👤 Pratik Gangwani

Senataxin is an RNA:DNA helicase that plays an important role in the resolution of RNA:DNA hybrids (R-loops) formed during transcription. R-loops are involved in the regulation of biological processes such as immunoglobulin class switching, gene expression and DNA repair. Excessive accumulation of R-loops results in DNA damage and loss of genomic integrity. Senataxin is critical for maintaining optimal levels of R-loops to prevent DNA damage and acts as a genome guardian. Within the nucleus, senataxin interacts with various RNA processing factors and DNA damage response and repair proteins. Senataxin interactors include survival motor neuron and zinc finger protein 1, with whom it co-localizes in sub-nuclear bodies. Despite its ubiquitous expression, mutations in senataxin specifically affect neurons and result in distinct neurodegenerative diseases such as amyotrophic lateral sclerosis type 4 and ataxia with oculomotor apraxia type 2, which are attributed to the gain-of-function and the loss-of-function mutations in senataxin, respectively. In addition, low levels of senataxin (loss-of-function) in spinal muscular atrophy result in the accumulation of R-loops causing DNA damage and motor neuron degeneration. Senataxin may play multiple functions in diverse cellular processes; however, its emerging role in R-loop resolution and maintenance of genomic integrity is gaining attention in the field of neurodegenerative diseases. In this review, we highlight the role of senataxin in R-loop resolution and its potential as a therapeutic target to treat neurodegenerative diseases. Show less

Defects in DNA repair pathways are a major cause of DNA damage accumulation leading to genomic instability and neurodegeneration. Efficient DNA damage repair is critical to maintain genomicstability and support cell function and viability. DNA damage results in the activation of cell death pathways, causing neuronal death in an expanding spectrum of neurological disorders, such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), and spinal muscular atrophy (SMA). SMA is a neurodegenerative disorder caused by mutations in the Show less

Mutation in the senataxin (SETX) gene causes an autosomal dominant neuromuscular disorder, amyotrophic lateral sclerosis 4 (ALS4), characterized by degeneration of motor neurons, muscle weakness and atrophy. SETX is an RNA-DNA helicase that mediates resolution of co-transcriptional RNA:DNA hybrids (R-loops). The process of R-loop resolution is essential for the normal functioning of cells, including neurons. The molecular basis of ALS4 pathogenesis and the mechanism of R-loop resolution are unclear. We report that the zinc finger protein ZPR1 binds to RNA:DNA hybrids, recruits SETX onto R-loops and is critical for R-loop resolution. ZPR1 deficiency disrupts the integrity of R-loop resolution complexes containing SETX and causes increased R-loop accumulation throughout gene transcription. We uncover that SETX is a downstream target of ZPR1 and that overexpression of ZPR1 can rescue R-loop resolution complexe assembly in SETX-deficient cells but not vice versa. To uncover the mechanism of R-loop resolution, we examined the function of SETX-ZPR1 complexes using two genetic motor neuron disease models with altered R-loop resolution. Notably, chronic low levels of SETX-ZPR1 complexes onto R-loops result in a decrease of R-loop resolution activity causing an increase in R-loop levels in spinal muscular atrophy. ZPR1 overexpression increases recruitment of SETX onto R-loops, decreases R-loops and rescues the spinal muscular atrophy phenotype in motor neurons and patient cells. Strikingly, interaction of SETX with ZPR1 is disrupted in ALS4 patients that have heterozygous SETX (L389S) mutation. ZPR1 fails to recruit the mutant SETX homodimer but recruits the heterodimer with partially disrupted interaction between SETX and ZPR1. Interestingly, disruption of SETX-ZPR1 complexes causes increase in R-loop resolution activity leading to fewer R-loops in ALS4. Modulation of ZPR1 levels regulates R-loop accumulation and rescues the pathogenic R-loop phenotype in ALS4 patient cells. These findings originate a new concept, 'opposite alterations in a cell biological activity (R-loop resolution) result in similar pathogenesis (neurodegeneration) in different genetic motor neuron disorders'. We propose that ZPR1 collaborates with SETX and may function as a molecular brake to regulate SETX-dependent R-loop resolution activity critical for the normal functioning of motor neurons. Show less

