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, 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 subset of previously unreported 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.

An iPSC model for POLR3A-associated spastic ataxia: Generation of three unrelated patient cell lines.

Spastic Ataxias (SA) are a group of neurodegenerative disorders with combined pyramidal and cerebellar system affection, leading to an overlap phenotype between Hereditary Spastic Paraplegias (HSP) and Cerebellar Ataxias (CA). Here we describe the generation of iPSCs from three unrelated patients with an ultra-rare subtype of SA caused by compound heterozygous mutations in POLR3A, that encodes the largest subunit of RNA polymerase III. iPSCs were reprogrammed from normal human dermal fibroblasts (NHDFs) using episomal reprogramming with integration-free plasmid vectors: HIHRSi004-A, derived from a 44 year-old male carrying the mutations c.1909 + 22G > A/c.3944_3945delTG, HIHRSi005-A obtained from a 66 year-old male carrying the mutations c.1909 + 22G > A/c.1531C > T, and HIHRSi006-A from a 27 year-old male carrying the mutations c.1909 + 22G > A/c.2472_2472delC (ENST00000372371.8).

The kinesin motor KIF1C is a putative transporter of the exon junction complex in neuronal cells.

Neurons critically depend on regulated RNA localization and tight control of spatio-temporal gene expression to maintain their morphological and functional integrity. Mutations in the kinesin motor protein gene KIF1C cause Hereditary Spastic Paraplegia, an autosomal recessive disease leading to predominant degeneration of the long axons of central motoneurons. In this study we aimed to gain insight into the molecular function of KIF1C and understand how KIF1C dysfunction contributes to motoneuron degeneration. We used affinity proteomics in neuronally differentiated neuroblastoma cells (SH-SY5Y) to identify the protein complex associated with KIF1C in neuronal cells; candidate interactions were then validated by immunoprecipitation and mislocalization of putative KIF1C cargoes was studied by immunostainings. We found KIF1C to interact with all core components of the exon junction complex (EJC); expression of mutant KIF1C in neuronal cells leads to loss of the typical localization distally in neurites. Instead, EJC core components accumulate in the pericentrosomal region, here co-localizing with mutant KIF1C. These findings suggest KIF1C as a neuronal transporter of the EJC. Interestingly, the binding of KIF1C to the EJC is RNA-mediated, as treatment with RNAse prior to immunoprecipitation almost completely abolishes the interaction. Silica-based solid-phase extraction of UV-crosslinked RNA-protein complexes furthermore supports direct interaction of KIF1C with RNA, as recently also demonstrated for kinesin heavy chain. Taken together, our findings are consistent with a model where KIF1C transports mRNA in an EJC-bound and therefore transcriptionally silenced state along neurites, thus providing the missing link between the EJC and mRNA localization in neurons.

Generation of the CRISPR/Cas9-mediated KIF1C knock-out human iPSC line HIHRSi003-A-1.

Bi-allelic loss-of-function mutations in the gene encoding the motor protein KIF1C are associated with Hereditary Spastic Paraplegia (HSP) type SPG58, a slowly progressive neurodegenerative motoneuron disease. The biological role of KIF1C is incompletely understood. We used a protein-based CRISPR/Cas9 genome editing approach to generate a homozygous KIF1C knock-out iPSC line (HIHRSi003-A-1) from a healthy control. This iPSC-KIF1C-/- line and the corresponding isogenic control are a useful model to study the physiological function of KIF1C and the pathophysiological consequences of KIF1C dysfunction in human disease.

Bi-allelic variants in RNF170 are associated with hereditary spastic paraplegia.

Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions.

Generation of two iPSC lines derived from two unrelated patients with Gaucher disease.

Gaucher disease is the most common autosomal recessive lysosomal storage disorder, caused by mutations in the ß-glucocerebrosidase gene GBA. Here we describe generation of iPSC from skin-derived fibroblasts from two unrelated individuals with neuronopathic forms of Gaucher disease. The donor for line iPSC-GBA-1, a 21 month old girl, carried the recurring GBA mutation c.1448 T > C, p.Leu483Pro at homozygous state; fibroblasts for line iPS-GBA-2 were obtained from a 4 year old girl compound heterozygous for the GBA mutations c.667 T > C, p.Trp223Arg and c.1226A > G, p.Asn409Ser. iPSCs were developed using integration free episomal vectors (OCT4, KLF4; L-MYC, SOX2 (OSKM) and LIN28). Resource table.

Calpastatin ablation aggravates the molecular phenotype in cell and animal models of Huntington disease.

Deciphering the molecular pathology of Huntington disease is of particular importance, not only for a better understanding of this neurodegenerative disease, but also to identify potential therapeutic targets. The polyglutamine-expanded disease protein huntingtin was shown to undergo proteolysis, which results in the accumulation of toxic and aggregation-prone fragments. Amongst several classes of proteolytic enzymes responsible for huntingtin processing, the group of calcium-activated calpains has been found to be a significant mediator of the disease protein toxicity. To confirm the impact of calpain-mediated huntingtin cleavage in Huntington disease, we analysed the effect of depleting or overexpressing the endogenous calpain inhibitor calpastatin in HEK293T cells transfected with wild-type or polyglutamine-expanded huntingtin. Moreover, we crossbred huntingtin knock-in mice with calpastatin knockout animals to assess its effect not only on huntingtin cleavage and aggregation but also additional molecular markers. We demonstrated that a reduced or ablated expression of calpastatin triggers calpain overactivation and a consequently increased mutant huntingtin cleavage in cells and in vivo. These alterations were accompanied by an elevated formation of predominantly cytoplasmic huntingtin aggregates. On the other hand, overexpression of calpastatin in cells attenuated huntingtin fragmentation and aggregation. In addition, we observed an enhanced cleavage of DARPP-32, p35 and synapsin-1 in neuronal tissue upon calpain overactivation. Our results corroborate the important role of calpains in the molecular pathogenesis of Huntington disease and endorse targeting these proteolytic enzymes as a therapeutic approach.

A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders.