Cajal Bodies Research Articles

Regulation of Cellular Ribonucleoprotein Granules: From Assembly to Degradation via Post-translational Modification.

Cells possess membraneless ribonucleoprotein (RNP) granules, including stress granules, processing bodies, Cajal bodies, or paraspeckles, that play physiological or pathological roles. RNP granules contain RNA and numerous RNA-binding proteins, transiently formed through the liquid–liquid phase separation. The assembly or disassembly of numerous RNP granules is strongly controlled to maintain their homeostasis and perform their cellular functions properly. Normal RNA granules are reversibly assembled, whereas abnormal RNP granules accumulate and associate with various neurodegenerative diseases. This review summarizes current studies on the physiological or pathological roles of post-translational modifications of various cellular RNP granules and discusses the therapeutic methods in curing diseases related to abnormal RNP granules by autophagy.

Coilin as a regulator of NF-kB mediated inflammation in preeclampsia.

ABSTRACTThe nuclear factor-Kappa B (NF-κB) pathway is a crucial mediator of inflammatory signaling. Aberrant activation of NF-κB is associated with several disorders including preeclampsia (PE). Many regulators of the NF-κB pathway have been identified, including microRNAs (miRNAs). Specifically, miR-517-3p targets mRNA encoding TNFAIP3 Interacting Protein 1 (TNIP1), an inhibitor of NF-κB signaling. Activation of NF-κB increases production of the cytokine TNF superfamily member 15 (TNFSF15), leading to the upregulation of anti-angiogenic soluble vascular endothelial growth factor receptor 1 (sFlt-1). We have previously observed that Cajal bodies (CBs), subnuclear domains, are associated with the chromosome 19 miRNA gene cluster (C19MC), which encodes miR-517-3p. We have also found that coilin, the CB marker protein, is a positive regulator of miRNA biogenesis. Here we report that coilin is a regulator of miR-517-3p, sFlt-1, TNIP1, TNFSF15 and NF-κB activation, and this regulation is influenced by hypoxia. We also report that coilin and CBs are induced in the reduced uterine perfusion pressure (RUPP) rat model of PE. Collectively, the data presented here implicate coilin as a novel regulator of NF-κB activation and sFlt-1 upregulation.

Analysis of the Arabidopsis coilin mutant reveals a positive role of AtCOILIN in plant immunity.

Biogenesis of ribonucleoproteins occurs in dynamic subnuclear compartments called Cajal bodies (CBs). COILIN is a critical scaffolding component essential for CB formation, composition, and activity. We recently showed that Arabidopsis (Arabidopsis thaliana) AtCOILIN is phosphorylated in response to bacterial elicitor treatment. Here, we further investigated the role of AtCOILIN in plant innate immunity. Atcoilin mutants are compromised in defense responses to bacterial pathogens. Besides confirming a role of AtCOILIN in alternative splicing (AS), Atcoilin showed differential expression of genes that are distinct from those of AS, including factors involved in RNA biogenesis, metabolism, plant immunity, and phytohormones. Atcoilin mutant plants have reduced levels of defense phytohormones. As expected, the mutant plants were more sensitive to the necrotrophic fungal pathogen Botrytis cinerea. Our findings reveal an important role for AtCOILIN in innate plant immunity.

The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3′-end processing factors for RDH genes are enriched; however, this association’s role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs’ association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)–Cap binding complex (CBC)–Negative elongation factor (NELF), components of CBs, interferes with CBs’ association with HLBs and 3′ Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator’s involvement in CBs’ association with HLBs to facilitate 3′ Pol II pausing and subsequent 3′-end processing of RDH genes by supplying 3′-end processing factors.

