Neuronal Circuit Formation Research Articles

Unraveling the socio-cognitive consequences of KCC2 disruption in zebrafish: implications for neurodevelopmental disorders and therapeutic interventions.

During postnatal brain development, maintaining a delicate balance between excitation and inhibition (E/I) is essential for the precise formation of neuronal circuits. The K+/cl- cotransporter 2 (KCC2) is instrumental in this process, and its dysregulation is implicated in various neurological disorders. This study utilized zebrafish (Danio rerio) to investigate the socio-cognitive consequences of KCC2 disruption. Through CRISPR-Cas9 technology, biallelic kcc2a knockout zebrafish larvae were generated, revealing behavioral abnormalities, including impaired social interactions and memory deficits. Molecular analyses unveiled alterations in key genes associated with the GABAergic and glutamatergic systems, potentially contributing to E/I imbalance. Additionally, KCC2 disruption influenced the expression of oxytocin and BDNF, crucial regulators of social behaviors, synaptic plasticity, and memory formation. The study also explored the therapeutic potential of KCC2 modulation using pharmaceuticals, showing the rescuing effects of CLP-290 and LIT-001 on social abnormalities. However, the selective impact of LIT-001 on social behaviors, not memory, highlights the complexity of neurobehavioral modulation. In summary, this study sheds light on the pivotal role of KCC2 in shaping socio-cognitive functions and suggests potential therapeutic avenues for KCC2-related neurological disorders.

The BDNF-ERK/MAPK axis reduces phosphatase and actin regulator1, 2 and 3 (PHACTR1, 2 and 3) mRNA expressions in cortical neurons.

Actin rearrangement and phosphorylation-dephosphorylation in the nervous system contribute to plastic alteration of neuronal structure and function. Phosphatase and actin regulator (PHACTR) family members are actin- and protein phosphatase 1 (PP1)-binding proteins. Because some family members act as regulators of neuronal morphology, studying the regulatory mechanisms of PHACTR is valuable for understanding the basis of neuronal circuit formation. Although expression patterns of PHACTR family molecules (PHACTR1-4) vary across distinct brain areas, little is known about the extracellular ligands that influence their mRNA levels. In this study, we focused on an important neurotrophin, brain-derived neurotrophic factor (BDNF), and examined its effect on mRNA expression of PHACTR family member in cortical neurons. PHACTR1-3, but not PHACTR4, were affected by stimulation of primary cultured cortical neurons with BDNF; namely, sustained downregulation of their mRNA levels was observed. The observed downregulation was blocked by an inhibitor of the extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, U0126, suggesting that ERK/MAPK plays an inhibitory role for gene induction of PHACTR1-3. These findings aid the elucidation of how BDNF regulates actin- and PP1-related neuronal functions.

Nogo Receptor Antagonist LOTUS Promotes Neurite Outgrowth through Its Interaction with Teneurin-4.

Neurite outgrowth is a crucial process for organizing neuronal circuits in neuronal development and regeneration after injury. Regenerative failure in the adult mammalian central nervous system (CNS) is attributed to axonal growth inhibitors such as the Nogo protein that commonly binds to Nogo receptor-1 (NgR1). We previously reported that lateral olfactory tract usher substance (LOTUS) functions as an endogenous antagonist for NgR1 in forming neuronal circuits in the developing brain and improving axonal regeneration in the adult injured CNS. However, another molecular and cellular function of LOTUS remains unknown. In this study, we found that cultured retinal explant neurons extend their neurites on the LOTUS-coating substrate. This action was also observed in cultured retinal explant neurons derived from Ngr1-deficient mouse embryos, indicating that the promoting action of LOTUS on neurite outgrowth may be mediated by unidentified LOTUS-binding protein(s). We therefore screened the binding partner(s) of LOTUS by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS analysis and pull-down assay showed that LOTUS interacts with Teneurin-4 (Ten-4), a cell adhesion molecule. RNAi knockdown of Ten-4 inhibited neurite outgrowth on the LOTUS substrate in retinoic acid (RA)-treated Neuro2A cells. Furthermore, a soluble form of Ten-4 attenuates the promoting action on neurite outgrowth in cultured retinal explant neurons on the LOTUS substrate. These results suggest that LOTUS promotes neurite outgrowth by interacting with Ten-4. Our findings may provide a new molecular mechanism of LOTUS to contribute to neuronal circuit formation in development and to enhance axonal regeneration after CNS injury.