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous mutation or deletion of the survival motor neuron 1 (SMN1) gene. A second copy, SMN2, is similar to SMN1 but produces ∼10% SMN protein because of a single-point mutation that causes splicing defects. Chronic low levels of SMN cause accumulation of co-transcriptional R-loops and DNA damage leading to genomic instability and neurodegeneration in SMA. Severity of SMA disease correlates inversely with SMN levels. SMN2 is a promising target to produce higher levels of SMN by enhancing its expression. Mechanisms that regulate expression of SMN genes are largely unknown. We report that zinc finger protein ZPR1 binds to RNA polymerase II, interacts in vivo with SMN locus and upregulates SMN2 expression in SMA mice and patient cells. Modulation of ZPR1 levels directly correlates and influences SMN2 expression levels in SMA patient cells. ZPR1 overexpression in vivo results in a systemic increase of SMN levels and rescues severe to moderate disease in SMA mice. ZPR1-dependent rescue improves growth and motor function and increases the lifespan of male and female SMA mice. ZPR1 reduces neurodegeneration in SMA mice and prevents degeneration of cultured primary spinal cord neurons derived from SMA mice. Further, we show that the low levels of ZPR1 associated with SMA pathogenesis cause accumulation of co-transcriptional RNA-DNA hybrids (R-loops) and DNA damage leading to genomic instability in SMA mice and patient cells. Complementation with ZPR1 elevates senataxin levels, reduces R-loop accumulation and rescues DNA damage in SMA mice, motor neurons and patient cells. In conclusion, ZPR1 is critical for preventing accumulation of co-transcriptional R-loops and DNA damage to avert genomic instability and neurodegeneration in SMA. ZPR1 enhances SMN2 expression and leads to SMN-dependent rescue of SMA. ZPR1 represents a protective modifier and a therapeutic target for developing a new method for the treatment of SMA. Show less

Spinal muscular atrophy (SMA) is caused by the low levels of survival motor neuron (SMN) protein and is characterized by motor neuron degeneration and muscle atrophy. Respiratory failure causes death in SMA but the underlying molecular mechanism is unknown. The zinc finger protein ZPR1 interacts with SMN. ZPR1 is down regulated in SMA patients. We report that ZPR1 functions downstream of SMN to regulate HoxA5 levels in phrenic motor neurons that control respiration. Spatiotemporal inactivation of Zpr1 gene in motor neurons down-regulates HoxA5 and causes defects in the function of phrenic motor neurons that results in respiratory failure and perinatal lethality in mice. Modulation in ZPR1 levels directly correlates and influences levels of HoxA5 transcription. In SMA mice, SMN-deficiency causes down-regulation of ZPR1 and HoxA5 that result in degeneration of phrenic motor neurons. Identification of ZPR1 and HoxA5 as potential targets provides a paradigm for developing strategies to treat respiratory distress in SMA. Show less

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA. Show less

Spinal muscular atrophy (SMA) is caused by mutation of the Survival Motor Neurons 1 (SMN1) gene and is characterized by degeneration of spinal motor neurons. The severity of SMA is primarily influenced by the copy number of the SMN2 gene. Additional modifier genes that lie outside the SMA locus exist and one gene that could modify SMA is the Zinc Finger Protein (ZPR1) gene. To test the significance of ZPR1 downregulation in SMA, we examined the effect of reduced ZPR1 expression in mice with mild and severe SMA. We report that the reduced ZPR1 expression causes increase in the loss of motor neurons, hypermyelination in phrenic nerves, increase in respiratory distress and disease severity and reduces the lifespan of SMA mice. The deficiency of SMN-containing sub-nuclear bodies correlates with the severity of SMA. ZPR1 is required for the accumulation of SMN in sub-nuclear bodies. Further, we report that ZPR1 overexpression increases levels of SMN and promotes accumulation of SMN in sub-nuclear bodies in SMA patient fibroblasts. ZPR1 stimulates neurite growth and rescues axonal growth defects in SMN-deficient spinal cord neurons from SMA mice. These data suggest that the severity of disease correlates negatively with ZPR1 levels and ZPR1 may be a protective modifier of SMA. Show less

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Most SMA cases are associated with the low levels of SMN owing to deletion of Survival Motor Neuron 1 (SMN1). SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to predominant skipping of exon 7. Hence, correction of aberrant splicing of SMN2 exon 7 holds the potential for cure of SMA. Here we report an 8-mer antisense oligonucleotide (ASO) to have a profound stimulatory response on correction of aberrant splicing of SMN2 exon 7 by binding to a unique GC-rich sequence located within intron 7 of SMN2. We confirm that the splicing-switching ability of this short ASO comes with a high degree of specificity and reduced off-target effect compared to larger ASOs targeting the same sequence. We further demonstrate that a single low nanomolar dose of this 8-mer ASO substantially increases the levels of SMN and a host of factors including Gemin 2, Gemin 8, ZPR1, hnRNP Q and Tra2-beta1 known to be down-regulated in SMA. Our findings underscore the advantages and unmatched potential of very short ASOs in splicing modulation in vivo. Show less

