TNF-NF-κB-p53 axis restricts in vivo survival of hPSC-derived dopamine neurons.

Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis.

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype.

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZATM, has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

SMN regulates axonal local translation via miR-183/mTOR pathway.

Reduced expression of SMN protein causes spinal muscular atrophy (SMA), a neurodegenerative disorder leading to motor neuron dysfunction and loss. However, the molecular mechanisms by which SMN regulates neuronal dysfunction are not fully understood. Here, we report that reduced SMN protein level alters miRNA expression and distribution in neurons. In particular, miR-183 levels are increased in neurites of SMN-deficient neurons. We demonstrate that miR-183 regulates translation of mTor via direct binding to its 3' UTR. Interestingly, local axonal translation of mTor is reduced in SMN-deficient neurons, and this can be recovered by miR-183 inhibition. Finally, inhibition of miR-183 expression in the spinal cord of an SMA mouse model prolongs survival and improves motor function of Smn-mutant mice. Together, these observations suggest that axonal miRNAs and the mTOR pathway are previously unidentified molecular mechanisms contributing to SMA pathology.

PLS3 mutations in X-linked osteoporosis with fractures.

Plastin 3 (PLS3), a protein involved in the formation of filamentous actin (F-actin) bundles, appears to be important in human bone health, on the basis of pathogenic variants in PLS3 in five families with X-linked osteoporosis and osteoporotic fractures that we report here. The bone-regulatory properties of PLS3 were supported by in vivo analyses in zebrafish. Furthermore, in an additional five families (described in less detail) referred for diagnosis or ruling out of osteogenesis imperfecta type I, a rare variant (rs140121121) in PLS3 was found. This variant was also associated with a risk of fracture among elderly heterozygous women that was two times as high as that among noncarriers, which indicates that genetic variation in PLS3 is a novel etiologic factor involved in common, multi-factorial osteoporosis.

Plastin 3 ameliorates spinal muscular atrophy via delayed axon pruning and improves neuromuscular junction functionality.

F-actin bundling plastin 3 (PLS3) is a fully protective modifier of the neuromuscular disease spinal muscular atrophy (SMA), the most common genetic cause of infant death. The generation of a conditional PLS3-over-expressing mouse and its breeding into an SMA background allowed us to decipher the exact biological mechanism underlying PLS3-mediated SMA protection. We show that PLS3 is a key regulator that restores main processes depending on actin dynamics in SMA motor neurons (MNs). MN soma size significantly increased and a higher number of afferent proprioceptive inputs were counted in SMAPLS3 compared with SMA mice. PLS3 increased presynaptic F-actin amount, rescued synaptic vesicle and active zones content, restored the organization of readily releasable pool of vesicles and increased the quantal content of the neuromuscular junctions (NMJs). Most remarkably, PLS3 over-expression led to a stabilization of axons which, in turn, resulted in a significant delay of axon pruning, counteracting poor axonal connectivity at SMA NMJs. These findings together with the observation of increased endplate and muscle fiber size upon MN-specific PLS3 over-expression suggest that PLS3 significantly improves neuromuscular transmission. Indeed, ubiquitous over-expression moderately improved survival and motor function in SMA mice. As PLS3 seems to act independently of Smn, PLS3 might be a potential therapeutic target not only in SMA but also in other MN diseases.

Lipid accumulation drives cellular senescence in dopaminergic neurons.

Parkinson's disease (PD) is an age-related movement disorder caused by the loss of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc) of the midbrain, however, the underlying cause(s) of this DA neuron loss in PD is unknown and there are currently no effective treatment options to prevent or slow neuronal loss or the progression of related symptoms. It has been shown that both environmental factors as well as genetic predispositions underpin PD development and recent research has revealed that lysosomal dysfunction and lipid accumulation are contributors to disease progression, where an age-related aggregation of alpha-synuclein as well as lipids have been found in PD patients. Interestingly, the most common genetic risk factor for PD is Glucosylceramidase Beta 1 (GBA), which encodes a lysosomal glucocerebrosidase (GCase) that cleaves the beta-glucosidic linkage of lipids known as glucocerebrosides (GluCer). We have recently discovered that artificial induction of GluCer accumulation leads to cellular senescence of DA neurons, suggesting that lipid aggregation plays a crucial role in the pathology of PD by driving senescence in these vulnerable DA neurons. Here, we discuss the relevance of the age-related aggregation of lipids as well as the direct functional link between general lipid aggregation, cellular senescence, and inflammaging of DA neurons. We propose that the expression of a cellular senescence phenotype in the most vulnerable neurons in PD can be triggered by lysosomal impairment and lipid aggregation. Importantly, we highlight additional data that perilipin (PLIN2) is significantly upregulated in senescent DA neurons, suggesting an overall enrichment of lipid droplets (LDs) in these cells. These findings align with our previous results in dopaminergic neurons in highlighting a central role for lipid accumulation in the senescence of DA neurons. Importantly, general lipid droplet aggregation and global lysosomal impairment have been implicated in many neurodegenerative diseases including PD. Taken together, our data suggest a connection between age-related lysosomal impairment, lipid accumulation, and cellular senescence in DA neurons that in turn drives inflammaging in the midbrain and ultimately leads to neurodegeneration and PD.

