iPSC-derived Neurons Research Articles

Development of a Novel Microphysiological System for Peripheral Neurotoxicity Prediction Using Human iPSC-Derived Neurons with Morphological Deep Learning

A microphysiological system (MPS) is an in vitro culture technology that reproduces the physiological microenvironment and functionality of humans and is expected to be applied for drug screening. In this study, we developed an MPS for the structured culture of human iPSC-derived sensory neurons and then predicted drug-induced neurotoxicity by morphological deep learning. Using human iPSC-derived sensory neurons, after the administration of representative anti-cancer drugs, the toxic effects on soma and axons were evaluated by an AI model with neurite images. Significant toxicity was detected in positive drugs and could be classified by different effects on soma or axons, suggesting that the current method provides an effective evaluation of chemotherapy-induced peripheral neuropathy. The results of neurofilament light chain expression changes in the MPS device also agreed with clinical reports. Therefore, the present MPS combined with morphological deep learning is a useful platform for in vitro peripheral neurotoxicity assessment.

CNSC-20. MONOSYNAPTIC TRACING DEFINES BRAIN-WIDE CIRCUIT CONNECTIVITY OF HUMAN GLIOBLASTOMA

Abstract Glioblastoma (GBM), a deadly brain cancer, infiltrates the brain and can be synaptically innervated by neurons. Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic in nature. The extent of integration of GBM cells into the brain-wide neuronal circuitry is therefore not well understood. We report the application of transsynaptic viral tracing approaches to study the neuronal connectome of GBM. We applied rabies virus-mediated retrograde tracing and herpes simplex virus (HSV)-mediated anterograde tracing approaches to characterize presynaptic partners in vivo in the adult mouse brain and ex vivo with iPSC-derived neurons, cortical organoid-GBM assembloids, and human surgical specimens. After transplantation into adult mice, GBM cells derived from multiple patients rapidly integrated into brain-wide neuronal circuits. GBM exhibited increased connectivity rates compared to transplanted neural progenitor cells, highlighting functional connectivity as a hallmark of malignant cells. Beyond glutamatergic inputs, we identified neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. We validated these long-range cholinergic neuron-to-glioma synapses signaling through the metabotropic CHRM3 receptor by high-resolution microscopy, anterograde monosynaptic HSV tracing, calcium imaging, and patch-clamp electrophysiology. To interrogate the functional effects of acetylcholine (ACh) on GBM, we performed calcium imaging and RNA sequencing analyses, which showed that ACh stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via CHRM3. Importantly, CHRM3 activation promoted GBM cell invasion, whereas CHRM3 downregulation suppressed GBM cell invasion in vitro and in vivo, suggesting CHRM3 as a potential therapeutic target for this disease. Together, these results reveal the capacity of human GBM cells to robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain. They also support a model wherein rapid synapse formation onto GBM cells may promote a long-lasting increase in tumor cell fitness.

CNSC-64. BEYOND THE LOCAL TUMOR MICROENVIRONMENT: CHARACTERIZING CHANGES TO NEURONAL ACTIVITY INDUCED BY PEDIATRIC HIGH-GRADE GLIOMA

