Frontiers in Molecular Neuroscience

Neuronal function and pathology are deeply influenced by the distinct molecular profiles of the axon and soma. Traditional studies have often overlooked these differences due to the technical challenges of compartment specific analysis. In this study, we employ a robust RNA-sequencing (RNA-seq) approach, using microfluidic devices, to generate high-quality axonal transcriptomes from iPSC-derived cortical neurons (CNs). We achieve high specificity of axonal fractions, ensuring sample purity without contamination. Comparative analysis revealed a unique and specific transcriptional landscape in axonal compartments, characterized by diverse transcript types, including protein-coding mRNAs, RNAs encoding ribosomal proteins (RPs), mitochondrial-encoded RNAs, and long non-coding RNAs (lncRNAs). Previous works have reported the existence of transcription factors (TFs) in the axon. Here, we detect a set of TFs specific to the axon and indicative of their active participation in transcriptional regulation. To investigate transcripts and pathways essential for central motor neuron (MN) degeneration and maintenance we analyzed KIF1C-knockout (KO) CNs, modeling hereditary spastic paraplegia (HSP), a disorder associated with prominent length-dependent degeneration of central MN axons. We found that several key factors crucial for survival and health were absent in KIF1C-KO axons, highlighting a possible role of these also in other neurodegenerative diseases. Taken together, this study underscores the utility of microfluidic devices in studying compartment-specific transcriptomics in human neuronal models and reveals complex molecular dynamics of axonal biology. The impact of KIF1C on the axonal transcriptome not only deepens our understanding of MN diseases but also presents a promising avenue for exploration of compartment specific disease mechanisms.

Chronic pain arises when dorsal root ganglion (DRG) neurons become sensitised to noxious inputs, a process driven by inflammatory mediators such as prostaglandin E2 (PGE2). Local translation of axonal mRNAs is a key regulator of nociceptor plasticity, yet how axonal transcriptome dynamics contribute to inflammatory sensitisation remains unclear. Using compartmentalised culture systems and RNA-sequencing, we defined axonal and somatic transcriptomes in embryonic (E16.5) and adult (W8) DRG neurons and assessed their remodelling after PGE2 exposure. We identify a conserved core axonal transcriptome spanning embryonic to adult stages, prominently enriched for ribosomal and mitochondrial functions, consistent with sustained translational and metabolic demands. PGE2 elicited compartment-specific reprogramming: pathways related to sensory processing and pain were upregulated in axons but downregulated in somata. Functionally, prolonged axonal PGE2 exposure enhanced capsaicin-evoked Ca{superscript 2} responses and drove retrograde sensitisation of neuronal somata. Integrating transcriptomics with functional assays, we pinpointed Tnfrsf12a (Fn14), a cytokine receptor linked to regeneration and neuropathic pain, as a PGE2-induced axonal mRNA. Crucially, local axonal knockdown of Tnfrsf12a significantly reduced neuronal excitability, providing proof-of-concept that axonally enriched transcripts can be targeted to modulate sensitisation. These findings position conserved axonal transcriptome programmes as drivers of peripheral sensitisation and establish Tnfrsf12a as a therapeutic candidate for inflammatory pain.

In this study, we identified active enhancers in the mouse cerebellum at embryonic and postnatal stages establishing the first catalog of enhancers active during embryonic cerebellum development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Our study provides insight into the dynamics of gene expression regulation by enhancers in the developing brain and delivers a rich resource of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.

The endocannabinoid system (ECS) has a widespread role in the development and function of the central nervous system (CNS). Cannabinoid receptors like CB1 and CB2 can be activated with exogenous cannabinoids most popularly known as tetrahydrocannabinol (THC) or cannabis and cannabidiol (CBD). The components of the ECS are expressed early in fetal development, and prenatal exposure to cannabis can lead to structural changes in white matter. White matter is composed of neuronal axons ensheathed in myelin, a lipid-rich insulation that facilitates saltatory conduction and maintains axon integrity. In the CNS, myelin is made by specialized glial cells called oligodendrocytes (OLs), which in addition to neurons also express components of the ECS. However, while several studies have focused on how the ECS regulates neuronal development, there is a limited understanding of its impact on OL development or myelin formation. Therefore, our current study set out to understand how pharmacological activation of the ECS alters OL differentiation and myelin formation in vivo. We administered WIN 55,212-2 (WIN 55), a CB1 and CB2 agonist, to larval zebrafish and longitudinally analyzed OL development and myelination in vivo. Interestingly, we observed an increase in non-axonal ensheathments in the spinal cord, which appeared to be surrounding neuronal cell bodies. These non-axonal ensheathments were dependent on CB1, as the addition of WIN 55 in a global CB1 mutant prevented this phenotype. Furthermore, this ectopic cell body ensheathment occurred independently from normal myelination processes, as individual OLs did not exhibit changes in the number of myelin sheaths, sheath length, or total myelin output. This study shows that activation of CB receptors in vivo leads to increased non-axonal ensheathment without significantly changing OL differentiation or normal myelin formation. Future studies can further investigate the pathways that drive this phenotype to better understand how exogenous cannabinoid activation can regulate the precision of oligodendrocyte ensheathment.

