine abnormalities is well known, but it is unclear whether the deficits relate to specific neuron types and brain regions most relevant to ASD. Recent genetic studies have identified a convergence of ASD risk genes in deep layer pyramidal neurons of the prefrontal cortex. Here, we use retrograde recombinant adeno-associated viruses to label specifically two major layer V pyramidal neuron types of the medial prefrontal cortex: the commissural neurons, which put the two cerebral hemispheres in direct communication, and the corticopontine neurons, which transmit information outside the cortex. We compare the basal dendritic spines on commissural and corticopontine neurons in WT and KO mice for the ASD risk gene Itgb3, which encodes for the cell adhesion molecule ß3 integrin selectively enriched in layer V pyramidal neurons. Regardless of the genotype, corticopontine neurons had a higher ratio of stubby to mushroom spines than commissural neurons. ß3 integrin affected selectively spine length in corticopontine neurons. Ablation of ß3 integrin resulted in corticopontine neurons lacking long (> 2 µm) thin dendritic spines. These findings suggest that a deficiency in ß3 integrin expression compromises specifically immature spines on corticopontine neurons, thereby reducing the cortical territory they can sample. Because corticopontine neurons receive extensive local and long-range excitatory inputs before relaying information outside the cortex, specific alterations in dendritic spines of corticopontine neurons may compromise the computational output of the full cortex, thereby contributing to ASD pathophysiology.

The tetraspanin TSPAN5 regulates AMPAR exocytosis by interacting with the AP4 complex.

Intracellular trafficking of AMPA receptors is a tightly regulated process which involves several adaptor proteins, and is crucial for the activity of excitatory synapses both in basal conditions and during synaptic plasticity. We found that, in rat hippocampal neurons, an intracellular pool of the tetraspanin TSPAN5 promotes exocytosis of AMPA receptors without affecting their internalisation. TSPAN5 mediates this function by interacting with the adaptor protein complex AP4 and Stargazin and possibly using recycling endosomes as a delivery route. This work highlights TSPAN5 as a new adaptor regulating AMPA receptor trafficking.

CRISPR-mediated activation of autism gene Itgb3 restores cortical network excitability via mGluR5 signaling.

Many mutations in autism spectrum disorder (ASD) affect a single allele, indicating a key role for gene dosage in ASD susceptibility. Recently, haplo-insufficiency of ITGB3, the gene encoding the extracellular matrix receptor ß3 integrin, was associated with ASD. Accordingly, Itgb3 knockout (KO) mice exhibit autism-like phenotypes. The pathophysiological mechanisms of Itgb3 remain, however, unknown, and the potential of targeting this gene for developing ASD therapies uninvestigated. By combining molecular, biochemical, imaging, and pharmacological analyses, we establish that Itgb3 haplo-insufficiency impairs cortical network excitability by promoting extra-synaptic over synaptic signaling of the metabotropic glutamate receptor mGluR5, which is similarly dysregulated in fragile X syndrome, the most frequent monogenic form of ASD. To assess the therapeutic potential of regulating Itgb3 gene dosage, we implemented CRISPR activation and compared its efficacy with that of a pharmacological rescue strategy for fragile X syndrome. Correction of neuronal Itgb3 haplo-insufficiency by CRISPR activation rebalanced network excitability as effectively as blockade of mGluR5 with the selective antagonist MPEP. Our findings reveal an unexpected functional interaction between two ASD genes, thereby validating the pathogenicity of ITGB3 haplo-insufficiency. Further, they pave the way for exploiting CRISPR activation as gene therapy for normalizing gene dosage and network excitability in ASD.

CRISPR-Mediated Activation of αV Integrin Subtypes Promotes Neuronal Differentiation of Neuroblastoma Neuro2a Cells.