Defining and characterizing neuronal senescence, 'neurescence', as GX arrested cells.

Cellular senescence is a cell state characterized by resistance to apoptosis and stable cell cycle arrest. Senescence was first observed in mitotic cells in vitro. Recent evidence from in vivo studies and human tissue indicates that postmitotic cells, including neurons, may also become senescent. The quiescent cell state of neurons and inconsistent descriptions of neuronal senescence across studies, however, have caused confusion in this burgeoning field. We summarize evidence demonstrating that exit from G0 quiescence may protect neurons against apoptosis and predispose them toward senescence. Additionally, we propose the term 'neurescent' for senescent neurons and introduce the cell state, GX, to describe cell cycle arrest achieved by passing through G0 quiescence. Criteria are provided to identify neurescent cells, distinguish them from G0 quiescent neurons, and compare neurescent phenotypes with classic replicative senescence.

Therapeutic targeting of senescent cells in the CNS.

Senescent cells accumulate throughout the body with advanced age, diseases and chronic conditions. They negatively impact health and function of multiple systems, including the central nervous system (CNS). Therapies that target senescent cells, broadly referred to as senotherapeutics, recently emerged as potentially important treatment strategies for the CNS. Promising therapeutic approaches involve clearing senescent cells by disarming their pro-survival pathways with 'senolytics'; or dampening their toxic senescence-associated secretory phenotype (SASP) using 'senomorphics'. Following the pioneering discovery of first-generation senolytics dasatinib and quercetin, dozens of additional therapies have been identified, and several promising targets are under investigation. Although potentially transformative, senotherapies are still in early stages and require thorough testing to ensure reliable target engagement, specificity, safety and efficacy. The limited brain penetrance and potential toxic side effects of CNS-acting senotherapeutics pose challenges for drug development and translation to the clinic. This Review assesses the potential impact of senotherapeutics for neurological conditions by summarizing preclinical evidence, innovative methods for target and biomarker identification, academic and industry drug development pipelines and progress in clinical trials.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilize human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

Reversible molecular pathology of skeletal muscle in spinal muscular atrophy.

Low levels of full-length survival motor neuron (SMN) protein cause the motor neuron disease, spinal muscular atrophy (SMA). Although motor neurons undoubtedly contribute directly to SMA pathogenesis, the role of muscle is less clear. We demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic severe SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomic data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. Robust upregulation of voltage-dependent anion-selective channel protein 2 (Vdac2) and downregulation of parvalbumin in severe SMA mice was confirmed in a milder SMA mouse model and in human patient muscle biopsies. Molecular pathology of skeletal muscle was ameliorated in mice treated with the FDA-approved histone deacetylase inhibitor, suberoylanilide hydroxamic acid. We conclude that intrinsic pathology of skeletal muscle is an important and reversible event in SMA and also suggest that muscle proteins have the potential to act as novel biomarkers in SMA.

TNF-NFkB-p53 axis restricts in vivo survival of hPSC-derived dopamine neuron.