Excitatory and inhibitory synapses show a tight subcellular correlation that weakens over development

Neurons receive correlated levels of excitation and inhibition, a feature that is important for proper brain function. However, how this relationship between excitatory and inhibitory inputs is established during the dynamic period of circuit wiring remains unexplored. Using multiple techniques, including in utero electroporation, electron microscopy, and electrophysiology, we reveal a tight correlation in the distribution of excitatory and inhibitory synapses along the dendrites of developing CA1 hippocampal neurons. This correlation was present within short dendritic stretches (<20μm) and, surprisingly, was most pronounced during early development, sharply declining with maturity. The tight matching between excitation and inhibition was unexpected, as inhibitory synapses lacked an active zone when formed and exhibited compromised evoked release. We propose that inhibitory synapses form as a stabilizing scaffold to counterbalance growing excitation levels. This relationship diminishes over time, suggesting a critical role for a subcellular balance in early neuronal function and circuit formation.

The role of microglial TREM2 in development: A path toward neurodegeneration?

The nervous and the immune systems undergo a continuous cross talk, starting from early development and continuing throughout adulthood and aging. Defects in this cross talk contribute to neurodevelopmental and neurodegenerative diseases. Microglia are the resident immune cells in the brain that are primarily involved in this bidirectional communication. Among the microglial genes, trem2 is a key player, controlling the functional state of microglia and being at the forefront of many processes that require interaction between microglia and other brain components, such as neurons and oligodendrocytes. The present review focuses on the early developmental window, describing the early brain processes in which TREM2 is primarily involved, including the modulation of synapse formation and elimination, the control of neuronal bioenergetic states as well as the contribution to myelination processes and neuronal circuit formation. By causing imbalances during these early maturation phases, dysfunctional TREM2 may have a striking impact on the adult brain, making it a more sensitive target for insults occurring during adulthood and aging.

Contribution of the serotonergic system to developmental brain abnormalities in autism spectrum disorder.

This review highlights a key role of the serotonergic system in brain development and in distortions of normal brain development in early stages of fetal life resulting in cascades of abnormalities, including defects of neurogenesis, neuronal migration, neuronal growth, differentiation, and arborization, as well as defective neuronal circuit formation in the cortex, subcortical structures, brainstem, and cerebellum of autistic subjects. In autism, defects in regulation of neuronal growth are the most frequent and ubiquitous developmental changes associated with impaired neuron differentiation, smaller size, distorted shape, loss of spatial orientation, and distortion of cortex organization. Common developmental defects of the brain in autism include multiregional focal dysplastic changes contributing to local neuronal circuit distortion, epileptogenic activity, and epilepsy. There is a discrepancy between more than 500 reports demonstrating the contribution of the serotonergic system to autism's behavioral anomalies, highlighted by lack of studies of autistic subjects' brainstem raphe nuclei, the center of brain serotonergic innervation, and of the contribution of the serotonergic system to the diagnostic features of autism spectrum disorder (ASD). Discovery of severe fetal brainstem auditory system neuronal deficits and other anomalies leading to a spectrum of hearing deficits contributing to a cascade of behavioral alterations, including deficits of social and verbal communication in individuals with autism, is another argument to intensify postmortem studies of the type and topography of, and the severity of developmental defects in raphe nuclei and their contribution to abnormal brain development and to the broad spectrum of functional deficits and comorbid conditions in ASD.

Perinatal Western-style diet exposure associated with decreased microglial counts throughout the arcuate nucleus of the hypothalamus in Japanese macaques.