Abstract Bidirectional electrochemical signaling between neurons and tumor cells is emerging as a critical regulator of pediatric high-grade glioma (HGG) pathology. Neuron-tumor interactions are known to induce local neuronal hyperactivity. However, recent work tracing the projections of these neuron-tumor networks revealed that HGG neuronal recruitment extends beyond the local tumor microenvironment (TME). These findings suggest that tumor-induced hyperexcitability may spread beyond the local TME and drive brain-wide circuit-level changes. To elucidate how non-tumor innervated brain regions are affected by TME hyperactivity we first characterized neuronal hyperactivity induced by a cortical patient-derived pediatric high-grade cell line in vitro on human iPSC-derived neurons. In vitro calcium imaging experiments demonstrated an increase in the number of calcium fluctuations in glioma co-cultured neurons. In parallel, multi-electrode recordings revealed increased population spiking activity and amplitude in neurons co-cultured with glioma cells. We then xenografted these HGG cells into the prefrontal cortex of mice and performed whole brain-clearing and cFos staining on tumor-bearing vs saline-injected control samples. As cFos is an immediate early gene activated by neuronal activity, we utilized the density of cFos expression as a readout-out of neuronal activation in our atlas-aligned segmented brain section analyses. We expectedly observed increased cFos expression within the ipsilateral primary and secondary motor region, which collectively comprise the local TME. Interestingly, we additionally observed elevated cFos in non-tumor bearing brain regions on both the ipsilateral and contralateral hemispheres, largely in layer 2/3 cortical neurons. These neurons form extensive cortico-cortical projections, and we coincidingly observed increased cFos expression in known cortical projection targets of the prefrontal xenograft location, such as somatosensory, visual, and retrosplenial cortices. Our findings suggest that tumor-induced neuronal hyperactivity extends beyond the local TME in a brain-wide manner and potentially follows established neuronal circuits, rendering these connected regions vulnerable to alterations in neuronal activity.

Mitochondrial dynamics and bioenergetics in Alzheimer's induced pluripotent stem cell-derived neurons.

Mitochondrial (MT) dysfunction is a hallmark of Alzheimer's Disease (AD), but the scope and severity of these specific deficits across forms of AD are not well characterized. We designed a high-throughput, longitudinal, phenotypic assay to track MT dynamics and bioenergetics in glutamatergic iPSC-derived human neurons possessing mutations in presenilin 1 (PSEN1), presenilin 2 (PSEN2) and the amyloid beta precursor protein (APP). Each gene set was comprised of iPSC-derived neurons from an AD patient as well as two to three engineered mutations with appropriate isogenic and age matched controls. These iPSC-derived neurons were imaged every other day beginning at DIV10 to assess how MT length and content change over a ten day time course using a mitochondrially targeted reporter. A second cytosolic reporter allowed for visualization of neurites. Bioenergetics assays, focusing on MT respiration and individual electron transport chain (ETC) complexes, were also surveyed over this time course. Mutations in all three genes altered MT function measured by basal, ATP-linked, and maximal oxygen consumption rate; and spare respiratory capacity, with PSEN1/PSEN2 alleles being more severe than APP mutations. Electron flow through Complexes I-IV was decreased in PSEN1/PSEN2 mutations but; in contrast, APP alleles had only modest impairments of Complexes I and II. We measured aspects of MT dynamics, including fragmentation, and neurite degeneration, both of which were dramatic in PSEN1/PSEN2 alleles, but essentially absent in APP alleles. The marked differences in MT pathology may occur from the distinct ways amyloids are processed into amyloid beta peptides (Aβ) and may correlate with the disease severity.

Microglia depletion reduces human neuronal APOE4-related pathologies in a chimeric Alzheimer's disease model.

Despite strong evidence supporting the important roles of both apolipoprotein E4 (APOE4) and microglia in Alzheimer's disease (AD) pathogenesis, the effects of microglia on neuronal APOE4-related AD pathogenesis remain elusive. To examine such effects, we utilized microglial depletion in a chimeric model with induced pluripotent stem cell (iPSC)-derived human neurons in mouse hippocampus. Specifically, we transplanted homozygous APOE4, isogenic APOE3, and APOE-knockout (APOE-KO) iPSC-derived human neurons into the hippocampus of human APOE3 or APOE4 knockin mice and then depleted microglia in half of the chimeric mice. We found that both neuronal APOE and microglial presence were important for the formation of Aβ and tau pathologies in an APOE isoform-dependent manner (APOE4>APOE3). Single-cell RNA sequencing analysis identified two pro-inflammatory microglial subtypes with elevated MHC-II gene expression enriched in chimeric mice with human APOE4 neuron transplants. These findings highlight the concerted roles of neuronal APOE, especially APOE4, and microglia in AD pathogenesis.