Neuronal differentiation is a complex process whose dysfunction can lead to brain disorders. The development of new tools to target specific steps in the neuronal differentiation process is of paramount importance for a better understanding of the molecular mechanisms involved, and ultimately for developing effective therapeutic strategies for neurodevelopmental disorders. Through their interactions with extracellular matrix proteins, the cell adhesion molecules of the integrin family play essential roles in the formation of functional neuronal circuits by regulating cell migration, neurite outgrowth, dendritic spine formation and synaptic plasticity. However, how different integrin receptors contribute to the successive phases of neuronal differentiation remains to be elucidated. Here, we implemented a CRISPR activation system to enhance the endogenous expression of specific integrin subunits in an in vitro model of neuronal differentiation, the murine neuroblastoma Neuro2a cell line. By combining CRISPR activation with morphological and RT-qPCR analyses, we show that integrins of the αV family are powerful inducers of neuronal differentiation. Further, we identify a subtype-specific role for αV integrins in controlling neurite outgrowth. While αVß3 integrin initiates neuronal differentiation of Neuro2a cells under proliferative conditions, αVß5 integrin appears responsible for promoting a complex arborization in cells already committed to differentiation. Interestingly, primary neurons exhibit a complementary expression pattern for ß3 and ß5 integrin subunits during development. Our findings reveal the existence of a developmental switch between αV integrin subtypes during differentiation and suggest that a timely controlled modulation of the expression of αV integrins by CRISPRa provides a means to promote neuronal differentiation.

Kidins220/ARMS modulates brain morphology and anxiety-like traits in adult mice.

Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a transmembrane scaffold protein that participates in fundamental aspects of neuronal physiology including cell survival, differentiation, and synaptic plasticity. The Kidins220 constitutive knockout line displays developmental defects in the nervous and cardiovascular systems that lead to embryonic lethality, which has so far precluded the study of this protein in the adult. Moreover, Kidins220 mRNA is tightly regulated by alternative splicing, whose impact on nervous system physiology has not yet been addressed in vivo. Here, we have asked to what extent the absence of Kidins220 splicing and the selective knockout of Kidins220 impact on adult brain homeostasis. To answer this question, we used a floxed line that expresses only the full-length, non-spliced Kidins220 mRNA, and a forebrain-specific, CaMKII-Cre driven Kidins220 conditional knockout (cKO) line. Kidins220 cKO brains are characterized by enlarged ventricles in the absence of cell death, and by deficient dendritic arborization in several cortical regions. The deletion of Kidins220 leads to behavioral changes, such as reduced anxiety-like traits linked to alterations in TrkB-BDNF signaling and sex-dependent alterations of hippocampal-dependent spatial memory. Kidins220 floxed mice present similarly enlarged brain ventricles and increased associative memory. Thus, both the absolute levels of Kidins220 expression and its splicing pattern are required for the correct brain development and related expression of behavioral phenotypes. These findings are relevant in light of the increasing evidence linking mutations in the human KIDINS220 gene to the onset of severe neurodevelopmental disorders.

A developmental stage- and Kidins220-dependent switch in astrocyte responsiveness to brain-derived neurotrophic factor.

Astroglial cells are key to maintain nervous system homeostasis. Neurotrophins are known for their pleiotropic effects on neuronal physiology but also exert complex functions to glial cells. Here, we investigated (i) the signaling competence of mouse embryonic and postnatal primary cortical astrocytes exposed to brain-derived neurotrophic factor (BDNF) and, (ii) the role of kinase D-interacting substrate of 220 kDa (Kidins220), a transmembrane scaffold protein that mediates neurotrophin signaling in neurons. We found a shift from a kinase-based response in embryonic cells to a response predominantly relying on intracellular Ca2+ transients [Ca2+]i within postnatal cultures, associated with a decrease in the synthesis of full-length BDNF receptor TrkB, with Kidins220 contributing to the BDNF-activated kinase and [Ca2+]i pathways. Finally, Kidins220 participates in the homeostatic function of astrocytes by controlling the expression of the ATP-sensitive inward rectifier potassium channel 10 (Kir4.1) and the metabolic balance of embryonic astrocytes. Overall, our data contribute to the understanding of the complex role played by astrocytes within the central nervous system, and identify Kidins220 as a novel actor in the increasing number of pathologies characterized by astrocytic dysfunctions. This article has an associated First Person interview with the first authors of the paper.

Integrin adhesion in brain assembly: From molecular structure to neuropsychiatric disorders.

