Regulatory role of endoplasmic reticulum resident chaperone protein ERp29 in anti-murine ß-coronavirus host cell response.

Gap junctional intercellular communication (GJIC) involving astrocytes is important for proper CNS homeostasis. As determined in our previous studies, trafficking of the predominant astrocyte GJ protein, Connexin43 (Cx43), is disrupted in response to infection with a neurotropic murine ß-coronavirus (MHV-A59). However, how host factors are involved in Cx43 trafficking and the infection response is not clear. Here, we show that Cx43 retention due to MHV-A59 infection was associated with increased ER stress and reduced expression of chaperone protein ERp29. Treatment of MHV-A59-infected astrocytes with the chemical chaperone 4-sodium phenylbutyrate increased ERp29 expression, rescued Cx43 transport to the cell surface, increased GJIC, and reduced ER stress. We obtained similar results using an astrocytoma cell line (delayed brain tumor) upon MHV-A59 infection. Critically, delayed brain tumor cells transfected to express exogenous ERp29 were less susceptible to MHV-A59 infection and showed increased Cx43-mediated GJIC. Treatment with Cx43 mimetic peptides inhibited GJIC and increased viral susceptibility, demonstrating a role for intercellular communication in reducing MHV-A59 infectivity. Taken together, these results support a therapeutically targetable ERp29-dependent mechanism where ß-coronavirus infectivity is modulated by reducing ER stress and rescuing Cx43 trafficking and function.

Amyloid-ß regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes.

Altered expression and function of astroglial gap junction protein connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). Although earlier studies have examined the effect of increased ß-amyloid (Aß) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aß modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aß can alter Cx43 gap junctions. We show that Aß25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aß25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aß25-35-exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aß-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aß25-35-exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aß25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aß can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aß modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

APP overexpression in the absence of NPC1 exacerbates metabolism of amyloidogenic proteins of Alzheimer's disease.

Amyloid-ß (Aß) peptides originating from ß-amyloid precursor protein (APP) are critical in Alzheimer's disease (AD). Cellular cholesterol levels/distribution can regulate production and clearance of Aß peptides, albeit with contradictory outcomes. To better understand the relationship between cholesterol homeostasis and APP/Aß metabolism, we have recently generated a bigenic ANPC mouse line overexpressing mutant human APP in the absence of Niemann-Pick type C-1 protein required for intracellular cholesterol transport. Using this unique bigenic ANPC mice and complementary stable N2a cells, we have examined the functional consequences of cellular cholesterol sequestration in the endosomal-lysosomal system, a major site of Aß production, on APP/Aß metabolism and its relation to neuronal viability. Levels of APP C-terminal fragments (α-CTF/ß-CTF) and Aß peptides, but not APP mRNA/protein or soluble APPα/APPß, were increased in ANPC mouse brains and N2a-ANPC cells. These changes were accompanied by reduced clearance of peptides and an increased level/activity of γ-secretase, suggesting that accumulation of APP-CTFs is due to decreased turnover, whereas increased Aß levels may result from a combination of increased production and decreased turnover. APP-CTFs and Aß peptides were localized primarily in early-/late-endosomes and to some extent in lysosomes/autophagosomes. Cholesterol sequestration impaired endocytic-autophagic-lysosomal, but not proteasomal, clearance of APP-CTFs/Aß peptides. Moreover, markers of oxidative stress were increased in vulnerable brain regions of ANPC mice and enhanced ß-CTF/Aß levels increased susceptibility of N2a-ANPC cells to H2O2-induced toxicity. Collectively, our results show that cellular cholesterol sequestration plays a key role in APP/Aß metabolism and increasing neuronal vulnerability to oxidative stress in AD-related pathology.

Mutant human APP exacerbates pathology in a mouse model of NPC and its reversal by a ß-cyclodextrin.

