MYO1H is a novel candidate gene for autosomal dominant pure hereditary spastic paraplegia.

In this study, we aimed to determine the genetic basis of a Turkish family related to hereditary spastic paraplegia (HSP) by exome sequencing. HSP is a progressive neurodegenerative disorder and displays genetic and clinical heterogeneity. The major symptoms are muscle weakness and spasticity, especially in the lower extremities. We studied seven affected and seven unaffected family members, as well as a clinically undetermined member, to identify the disease-causing gene. Exome sequencing was performed for four affected and two unaffected individuals. The variants were firstly filtered for HSP-associated genes, and we found a common variant in the ZFYVE27 gene, which has been previously implied for association with HSP. Due to the incompletely penetrant segregation pattern of the ZFYVE27 variant, revealed by Sanger sequencing, with the disease in this family, filtering was re-performed according to the mode of inheritance and allelic frequencies. The resulting 14 rare variants were further evaluated in terms of their cellular functions, and three candidate variants in ATAD3C, VPS16, and MYO1H genes were selected as possible causative variants, which were analyzed for their familial segregation. ATAD3C and VPS16 variants were eliminated due to incomplete penetrance. Eventually, the MYO1H variant NM_001101421.3:c.2972_2974del (p.Glu992del, rs372231088) was found as the possible disease-causing deletion for HSP in this family. This is the first study reporting the possible role of a MYO1H variant in HSP pathogenesis. Further studies on the cellular roles of Myo1h protein are needed to validate the causality of MYO1H gene at the onset of HSP.

Cyclin-dependent kinase inhibitors, roscovitine and purvalanol, induce apoptosis and autophagy related to unfolded protein response in HeLa cervical cancer cells.

Roscovitine (Rosc) and purvalanol (Pur) are competitive inhibitors of cyclin-dependent kinases (CDKs) by targeting their ATP-binding pockets. Both drugs are shown to be effective to decrease cell viability and dysregulate the ratio of pro- and anti-apoptotic Bcl-2 family members, which finally led to apoptotic cell death in different cancer cell lines in vitro. It was well established that Bcl-2 family members have distinct roles in the regulation of other cellular processes such as endoplasmic reticulum (ER) stress. The induction of ER stress has been shown to play critical role in cell death/survival decision via autophagy or apoptosis. In this study, our aim was to investigate the molecular targets of CDK inhibitors on ER stress mechanism related to distinct cell death types in time-dependent manner in HeLa cervical cancer cells. Our results showed that Rosc and Pur decreased the cell viability, cell growth and colony formation, induced ER stress-mediated autophagy or apoptosis in time-dependent manner. Thus, we conclude that exposure of cells to CDK inhibitors induces unfolded protein response and ER stress leading to autophagy and apoptosis processes in HeLa cervical cancer cells.

Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

Microtubule-severing protein Spastin has been shown to co-localize with actin in migratory glioblastoma cells and is linked to glioblastomas' migration and invasion capacity. However, the effectiveness of Spastin in glioblastoma migration and the molecular mechanism underpinning the orientation of Spastin towards actin filaments remain unknown. Here, we demonstrated that Spastin plays an active role in glioblastoma migration by showing a reduced migratory potential of T98G glioblastoma cells using real-time cell analysis (RTCA) in Spastin-depleted cells. Pull-down assays revealed that a cis-trans isomerase Pin1 interacts with Spastin through binding to the phosphorylated Pin1 recognition motifs in the microtubule-binding domain (MBD), and immunocytochemistry analysis showed that interaction with Pin1 directs Spastin to actin filaments in extended cell regions. Consequently, by utilizing RTCA, we proved that the migration and invasion capacity of T98G glioblastoma cells significantly increased with the overexpression of Spastin, of which the Pin1 recognition motifs in MBD are constitutively phosphorylated, while the overexpression of phospho-mutant form did not have a significant effect on migration and invasion of T98G glioblastoma cells. These findings demonstrate that Pin1 is a novel interaction partner of Spastin, and their interaction drives Spastin to actin filaments, allowing Spastin to contribute to the glioblastomas' migration and invasion abilities.

miR96- and miR182-driven regulation of cytoskeleton results in inhibition of glioblastoma motility.

Glioblastoma multiforme (GBM) is one of the most common forms of brain tumor. As an excessively invasive tumor type, GBM cannot be fully cured due to its invasion ability into healthy brain tissues. Therefore, molecular mechanisms behind GBM migration and invasion need to be deeply investigated for the development of effective GBM treatments. Cellular motility and invasion are strictly associated with the cytoskeleton, especially with actins and tubulins. Palladin, an actin-binding protein, tightly bundles actins during initial invadopodia and contraction fiber formations, which are essential for cellular motility. Spastin, a microtubule-binding protein, cuts microtubules into small pieces and acts on invadopodia elongation and cellular trafficking of invadopodia-associated proteins. Regulation of proteins such as spastin and palladin involved in dynamic reorganization of the cytoskeleton, are rapidly carried out by microRNAs at the posttranscriptional level. Therefore, determining possible regulatory miRNAs of spastin and palladin is critical to elucidate GBM motility. miR96 and miR182 down-regulate SPAST and PALLD at both transcript and protein levels. Over-expression of miR96 and miR182 resulted in inhibition of the motility. However, over-expression of spastin and palladin induced the motility. Spastin and palladin rescue of miR96- or miR182-transfected U251 MG cells resulted in diminished effects of the miRNAs and rescued the motility. Our results demonstrate that miR96 and miR182 over-expressions inhibit GBM motility by regulating cytoskeleton through spastin and palladin. These findings suggest that miR96 and miR182 should be investigated in more detail for their potential use in GBM therapy.

Novel mutations in ATP13A2 associated with mixed neurological presentations and iron toxicity due to nonsense-mediated decay.

BACKGROUND: Kufor-Rakeb Syndrome (KRS) is an autosomal recessive disease characterized by Parkinsonism, pyramidal signs, dementia, and supranuclear gaze palsy. KRS is caused by mutations in ATP13A2producing a transmembrane protein responsible for the regulation of intracellular inorganic cations. OBJECTIVE: Two siblings born to a Turkish family of consanguineous marriage had mixed neurological presentations with the presence of hypointense images on T2-weighted MRI and were pre-diagnosed as having autosomal recessive spastic paraparesis or ataxia.We aimed to identify the disease-causing mutation by whole-exome sequencing and elucidate the underlying molecular mechanism of the causative mutation. METHODS: Prussian blue staining was conducted for the detection of cellular iron accumulation. Disease-causing mutation inATP13A2was detected by whole-exome sequencing. Expression levels of ATP13A2 mRNA and protein were assessed by qRT-PCR and Western Blot. RESULTS: Iron deposits in the patients' fibroblasts were detected by Prussian blue staining. Novel homozygous mutation c.1422_1423del:p.P474fs was detected intheATP13A2. As this mutation caused a premature termination codon (PTC), the expression of mutant ATP13A2 mRNA through qRT-PCR analysis was found to be degraded by nonsense-mediated decay and this prevented the expression of ATP13A2 protein in the patients' fibroblasts. CONCLUSIONS: Novel frameshift mutation causing a PTC in ATP13A2 lead to degradation of ATP13A2 mRNA by NMD. Iron accumulation due to the absence of ATP13A2 protein in the patient's fibroblasts and hypointense areas on T2-weighted images may expand the spectrum of KRS to consider it as neurodegeneration with brain iron accumulation disorders.