Identification of Signaling Pathways and Phase Separating Domains that Drive Cajal Body Formation

Cajal bodies (CBs) are membraneless subnuclear domains that contribute to the biogenesis and maturation of ribonucleoproteins (RNPs) which take part in fundamental cellular activities such as translation, pre‐mRNA splicing, and telomere maintenance. Partitioning of CBs occurs through liquid‐liquid phase separation (LLPS) where the CB marker protein, coilin, is thought to scaffold the CB. However, the formation of CBs and the regulation of CB number has not been fully elucidated. Disease states such as cancer and preeclampsia have altered CB expression, thus additional insight into CB condensation would provide an important cellular model system that could be used to investigate these and other diseases. We hypothesize that the post‐translational modification (PTM) of coilin regulates CB formation and number by influencing the LLPS of coilin via residues in its intrinsically disordered region (IDR). Here we show, for the first time, a cycloheximide inducible model of CB formation in fibroblasts without increasing coilin concentration. We are also able to induce CB formation by inhibiting kinase GSK3β with indirubin, as well as abolish CB formation by inhibiting phosphatases PP1 and PP2A with okadaic acid. Lastly, we have identified two phase‐separating domains in coilin’s IDR using the phase separation predicting software ParSe. Deletion of each domain prevents CB formation in HeLa cells but does not prevent coilin’s self‐interaction. Collectively, these data will be able to help us understand the role of coilin’s scaffolding capabilities via LLPS mediated CB condensation, as well as provide an important cellular model system that can be used to investigate CB functionality in disease states where CB formation is dysregulated.

Coilin Modulates Nuclear Organization by Promoting Protein SUMOylation

Coilin was first identified as the marker protein for Cajal Bodies (CBs). However, CBs are only abundant in neuronal, embryonic, and cancer cells, while primary cells have few CBs. Even in cells with CBs, 70% of coilin is nucleoplasmic, not found in CBs. CBs are subnuclear domains that function in small nuclear ribonucleoprotein (snRNP) biogenesis as well as production of telomerase RNP, expression and 3’end processing of small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and small CB‐associated RNAs (scaRNAs). The function of nucleoplasmic coilin is not well understood. We have previously shown that coilin promotes miRNA biogenesis by promoting phosphorylation of the Microprocessor component, DGCR8, at its S377 residue in both cells with and without CBs. We have since identified 7 additional serine residues of DGCR8 with decreased phosphorylation in coilin knockdown through phosphoproteomic analysis of HeLa cells. In addition to phosphorylation, the addition of a small ubiquitin‐like modifier (SUMO), or SUMOylation, of DGCR8 also increases its stability. Because of coilin’s role in the promotion of one post‐translational modification, here we aimed to test the hypothesis that coilin knockdown alters protein SUMOylation and results in altered nuclear organization. We conducted a series of siRNA knockdowns, Western blots, and immunoprecipitation experiments. Here we show, for the first time, that coilin knockdown results in global decrease of protein SUMOylation in cells with (HeLa) and without CBs (WI‐38), suggesting that regulation of protein SUMOylation may be a function of nucleoplasmic coilin. Specifically, we found that coilin knockdown results in decreased SUMOylation of DGCR8 and Speckled protein 100kDa (Sp100), a major component of the promyelocytic leukemia nuclear body (PML‐NB). Sp100 is vital to PML‐NB formation, and decreased SUMOylation of Sp100 decreases its stability. This information led us to the second part of our hypothesis. We used immunofluorescence and found that coilin knockdown results in decreased numbers of PML‐NBs per cell. To summarize, we have identified coilin as a regulator of protein phosphorylation, SUMOylation, and nuclear organization.

Zebrafish as a Model for Evaluating Coilin‐Mediated microRNA Biogenesis

Cajal bodies (CBs) are nuclear bodies that are hosts for the modification and assembly of ribonucleoproteins (RNPs). The nuclear protein coilin is known as the CB marker protein as it is the scaffold for CB assembly and vital for structural integrity. However, there is still not much understood about the function of coilin. Recent studies by our lab implicate coilin in the biogenesis of microRNAs (miRNAs). Currently, there are sparse options in animal models for studying coilin and CB function that account for limitations such as off‐target effects of coilin depletion or incomplete protein knockdown. Due to this, we sought to develop novel coilin mutations in zebrafish to advance functional studies of this protein and CBs. In considering how we would approach this, we opted for the innovative CRISPR/Cas9 technology as an optimal strategy and hypothesized that the implementation of this genome editing technology would allow us to develop multiple lines of coilin‐modified zebrafish for studies investigating coilin’s role in miRNA biogenesis. We selected a suitable Protospacer Adjacent Motif (PAM) sequence and designed guide RNAs for our mutations of interest in silico, which we then generated in vitro, and injected into fertilized zebrafish embryos at the one‐cell stage. An initial screening for insertions or deletions (indels) took place at 72 hours post fertilization to determine mutation frequency and once again when the zebrafish reached adulthood. Through our efforts, we have managed to obtain zebrafish that are heterozygous for coilin‐null mutations and are currently in the midst of screening for additional lines of coilin mutations which could serve to characterize the functional domains of coilin in vivo. Utilizing CRISPR/Cas9 technology, we have generated the first coilin‐null mutation in a zebrafish line allowing our lab to expand our investigations into the role of coilin and CBs in the regulatory network of the nucleus.

