👤 S Chattopadhyay

ApoB is an essential structural protein for the assembly and secretion of triglyceride-rich lipoproteins and therefore remains a potential target to lower plasma cholesterol levels in hypercholesterolemia patients. To understand the global consequences of APOB gene deficiency, we employed CRISPR-Cas9 system to generate apoB-deficient human hepatoma Huh-7 cells (Ako cells). ApoB was not detectable in the cells and media of the Ako cells. ApoB deficiency had no effect on microsomal triglyceride transfer protein expression and activity. These cells supported apoB48 secretion when transfected with plasmids for the expression of apoB48 suggesting that these cells retain all the lipoprotein assembly and secretion machinery except for apoB expression. APOB gene deficiency had no significant effect on cellular lipid levels, cell growth, and ER stress markers. Proteome analysis of secreted proteins revealed that the most upregulated protein was the vitamin D binding protein, while the most downregulated protein was apoB in Ako cells compared to control cells. This analysis also identified coagulation as an upregulated pathway. Total RNA transcriptome analysis identified DNA replication and complement and coagulation pathways as the most upregulated pathways in Ako cells. Further detailed studies are needed to establish how apoB regulates these pathways. These Ako cells may be useful in studying structure-function analysis of apoB peptides and to address the cellular consequences of disruptions in lipoprotein assembly and secretion. Show less

Multiple myeloma (MM) is an incurable disease characterized by the presence of malignant plasma cells in the bone marrow that secrete specific monoclonal immunoglobulins into the blood. Obesity has been associated with the risk of developing solid and hematological cancers, but its role as a risk factor for MM needs to be further explored. Here, we evaluated whether 32 genome-wide association study (GWAS)-identified variants for obesity were associated with the risk of MM in 4189 German subjects from the German Multiple Myeloma Group (GMMG) cohort (2121 MM cases and 2068 controls) and 1293 Spanish subjects (206 MM cases and 1087 controls). Results were then validated through meta-analysis with data from the UKBiobank (554 MM cases and 402,714 controls) and FinnGen cohorts (914 MM cases and 248,695 controls). Finally, we evaluated the correlation of these single nucleotide polymorphisms (SNPs) with cQTL data, serum inflammatory proteins, steroid hormones, and absolute numbers of blood-derived cell populations ( Show less

Ashwagandha extracts play a significant role in traditional Indian medicine to help treat a wide range of disorders from amnesia, erectile dysfunction, neurodegenerative and cardiovascular diseases, cancer, stress, anxiety, and many more. Ashwagandha root is enriched with bioactive plant metabolites of which withanolides are the most important ones. The concentration and constitution of withanolides primarily determine ashwagandha's potency and pharmacology. Various factors modulate the withanolide constitution in the plant-derived extracts, rendering inconsistent therapeutic efficacy. Standardisation of the extraction protocol and a better understanding of the pharmacology mechanism of different extracts with varied withanolide constitutions is therefore critical for developing reliable, repeatable, and effective ashwagandha-based treatment. Here, we work toward defining indication mechanisms for two varieties of ashwagandha extract-ASHWITH (ASH-Ext1) and Regenolide (ASH-Ext2)-with different proprietary withanolide proportions. ASH-Ext1 was studied for antioxidant signaling modulation using HEK293, HeLa, and A549 cells, and ASH-Ext2 was studied for subcellular drug targets associated with the reactivation and longevity of human hair follicles, using primary human hair follicle dermal papilla cells (HFDPCs). Study findings support the antioxidant activity and Nrf2 signaling modulation by ASH-Ext1 in various cell models. Of note, ASH-Ext2 was found to increase The results of drug target modulation show us that the withanolide constitution associated with different extraction protocols influences the pharmacological potential of the extract significantly and points to the value of standardisation not only of total withanolide content but also of internal withanolide proportions. Show less

Elicitation of the tumor-eliminating immune response is a major challenge, as macrophages- constituting a major component of solid tumor mass- play important roles in development, maintenance and tumor regression. The macrophage-expressed Toll-Like Receptors (TLRs) enhance macrophage function and their ability to activate T cells via secretion of cytokines, which may help in tumor regression. IL-27, a member of the IL-12 family of cytokines, is shown to exhibit anti-tumor and anti-angiogenic activities. Herein, we developed B16BL6 melanoma model in C75BL/6 mouse to dissect the crosstalk between TLRs and IL-27 in tumors. We report existence of a novel TLR- IL-27 feed-forward loop, whereby TLRs and IL-27 up-regulated each other's expression, which we found perturbed during melanoma tumorigenesis. Intra-tumoral injection of Imiquimod, a TLR7/8 ligand, reduced the tumor burden; the anti-tumor effect was reversed upon IL-27 and IL-27R silencing by intra-tumorally administered, lentivirally expressed IL-27 and IL-27R shRNA. The reduced tumor growth was accompanied by significantly fewer Treg cells but increased IFN-γ and granzyme B expression by CD8 Show less