Ongoing, first-in-human clinical trials illustrate the feasibility and translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, a major unresolved challenge in the field is the extensive cell death following transplantation with <10% of grafted dopamine neurons surviving. Here, we performed a pooled CRISPR/Cas9 screen to enhance survival of postmitotic dopamine neurons in vivo . We identified p53-mediated apoptotic cell death as major contributor to dopamine neuron loss and uncovered a causal link of TNFa-NFκB signaling in limiting cell survival. As a translationally applicable strategy to purify postmitotic dopamine neurons, we performed a cell surface marker screen that enabled purification without the need for genetic reporters. Combining cell sorting with adalimumab pretreatment, a clinically approved and widely used TNFa inhibitor, enabled efficient engraftment of postmitotic dopamine neurons leading to extensive re-innervation and functional recovery in a preclinical PD mouse model. Thus, transient TNFa inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic human PSC-derived dopamine neurons in PD. Highlights: In vivo CRISPR-Cas9 screen identifies p53 limiting survival of grafted human dopamine neurons. TNFα-NFκB pathway mediates p53-dependent human dopamine neuron deathCell surface marker screen to enrich human dopamine neurons for translational use. FDA approved TNF-alpha inhibitor rescues in vivo dopamine neuron survival with in vivo function.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilized human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence dependent on S100A9 and stress factors. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

SAHA ameliorates the SMA phenotype in two mouse models for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder determined by functional impairment of alpha-motor neurons within the spinal cord. SMA is caused by functional loss of the survival motor neuron gene 1 (SMN1), whereas disease severity is mainly influenced by the number of SMN2 copies. SMN2, which produces only low levels of full-length mRNA/protein, can be modulated by small molecules and drugs, thus offering a unique possibility for SMA therapy. Here, we analysed suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, as potential drug in two severe SMA mouse models each carrying two SMN2 transgenes: US-SMA mice with one SMN2 per allele (Smn(-/-);SMN2(tg/tg)) and Taiwanese-SMA mice with two SMN2 per allele (Smn(-/-);SMN2(tg/wt)), both on pure FVB/N background. The US-SMA mice were embryonically lethal with heterozygous males showing significantly reduced fertility. SAHA treatment of pregnant mothers rescued the embryonic lethality giving rise to SMA offspring. By using a novel breeding strategy for the Taiwanese model (Smn(-/-);SMN2(tg/tg) x Smn(-/+) mice), we obtained 50% SMA offspring that survive approximately 10 days and 50% control carriers in each litter. Treatment with 25 mg/kg twice daily SAHA increased lifespan of SMA mice by 30%, significantly improved motor function abilities, reduced degeneration of motor neurons within the spinal cord and increased the size of neuromuscular junctions and muscle fibers compared with vehicle-treated SMA mice. SMN RNA and protein levels were significantly elevated in various tissues including spinal cord and muscle. Hence, SAHA, which lessens the progression of SMA, might be suitable for SMA therapy.

Deficiency of the splicing factor Sfrs10 results in early embryonic lethality in mice and has no impact on full-length SMN/Smn splicing.

The SR-like splicing factor SFRS10 (Htra2-beta1) is well known to influence various alternatively spliced exons without being an essential splicing factor. We have shown earlier that SFRS10 binds SMN1/SMN2 RNA and restores full-length (FL)-SMN2 mRNA levels in vitro. As SMN1 is absent in patients with spinal muscular atrophy (SMA), the level of FL-SMN2 determines the disease severity. Correct splicing of SMN2 can be facilitated by histone deacetylase inhibitors (HDACis) via upregulation of SFRS10. As HDACis are already used in SMA clinical trials, it is crucial to identify the spectrum of alternatively spliced transcripts modulated by SFRS10, because elevated SFRS10 levels may influence or misregulate also other biological processes. To address this issue, we generated a conditional Sfrs10 allele in mice using the Cre/loxP system. The ubiquitous homozygous deletion of Sfrs10, however, resulted in early embryonic lethality around E7.5, indicating an essential role of Sfrs10 during mouse embryogenesis. Deletion of Sfrs10 with recombinant Cre in murine embryonic fibroblasts (MEFs) derived from Sfrs10(fl/fl) embryos increased the low levels of SmnDelta7 3-4-fold, without affecting FL-Smn levels. The weak influence of Sfrs10 on Smn splicing was further proven by a Hb9-Cre driven motor neuron-specific deletion of Sfrs10 in mice, which developed normally without revealing any SMA phenotype. To assess the role of Sfrs10 on FL-SMN2 splicing, we established MEFs from Smn(-/-);SMN2(tg/tg);Sfrs10(fl/fl) embryos. Surprisingly, deletion of Sfrs10 by recombinant Cre showed no impact on SMN2 splicing but increased SMN levels. Our findings highlight the complexity by which alternatively spliced exons are regulated in vivo.