Niemann-Pick type C (NPC) disease, an autosomal recessive disorder caused primarily by loss-of-function mutations in NPC1 gene, is characterized neuropathologically by intracellular cholesterol accumulation, gliosis and neuronal loss in selected brain regions. Recent studies have shown that NPC disease exhibits intriguing parallels with Alzheimer's disease (AD), including the presence of tau-positive neurofibrillary tangles (NFTs) and ß-amyloid (Aß)-related peptides in vulnerable brain regions. Since enhanced cholesterol level, which acts as a risk factor for AD, can increase Aß production by regulating amyloid precursor protein (APP) metabolism, it is possible that APP overexpression can influence cholesterol-regulated NPC pathology. We have addressed this issue in a novel bigenic mice (ANPC) generated by crossing heterozygous Npc1-deficient mice with mutant human APP transgenic mice. These mice exhibited decreased lifespan, early object memory and motor impairments, and exacerbated glial pathology compared with other littermates. Neurodegeneration observed in the cerebellum of ANPC mice was found to be accelerated along with a selective increase in the phosphorylation/cleavage of tau protein. Additionally, enhanced levels/activity of cytosolic cathepsin D together with cytochrome c and Bcl-2-associated X protein suggest a role for the lysosomal enzyme in the caspase-induced degeneration of neurons in ANPC mice. The reversal of cholesterol accretion by 2-hydroxypropyl-ß-cyclodextrin (2-HPC) treatment increased longevity and attenuated behavioral/pathological abnormalities in ANPC mice. Collectively, our results reveal that overexpression of APP in Npc1-deficient mice can negatively influence longevity and a wide spectrum of behavioral/neuropathological abnormalities, thus raising the possibility that APP and NPC1 may interact functionally to regulate the development of AD and NPC pathologies.

Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration.

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

Baseline cell proliferation rates and response to UV differ in lymphoblastoid cell lines derived from healthy individuals of extreme constitution types.

Differences in human phenotypes and susceptibility to complex diseases are an outcome of genetic and environmental interactions. This is evident in diseases that progress through a common set of intermediate patho-endophenotypes. Precision medicine aims to delineate molecular players for individualized and early interventions. Functional studies of lymphoblastoid cell line (LCL) model of phenotypically well-characterized healthy individuals can help deconvolute and validate these molecular mechanisms. In this study, LCLs are developed from eight healthy individuals belonging to three extreme constitution types, deep phenotyped on the basis of Ayurveda. LCLs were characterized by karyotyping and immunophenotyping. Growth characteristics and response to UV were studied in these LCLs. Significant differences in cell proliferation rates were observed between the contrasting groups such that one type (Kapha) proliferates significantly slower than the other two (Vata, Pitta). In response to UV, one of the fast growing groups (Vata) shows higher cell death but recovers its numbers due to an inherent higher rates of proliferation. This study reveals that baseline differences in cell proliferation could be a key to understanding the survivability of cells under UV stress. Variability in baseline cellular phenotypes not only explains the cellular basis of different constitution types but can also help set priors during the design of an individualized therapy with DNA damaging agents. This is the first study of its kind that shows variability of intermediate patho-phenotypes among healthy individuals with potential implications in precision medicine.

Loss of Cx43-Mediated Functional Gap Junction Communication in Meningeal Fibroblasts Following Mouse Hepatitis Virus Infection.

Mouse hepatitis virus (MHV) infection causes meningoencephalitis by disrupting the neuro-glial and glial-pial homeostasis. Recent studies suggest that MHV infection alters gap junction protein connexin 43 (Cx43)-mediated intercellular communication in brain and primary cultured astrocytes. In addition to astrocytes, meningeal fibroblasts also express high levels of Cx43. Fibroblasts in the meninges together with the basal lamina and the astrocyte endfeet forms the glial limitans superficialis as part of the blood-brain barrier (BBB). Alteration of glial-pial gap junction intercellular communication (GJIC) in MHV infection has the potential to affect the integrity of BBB. Till date, it is not known if viral infection can modulate Cx43 expression and function in cells of the brain meninges and thus affect BBB permeability. In the present study, we have investigated the effect of MHV infection on Cx43 localization and function in mouse brain meningeal cells and primary meningeal fibroblasts. Our results show that MHV infection reduces total Cx43 levels and causes its intracellular retention in the perinuclear compartments reducing its surface expression. Reduced trafficking of Cx43 to the cell surface in MHV-infected cells is associated with loss functional GJIC. Together, these data suggest that MHV infection can directly affect expression and cellular distribution of Cx43 resulting in loss of Cx43-mediated GJIC in meningeal fibroblasts, which may be associated with altered BBB function observed in acute infection.