Integrins are extracellular matrix receptors that mediate biochemical and mechanical bi-directional signals between the extracellular and intracellular environment of a cell thanks to allosteric conformational changes. In the brain, they are found in both neurons and glial cells, where they play essential roles in several aspects of brain development and function, such as cell migration, axon guidance, synaptogenesis, synaptic plasticity and neuro-inflammation. Although there are many successful examples of how regulating integrin adhesion and signaling can be used for therapeutic purposes, for example for halting tumor progression, this is not the case for the brain, where the growing evidence of the importance of integrins for brain pathophysiology has not translated yet into medical applications. Here, we review recent literature showing how alterations in integrin structure, expression and signaling may be involved in the etiology of autism spectrum disorder, epilepsy, schizophrenia, addiction, depression and Alzheimer's disease. We focus on common mechanisms and recurrent signaling pathways, trying to bridge studies on the genetics and molecular structure of integrins with those on synaptic physiology and brain pathology. Further, we discuss integrin-targeting strategies and their potential benefits for therapeutic purposes in neuropsychiatric disorders.

Targeting Alternative Splicing as a Potential Therapy for Episodic Ataxia Type 2.

Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder characterized by paroxysmal attacks of ataxia, vertigo, and nausea that usually last hours to days. It is caused by loss-of-function mutations in CACNA1A, the gene encoding the pore-forming α1 subunit of P/Q-type voltage-gated Ca2+ channels. Although pharmacological treatments, such as acetazolamide and 4-aminopyridine, exist for EA2, they do not reduce or control the symptoms in all patients. CACNA1A is heavily spliced and some of the identified EA2 mutations are predicted to disrupt selective isoforms of this gene. Modulating splicing of CACNA1A may therefore represent a promising new strategy to develop improved EA2 therapies. Because RNA splicing is dysregulated in many other genetic diseases, several tools, such as antisense oligonucleotides, trans-splicing, and CRISPR-based strategies, have been developed for medical purposes. Here, we review splicing-based strategies used for genetic disorders, including those for Duchenne muscular dystrophy, spinal muscular dystrophy, and frontotemporal dementia with Parkinsonism linked to chromosome 17, and discuss their potential applicability to EA2.

Emerging Roles of Activity-Dependent Alternative Splicing in Homeostatic Plasticity.

Homeostatic plasticity refers to the ability of neuronal networks to stabilize their activity in the face of external perturbations. Most forms of homeostatic plasticity ultimately depend on changes in the expression or activity of ion channels and synaptic proteins, which may occur at the gene, transcript, or protein level. The most extensively investigated homeostatic mechanisms entail adaptations in protein function or localization following activity-dependent posttranslational modifications. Numerous studies have also highlighted how homeostatic plasticity can be achieved by adjusting local protein translation at synapses or transcription of specific genes in the nucleus. In comparison, little attention has been devoted to whether and how alternative splicing (AS) of pre-mRNAs underlies some forms of homeostatic plasticity. AS not only expands proteome diversity but also contributes to the spatiotemporal dynamics of mRNA transcripts. Prominent in the brain where it can be regulated by neuronal activity, it is a flexible process, tightly controlled by a multitude of factors. Given its extensive use and versatility in optimizing the function of ion channels and synaptic proteins, we argue that AS is ideally suited to achieve homeostatic control of neuronal output. We support this thesis by reviewing emerging evidence linking AS to various forms of homeostatic plasticity: homeostatic intrinsic plasticity, synaptic scaling, and presynaptic homeostatic plasticity. Further, we highlight the relevance of this connection for brain pathologies.

Kidins220/ARMS controls astrocyte calcium signaling and neuron-astrocyte communication.

Through their ability to modulate synaptic transmission, glial cells are key regulators of neuronal circuit formation and activity. Kidins220/ARMS (kinase-D interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning) is one of the key effectors of the neurotrophin pathways in neurons where it is required for differentiation, survival, and plasticity. However, its role in glial cells remains largely unknown. Here, we show that ablation of Kidins220 in primary cultured astrocytes induced defects in calcium (Ca2+) signaling that were linked to altered store-operated Ca2+ entry and strong overexpression of the transient receptor potential channel TRPV4. Moreover, Kidins220-/- astrocytes were more sensitive to genotoxic stress. We also show that Kidins220 expression in astrocytes is required for the establishment of proper connectivity of cocultured wild-type neurons. Altogether, our data reveal a previously unidentified role for astrocyte-expressed Kidins220 in the control of glial Ca2+ dynamics, survival/death pathways and astrocyte-neuron communication.