Polyethyleneimine facilitates the growth and electrophysiological characterization of iPSC-derived motor neurons.

Induced pluripotent stem cell (iPSC) technology, in combination with electrophysiological characterization via multielectrode array (MEA), has facilitated the utilization of iPSC-derived motor neurons (iPSC-MNs) as highly valuable models for underpinning pathogenic mechanisms and developing novel therapeutic interventions for motor neuron diseases (MNDs). However, the challenge of MN adherence to the MEA plate and the heterogeneity presented in iPSC-derived cultures raise concerns about the reproducibility of the findings obtained from these cellular models. We discovered that one novel factor modulating the electrophysiological activity of iPSC-MNs is the extracellular matrix (ECM) used in the coating to support in vitro growth, differentiation and maturation of iPSC-MNs. The current study showed that two coating conditions, namely, Poly-L-ornithine/Matrigel (POM) and Polyethyleneimine (PEI) strongly promoted attachment of iPSC-MNs on MEA culture dishes compared to three other coating conditions, and both facilitated the maturation of iPSC-MNs as characterized by the detection of extensive electrophysiological activities from the MEA plates. POM coating accelerated the maturation of the iPSC-MNs for up to 5weeks, which suits modeling of neurodevelopmental disorders. However, the application of PEI resulted in more even distribution of the MNs on the culture dish and reduced variability of electrophysiological signals from the iPSC-MNs in 7-week cultures, which permitted the detection of enhanced excitability in iPSC-MNs from patients with amyotrophic lateral sclerosis (ALS). This study provides a comprehensive comparison of five coating conditions and offers POM and PEI as favorable coatings for in vitro modeling of neurodevelopmental and neurodegenerative disorders, respectively.

Nifuroxazide rescues the deleterious effects due to CHCHD10-associated MICOS defects in disease models.

The identification of a point mutation (p.Ser59Leu) in the CHCHD10 gene was the first genetic evidence that mitochondrial dysfunction can trigger motor neuron disease. Since then, we have shown that this mutation leads to the disorganization of the MItochondrial contact site and Cristae Organizing System (MICOS) complex that maintains the mitochondrial cristae structure. Here, we generated yeast mutant strains mimicking MICOS instability and used them to test the ability of more than 1600 compounds from 2 repurposed libraries to rescue the growth defect of those cells. Among the hits identified, we selected nifuroxazide, a broad-spectrum antibacterial molecule. We show that nifuroxazide rescues mitochondrial network fragmentation and cristae abnormalities in CHCHD10S59L/+ patient fibroblasts. This molecule also decreases caspase-dependent death of human CHCHD10S59L/+ iPSC-derived motor neurons. Its benefits involve KIF5B-mediated mitochondrial transport enhancement, evidenced by increased axonal movement and syntaphilin degradation in patient-derived motor neurons. Our findings strengthen the MICOS-mitochondrial transport connection. Nifuroxazide and analogues emerge as potential therapeutics for MICOS-related disorders like motor neuron disease. Its impact on syntaphilin hints at broader neurological disorder applicability for nifuroxazide.

A de novo Missense Mutation in PPP2R5D Alters Dopamine Pathways and Morphology of iPSC-derived Midbrain Neurons.

Induced pluripotent stem cell (iPSC) models of neurodevelopmental disorders (NDDs) have promoted an understanding of commonalities and differences within or across patient populations by revealing the underlying molecular and cellular mechanisms contributing to disease pathology. Here, we focus on developing a human model for PPP2R5D-related NDD, called Jordan syndrome, which has been linked to Early-Onset Parkinson's Disease (EOPD). Here we sought to understand the underlying molecular and cellular phenotypes across multiple cell states and neuronal subtypes in order to gain insight into Jordan syndrome pathology. Our work revealed that iPSC-derived midbrain neurons from Jordan syndrome patients display significant differences in dopamine-associated pathways and neuronal architecture. We then evaluated a CRISPR-based approach for editing heterozygous dominant G-to-A mutations at the transcript level in patient-derived neural stem cells. Our findings show site-directed RNA editing is influenced by sgRNA length and cell type. These studies support the potential for a CRISPR RNA editor system to selectively edit mutant transcripts harboring G-to-A mutations in neural stem cells while providing an alternative editing technology for those suffering from NDDs.