Evaluation of genetic markers linked to hemophilia A locus: an Indian experience.

Hemophilia A is an X-linked recessive bleeding disorder caused by defects in factor VIII gene (F8). Our study examines variations of single nucleotide polymorphism (SNP) in F8 in the Indian population and establishes the utility of a combination of SNP and microsatellite markers for the successful identification of carriers in the affected families.

Alterations in gene expression in mutant amyloid precursor protein transgenic mice lacking Niemann-Pick type C1 protein.

Niemann-Pick type C (NPC) disease, a rare autosomal recessive disorder caused mostly by mutation in NPC1 gene, is pathologically characterized by the accumulation of free cholesterol in brain and other tissues. This is accompanied by gliosis and loss of neurons in selected brain regions, including the cerebellum. Recent studies have shown that NPC disease exhibits intriguing parallels with Alzheimer's disease, including the presence of neurofibrillary tangles and increased levels of amyloid precursor protein (APP)-derived ß-amyloid (Aß) peptides in vulnerable brain neurons. To evaluate the role of Aß in NPC disease, we determined the gene expression profile in selected brain regions of our recently developed bigenic ANPC mice, generated by crossing APP transgenic (Tg) mice with heterozygous Npc1-deficient mice. The ANPC mice exhibited exacerbated neuronal and glial pathology compared to other genotypes [i.e., APP-Tg, double heterozygous (Dhet), Npc1-null and wild-type mice]. Analysis of expression profiles of 86 selected genes using real-time RT-PCR arrays showed a wide-spectrum of alterations in the four genotypes compared to wild-type controls. The changes observed in APP-Tg and Dhet mice are limited to only few genes involved mostly in the regulation of cholesterol metabolism, whereas Npc1-null and ANPC mice showed alterations in the expression profiles of a number of genes regulating cholesterol homeostasis, APP metabolism, vesicular trafficking and cell death mechanism in both hippocampus and cerebellum compared to wild-type mice. Intriguingly, ANPC and Npc1-null mice, with some exceptions, exhibited similar changes, although more genes were differentially expressed in the affected cerebellum than the relatively spared hippocampus. The altered gene profiles were found to match with the corresponding protein levels. These results suggest that lack of Npc1 protein can alter the expression profile of selected transcripts as well as proteins, and APP overexpression influences cerebral pathology by enhancing changes triggered by Npc1 deficiency in the bigenic line.

Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder caused by defects in the ATPase, Cu(2+) transporting, beta-polypeptide gene (ATP7B) resulting in accumulation of copper in liver and brain. WD can be thwarted if detected at a presymptomatic stage, but occasional recombination during carrier detection with dinucleotide repeat markers flanking the WD locus may lead to faulty diagnosis. We examined the use of intragenic single-nucleotide polymorphism (SNP) markers to avoid this limitation. METHODS: We prepared genomic DNA from the peripheral blood of Indian WD patients. By use of PCR, we amplified the exons and flanking regions of the WD gene and then performed sequencing to identify the nucleotide variants. We genotyped the SNPs in 1871 individuals by use of the Sequenom mass array system. We made linkage disequilibrium plots using Haploview software. RESULTS: We identified 1 mutation accounting for 11% (19 of 174) of WD chromosomes among patients in addition to 4 prevalent mutations characterized previously. Among 24 innocuous allelic variants identified, we selected 3 SNPs found to have high heterozygosity (>0.40) for the detection of mutant WD chromosomes. On analyzing these SNPs in 28 test individuals, who were sibs to 17 unrelated WD patients, we obtained unequivocal genotyping in 25 cases (approximately 89%). The remaining 3 cases were genotyped by dinucleotide repeat marker (D13S133). CONCLUSION: Sets of SNP markers are highly heterozygous across most world populations and could be used in combination with analysis of prevalent mutations as a comprehensive strategy for determining presymptomatic and carrier sibs of WD patients.