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

The RhoGEF DOCK10 is essential for dendritic spine morphogenesis.

By regulating actin cytoskeleton dynamics, Rho GTPases and their activators RhoGEFs are implicated in various aspects of neuronal differentiation, including dendritogenesis and synaptogenesis. Purkinje cells (PCs) of the cerebellum, by developing spectacular dendrites covered with spines, represent an attractive model system in which to decipher the molecular signaling underlying these processes. To identify novel regulators of dendritic spine morphogenesis among members of the poorly characterized DOCK family of RhoGEFs, we performed gene expression profiling of fluorescence-activated cell sorting (FACS)-purified murine PCs at various stages of their postnatal differentiation. We found a strong increase in the expression of the Cdc42-specific GEF DOCK10. Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs. Accordingly, in mouse hippocampal neurons, depletion of DOCK10 or expression of a DOCK10 GEF-dead mutant led to a strong decrease in spine density and size. Conversely, overexpression of DOCK10 led to increased spine formation. We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1. Our global approach thus identifies an unprecedented function for DOCK10 as a novel regulator of dendritic spine morphogenesis via a Cdc42-mediated pathway.

Purkinje cell maturation participates in the control of oligodendrocyte differentiation: role of sonic hedgehog and vitronectin.

Oligodendrocyte differentiation is temporally regulated during development by multiple factors. Here, we investigated whether the timing of oligodendrocyte differentiation might be controlled by neuronal differentiation in cerebellar organotypic cultures. In these cultures, the slices taken from newborn mice show very few oligodendrocytes during the first week of culture (immature slices) whereas their number increases importantly during the second week (mature slices). First, we showed that mature cerebellar slices or their conditioned media stimulated oligodendrocyte differentiation in immature slices thus demonstrating the existence of diffusible factors controlling oligodendrocyte differentiation. Using conditioned media from different models of slice culture in which the number of Purkinje cells varies drastically, we showed that the effects of these differentiating factors were proportional to the number of Purkinje cells. To identify these diffusible factors, we first performed a transcriptome analysis with an Affymetrix array for cerebellar cortex and then real-time quantitative PCR on mRNAs extracted from fluorescent flow cytometry sorted (FACS) Purkinje cells of L7-GFP transgenic mice at different ages. These analyses revealed that during postnatal maturation, Purkinje cells down-regulate Sonic Hedgehog and up-regulate vitronectin. Then, we showed that Sonic Hedgehog stimulates the proliferation of oligodendrocyte precursor cells and inhibits their differentiation. In contrast, vitronectin stimulates oligodendrocyte differentiation, whereas its inhibition with blocking antibodies abolishes the conditioned media effects. Altogether, these results suggest that Purkinje cells participate in controlling the timing of oligodendrocyte differentiation in the cerebellum through the developmentally regulated expression of diffusible molecules such as Sonic Hedgehog and vitronectin.

A Trio-RhoA-Shroom3 pathway is required for apical constriction and epithelial invagination.

Epithelial invagination is a common feature of embryogenesis. An example of invagination morphogenesis occurs during development of the early eye when the lens placode forms the lens pit. This morphogenesis is accompanied by a columnar-to-conical cell shape change (apical constriction or AC) and is known to be dependent on the cytoskeletal protein Shroom3. Because Shroom3-induced AC can be Rock1/2 dependent, we hypothesized that during lens invagination, RhoA, Rock and a RhoA guanine nucleotide exchange factor (RhoA-GEF) would also be required. In this study, we show that Rock activity is required for lens pit invagination and that RhoA activity is required for Shroom3-induced AC. We demonstrate that RhoA, when activated and targeted apically, is sufficient to induce AC and that RhoA plays a key role in Shroom3 apical localization. Furthermore, we identify Trio as a RhoA-GEF required for Shroom3-dependent AC in MDCK cells and in the lens pit. Collectively, these data indicate that a Trio-RhoA-Shroom3 pathway is required for AC during lens pit invagination.