Genome sequencing analysis identifies new loci associated with Lewy body dementia and provides insights into its genetic architecture.

The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia, and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer's disease and Parkinson's disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition.

Elevated Mortalin correlates with poor outcome in hepatocellular carcinoma.

Although several lines of evidence existed suggesting that Mortalin was linked with survival in malignant tumors; it has been barely described regarding the prognostic involvement of its expression in hepatocellular carcinoma (HCC). Herein, to understand the prognostic meaning of Mortalin expression, Immunohistochemistry was undertaken to observe the immunohistochemical characteristics of Mortalin on HCC tissue microarray consisting of 90 cases of HCC and its paired normal control dots, followed by detailed statistical analysis with the accompanying clinicopathological variables available, including gender, age, tumor size, differentiation, cirrhosis, vascular invasion, clinical stage, T classification and intrahepatic metastases. Meanwhile, Kaplan-Meier survival curve was plotted to analyze the prognostic difference for patients with high and low expression of Mortalin. It was exhibited that Mortalin was over-expressed in HCC tissues relative to paired normal control and elevated Mortalin significantly correlated with vascular invasion, clinical stage and intrahepatic metastasis. Kaplan-Meier survival analysis revealed that Mortalin was remarkably associated with overall survival and disease-free survival. Multivariate Cox regression analysis showed that expression of Mortalin was an independent prognostic factor in HCC. Collectively, the data we provided here support the prognostic prediction value of Mortalin in HCC.

Leucine-rich repeat kinase 2 controls protein kinase A activation state through phosphodiesterase 4.

BACKGROUND: Evidence indicates a cross-regulation between two kinases, leucine-rich repeat kinase 2 (LRRK2) and protein kinase A (PKA). In neurons, LRRK2 negatively regulates PKA activity in spiny projecting neurons during synaptogenesis and in response to dopamine D1 receptor activation acting as an A-anchoring kinase protein (AKAP). In microglia cells, we showed that LRRK2 kinase activity negatively regulates PKA, impacting NF-κB p50 signaling and the inflammatory response. Here, we explore the molecular mechanism underlying the functional interaction between LRRK2 and PKA in microglia. METHODS: To understand which step of PKA signaling is modulated by LRRK2, we used a combination of in vitro and ex vivo systems with hyperactive or inactive LRRK2 as well as different readouts of PKA signaling. RESULTS: We confirmed that LRRK2 kinase activity acts as a negative regulator of PKA activation state in microglia. Specifically, we found that LRRK2 controls PKA by affecting phosphodiesterase 4 (PDE4) activity, modulating cAMP degradation, content, and its dependent signaling. Moreover, we showed that LRRK2 carrying the G2019S pathological mutation downregulates PKA activation causing a reduction of PKA-mediated NF-κB inhibitory signaling, which results, in turn, in increased inflammation in LRRK2 G2019S primary microglia upon α-synuclein pre-formed fibrils priming. CONCLUSIONS: Overall, our findings indicate that LRRK2 kinase activity is a key regulator of PKA signaling and suggest PDE4 as a putative LRRK2 effector in microglia. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD.

Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

Hexokinase activity is required for recruitment of parkin to depolarized mitochondria.

Autosomal recessive parkinsonism genes contribute to maintenance of mitochondrial function. Two of these, PINK1 and parkin, act in a pathway promoting autophagic removal of depolarized mitochondria. Although recruitment of parkin to mitochondria is PINK1-dependent, additional components necessary for signaling are unclear. We performed a screen for endogenous modifiers of parkin recruitment to depolarized mitochondria and identified hexokinase 2 (HK2) as a novel modifier of depolarization-induced parkin recruitment. Hexose kinase activity was required for parkin relocalization, suggesting the effects are shared among hexokinases including the brain-expressed hexokinase 1 (HK1). Knockdown of both HK1 and HK2 led to a stronger block in parkin relocalization than either isoform alone, and expression of HK2 in primary neurons promoted YFP-parkin recruitment to depolarized mitochondria. Mitochondrial parkin recruitment was attenuated with AKT inhibition, which is known to modulate HK2 activity and mitochondrial localization. We, therefore, propose that Akt-dependent recruitment of hexokinases is a required step in the recruitment of parkin prior to mitophagy.