ZNF804A was amongst the first genes robustly associated with schizophrenia based on findings from large-scale genomic studies. Previous research has implicated ZNF804A in the regulation of gene expression and synaptic function, but the role of this gene in neurodevelopment and in schizophrenia pathogenesis remains unclear. To study its function during neurodevelopment, we generated isogenic human induced pluripotent stem cells (hiPSCs) with reduced ZNF804A expression, differentiated them into developing cortical glutamatergic neurons and studied their transcriptomic, synaptic and protein signatures. Mutant neurons showed modest evidence changes in gene expression. However, high-content confocal imaging revealed increased excitatory synapse density in mutant neurons. Cell-compartment specific proteomic analysis further revealed that mutant neurons had higher levels of ribosomal and translational proteins within neurites, and high-content imaging confirmed increased local protein synthesis efficiency. Overall, these results demonstrate that in human developing cortical glutamatergic neurons, ZNF804A regulates excitatory synapse formation potential via increased local protein translation.

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes have been identified as robust susceptibility genes in large-scale genome-wide association studies of schizophrenia. The ZNF804A and NT5C2 proteins are highly expressed in developing and mature cortical neurons. ZNF804A has been implicated in regulating the development of neuronal morphology; it localises to synapses and is required for activity-dependent modifications of dendritic spines. NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and implicated in influencing protein synthesis in neural progenitor cells. But despite these findings, a better understanding of the role these proteins play in regulating neuronal function is needed. A recent yeast two-hybrid screen has identified ZNF804A and NT5C2 as potential interacting proteins, but whether this occurs in situ; and moreover, in cortical neurons, is unknown. Here we show that ZNF804A and Nt5C2 colocalise and interact in hEK293T cells. Furthermore, their rodent homolouges, ZFP804A and NT5C2, specifically colocalise at synapses and form a protein complex in cortical neurons. Knockdown of Zfp804A or Nt5c2 resulted in a significant decrease in synaptic expression of both proteins, suggesting that both proteins are required for the synaptic targeting of each other. Taken together, these data indicate that ZNF804A/ZFP804A and NT5C2 interact together in cortical neurons and indicate that these GWAS risk factors may function as a complex to regulate neuronal function.

Spiral ganglion neurons (SGNs) are the primary auditory afferents in the inner ear. These neurons degenerate in response to a number of conditions, including auditory neuropathies, concussions, and aging. Research to assess the extent of degeneration and to test the efficacy of protective or rehabilitative strategies requires quantification of SGNs from tissue sections. However, manual counting of SGNs can be arduous and time-consuming due to dense crowding and the lack of reliable nuclear-specific labels. SGNs receive afferent input via GluA2-containing AMPA receptors. As the Gria2 transcripts that code for GluA2 must undergo RNA editing to ensure calcium impermeability, we hypothesized that SGNs would express high levels of the adenosine deaminase acting on RNA (ADAR) enzyme ADARB1. Here we confirm enriched expression of Adarb1 in SGNs via in situ hybridization and show that anti-ADARB1 antibodies robustly label the nuclei of both type I and type II SGNs in cochlear sections from young and aged mice. Neuronal specificity was confirmed using antibodies against neurofilament heavy chain (NFH), human antigen D (HuD), GATA binding protein 3 (GATA3), and SRY-box 2 (SOX2). A blinded investigator manually counted SGNs via NFH staining, and these were compared to automated counts of ADARB1-positive nuclei using the analyze particles function in ImageJ. A concordance correlation coefficient and Bland-Altman analysis demonstrated strong agreement between the manual and automated counts. Additionally, immunolabeling of ADARB1 in macaque and human temporal bone sections confirm robust labeling of SGN nuclei, suggesting broad utility of ADARB1 immunolabeling for automated counts of SGNs across species.

Onset of many neurodegenerative and neuromuscular diseases usually starts in adulthood; however, recent advances point toward neurodevelopmental changes as drivers of late neurodegeneration. How early neuropathological features occur in these conditions remains unclear, which is critical for timely therapeutic intervention. Here, we provide evidence that neurodevelopmental axonal defects initiate a motor phenotype in a zebrafish model of spinocerebellar ataxia type 37 (SCA37), a degenerative hereditary condition caused by an ATTTC repeat in the DAB1 gene. We investigated neuronal defects triggered by the embryonic AUUUC repeat RNA and their effects later in life by transiently expressing this RNA in embryos and analyzing innervation and motor function. We found abnormalities in motor neuron axonal outgrowth and muscle innervation. We also discovered disrupted embryonic motor activity and reduced locomotor distance and velocity in late adult zebrafish, demonstrating motor impairment. Moreover, we showed that NOVA2 expression rescues axonal defects, indicating dysfunction of NOVA2-regulated neurodevelopmental processes. Overall, our results establish embryonic expression of the AUUUC repeat RNA as a driver of axonal and synaptic abnormalities, interfering with neuronal circuits and culminating in adult motor dysfunction.

Hereditary spastic paraplegias (HSPs) are a diverse set of neurological disorders characterized by progressive spasticity and weakness in the lower limbs caused by damage to the axons of the corticospinal tract. More than 88 genetic mutations have been associated with HSP, yet the mechanisms underlying these disorders are little understood. We studied the pathogenesis of one form of HSP known as spastic paraplegia 15 (SPG15). This disorder is caused by mutations in the ZFYVE26 gene, which codes for a protein called SPASTIZIN. We show that, in zebrafish, the significant reduction of Spastizin caused degeneration of Mauthner (M)-cells. M-cell degeneration is associated with axon demyelination in the spinal cord and impaired locomotion in the spastizin mutants. Our findings reveal that the mutation not only compromises axonal integrity but also affects the structural molecules of the myelin sheath, laying the foundation for degeneration and advancing our understanding of the intricate mechanisms underlying HSPs.

Pigment epithelial-derived factor (PEDF) is a multifunctional protein produced predominantly by the retinal pigment epithelium and expressed in many tissues, including the brain, highlighting its participation in crucial processes, such as neuroprotection and angiogenesis. Some neurodevelopmental disorders, such as ASD, are characterized by neurodevelopmental abnormalities, including altered neurite formation, spine formation, and neuronal activities. Many efforts have been made to resolve NDDs, but until now, some symptoms remain untargeted. PEDF is involved in many steps of neurodevelopment. The treatment of PEDF peptide might improve the outcome of NDD symptoms by altering neuronal morphologies. We used PEDF peptides that contain different functional domains to study the effect of administering PEDF peptides on neuronal morphology in a prenatal valproic acid (VPA)-exposed mouse model. We identified that the treatment with PEDF peptides rectified the abnormalities in neurite formation and spine formation in VPA-exposed cortical neurons. In vitro calcium imaging showed abnormalities in the spontaneous activity in VPA-exposed cortical neurons. Treatment of a short PEDF peptide normalized intracellular calcium response to the control level. Accordingly, PEDF peptides have the prospect of serving as potential treatments for patients with neurodevelopmental disorders, such as ASD.

T-type/Cav3 calcium channels are key in neuronal excitability and pain processing with Cav3.2 being the prominent isoform in primary sensory neurons of the dorsal root ganglion (DRG). Its pharmacological inhibition or gene silencing induces analgesia in several preclinical models of inflammatory and neuropathic pain. However, the presence of Cav3.2, encoded by the CACNA1H gene, in human DRG neurons remains unresolved. Using RNA in-situ hybridization and electrophysiological recordings, we show that human DRGs express Cav3.2 in a subset of neurons positive for the neurotrophic factor receptor TrkB (NTRK2 gene). The Cav3.2 current exhibits typical biophysical and pharmacological properties, including inhibition by a low concentration of nickel and by Z944, a specific T-type calcium channel blocker in advanced clinical development. Conversely, ABT-639, a T-type calcium channel inhibitor that failed in Phase 2 trials for pain relief, does not inhibit Cav3.2 currents in human DRG neurons. Importantly, Cav3.2 currents are prominent in neurons from female organ donors, supporting the presence of sex differences in pain mechanisms in humans. These findings underscore the potential of continued exploration of Cav3.2 as a therapeutic target for pain treatment and highlight a specific subset of human neurons that likely rely on this channel to modulate their excitability.