Validation of a functional human AD model with four AD therapeutics utilizing patterned ipsc-derived cortical neurons integrated with microelectrode arrays

Preclinical methods are needed for screening potential Alzheimer’s disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. Long-term potentiation (LTP), which is a correlate of learning and memory, was induced in mature human iPSC-derived cortical neurons cultured on microelectrode arrays utilizing circuit patterns connecting two adjacent electrodes. We demonstrated an LTP system that modeled the MCI and pre-MCI stages of Alzheimer’s and validated this functional system utilizing four AD therapeutics, which was also verified utilizing patch-clamp electrophysiology. LTP was induced by tetanic electrical stimulation, and LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aβ42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aβ42-induced LTP impairment. The results illustrate the utility of the system as a validated platform to model MCI AD pathology, and potentially for the pre-MCI phase before significant neuronal death. This system also has the potential to become an ideal platform for high-content therapeutic screening for other neurodegenerative diseases.

Direct and indirect regulation of β-glucocerebrosidase by the transcription factors USF2 and ONECUT2

Mutations in GBA1 encoding the lysosomal enzyme β-glucocerebrosidase (GCase) are among the most prevalent genetic susceptibility factors for Parkinson’s disease (PD), with 10–30% of carriers developing the disease. To identify genetic modifiers contributing to the incomplete penetrance, we examined the effect of 1634 human transcription factors (TFs) on GCase activity in lysates of an engineered human glioblastoma line homozygous for the pathogenic GBA1 L444P variant. Using an arrayed CRISPR activation library, we uncovered 11 TFs as regulators of GCase activity. Among these, activation of MITF and TFEC increased lysosomal GCase activity in live cells, while activation of ONECUT2 and USF2 decreased it. While MITF, TFEC, and USF2 affected GBA1 transcription, ONECUT2 might control GCase trafficking. The effects of MITF, TFEC, and USF2 on lysosomal GCase activity were reproducible in iPSC-derived neurons from PD patients. Our study provides a systematic approach to identifying modulators of GCase activity and deepens our understanding of the mechanisms regulating GCase.

CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB-specific cytoplasmic degradation. By adding a neuronal-specific NLS, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo postmitotic cell models.

Localized synthesis of molecular chaperones sustains neuronal proteostasis.

Neurons are challenged to maintain proteostasis in neuronal projections, particularly with the physiological stress at synapses to support intercellular communication underlying important functions such as memory and movement control. Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. Using high-resolution fluorescent microscopy, we discovered that neurons localize a subset of chaperone mRNAs to their dendrites, particularly more proximal regions, and increase this asymmetric localization following proteotoxic stress through microtubule-based transport from the soma. The most abundant chaperone mRNA in dendrites encodes the constitutive heat shock protein 70, HSPA8. Proteotoxic stress in cultured neurons, induced by inhibiting proteasome activity or inducing oxidative stress, enhanced transport of Hspa8 mRNAs to dendrites and the percentage of mRNAs engaged in translation on mono and polyribosomes. Knocking down the ALS-related protein Fused in Sarcoma (FUS) and a dominant mutation in the heterogenous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) impaired stress-mediated localization of Hspa8 mRNA to dendrites in cultured murine motor neurons and human iPSC-derived neurons, respectively, revealing the importance of these RNA-binding proteins in maintaining proteostasis. These results reveal the increased dendritic localization and translation of the constitutive HSP70 Hspa8 mRNA as a crucial neuronal stress response to uphold proteostasis and prevent neurodegeneration.

