ALBERT

Description: Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin–proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy.

Description: Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study used genome-wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That study minimized the importance of cell type-specific differences in CpG methylation, claiming these are restricted to localized genomic regions, and instead emphasized that widespread and highly conserved differences in non-CpG methylation distinguish neurons and glia. We reanalyzed the data from that study and came to markedly different conclusions. In particular, we found widespread cell type-specific differences in CpG methylation, with a genome-wide tendency for neuronal CpG-hypermethylation punctuated by regions of glia-specific hypermethylation. Alarmingly, our analysis indicated that the majority of genes identified by the primary study as exhibiting cell type-specific CpG methylation differences were misclassified. To verify the accuracy of our analysis, we isolated neuronal and glial DNA from mouse cortex and performed quantitative bisulfite pyrosequencing at nine loci. The pyrosequencing results corroborated our analysis, without exception. Most interestingly, we found that gene-associated neuron vs. glia CpG methylation differences are highly conserved across human and mouse, and are very likely to be functional. In addition to underscoring the importance of independent verification to confirm the conclusions of genome-wide epigenetic analyses, our data indicate that CpG methylation plays a major role in neuroepigenetics, and that the mouse is likely an excellent model in which to study the role of DNA methylation in human neurodevelopment and disease.

Description: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure. To develop effective treatments for this devastating disease, an appropriate strategy for targeting the molecule responsible for the pathogenesis of ALS is needed. We previously reported that mutant SOD1 protein causes motor neuron death through activation of ASK1, a mitogen-activated protein kinase kinase kinase. Additionally, we recently developed K811 and K812, which are selective inhibitors for ASK1. Here, we report the effect of K811 and K812 in a mouse model of ALS (SOD1 G93A transgenic mice). Oral administration of K811 or K812 significantly extended the life span of SOD1 G93A transgenic mice (1.06 and 1.08% improvement in survival). Moreover, ASK1 activation observed in the lumbar spinal cord of mice at the disease progression stage was markedly decreased in the K811- and K812-treated groups. In parallel, immunohistochemical analysis revealed that K811 and K812 treatment inhibited glial activation in the lumbar spinal cord of SOD1 G93A transgenic mice. These results reinforce the importance of ASK1 as a therapeutic target for ALS treatment.

Description: Dilated cardiomyopathy (DCM) is a leading cause of heart failure. In families with autosomal-dominant DCM, heterozygous missense mutations were identified in RNA-binding motif protein 20 ( RBM20 ), a spliceosome protein induced during early cardiogenesis. Dermal fibroblasts from two unrelated patients harboring an RBM20 R636S missense mutation were reprogrammed to human induced pluripotent stem cells (hiPSCs) and differentiated to beating cardiomyocytes (CMs). Stage-specific transcriptome profiling identified differentially expressed genes ranging from angiogenesis regulator to embryonic heart transcription factor as initial molecular aberrations. Furthermore, gene expression analysis for RBM20- dependent splice variants affected sarcomeric ( TTN and LDB3 ) and calcium (Ca 2+ ) handling ( CAMK2D and CACNA1C ) genes. Indeed, RBM20 hiPSC-CMs exhibited increased sarcomeric length ( RBM20 : 1.747 ± 0.238 µm versus control: 1.404 ± 0.194 µm; P 〈 0.0001) and decreased sarcomeric width ( RBM20 : 0.791 ± 0.609 µm versus control: 0.943 ± 0.166 µm; P 〈 0.0001). Additionally, CMs showed defective Ca 2+ handling machinery with prolonged Ca 2+ levels in the cytoplasm as measured by greater area under the curve ( RBM20 : 814.718 ± 94.343 AU versus control: 206.941 ± 22.417 AU; P 〈 0.05) and higher Ca 2+ spike amplitude ( RBM20 : 35.281 ± 4.060 AU versus control:18.484 ± 1.518 AU; P 〈 0.05). β-adrenergic stress induced with 10 µ m norepinephrine demonstrated increased susceptibility to sarcomeric disorganization ( RBM20 : 86 ± 10.5% versus control: 40 ± 7%; P 〈 0.001). This study features the first hiPSC model of RBM20 familial DCM. By monitoring human cardiac disease according to stage-specific cardiogenesis, this study demonstrates RBM20 familial DCM is a developmental disorder initiated by molecular defects that pattern maladaptive cellular mechanisms of pathological cardiac remodeling. Indeed, hiPSC-CMs recapitulate RBM20 familial DCM phenotype in a dish and establish a tool to dissect disease-relevant defects in RBM20 splicing as a global regulator of heart function.

Description: Synucleins belong to a family of intrinsically unstructured proteins that includes alpha-synuclein (aSyn), beta-synuclein (bSyn) and gamma-synuclein (gSyn). aSyn is the most studied member of the synuclein family due to its central role in genetic and sporadic forms of Parkinson's disease and other neurodegenerative disorders known as synucleionopathies. In contrast, bSyn and gSyn have been less studied, but recent reports also suggest that, unexpectedly, these proteins may also cause neurotoxicity. Here, we explored the yeast toolbox to investigate the cellular effects of bSyn and gSyn. We found that bSyn is toxic and forms cytosolic inclusions that are similar to those formed by aSyn. Moreover, we found that bSyn shares similar toxicity mechanisms with aSyn, including vesicular trafficking impairment and induction of oxidative stress. We demonstrate that co-expression of aSyn and bSyn exacerbates cytotoxicity, due to increased dosage of toxic synuclein forms, and that they are able to form heterodimers in both yeast and in human cells. In contrast, gSyn is not toxic and does not form inclusions in yeast cells. Altogether, our findings shed light into the question of whether bSyn can exert toxic effects and confirms the occurrence of aSyn/bSyn heterodimers, opening novel perspectives for the development of novel strategies for therapeutic intervention in synucleinopathies.