Perinatal exposure to a high-fat, high-sugar Western-style diet (WSD) is associated with altered neural circuitry in the melanocortin system. This association may have an underlying inflammatory component, as consumption of a WSD during pregnancy can lead to an elevated inflammatory environment. Our group previously demonstrated that prenatal WSD exposure was associated with increased markers of inflammation in the placenta and fetal hypothalamus in Japanese macaques. In this follow-up study, we sought to determine whether this heightened inflammatory state persisted into the postnatal period, as prenatal exposure to inflammation has been shown to reprogram offspring immune function and long-term neuroinflammation would present a potential means for prolonged disruptions to microglia-mediated neuronal circuit formation. Neuroinflammation was approximated in 1-yr-old offspring by counting resident microglia and peripherally derived macrophages in the region of the hypothalamus examined in the fetal study, the arcuate nucleus (ARC). Microglia and macrophages were immunofluorescently stained with their shared marker, ionized calcium-binding adapter molecule 1 (Iba1), and quantified in 11 regions along the rostral-caudal axis of the ARC. A mixed-effects model revealed main effects of perinatal diet (P = 0.011) and spatial location (P = 0.003) on Iba1-stained cell count. Perinatal WSD exposure was associated with a slight decrease in the number of Iba1-stained cells, and cells were more densely located in the center of the ARC. These findings suggest that the heightened inflammatory state experienced in utero does not persist postnatally. This inflammatory response trajectory could have important implications for understanding how neurodevelopmental disorders progress.NEW & NOTEWORTHY Prenatal Western-style diet exposure is associated with increased microglial activity in utero. However, we found a potentially neuroprotective reduction in microglia count during early postnatal development. This trajectory could inform the timing of disruptions to microglia-mediated neuronal circuit formation. Additionally, this is the first study in juvenile macaques to characterize the distribution of microglia along the rostral-caudal axis of the arcuate nucleus of the hypothalamus. Nearby neuronal populations may be greater targets during inflammatory insults.

Homeodomain proteins hierarchically specify neuronal diversity and synaptic connectivity.

How our brain generates diverse neuron types that assemble into precise neural circuits remains unclear. Using Drosophila lamina neuron types (L1-L5), we show that the primary homeodomain transcription factor (HDTF) brain-specific homeobox (Bsh) is initiated in progenitors and maintained in L4/L5 neurons to adulthood. Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates while repressing the HDTF Zfh1 to prevent ectopic L1/L3 fates (control: L1-L5; Bsh-knockdown: L1-L3), thereby generating lamina neuronal diversity for normal visual sensitivity. Subsequently, in L4 neurons, Bsh and Ap function in a feed-forward loop to activate the synapse recognition molecule DIP-β, thereby bridging neuronal fate decision to synaptic connectivity. Expression of a Bsh:Dam, specifically in L4, reveals Bsh binding to the DIP-β locus and additional candidate L4 functional identity genes. We propose that HDTFs function hierarchically to coordinate neuronal molecular identity, circuit formation, and function. Hierarchical HDTFs may represent a conserved mechanism for linking neuronal diversity to circuit assembly and function.

Homeodomain proteins hierarchically specify neuronal diversity and synaptic connectivity

How our brain generates diverse neuron types that assemble into precise neural circuits remains unclear. Using Drosophila lamina neuron types (L1-L5), we show that the primary homeodomain transcription factor (HDTF) brain-specific homeobox (Bsh) is initiated in progenitors and maintained in L4/L5 neurons to adulthood. Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates while repressing the HDTF Zfh1 to prevent ectopic L1/L3 fates (control: L1-L5; Bsh-knockdown: L1-L3), thereby generating lamina neuronal diversity for normal visual sensitivity. Subsequently, in L4 neurons, Bsh and Ap function in a feed-forward loop to activate the synapse recognition molecule DIP-β, thereby bridging neuronal fate decision to synaptic connectivity. Expression of a Bsh:Dam, specifically in L4, reveals Bsh binding to the DIP-β locus and additional candidate L4 functional identity genes. We propose that HDTFs function hierarchically to coordinate neuronal molecular identity, circuit formation, and function. Hierarchical HDTFs may represent a conserved mechanism for linking neuronal diversity to circuit assembly and function.