Age-Related Midbrain Inflammation and Senescence in Parkinson's Disease.

Immune responses are arising as a common feature of several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), and Amyotrophic Lateral Sclerosis (ALS), but their role as either causative or consequential remains debated. It is evident that there is local inflammation in the midbrain in PD patients even before symptom onset, but the underlying mechanisms remain elusive. In this mini-review, we discuss this midbrain inflammation in the context of PD and argue that cellular senescence may be the cause for this immune response. We postulate that to unravel the relationship between inflammation and senescence in PD, it is crucial to first understand the potential causative roles of various cell types of the midbrain and determine how the possible paracrine spreading of senescence between them may lead to observed local immune responses. We hypothesize that secretion of pro-inflammatory factors by senescent cells in the midbrain triggers neuroinflammation resulting in immune cell-mediated killing of midbrain dopaminergic (DA) neurons in PD.

LBH589 induces up to 10-fold SMN protein levels by several independent mechanisms and is effective even in cells from SMA patients non-responsive to valproate.

Histone deacetylase inhibitors (HDACi) are potential candidates for therapeutic approaches in cancer and neurodegenerative diseases such as spinal muscular atrophy (SMA)--a common autosomal recessive disorder and frequent cause of early childhood death. SMA is caused by homozygous absence of SMN1. Importantly, all SMA patients carry a nearly identical copy gene, SMN2, that produces only minor levels of correctly spliced full-length transcripts and SMN protein. Since an increased number of SMN2 copies strongly correlates with a milder SMA phenotype, activation or stabilization of SMN2 is considered as a therapeutic strategy. However, clinical trials demonstrated effectiveness of the HDACi valproate (VPA) and phenylbutyrate only in <50% of patients; therefore, identification of new drugs is of vital importance. Here we characterize the novel hydroxamic acid LBH589, an HDACi already widely used in cancer clinical trials. LBH589 treatment of human SMA fibroblasts induced up to 10-fold elevated SMN levels, the highest ever reported, accompanied by a markedly increased number of gems. FL-SMN2 levels were increased 2-3-fold by transcription activation via SMN2 promoter H3K9 hyperacetylation and restoration of correct splicing via elevated hTRA2-beta1 levels. Furthermore, LBH589 stabilizes SMN by reducing its ubiquitinylation as well as favouring incorporation into the SMN complex. Cytotoxic effects were not detectable at SMN2 activating concentrations. Notably, LBH589 also induces SMN2 expression in SMA fibroblasts inert to VPA, in human neural stem cells and in the spinal cord of SMN2-transgenic mice. Hence, LBH589, which is active already at nanomolar doses, is a highly promising candidate for SMA therapy.

Survival motor neuron gene 2 silencing by DNA methylation correlates with spinal muscular atrophy disease severity and can be bypassed by histone deacetylase inhibition.

Spinal muscular atrophy (SMA), a common neuromuscular disorder, is caused by homozygous absence of the survival motor neuron gene 1 (SMN1), while the disease severity is mainly influenced by the number of SMN2 gene copies. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We demonstrate that the SMN2 gene is subject to gene silencing by DNA methylation. SMN2 contains four CpG islands which present highly conserved methylation patterns and little interindividual variations in SMN1-deleted SMA patients. The comprehensive analysis of SMN2 methylation in patients suffering from severe versus mild SMA carrying identical SMN2 copy numbers revealed a correlation of CpG methylation at the positions -290 and -296 with the disease severity and the activity of the first transcriptional start site of SMN2 at position -296. These results provide first evidence that SMN2 alleles are functionally not equivalent due to differences in DNA methylation. We demonstrate that the methyl-CpG-binding protein 2, a transcriptional repressor, binds to the critical SMN2 promoter region in a methylation-dependent manner. However, inhibition of SMN2 gene silencing conferred by DNA methylation might represent a promising strategy for pharmacologic SMA therapy. We identified histone deacetylase (HDAC) inhibitors including vorinostat and romidepsin which are able to bypass SMN2 gene silencing by DNA methylation, while others such as valproic acid and phenylbutyrate do not, due to HDAC isoenzyme specificities. These findings indicate that DNA methylation is functionally important regarding SMA disease progression and pharmacological SMN2 gene activation which might have implications for future SMA therapy regimens.