Role of mitofusin 2 (Mfn2) in controlling cellular proliferation.

It has been reported that Mitofusin2 (Mfn2) inhibits cell proliferation when overexpressed. We wanted to study the role of endogenous Mfn2 in cell proliferation, along with the structural features of Mfn2 that influence its mitochondrial localization and control of cell proliferation. Mfn2-knockdown clones of a B-cell lymphoma cell line BJAB exhibited an increased rate of cell proliferation. A 2-fold increase in cell proliferation was also observed in Mfn2-knockout mouse embryonic fibroblast (MEF) cells as compared with the control wild-type cells, and the proliferative advantage of the knockout MEF cells was blocked on reintroduction of the Mfn2 gene. Mfn2 exerts its antiproliferative effect by acting as an effector molecule of Ras, resulting in the inhibition of the Ras-Raf-ERK signaling pathway. Furthermore, both the N-terminal (aa 1-264) and the C-terminal (aa 265-757) fragments of Mfn2 blocked cell proliferation through distinct mechanisms: the N-terminal-mediated inhibition was due to its interaction with Raf-1, whereas the C-terminal fragment of Mfn2 inhibited cell proliferation by interacting with Ras. The inhibition of proliferation by the N-terminal fragment was independent of its mitochondrial localization. Collectively, our data provide new insights regarding the role of Mfn2 in controlling cellular proliferation.

Age-associated miRNA alterations in skeletal muscle from rhesus monkeys reversed by caloric restriction.

The levels of microRNAs (miRNAs) are altered under different conditions such as cancer, senescence, and aging. Here, we have identified differentially expressed miRNAs in skeletal muscle from young and old rhesus monkeys using RNA sequencing. In old muscle, several miRNAs were upregulated, including miR-451, miR-144, miR-18a and miR-15a, while a few miRNAs were downregulated, including miR-181a and miR-181b. A number of novel miRNAs were also identified, particularly in old muscle. We also examined the impact of caloric restriction (CR) on miRNA abundance by reverse transcription (RT) followed by real-time, quantitative (q)PCR analysis and found that CR rescued the levels of miR-181b and chr1:205580546, and also dampened the age-induced increase in miR-451 and miR-144 levels. Our results reveal that there are changes in expression of known and novel miRNAs with skeletal muscle aging and that CR may reverse some of these changes to a younger phenotype.

Exome sequencing reveals VCP mutations as a cause of familial ALS.

Using exome sequencing, we identified a p.R191Q amino acid change in the valosin-containing protein (VCP) gene in an Italian family with autosomal dominantly inherited amyotrophic lateral sclerosis (ALS). Mutations in VCP have previously been identified in families with Inclusion Body Myopathy, Paget disease, and Frontotemporal Dementia (IBMPFD). Screening of VCP in a cohort of 210 familial ALS cases and 78 autopsy-proven ALS cases identified four additional mutations including a p.R155H mutation in a pathologically proven case of ALS. VCP protein is essential for maturation of ubiquitin-containing autophagosomes, and mutant VCP toxicity is partially mediated through its effect on TDP-43 protein, a major constituent of ubiquitin inclusions that neuropathologically characterize ALS. Our data broaden the phenotype of IBMPFD to include motor neuron degeneration, suggest that VCP mutations may account for ∼1%-2% of familial ALS, and provide evidence directly implicating defects in the ubiquitination/protein degradation pathway in motor neuron degeneration.

S-nitrosylation regulates nuclear translocation of chloride intracellular channel protein CLIC4.

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca(2+)-induced differentiation, stress-induced apoptosis, and modulating TGF-beta signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin alpha and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor alpha-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor alpha-induced nitrosylation and association with importin alpha and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways.

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.

Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism.

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.