Extracellular molecular signals shaping dendrite architecture during brain development.

Proper growth and branching of dendrites are crucial for adequate central nervous system (CNS) functioning. The neuronal dendritic geometry determines the mode and quality of information processing. Any defects in dendrite development will disrupt neuronal circuit formation, affecting brain function. Besides cell-intrinsic programmes, extrinsic factors regulate various aspects of dendritic development. Among these extrinsic factors are extracellular molecular signals which can shape the dendrite architecture during early development. This review will focus on extrinsic factors regulating dendritic growth during early neuronal development, including neurotransmitters, neurotrophins, extracellular matrix proteins, contact-mediated ligands, and secreted and diffusible cues. How these extracellular molecular signals contribute to dendritic growth has been investigated in developing nervous systems using different species, different areas within the CNS, and different neuronal types. The response of the dendritic tree to these extracellular molecular signals can result in growth-promoting or growth-limiting effects, and it depends on the receptor subtype, receptor quantity, receptor efficiency, the animal model used, the developmental time windows, and finally, the targeted signal cascade. This article reviews our current understanding of the role of various extracellular signals in the establishment of the architecture of the dendrites.

&lt;i&gt;In silico&lt;/i&gt; prediction of the transcription factor-enhancer interaction as a first stage of axonal growth regulation

The development of neurodegenerative diseases is associated with proper neuronal circuit formation, axonal guidance. The DCC receptor (deleted in colorectal cancer / colorectal cancer suppressor) and SHH (sonic hedgehog protein) are among the key regulators of axonal guidance.Aim. Interaction prediction of specific enhancer regions of DCC and SHH genes with respectively annotated transcription factors.Materials and methods. An in silico study was performed. The iEnhancer-2L and ES-ARCNN algorithms were selected to estimate enhancer sequence strength. The interaction between transcription factor and enhancer sequence was assessed using the molecular docking method. The enhancer sequence of DCC and SHH protein genes were taken from the NCBI open-source database in FASTA format. Ensembl database was used for enhancer mapping, GeneCards was used for screening and selection of potentially appropriate enhancers and transcription factors associated with these enhancers. The structures of transcription factors as well as their DNA-binding domains were taken from the UniProtKB/Swiss-prot database. An HDOCK scoring function was used as a metric for assessing the possibility of interaction of the target gene transcription factor with associated enhancer sequence.Results. The results showed that the interactions of transcription factor NANOG with the DCC gene enhancer sequence and the interaction of transcription factor CEBPA with the SHH gene enhancer sequence predicted by molecular docking method are potentially possible. The iEnhancer-2L and ES-ARCNN algorithms predicted the enhancer sequence of the SHH gene as strong one. The enhancer sequence of the DCC gene was estimated as strong in the iEnhancer-2L algorithm and as weak in ES-ARCNN. Binding of the DCC gene enhancer sequence to the transcription factor NANOG at 1–206 bp and 686–885 bp sites is the most probable, binding of the SHH gene enhancer sequence to the transcription factor CEBPA at 1–500 bp (HDOCK limitation of 500 bp) is possible.Conclusion. In silico techniques applied in this study demonstrated satisfactory results of predicting the interaction of the transcription factor with the enhancer sequence. Limitations of the current techniques is the lack of consideration of specific transcription factor binding sites. This drawback can be eliminated by implementing an ab initio molecular dynamics simulations into the present pipeline.

Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

LTK and ALK promote neuronal polarity and cortical migration by inhibiting IGF1R activity

The establishment of axon‐dendrite polarity is fundamental for radial migration of neurons, cortical patterning, and formation of neuronal circuits. Here, we show that the receptor tyrosine kinases, Ltk and Alk, are required for proper neuronal polarization. In isolated primary mouse embryonic neurons, the loss of Ltk and/or Alk causes a multiple axon phenotype. In mouse embryos and newborn pups, the absence of Ltk and Alk delays neuronal migration and subsequent cortical patterning. In adult cortices, neurons with aberrant neuronal projections are evident and axon tracts in the corpus callosum are disrupted. Mechanistically, we show that the loss of Alk and Ltk increases the cell‐surface expression and activity of the insulin‐like growth factor 1 receptor (Igf‐1r), which activates downstream PI3 kinase signaling to drive the excess axon phenotype. Our data reveal Ltk and Alk as new regulators of neuronal polarity and migration whose disruption results in behavioral abnormalities.

New Biosensor for Determination of Neuropilin-1 with Detection by Surface Plasmon Resonance Imaging.

Neuropilin-1 is transmembrane protein with soluble isoforms. It plays a pivotal role in both physiological and pathological processes. NRP-1 is involved in the immune response, formation of neuronal circuits, angiogenesis, survival and migration of cells. The specific SPRI biosensor for the determination of neuropilin-1 was constructed using mouse monoclonal antibody that captures unbound NRP-1 form body fluids. The biosensor exhibits linearity of the analytical signal between 0.01 and 2.5 ng/mL, average precision value 4.7% and recovery between 97% and 104%. The detection limit is 0.011 ng/mL, and the limit of quantification is 0.038 ng/mL. The biosensor was validated by parallel determination of NRP-1 in serum and saliva samples using the ELISA test, with good agreement of the results.

Developmental stage-specific exposure and neurotoxicity evaluation of low-dose clothianidin during neuronal circuit formation.

Neonicotinoid pesticides (NN) were recently reported to exhibit adverse effects in higher vertebrates. Moreover, NNs are routinely transferred from mother to offspring, raising concerns about their effects on future generations. The fetal and neonatal periods are the most critical to the formation of neural circuits in the brain through neurogenesis and differentiation, neuronal migration, axon guidance, and synaptogenesis. NN exposure throughout the fetal and neonatal periods was found to affect the neurobehavior of the offspring, but the stage-specific neurobehavioral effects are unclear. We exposed fetal and neonatal mice to a no-observed-adverse-effect level (NOAEL) of clothianidin (CLO) for 4 days during each of four developmental stages: neurite proliferation and differentiation (fetal days 9-12, CLO-1), neurite outgrowth (fetal days 15-18, CLO-2), synapse formation and astrocyte differentiation (days 1-4 after birth, CLO-3), and synapse remodeling (days 11-14 after birth, CLO-4). CLO's neurobehavioral effects were evaluated in juveniles and adults, revealing that CLO-1 and CLO-2 caused behavioral abnormalities in adult mice. CLO-3 significantly increased locomotor activity and decreased juvenile neurons in the hippocampal dentate gyrus in adulthood. Comprehensive gene analysis of CLO-3 revealed high expression of genes related to neurite outgrowth and axonal branching in the hippocampus in juveniles and adults. These results revealed developmental stage-specific effects of a NOAEL of CLO in the fetal and neonatal periods, suggesting that the susceptibility of the fetus and neonate to CLO varies by developmental stage.

NSC Physiological Features in Spinal Muscular Atrophy: SMN Deficiency Effects on Neurogenesis

While the U.S. Food and Drug Administration and the European Medicines Evaluation Agency have recently approved new drugs to treat spinal muscular atrophy 1 (SMA1) in young patients, they are mostly ineffective in older patients since many motor neurons have already been lost. Therefore, understanding nervous system (NS) physiology in SMA patients is essential. Consequently, studying neural stem cells (NSCs) from SMA patients is of significant interest in searching for new treatment targets that will enable researchers to identify new pharmacological approaches. However, studying NSCs in these patients is challenging since their isolation damages the NS, making it impossible with living patients. Nevertheless, it is possible to study NSCs from animal models or create them by differentiating induced pluripotent stem cells obtained from SMA patient peripheral tissues. On the other hand, therapeutic interventions such as NSCs transplantation could ameliorate SMA condition. This review summarizes current knowledge on the physiological properties of NSCs from animals and human cellular models with an SMA background converging on the molecular and neuronal circuit formation alterations of SMA fetuses and is not focused on the treatment of SMA. By understanding how SMA alters NSC physiology, we can identify new and promising interventions that could help support affected patients.