Exploring P2X7 receptor antagonism as a therapeutic target for neuroprotection in an hiPSC motor neuron model.

ATP is present in negligible concentrations in the interstitium of healthy tissues but accumulates to significantly higher concentrations in an inflammatory microenvironment. ATP binds to 2 categories of purine receptors on the surface of cells, the ionotropic P2X receptors and metabotropic P2Y receptors. Included in the family of ionotropic purine receptors is P2X7 (P2X7R), a non-specific cation channel with unique functional and structural properties that suggest it has distinct roles in pathological conditions marked by increased extracellular ATP. The role of P2X7R has previously been explored in microglia and astrocytes within the context of neuroinflammation, however the presence of P2X7R on human motor neurons and its potential role in neurodegenerative diseases has not been the focus of the current literature. We leveraged the use of human iPSC-derived spinal motor neurons (hiPSC-MN) as well as human and rodent tissue to demonstrate the expression of P2X7R on motor neurons. We extend this observation to demonstrate that these receptors are functionally active on hiPSC-MN and that ATP can directly induce death via P2X7R activation in a dose dependent manner. Finally, using a highly specific P2X7R blocker, we demonstrate how modulation of P2X7R activation on motor neurons is neuroprotective and could provide a unique pharmacologic target for ATP-induced MN death that is distinct from the role of ATP as a modulator of neuroinflammation.

Subtype-specific neurons from patient iPSCs display distinct neuropathological features of Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by massive neuronal loss in the brain. Both cortical glutamatergic neurons and basal forebrain cholinergic neurons (BFCNs) in the AD brain are selectively vulnerable. The degeneration and dysfunction of these two subtypes of neurons are closely associated with the cognitive decline of AD patients. The determination of cellular and molecular mechanisms involved in AD pathogenesis, especially in the early stage, will largely facilitate the understanding of this disease and the development of proper intervention strategies. However, due to the inaccessibility of living neurons in the brains of patients, it remains unclear how cortical glutamatergic neurons and BFCNs respond to pathological stress in the early stage of AD. In this study, we established in vitro differentiation systems that can efficiently differentiate patient-derived iPSCs into BFCNs. We found that AD-BFCNs secreted less Aβ peptide than cortical glutamatergic neurons did, even though the Aβ42/Aβ40 ratio was comparable to that of cortical glutamatergic neurons. To further mimic the neurotoxic niche in AD brain, we treated iPSC-derived neurons with Aβ42 oligomer (AβO). BFCNs are less sensitive to AβO induced tau phosphorylation and expression than cortical glutamatergic neurons. However, AβO could trigger apoptosis in both AD-cortical glutamatergic neurons and AD-BFCNs. In addition, AD iPSC-derived BFCNs and cortical glutamatergic neurons exhibited distinct electrophysiological firing patterns and elicited different responses to AβO treatment. These observations revealed that subtype-specific neurons display distinct neuropathological changes during the progression of AD, which might help to understand AD pathogenesis at the cellular level.

Alzheimer's disease induced neurons bearing PSEN1 mutations exhibit reduced excitability.

Alzheimer's disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined iPSC-derived neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding disease pathogenesis.

Widespread transposable element dysregulation in human aging brains with Alzheimer's disease.

