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.

Mutations in SEC24D, encoding a component of the COPII machinery, cause a syndromic form of osteogenesis imperfecta.

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205(∗)) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish).

A paucisymptomatic neuromuscular disease mimicking type III 5q-SMA with complex rearrangements in the SMN gene.

Spinal muscular atrophy is an autosomal-recessive neuromuscular disorder, causing progressive proximal weakness and atrophy of the voluntary muscles. More than 96% of the spinal muscular atrophy patients show a homozygous absence of exons 7 and 8, or exon 7 only, in SMN1, the telomeric copy of the SMN gene. We report a young male patient with neurogenic symptoms and sparse muscle fiber atrophy, suggestive of a mild form of type III spinal muscular atrophy. He was found to be a carrier of intragenic mutations in both copies of the SMN gene, exhibiting a homozygous duplication of exons 7 and 8 in SMN1 and a homozygous deletion of exon 8 as well as a heterozygous deletion of exon 7 in SMN2. However, an intact full-length SMN1 complementary deoxyribonucleic acid was identified, and SMN protein levels in a muscle specimen were identical to that of a healthy control, formally excluding the diagnosis of spinal muscular atrophy III.

RAD51C deletion screening identifies a recurrent gross deletion in breast cancer and ovarian cancer families.

RAD51C is an integral part of the DNA double-strand repair through homologous recombination, and monoallelic mutations were found in ~1.3% of BRCA1/2-negative breast cancer (BC) and/or ovarian cancer (OC) families. Several studies confirmed the occurrence of RAD51C mutations predominantly in BC and/or OC families, although with varying frequencies, clearly establishing RAD51C as a cancer-predisposing gene. There is ongoing debate whether pathogenic RAD51C alterations increase the relative risk for BC in addition to that for OC, which was estimated to be 5.88 (95% confidence interval = 2.91 to 11.88; P = 7.65 × 10(-7)).

Histone acetylation as a potential therapeutic target in motor neuron degenerative diseases.

Among hereditary diseases, the group of motor neuron diseases (MNDs) includes some of the most devastating and rapidly progressive lethal conditions. Although degeneration of motor neurons is common to all of them, the phenotypic spectrum of MNDs is relatively broad and ranges from perinatal conditions like spinal muscular atrophy (SMA) to adult-onset diseases such as amyotrophic lateral sclerosis (ALS). While the understanding of the pathology of the diseases is constantly growing, the development of therapeutic approaches lags behind. In fact, there is no approved therapy for MNDs available at the moment. Recent findings demonstrated the existence of some patterns that are shared by several MNDs such as transcriptional dysregulation. In addition, conditions like SMA or certain types of Charcot-Marie-Tooth disease provide some defined targets which may be amenable to therapeutic approaches. Consequently, counteracting this dysregulation may be a valuable therapeutic option and ameliorate disease progression in MND patients. The feasibility of such an approach has been proven during the past years by the epigenetic treatment of various neoplastic entities with histone deacetylase inhibitors (HDACi). On these grounds, also epigenetic therapy of MNDs has become a promising option. So far, several HDACi have been tested in vitro and in animal models and some proceeded further and were evaluated in clinical trials. This review will summarize the advances of HDACi in MNDs and will give a perspective where the road will lead us.

Severe SMA mice show organ impairment that cannot be rescued by therapy with the HDACi JNJ-26481585.

Spinal muscular atrophy (SMA) is the leading genetic cause of early childhood death worldwide and no therapy is available today. Many drugs, especially histone deacetylase inhibitors (HDACi), increase SMN levels. As all HDACi tested so far only mildly ameliorate the SMA phenotype or are unsuitable for use in humans, there is still need to identify more potent drugs. Here, we assessed the therapeutic power of the pan-HDACi JNJ-26481585 for SMA, which is currently used in various clinical cancer trials. When administered for 64 h at 100 nM, JNJ-26481585 upregulated SMN levels in SMA fibroblast cell lines, including those from non-responders to valproic acid. Oral treatment of Taiwanese SMA mice and control littermates starting at P0 showed no overt extension of lifespan, despite mild improvements in motor abilities and weight progression. Many treated and untreated animals showed a very rapid decline or unexpected sudden death. We performed exploratory autopsy and histological assessment at different disease stages and found consistent abnormalities in the intestine, heart and lung and skeletal muscle vasculature of SMA animals, which were not prevented by JNJ-26481585 treatment. Interestingly, some of these features may be only indirectly caused by α-motoneuron function loss but may be major life-limiting factors in the course of disease. A better understanding of - primary or secondary - non-neuromuscular organ involvement in SMA patients may improve standard of care and may lead to reassessment of how to investigate SMA patients clinically.

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.