Microglia-dependent remodeling of neuronal circuits.

Microglia are tissue-resident macrophages responsible for the surveillance, neuronal support, and immune defense of the brain parenchyma. Recently, the role played by microglia in the formation and function of neuronal circuits has garnered substantial attention. During development, microglia have been shown to engulf neuronal precursors and participate in pruning mechanisms while, in the mature brain, they influence synaptic signaling, provide trophic support and shape synaptic plasticity. Recently, studies have unveiled different microglial characteristics associated with specific brain regions. This emerging view suggests that the maturation and function of distinct neuronal circuits may be potentially associated with the molecular identity microglia adopts across the brain. Here, we review and summarize the known role of these cells in the thalamus, hippocampus, cortex, and cerebellum. We focus on in vivo studies to highlight the characteristics of microglia that may be important in the remodeling of these neuronal circuits and in relation to neurodevelopmental and neuropsychiatric disorders.

Neuronal guidance genes in health and diseases.

Neurons migrate from their birthplaces to the destinations, and extending axons navigate to their synaptic targets by sensing various extracellular cues in spatiotemporally controlled manners. These evolutionally conserved guidance cues and their receptors regulate multiple aspects of neural development to establish the highly complex nervous system by mediating both short- and long-range cell-cell communications. Neuronal guidance genes (encoding cues, receptors, or downstream signal transducers) are critical not only for development of the nervous system but also for synaptic maintenance, remodeling, and function in the adult brain. One emerging theme is the combinatorial and complementary functions of relatively limited classes of neuronal guidance genes in multiple processes, including neuronal migration, axonal guidance, synaptogenesis, and circuit formation. Importantly, neuronal guidance genes also regulate cell migration and cell-cell communications outside the nervous system. We are just beginning to understand how cells integrate multiple guidance and adhesion signaling inputs to determine overall cellular/subcellular behavior and how aberrant guidance signaling in various cell types contributes to diverse human diseases, ranging from developmental, neuropsychiatric, and neurodegenerative disorders to cancer metastasis. We review classic studies and recent advances in understanding signaling mechanisms of the guidance genes as well as their roles in human diseases. Furthermore, we discuss the remaining challenges and therapeutic potentials of modulating neuronal guidance pathways in neural repair.

Interspecific variation in sex-specific gustatory organs in Drosophila.

Drosophila males use leg gustatory bristles to discriminate between male and female cuticular pheromones as an important part of courtship behavior. In Drosophila melanogaster, several male-specific gustatory bristles are present on the anterior surface of the first tarsal segment of the prothoracic leg, in addition to a larger set of gustatory bristles found in both sexes. These bristles are thought to be specialized for pheromone detection. Here, we report the number and location of sex-specific gustatory bristles in 27 other Drosophila species. Although some species have a pattern similar to D. melanogaster, others lack anterior male-specific bristles but have many dorsal male-specific gustatory bristles instead. Some species have both anterior and dorsal male-specific bristles, while others lack sexual dimorphism entirely. In several distantly related species, the number of gustatory bristles is much greater in males than in females due to a male-specific transformation of ancestrally mechanosensory bristles to a chemosensory identity. This variation in the extent and pattern of sexual dimorphism may affect the formation and function of neuronal circuits that control Drosophila courtship and contribute to the evolution of mating behavior.

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 αVβ3 integrin initiates neuronal differentiation of Neuro2a cells under proliferative conditions, αVβ5 integrin appears responsible for promoting a complex arborization in cells already committed to differentiation. Interestingly, primary neurons exhibit a complementary expression pattern for β3 and β5 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.