Differential Expression of Nuclear Body Component, Zc3h8, Disrupts DNA Double Strand Break Repair

The processes of DNA damage recognition and repair are paramount to maintaining genome integrity in the cell. Failure to do so results in genetic instability and the accumulation of mutations. Double strand breaks have the greatest potential to introduce mutations because rearrangements, non‐homologous end joining, deletions, or insertions may result. Unrepaired or incorrectly repaired DNA double strand breaks can lead to cell death, apoptosis, or disease such as cancer. We previously demonstrated that the C3H1 zinc finger‐containing protein Zc3h8 (also called Fliz1) promotes oncogenic phenotypes when overexpressed in cells, while oncogenic phenotypes are suppressed when expression is reduced. In mice, Zc3h8 expression affected tumorigenesis and tumor size in vivo. Higher expression of Zc3h8 led to more frequent tumors and larger tumors in mice. In humans Zc3h8 is overexpressed in 2‐6% of cancers and is a statistically significant prognostic indicator for poor rates of survival. The Zc3h8 protein has been localized to nuclear bodies including promyelocytic leukemia (PML) and Cajal Bodies where key cellular processes including transcription, translation, post‐translational modifications, and RNA processing are regulated. PML Bodies, in particular, have a role in DNA damage response and repair since P53 is located in PML Bodies along with DAXX, ATRX, and CK2. Since Zc3h8 co‐localized with DNA damage response and repair proteins in PML bodies and promotes oncogenic properties in cells, we investigated if Zc3h8 expression alters how cells respond to DNA damage such as double strand breaks and how this damage is mitigated in cells. Differential expression of Zc3h8 affected mouse mammary tumor cell’s ability to repair double strand breaks caused by the topoisomerase II inhibitor etoposide as indicated by the presence of γH2AX, a marker for unrepaired double strand breaks. Using a Comet assay, we found extensive DNA fragmentation in cells with elevated Zc3h8 expression despite high rates of proliferation and migration. Furthermore, Zc3h8 increases cellular tolerance for DNA damage and alters apoptosis. These phenotypes promote further mutation and may indicate a molecular cause for highly aggressive and progressive cancers with elevated Zc3h8 expression. These conclusions suggest a DNA damage repair and response pathway that is altered by Zc3h8 expression levels can affect multiple aspects of cellular behavior and promote oncogenic phenotypes.

TGS1 mediates 2,2,7-trimethyl guanosine capping of the human telomerase RNA to direct telomerase dependent telomere maintenance

Pathways that direct the selection of the telomerase-dependent or recombination-based, alternative lengthening of telomere (ALT) maintenance pathway in cancer cells are poorly understood. Using human lung cancer cells and tumor organoids we show that formation of the 2,2,7-trimethylguanosine (TMG) cap structure at the human telomerase RNA 5′ end by the Trimethylguanosine Synthase 1 (TGS1) is central for recruiting telomerase to telomeres and engaging Cajal bodies in telomere maintenance. TGS1 depletion or inhibition by the natural nucleoside sinefungin impairs telomerase recruitment to telomeres leading to Exonuclease 1 mediated generation of telomere 3′ end protrusions that engage in RAD51-dependent, homology directed recombination and the activation of key features of the ALT pathway. This indicates a critical role for 2,2,7-TMG capping of the RNA component of human telomerase (hTR) in enforcing telomerase-dependent telomere maintenance to restrict the formation of telomeric substrates conductive to ALT. Our work introduces a targetable pathway of telomere maintenance that holds relevance for telomere-related diseases such as cancer and aging.