Spiral ganglion neurons (SGNs) are the primary afferent neurons that convey sound information from the cochlea, but the epigenetic changes that occur during development are unknown. We identified the chromodomain helicase binding protein 4 (CHD4) expression in SGNs. CHD4 is an ATP-dependent chromatin remodeler. We employed the Neurog1 (Ngn1) CreERT2 Chd4 conditional knockout animals to investigate Chd4 function in SGNs. SGNs are classified as type I and II neurons with different innervation patterns. SGNs lacking CHD4 showed abnormal fasciculation of type I neurons along with improper pathfinding of type II fibers. CHD4 binding to chromatin from immortalized multipotent otic progenitor-derived neurons was used to identify candidate target genes in SGNs. Gene ontology analysis of CHD4 target genes revealed cellular processes involved in axon guidance, axonal fasciculation, and the ephrin receptor signaling pathway. Eph/ephrin signaling regulates various biological processes, including axon guidance. We confirmed increased Eph/ephrin transcripts in SGNs from Chd4 conditional knockout cochleae. The results implicate epigenetic changes in circuit wiring by modulating the expression of a subset of axon guidance molecules. The results also provide insights into neurodevelopmental diseases such as Sifrim-Hitz-Weiss syndrome (SIHIWES) and inform strategies for regenerating SGNs.

PTEN is a well-known tumor suppressor whose mutations are also strongly associated with Autism Spectrum Disorder (ASD). The activity and function of PTEN in neurons have been studied extensively in various settings, whereas animal models offer the best opportunity to experimentally modify and test the role of PTEN in neuronal development in vivo. On the molecular level, PTENs importance in the mTOR pathways suppression is well-known but many other interactions have been suggested and yet others likely remain undiscovered. Therefore, to systematically explore the regulatory landscape downstream of PTEN on the genetic level, we have set out to establish a singlecell RNA sequencing (scRNA-seq) workflow to measure gene expression changes as a result of knocking out the Pten gene in vivo. We were able to collect brain tissue from four conditionally knocked out mouse samples and dissociate them into single live cells, which were subsequently flow-sorted and a scRNA-seq library was prepared. All of these steps were completed in a single day, yielding sequencing data from hundreds of cells suitable for cell clustering, cell type identification, and differential expression measurements. Having demonstrated its feasibility, this approach promises to aid hypothesis generation and discovery in mapping the function of Pten as well other genes whose regulatory networks remain to be fully defined.

Neural injury triggers striking immune reactions from glial cells, including significant transcriptional and morphological changes, but it is unclear how these events are coordinated to mount an effective immune response. Here, we present a new variant of the Fluorescence assay to detect ribosome interactions with mRNA (FLARIM), which we term FLARIM v2.0, to visualize single immune gene transcripts and association with ribosomes in glia responding to neurodegeneration. Specifically, using an in vivo axotomy assay in Drosophila, we show that matrix metalloproteinase-1 (Mmp-1) mRNAs and associated ribosomes are detected in distal processes of reactive glia where they are actively engulfing degenerating axonal material, suggesting that local translation is an important component of the glial immune response to axotomy. This work also validates our enhanced FLARIM assay as a promising tool to investigate mechanisms of mRNA transport and translation in a wide range of in vitro and in vivo paradigms.

Neurons possess highly polarized morphology that require intricate molecular organization, partly facilitated by RNA localization. By localizing specific mRNA, neurons can modulate synaptic features through local translation and subsequent modification of protein concentrations in response to stimuli. The resulting activity-dependent modifications are essential for synaptic plasticity, and consequently, fundamental for learning and memory. Consequently, high-resolution characterization of the spatial distribution of dendritic transcripts and the spatial relationship across transcripts is critical for understanding the pathways and mechanisms underlying synaptic plasticity. In this study, we characterize the spatial distribution of six previously uncharacterized genes (Adap2, Colec12, Dtx3L, Kif5c, Nsmf, Pde2a) within the dendrites at a sub-micrometer scale, using single-molecule fluorescence in situ hybridization (smFISH). We found that spatial distributions of dendritically localized mRNA depended on both dendrite morphology and gene identity that cannot be recreated by diffusion alone, suggesting involvement of active mechanisms. Furthermore, our analysis reveals that dendritically localized mRNAs are likely co-transported and organized into clusters at larger spatial scales, indicating a more complex organization of mRNA within dendrites.

Identification of the neuronal types that form the specialized circuits controlling distinct behaviors has benefited greatly from the simplicity offered by zebrafish. Electrophysiological studies have shown that additional to connectivity, understanding of circuitry requires identification of functional specializations among individual circuit components, such as those that regulate levels of transmitter release and neuronal excitability. In this study we use single cell RNA sequencing (scRNAseq) to identify the molecular bases for functional distinctions between motoneuron types that are causal to their differential roles in swimming. The primary motoneuron (PMn) in particular, expresses high levels of a unique combination of voltage-dependent ion channel types and synaptic proteins termed functional cassettes. The ion channel types are specialized for promoting high frequency firing of action potentials and augmented transmitter release at the neuromuscular junction, both contributing to greater power generation. Our transcriptional profiling of spinal neurons further assigns expression of this cassette to specific interneuron types also involved in the central circuitry controlling high speed swimming and escape behaviors. Our analysis highlights the utility of scRNAseq in functional characterization of neuronal circuitry, in addition to providing a gene expression resource for studying cell type diversity.

