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.

Chronic low levels of survival motor neuron (SMN) protein cause spinal muscular atrophy (SMA). SMN is ubiquitously expressed, but the mechanisms underlying predominant neuron degeneration in SMA are poorly understood. We report that chronic low levels of SMN cause Senataxin (SETX)-deficiency, which results in increased RNA–DNA hybrids (R-loops) and DNA double-strand breaks (DSBs), and deficiency of DNA-activated protein kinase-catalytic subunit (DNA-PKcs), which impairs DSB repair. Consequently, DNA damage accumulates in patient cells, SMA mice neurons and patient spinal cord tissues. In dividing cells, DSBs are repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) pathways, but neurons predominantly use NHEJ, which relies on DNA-PKcs activity. In SMA dividing cells, HR repairs DSBs and supports cellular proliferation. In SMA neurons, DNA-PKcs-deficiency causes defects in NHEJ-mediated repair leading to DNA damage accumulation and neurodegeneration. Restoration of SMN levels rescues SETX and DNA-PKcs deficiencies and DSB accumulation in SMA neurons and patient cells. Moreover, SETX overexpression in SMA neurons reduces R-loops and DNA damage, and rescues neurodegeneration. Our findings identify combined deficiency of SETX and DNA-PKcs stemming downstream of SMN as an underlying cause of DSBs accumulation, genomic instability and neurodegeneration in SMA and suggest SETX as a potential therapeutic target for SMA.

Mutation of the Survival Motor Neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder that occurs in early childhood. Degeneration of spinal motor neurons caused by SMN deficiency results in progressive muscle atrophy and death in SMA. The molecular mechanism underlying neurodegeneration in SMA is unknown. No treatment is available to prevent neurodegeneration and reduce the burden of illness in SMA. We report that the c-Jun NH₂-terminal kinase (JNK) signaling pathway mediates neurodegeneration in SMA. The neuron-specific isoform JNK3 is required for neuron degeneration caused by SMN deficiency. JNK3 deficiency reduces degeneration of cultured neurons caused by low levels of SMN. Genetic inhibition of JNK pathway in vivo by Jnk3 knockout results in amelioration of SMA phenotype. JNK3 deficiency prevents the loss of spinal cord motor neurons, reduces muscle degeneration, improves muscle fiber thickness and muscle growth, improves motor function and overall growth and increases lifespan of mice with SMA that shows a systemic rescue of phenotype by a SMN-independent mechanism. JNK3 represents a potential (non-SMN) therapeutic target for the treatment of SMA.

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.

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.

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.