A point mutation in human coilin prevents Cajal body formation.

Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP-coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).

RNA is required for the integrity of multiple nuclear and cytoplasmic membrane‐less RNP granules

Numerous membrane‐less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super‐enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane‐less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules.

Arabidopsis AGO4 loses its Cajal body localization when heterologously expressed in Nicotiana benthamiana

ABSTRACT In plants, the RNA-directed DNA methylation (RdDM) pathway plays a major role in establishing DNA methylation. At least some components of the RdDM machinery, including the central component AGO4, are known to concentrate in a subnuclear compartment called the Cajal body in the model plant Arabidopsis thaliana. The molecular underpinnings of Cajal body localization, however, have remained elusive so far. Here, we found that Arabidopsis AGO4 (AtAGO4) fused to GFP does not present its typical Cajal body localization, when transiently expressed in Nicotiana benthamiana. Nevertheless, the endogenous AGO4 protein from N. benthamiana shows a clear accumulation in the Cajal body. Thus, our results suggest that the Cajal body localization of AtAGO4 requires specific molecular machinery that cannot be replaced by orthologues in N. benthamiana. This study presents an experimental system that could lead to mechanistic insights into the targeting of proteins to and localization in the Cajal body in plants.

Functional nuclear retention of pre-mRNA involving Cajal bodies during meiotic prophase in European larch (Larix decidua).

Gene regulation ensures that the appropriate genes are expressed at the proper time. Nuclear retention of incompletely spliced or mature mRNAs is emerging as a novel, previously underappreciated layer of posttranscriptional regulation. Studies on this phenomenon indicated that it exerts a significant influence on the regulation of gene expression by regulating export and translation delay, which allows the synthesis of specific proteins in response to a stimulus or at strictly controlled time points, for example, during cell differentiation or development. Here, we show that transcription in microsporocytes of European larch (Larix decidua) occurs in a pulsatile manner during prophase of the first meiotic division. Transcriptional activity was then silenced after each pulse. However, the transcripts synthesized were not exported immediately to the cytoplasm but were retained in the nucleoplasm and Cajal bodies (CBs). In contrast to the nucleoplasm, we did not detect mature transcripts in CBs, which only stored nonfully spliced transcripts with retained introns. Notably, the retained introns were spliced at precisely defined times, and fully mature mRNAs were released into the cytoplasm for translation. As similar processes have been observed during spermatogenesis in animals, our results illustrate an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.

Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient-induced pluripotent stem cell-derived retinal pigment epithelium cells.

IntroductionMutations in pre‐mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri‐snRNP complex, cause autosomal‐dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri‐snRNP proteins result in retina‐specific disorders, and so far, the underlying mechanism of splicing factors‐related RP is poorly understood.MethodsWe used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31‐adRP, unaffected individuals and a CRISPR/Cas9 isogenic control.ResultsTo fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle‐related pathways to be significantly affected. Strikingly, the patient‐derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin‐conjugated proteins including key visual cycle and other RP‐linked tri‐snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild‐type levels led to tri‐snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival.ConclusionsOur data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31‐initiated mis‐splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31‐adRP patients.

UHMK1-dependent phosphorylation of Cajal body protein coilin alters 5-FU sensitivity in colon cancer cells

Resistance to 5-fluorouracil (5-FU) in chemotherapy and recurrence of colorectal tumors is a serious concern that impedes improvements to clinical outcomes. In the present study, we found that conditioned medium (CM) derived from 5-FU-resistant HCT-8/FU cells reduced 5-FU chemosensitivity in HCT-8 colon cancer cells, with corresponding changes to number and morphology of Cajal bodies (CBs) as observable nuclear structures. We found that U2AF homology motif kinase 1 (UHMK1) altered CB disassembly and reassembly and regulated the phosphorylation of coilin, a major component of CBs. This subsequently resulted in a large number of variations in RNA alternative splicing that affected cell survival following 5-FU treatment, induced changes in intracellular phenotype, and transmitted preadaptive signals to adjacent cells in the tumor microenvironment (TME). Our findings suggest that CBs may be useful for indicating drug sensitivity or resistance in tumor cells in response to stress signals. The results also suggest that UHMK1 may be an important factor for maintaining CB structure and morphology by regulating splicing events, especially following cellular exposure to cytotoxic drugs.Graphical EzAUFb-Sx6xbWL9vLuNL_4Video