Transposable element (TE) dysregulation is associated with neuroinflammation in Alzheimer's disease (AD) brains. Yet, TE quantitative trait loci (teQTL) have not been well characterized in human aged brains with AD. We leveraged large-scale bulk and single-cell RNA sequencing, whole-genome sequencing (WGS), and xQTL from three human AD brain biobanks to characterize TE expression dysregulation and experimentally validate AD-associated TEs using CRISPR interference (CRISPRi) assays in human induced pluripotent stem cell (iPSC)-derived neurons. We identified 26,188 genome-wide significant TE expression QTLs (teQTLs) in human aged brains. Subsequent colocalization analysis of teQTLs with AD genetic loci identified AD-associated teQTLs and linked locus TEs. Using CRISPRi assays, we pinpointed a neuron-specific suppressive role of the activated short interspersed nuclear element (SINE; chr11:47608036-47608220) on expression of C1QTNF4 via reducing neuroinflammation in human iPSC-derived neurons. We identified widespread TE dysregulation in human AD brains and teQTLs offer a complementary analytic approach to identify likely AD risk genes. Widespread transposable element (TE) dysregulations are observed in human aging brains with degrees of neuropathology, apolipoprotein E (APOE) genotypes, and neuroinflammation in Alzheimer's disease (AD). A catalog of TE quantitative trait loci (teQTLs) in human aging brains was created using matched RNA sequencing and whole-genome sequencing data. CRISPR interference assays reveal that an upregulated intergenic TE from the MIR family (chr11: 47608036-47608220) suppresses expression of its nearest anti-inflammatory gene C1QTNF4 in human induced pluripotent stem cell-derived neurons.

How is Excitotoxicity Being Modelled in iPSC-Derived Neurons?

Excitotoxicity linked either to environmental causes (pesticide and cyanotoxin exposure), excitatory neurotransmitter imbalance, or to intrinsic neuronal hyperexcitability, is a pathological mechanism central to neurodegeneration in amyotrophic lateral sclerosis (ALS). Investigation of excitotoxic mechanisms using in vitro and in vivo animal models has been central to understanding ALS mechanisms of disease. In particular, advances in induced pluripotent stem cell (iPSC) technologies now provide human cell-based models that are readily amenable to environmental and network-based excitotoxic manipulations. The cell-type specific differentiation of iPSC, combined with approaches to modelling excitotoxicity that include editing of disease-associated gene variants, chemogenetics, and environmental risk-associated exposures make iPSC primed to examine gene-environment interactions and disease-associated excitotoxic mechanisms. Critical to this is knowledge of which neurotransmitter receptor subunits are expressed by iPSC-derived neuronal cultures being studied, how their activity responds to antagonists and agonists of these receptors, and how to interpret data derived from multi-parameter electrophysiological recordings. This review explores how iPSC-based studies have contributed to our understanding of ALS-linked excitotoxicity and highlights novel approaches to inducing excitotoxicity in iPSC-derived neurons to further our understanding of its pathological pathways.

Unraveling Axonal Transcriptional Landscapes: Insights from iPSC-Derived Cortical Neurons and Implications for Motor Neuron Degeneration.

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.

269P Human neuroblastoma cell-derived neurons and iPSC-derived neurons as models for neuromuscular disorders

269P Human neuroblastoma cell-derived neurons and iPSC-derived neurons as models for neuromuscular disorders

Altered mRNA transport and local translation in iNeurons with RNA binding protein knockdown.

Neurons rely on mRNA transport and local translation to facilitate rapid protein synthesis in processes far from the cell body. These processes allow precise spatial and temporal control of translation and are mediated by RNA binding proteins (RBPs), including those known to be associated with neurodegenerative diseases. Here, we use proteomics, transcriptomics, and microscopy to investigate the impact of RBP knockdown on mRNA transport and local translation in iPSC-derived neurons. We find thousands of transcripts enriched in neurites and that many of these transcripts are locally translated, possibly due to the shorter length of transcripts in neurites. Loss of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS)-associated RBPs TDP- 43 and hnRNPA1 lead to distinct alterations in the neuritic proteome and transcriptome. TDP-43 knockdown (KD) leads to increased neuritic mRNA and translation. In contrast, hnRNPA1 leads to increased neuritic mRNA, but not translation, and more moderate effects on local mRNA profiles, possibly due to compensation by hnRNPA3. These results highlight the crucial role of FTD/ALS-associated RBPs in mRNA transport and local translation in neurons and the importance of these processes in neuron health and disease.