Solid tumors elicit suppressive T cell responses which impair antigen-presenting cell (APC) functions. Such immune suppression results in uncontrolled tumor growth and mortality. Addressing APC dysfunction, dendritic cell (DC)-mediated anti-tumor vaccination was extensively investigated in both mice and humans. These studies never achieved full resistance to tumor relapse. Herein, we describe a repetitive RM-1 murine tumor rechallenge model for recurrence in humans. Using this newly developed model, we show that priming with tumor antigen-pulsed, Toll-like receptor (TLR)2 ligand-activated DCs elicits a host-protective anti-tumor immune response in C57BL/6 mice. Upon stimulation with the TLR2 ligand peptidoglycan (PGN), the tumor antigen-pulsed DCs induce complete resistance to repetitive tumor challenges. Intra-tumoral injection of PGN reduces tumor growth. The tumor resistance is accompanied by increased expression of interleukin (IL)-27, T-box transcription factor TBX21 (T-bet), IL-12, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, along with heightened cytotoxic T lymphocyte (CTL) functions. Mice primed four times with PGN-stimulated tumor antigen-pulsed DCs remain entirely resistant to repeat challenges with RM-1 tumor cells, suggesting complete prevention of relapse and recurrence of tumor. Adoptive transfer of T cells from these mice, which were fully protected from RM-1 rechallenge, confers anti-tumor immunity to syngeneic naive recipient mice upon RM-1 challenge. These observations indicate that PGN-activated DCs induce robust host-protective anti-tumor T cells that completely resist tumor growth and recurrence. Show less

Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor-clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures. Show less

Patients and a mouse model of Batten disease, the juvenile form of neuronal ceroid lipofuscinosis (JNCL), raise autoantibodies against GAD65 and other brain-directed antigens. Here we investigate the adaptive component of the neuroimmune response. Cln3(-/-) mice have autoantibodies to GAD65 in their cerebrospinal fluid and elevated levels of brain bound immunoglobulin G (IgG). IgG deposition was found within human JNCL autopsy material, a feature that became more evident with increased age in Cln3(-/-) mice. The lymphocyte infiltration present in human and murine JNCL occurred late in disease progression, and was not capable of central/intrathecal IgG production. In contrast, we found evidence for an early systemic immune dysregulation in Cln3(-/-) mice. In addition evidence for a size-selective breach in the blood-brain barrier integrity in these mice suggests that systemically produced autoantibodies can access the JNCL central nervous system and contribute to a progressive inflammatory response. Show less

Hook1 is a member of a family of microtubule-binding proteins. Studies on the Drosophila homolog of Hook1 have suggested a role in the maturation and trafficking of internalized proteins to the late endosome. A weak interaction between Hook1 and the lysosomal/late endosomal protein, CLN3, was recently reported. Mutations in CLN3 result in the neurological disorder Batten disease. Here we show a novel interaction between Hook1 and Ankyrin G, an adaptor protein that binds the spectrin-actin cytoskeleton and targets proteins to the peripheral membrane. Although we demonstrate co-localization of Hook1 and Ankyrin G, Hook1 also localizes to additional regions of the cell devoid of Ankyrin G where it likely interacts with other proteins. There is no disruption of the Hook1-Ankyrin G interaction or localization in tissue derived from a Cln3-knockout mouse despite a nearly threefold increase in the expression of Hook1. However, mutation of CLN3 could lead to alterations in the functioning and positioning of organelles and membrane proteins through this Hook1-Ankyrin G interaction. Show less