The spinal cord is the gateway for somatosensory and nociceptive information to the brain and a key locus for sensory-motor integration. Studies in mice have advanced our understanding of spinal cord circuitry, and transcriptomic studies have begun to characterize the human spinal cord; however, major gaps in knowledge persist. We conducted single-nucleus sequencing of lumbar spinal cord tissue from 11 adult organ donors and annotated spinal cord cell types with high resolution spatial transcriptomics. We identified 34 spatially and transcriptionally defined neuronal classes and detected sex-specific cell types and states across multiple glial populations. Electrophysiological recordings from dorsal horn neurons revealed firing patterns for neuronal subtypes and group I mGluR-dependent plasticity. Our work defines previously unknown aspects of human spinal cord molecular anatomy and physiology.

Approximately 60% of individuals with Rett syndrome (RTT) carry a nonsense variant in the MECP2 gene; thus, there is an unmet need to identify novel nonsense suppression compound(s) that can restore full length MeCP2 protein levels and function. Here, we characterized neuronal phenotypes in cultured cortical neurons from newborn knock-in mice harboring the MECP2 R255X variant. After 2 weeks in vitro, R255X mutant neurons showed smaller cell bodies, shorter dendrites, fewer dendritic branches, and a lower density of excitatory synapses when compared to wildtype (WT) neurons. Transduction of AAV9-MeCP2-GFP in R255X mutant neurons made these cellular phenotypes similar to those in WT neurons, including soma size, dendritic length and branching, and excitatory synapse density. As proof of principle for the potential clinical use of read-through compounds, cultured R255X mutant neurons treated with the aminoglycoside G418 for 72h in vitro showed cell body size and excitatory synapse density similar to WT neurons. We expect these combined approaches will identify effective compounds to suppress translation termination at a premature termination codon, which can be moved to further preclinical functional and behavioral studies in R255X MECP2 knock-in mice. Summary StatementExpression of wildtype MECP2 or treatment with G418 in vitro restored cell body size, dendritic length, and dendritic spine density in cortical neurons from R255X MECP2 knock-in mice to levels comparable to wildtype neurons.

MAPK8IP3-related neurodevelopmental disorders are a spectrum of rare conditions caused by de novo mutations in the MAPK8IP3 gene that encodes the JIP3 protein. These disorders are associated with a spectrum of neurodevelopmental symptoms that manifest in children and cause brain abnormalities, profound intellectual disabilities, movement disorders, and developmental delays. JIP3 is required for axonal transport of proteins and organelles between the soma and the synaptic terminal of neurons, a process critical for normal brain development and function. Homozygous loss-of-function mutations in JIP3 lead to impaired axonal transport and aggregation of cargo, which result in axonal swelling and stunted elongation. Despite these severe outcomes, disease mechanisms are poorly understood, and no current treatments are available. Here we conduct thorough morphological, behavioral, and motility phenotyping in the JIP3 knockout zebrafish and identify locomotor deficits and morphological abnormalities. To identify treatment options, we used insights from expert clinicians and the artificial intelligence tool, mediKanren, to identify drug candidates hypothesized to improve patient symptoms or compensate for the loss of JIP3 at the molecular level. We then prioritized drugs that are FDA-approved, safe for children, and readily available. These collective efforts identified amantadine and levodopa as candidate therapies and rescued motor phenotypes associated with JIP3 loss-of-function in zebrafish.

CRISPR-Cas9 editing is now the leading method for genome editing and is being advanced for the treatment of human disease. CRIPSR editing could have many applications for treatment of neurological diseases, including pain but traditional viral vector delivery approaches have neurotoxicity limiting their use. Overcoming these issues could open the door for genome editing treatments for diseases like intractable pain where the dorsal root ganglia (DRG) would be the desired target. To this end, we describe a simple method for viral-vector-independent transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. As proof of principle, we edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successful as demonstrated by the expression of the reporters as early as two days in vitro. CRISPR-Cas9 editing was confirmed at the genome level with insertion and deletion detection system T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for intractable pain. TeaserWe describe a non-viral transfection method for CRISPR-Cas9 gene editing in human dorsal root ganglion neurons.