Description: Identification of a systemically acting and universal small molecule therapy for Duchenne muscular dystrophy would be an enormous advance for this condition. Based on evidence gained from studies on mouse genetic models, we have identified tyrosine phosphorylation and degradation of β-dystroglycan as a key event in the aetiology of Duchenne muscular dystrophy. Thus, preventing tyrosine phosphorylation and degradation of β-dystroglycan presents itself as a potential therapeutic strategy. Using the dystrophic sapje zebrafish, we have investigated the use of tyrosine kinase and other inhibitors to treat the dystrophic symptoms in this model of Duchenne muscular dystrophy. Dasatinib, a potent and specific Src tyrosine kinase inhibitor, was found to decrease the levels of β-dystroglycan phosphorylation on tyrosine and to increase the relative levels of non-phosphorylated β-dystroglycan in sapje zebrafish. Furthermore, dasatinib treatment resulted in the improved physical appearance of the sapje zebrafish musculature and increased swimming ability as measured by both duration and distance of swimming of dasatinib-treated fish compared with control animals. These data suggest great promise for pharmacological agents that prevent the phosphorylation of β-dystroglycan on tyrosine and subsequent steps in the degradation pathway as therapeutic targets for the treatment of Duchenne muscular dystrophy.

Description: Neuroinflammation, immune reactivity and mitochondrial abnormalities are considered as causes and/or contributors to neuronal degeneration. Peroxisome proliferator-activated receptors (PPARs) regulate both inflammatory and multiple other pathways that are implicated in neurodegeneration. In the present study, we investigated the efficacy of fenofibrate (Tricor), a pan-PPAR agonist that activates PPAR-α as well as other PPARs. We administered fenofibrate to superoxide dismutase 1 (SOD1 G93A ) mice daily prior to any detectable phenotypes and then animal behavior, pathology and longevity were assessed. Treated animals showed a significant slowing of the progression of disease with weight loss attenuation, enhanced motor performance, delayed onset and survival extension. Histopathological analysis of the spinal cords showed that neuronal loss was significantly attenuated in fenofibrate-treated mice. Mitochondria were preserved as indicated by Cytochrome c immunostaining in the spinal cord, which maybe partly due to increased expression of the PPAR- co-activator 1-α. The total mRNA analysis revealed that neuroprotective and anti-inflammatory genes were elevated, while neuroinflammatory genes were down-regulated. This study demonstrates that the activation of PPAR-α action via fenofibrate leads to neuroprotection by both reducing neuroinflammation and protecting mitochondria, which leads to a significant increase in survival in SOD1 G93A mice. Therefore, the development of therapeutic strategies to activate PPAR-α as well as other PPARs may lead to new therapeutic agents to slow or halt the progression of amyotrophic lateral sclerosis.

Description: Several studies have shown that testis-specific gene antigen ( TSGA10 ) could be considered as a cancer testis antigen (CTA), except for one study which has identified it as a tumor suppressor gene. In order to exert its function, TSGA10 interacts closely with hypoxia inducible factor (HIF-1α) and since this interaction is still not completely defined, the exact role of TSGA10 in angiogenesis and invasion is also under question. The current study was conducted to investigate the function of TSGA10 gene and evaluate its potential effects on tumor angiogenesis and invasion. To do so, TSGA10 vector was designed for a stable transfection in HeLa cells, and then clonal selection was applied. The efficiency of transfection and the role of TSGA10 in abovementioned targets were evaluated by real-time PCR, western blot, zymography and ELISA tests in both normoxia and hypoxia. Invasion, migration and angiogenesis were assessed. Three-dimensional model of TSGA10 protein was accurately built in which TSGA10 docked to 2 domains of HIF-1α. Increased expression of TSGA10 correlated with decreased HIF-1α transcriptional activity and inhibited angiogenesis and HeLa cells invasion in normoxia as well as hypoxia. Docking analysis indicated that binding affinity of TSGA10 with TAD-C (CBP) domain of HIF-1α would be stronger than that with PAS-B domain. Our findings showed that overexpression of TSGA10 would induce disruption of HIF-1α axis and exert potent inhibitory effects on tumor angiogenesis and metastasis. Therefore, TSGA10 could be considered as a potent therapeutic candidate, prognostic factor and a cancer management tool.

Description: Zinc finger motifs are distributed amongst many eukaryotic protein families, directing nucleic acid–protein and protein–protein interactions. Zinc finger protein 106 (ZFP106) has previously been associated with roles in immune response, muscle differentiation, testes development and DNA damage, although little is known about its specific function. To further investigate the function of ZFP106, we performed an in-depth characterization of Zfp106 deficient mice ( Zfp106 –/– ), and we report a novel role for ZFP106 in motor and sensory neuronal maintenance and survival. Zfp106 –/– mice develop severe motor abnormalities, major deficits in muscle strength and histopathological changes in muscle. Intriguingly, despite being highly expressed throughout the central nervous system, Zfp106 –/– mice undergo selective motor and sensory neuronal and axonal degeneration specific to the spinal cord and peripheral nervous system. Neurodegeneration does not occur during development of Zfp106 –/– mice, suggesting that ZFP106 is likely required for the maintenance of mature peripheral motor and sensory neurons. Analysis of embryonic Zfp106 –/– motor neurons revealed deficits in mitochondrial function, with an inhibition of Complex I within the mitochondrial electron transport chain. Our results highlight a vital role for ZFP106 in sensory and motor neuron maintenance and reveal a novel player in mitochondrial dysfunction and neurodegeneration.