The pathogenic mechanisms underlying Batten disease are unclear. Patients uniformly possess autoantibodies against glutamic acid decarboxylase (GAD) that are predominantly reactive with a region of GAD (amino acids 1 to 20) distinct from subjects with autoimmune type 1 diabetes or stiff-person syndrome. Batten patients did not possess autoantibodies against other type 1 diabetes-associated autoantigens and human leukocyte antigen genotypes revealed no specific associations with this disease. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is one of the most common progressive neurodegenerative disorders of childhood, resulting from autosomal recessive inheritance of mutations in the CLN3 gene. Pathologically, Batten disease is characterized by lysosomal storage of autofluorescent material in all tissue types. Although characterized by seizures, mental retardation, and loss of motor skills, the first presenting symptom of Batten disease is vision loss. High-density oligonucleotide arrays were used to profile approximately 19,000 mRNAs in the eye of 10-week-old Cln3-knockout and normal mice, and the data were compared with that for the cerebellum in the same model as a means to identify gene expression changes that are specific to the eye. A detailed list was compiled of 285 functionally categorized genes that have altered expression in the eye of Cln3-knockout mice before the appearance of the characteristic lysosomal storage material. Furthermore, 18 genes were identified and 6 validated by semiquantitative RT-PCR that have altered expression in the eye, but not in the cerebellum of Cln3-knockout mice. The genes that have altered expression specific to the eye of the Cln3-knockout mouse may be of importance in understanding the function of CLN3 in different tissues. Downregulation of genes associated with energy production in the mitochondria appears to be specific to the eye. The CLN3 defect may result in altered mitochondrial function in eye but not other tissue. More detailed experimentation is needed to understand the contribution of these changes in expression to disease state, and whether these changes are specific for certain cell types within the eye. Show less

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1- and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identified reproducible changes in gene expression in the brains of both 10-week-old Cln1- and Cln3-knockout mice as compared to wild-type controls, and confirmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations affect the expression of different, non-overlapping sets of genes. The possible significance of these changes and the pathological mechanisms underlying NCL diseases are discussed. Show less

Btn2p is a novel coiled coil cytosolic protein in Saccharomyces cerevisiae. We report that Btn2p interacts with Yif1p, a component of a protein complex at the Golgi that functions in ER to Golgi transport. Deletion of Btn2p, btn2-delta, results in mis-localiztion of Yif1p to the vacuole. Therefore, Btn2p may have an apparent role in intracellular trafficking of proteins. Btn2p was originally identified as being up-regulated in a btn1-delta strain, which exhibits dysregulation of vacuolar pH, and this up-regulation of Btn2p was presumed to contribute to maintaining a stable vacuolar pH [Pearce et al. Nat. Genet. 22 (1999) 55]. We propose that up-regulation of Btn2p in btn1-delta is an indicator of altered trafficking within the cell, and as btn1-delta serves as a model for the lysosomal storage disorder Batten disease, that altered intracellular trafficking may contribute to some of the cellular pathological hallmarks of this disease. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is the most common progressive neurodegenerative disorder of childhood. The disease is inherited in an autosomal recessive manner and is the result of mutations in the CLN3 gene. One brain region severely affected in Batten disease is the cerebellum. Using a mouse model for Batten disease which shares pathological similarities to the disease in humans we have used oligonucleotide arrays to profile approximately 19000 mRNAs in the cerebellum. We have identified reproducible changes of twofold or more in the expression of 756 gene products in the cerebellum of 10-week-old Cln3-knockout mice as compared to wild-type controls. We have subsequently divided these genes with altered expression into 14 functional categories. We report a significant alteration in expression of genes associated with neurotransmission, neuronal cell structure and development, immune response and inflammation, and lipid metabolism. An apparent shift in metabolism toward gluconeogenesis is also evident in Cln3-knockout mice. Further experimentation will be necessary to understand the contribution of these changes in expression to a disease state. Detailed analysis of the functional consequences of altered expression of genes in the cerebellum of the Cln3-knockout mice may provide valuable clues in understanding the molecular basis of the pathological mechanisms underlying Batten disease. Show less

Btn2p, a novel coiled-coil protein, is up-regulated in btn1delta yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in btn1delta strains (D. A. Pearce, T. Ferea, S. A. Nosel, B. Das, and F. Sherman, Nat. Genet. 22:55-58, 1999). We now report that Btn2p interacts biochemically and functionally with Rsglp, a down-regulator of the Can1p arginine and lysine permease. Rsglp localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (btn2delta strains) fail to correctly localize Rsg1p. btn2delta strains, like rsg1delta strains, are sensitive for growth in the presence of the arginine analog canavanine. Furthermore, btn2delta strains, like rsg1delta strains, demonstrate an elevated rate of uptake of [14C]arginine, which leads to increased intracellular levels of arginine. Overexpression of BTN2 results in a decreased rate of arginine uptake. Collectively, these results indicate that altered levels of Btn2p can modulate arginine uptake through localization of the Can1p-arginine permease regulatory protein, Rsglp. Our original identification of Btn2p was that it is up-regulated in the btn1delta strain which serves as a model for the lysosomal storage disorder Batten disease. Btnlp is a vacuolar/lysosomal membrane protein, and btn1delta suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by btn2delta rsg1delta strains. We conclude that Btn2p interacts with Rsglp and modulates arginine uptake. Up-regulation of BTN2 expression in btn1delta strains may facilitate either a direct or indirect effect on intracellular arginine levels. Show less

Mutations in the CLN3 gene are responsible for the neurodegenerative disorder Batten disease; however, the molecular basis of this disease remains unknown. In studying a mouse model for Batten disease, we report the presence of an autoantibody to glutamic acid decarboxylase (GAD65) in cln3-knockout mice serum that associates with brain tissue but is not present in sera or brain of normal mice. The autoantibody to GAD65 has the ability to inhibit the activity of glutamic acid decarboxylase. Furthermore, brains from cln3-knockout mice have decreased activity of glutamic acid decarboxylase as a result of the inhibition of this enzyme by the autoantibody, resulting in brain samples from cln3-knockout mice having elevated levels of glutamate as compared with normal. This elevated glutamate in the brain of cln3-knockout mice co-localizes with presynaptic markers. The decreased activity of GAD65 and increased levels of glutamate may have a causative role in astrocytic hypertrophy evident in cln3-knockout mice, and in altered expression of genes involved in the synthesis and utilization of glutamate that underlie a shift from synthesis to utilization of glutamate. An autoantibody to GAD65 is also present in sera of 20 out of 20 individuals tested who have Batten disease. Postmortem tissue shows decreased reactivity to an anti-GAD65 antibody that may be due to loss of GAD65-positive neurons or due to the reactive epitope being blocked by the presence of the autoantibody. We propose that an autoimmune response to GAD65 may contribute to a preferential loss of GABAergic neurons associated with Batten disease. Show less

The neuronal ceroid lipofuscinoses (NCLs) are the most common neurodegenerative disorders of childhood. The CLN1, CLN2 and CLN3 genes are associated to the infantile, late infantile and juvenile forms of NCL, respectively. We have subcloned the cDNAs encoding CLN1, CLN2 and BTN1, the yeast homologue of human CLN3, into plasmid vectors to evaluate whether these proteins interact with other proteins co-expressed from either a cDNA library derived from human cerebellum or from yeast, respectively, using the two-hybrid system. We concluded that CLN1 most likely does not interact with any other proteins in vivo. Furthermore, it is unlikely that CLN2 interacts with other proteins in vivo, although this study utilized a cDNA encoding the CLN2 precursor and it is possible that interacting partners may be excluded by the nature of this protein structure. Finally, we conclude that proteins that interact with Btn1p and therefore CLN3 cannot be identified using the whole proteins in a two-hybrid system, due to the hydrophobic nature of this protein. By understanding the topology of CLN3, specific regions of CLN3 need to be tested by two-hybrid to identify any interacting partners. Show less

The BTN1 gene product of the yeast Saccharomyces cerevisiae is 39% identical and 59% similar to human CLN3, which is associated with the neurodegenerative disorder Batten disease. Furthermore, btn1-Delta strains have an elevated activity of the plasma membrane H(+)-ATPase due to an abnormally high vacuolar acidity during the early phase of growth. Previously, DNA microarray analysis revealed that btn1-Delta strains compensate for the altered plasma membrane H(+)-ATPase activity and vacuolar pH by elevating the expression of the two genes HSP30 and BTN2. We now show that deletion of either HSP30 or BTN2 in either BTN1(+) or btn1-Delta strains does not alter vacuolar pH but does lead to an increased activity of the vacuolar H(+)-ATPase. Deletion of BTN1, BTN2, or HSP30 does not alter cytosolic pH but diminishes pH buffering capacity and causes poor growth at low pH in a medium containing sorbic acid, a condition known to result in disturbed intracellular pH homeostasis. Btn2p was localized to the cytosol, suggesting a role in mediating pH homeostasis between the vacuole and plasma membrane H(+)-ATPase. Increased expression of HSP30 and BTN2 in btn1-Delta strains and diminished growth of btn1-Delta, hsp30-Delta, and btn2-Delta strains at low pH reinforce our view that altered pH homeostasis is the underlying cause of Batten disease. Show less

The neuronal ceroid lipofuscinoses (NCLs) are the most common neurodegenerative disorders of childhood. We have examined mRNA levels of the CLN1, CLN2, and CLN3 genes, which are associated with the infantile, late infantile, and juvenile forms of NCL in 64 different human tissues, and have grouped the results into gastrointestinal tract, central nervous system, glandular/secretory, muscle, and carcinoma tissue types. mRNA levels for CLN3 are highest in gastrointestinal tissue and are also high in glandular/secretory tissue, whereas mRNA levels for CLN1 and CLN2 do not appear to be preferentially elevated in any tissue type. The significance of extraneural expression of CLN3 is reviewed in the context of the function of the protein. Show less