Eukaryotic genomes encode a zinc finger protein (ZPR1) with tandem ZPR1 domains. In response to growth stimuli, ZPR1 assembles into complexes with eukaryotic translation elongation factor 1A (eEF1A) and the survival motor neurons protein. To gain insight into the structural mechanisms underlying the essential function of ZPR1 in diverse organisms, we determined the crystal structure of a ZPR1 domain tandem and characterized the interaction with eEF1A. The ZPR1 domain consists of an elongation initiation factor 2-like zinc finger and a double-stranded beta helix with a helical hairpin insertion. ZPR1 binds preferentially to GDP-bound eEF1A but does not directly influence the kinetics of nucleotide exchange or GTP hydrolysis. However, ZPR1 efficiently displaces the exchange factor eEF1Balpha from preformed nucleotide-free complexes, suggesting that it may function as a negative regulator of eEF1A activation. Structure-based mutational and complementation analyses reveal a conserved binding epitope for eEF1A that is required for normal cell growth, proliferation, and cell cycle progression. Structural differences between the ZPR1 domains contribute to the observed functional divergence and provide evidence for distinct modalities of interaction with eEF1A and survival motor neuron complexes. Show less

The zinc finger protein ZPR1 is present in both the cytoplasm and nucleoplasm. Cell cycle analysis demonstrates that ZPR1 undergoes major changes in subcellular distribution during proliferation. ZPR1 is diffusely localized throughout the cell during the G(1) and G(2)/M phases of the cell cycle. In contrast, ZPR1 redistributes to the nucleus during S phase and ZPR1 exhibits prominent co-localization with the survival motor neurons protein and the histone gene-specific transcription factor NPAT in subnuclear foci, including Cajal bodies that associate with histone gene clusters. ZPR1 deficiency causes disruption of survival motor neurons and NPAT localization within the nucleus, blocks S phase progression, and arrests cells in both the G(1) and G(2) phases of the cell cycle. These changes in subnuclear architecture and cell cycle progression may be caused by transcriptional defects in ZPR1-deficient cells, including decreased histone gene expression. Show less

Mutations that cause reduced expression of the full-length Survival Motor Neurons (SMN) protein are a major cause of spinal muscular atrophy (SMA), a disease characterized by degeneration of the alpha-motor neurons in the anterior horn of the spinal cord. The severity of SMA may be influenced by the actions of modifier genes. One potential modifier gene is represented by ZPR1, which is down-regulated in patients with SMA and encodes a zinc finger protein that interacts with complexes formed by SMN. To test the functional significance of ZPR1 gene down-regulation, we examined a mouse model with targeted ablation of the Zpr1 gene. We report that ZPR1-deficient mice exhibit axonal pathology and neurodegeneration. These data identify ZPR1 deficiency as a contributing factor in neurodegenerative disorders. Show less

Mutation of the survival motor neurons 1 (SMN1) gene causes motor neuron apoptosis and represents the major cause of spinal muscular atrophy in humans. Biochemical studies have established that the SMN protein plays an important role in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and that the SMN complex can interact with the zinc finger protein ZPR1. Here we report that targeted ablation of the Zpr1 gene in mice disrupts the subcellular localization of both SMN and spliceosomal snRNPs. Specifically, SMN localization to Cajal bodies and gems was not observed in cells derived from Zpr1-/- embryos and the amount of cytoplasmic snRNP detected in Zpr1-/- embryos was reduced compared with that in wild-type embryos. We found that Zpr1-/- mice die during early embryonic development, with reduced proliferation and increased apoptosis. These effects of Zpr1 gene disruption were confirmed and extended in studies of cultured motor neuron-like cells using small interfering RNA-mediated Zpr1 gene suppression; ZPR1 deficiency caused growth cone retraction, axonal defects, and apoptosis. Together, these data indicate that ZPR1 contributes to the regulation of SMN complexes and that it is essential for cell survival. Show less

The survival motor neurons (smn) gene in mice is essential for embryonic viability. In humans, mutation of the telomeric copy of the SMN1 gene causes spinal muscular atrophy, an autosomal recessive disease. Here we report that the SMN protein interacts with the zinc-finger protein ZPR1 and that these proteins colocalize in small subnuclear structures, including gems and Cajal bodies. SMN and ZPR1 redistribute from the cytoplasm to the nucleus in response to serum. This process is disrupted in cells from patients with Werdnig-Hoffman syndrome (spinal muscular atrophy type I) that have SMN1 mutations. Similarly, decreased ZPR1 expression prevents SMN localization to nuclear bodies. Our data show that ZPR1 is required for the localization of SMN in nuclear bodies. Show less

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation. Show less

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells. Show less