The creation of raspberry-like droplets and their coalescence dynamics: An ideal model for certain biological processes

HypothesisAlthough a raspberry-like configuration has been long observed in biological processes (e.g., the intimate association between Cajal bodies and B-snurposomes), studies on this morphology are very limited. Raspberry-like droplets created with multiple immiscible liquids are expected to provides an ideal model for such structures in biological systems, including their possible formation mechanism, phase behaviors, and coalescence dynamics. Experiments & SimulationsUsing three liquid phases, one surfactant and some colloidal particles, raspberry-like droplets containing one large central droplet and multiple protrusions embedded on its surface were successfully created. Confocal microscopy studies were carried out to track their formation and coalescence dynamics. A 2D phase-field model was applied to test the influence of the protrusions in the system. FindingsThe formation of this raspberry-like morphology involves a partial inversion process, which was predicted by Friberg et al. with numerical simulations but has never been demonstrated experimentally. A two-step coalescence was revealed, where the protrusions merge first and create a capillary bridge, which drives the droplets to coalesce. Increasing the viscosity of the continuous phase can help to prevent the destabilization. These fundamental features of raspberry-like droplets represent an important step toward producing multi-liquid materials with unique functionality, and can potentially illuminate some biological systems and processes.

Extreme value statistics and arcsine laws for heterogeneous diffusion processes.

Heterogeneous diffusion with a spatially changing diffusion coefficient arises in many experimental systems such as protein dynamics in the cell cytoplasm, mobility of cajal bodies, and confined hard-sphere fluids. Here, we showcase a simple model of heterogeneous diffusion where the diffusion coefficient D(x) varies in a power-law way, i.e., D(x)∼|x|^{-α} with the exponent α>-1. This model is known to exhibit anomalous scaling of the mean-squared displacement (MSD) of the form ∼t^{2/2+α} and weak ergodicity breaking in the sense that ensemble averaged and time averaged MSDs do not converge. In this paper, we look at the extreme value statistics of this model and derive, for all α, the exact probability distributions of the maximum spatial displacement M(t) and arg-maximum t_{m}(t) (i.e., the time at which this maximum is reached) till duration t. In the second part of our paper, we analyze the statistical properties of the residence time t_{r}(t) and the last-passage time t_{ℓ}(t) and compute their distributions exactly for all values of α. Our study unravels that the heterogeneous version (α≠0) displays many rich and contrasting features compared to that of the standard Brownian motion (BM). For example, while for BM (α=0), the distributions of t_{m}(t),t_{r}(t), and t_{ℓ}(t) are all identical (á la "arcsine laws" due to Lévy), they turn out to be significantly different for nonzero α. Another interesting property of t_{r}(t) is the existence of a critical α (which we denote by α_{c}=-0.3182) such that the distribution exhibits a local maximum at t_{r}=t/2 for α<α_{c} whereas it has minima at t_{r}=t/2 for α≥α_{c}. The underlying reasoning for this difference hints at the very contrasting natures of the process for α≥α_{c} and α<α_{c} which we thoroughly examine in our paper. All our analytical results are backed by extensive numerical simulations.

Liquid-liquid phase separation modulates membrane structure and hydration

Cells can condensate some of their intracellular components into compartments known as membraneless-organelles (also called liquid droplets or protein condensates). Among them are nucleoli, Cajal bodies, P-bodies, and stress granules, all existing as liquid protein-rich droplets within the cell and arising from the condensation of cellular material in a process termed liquid-liquid phase separation. Although termed “membraneless”, these droplets can come into contact, wet, and reshape membranous compartments.

An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

A Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated.

Altered action potential waveform and shorter axonal initial segment in hiPSC-derived motor neurons with mutations in VRK1

We recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching. We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient's Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients' hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.