A Toxicogenic Interaction between Intracellular Amyloid-ß and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid ß (Aß) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aß oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aß-ApoE interaction suppresses these changes and concomitantly reduces Aß toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aß oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Making NSC and Neurons from Patient-Derived Tissue Samples.

The human brain and mechanisms underlying its functioning has been a field of intense research due to its complexity, inaccessibility, and the large numbers of debilitating disorders affecting this organ. Model organisms have provided great insight into the functioning of the mammalian brain; however, there exist many features unique to humans which need detailed understanding. In this context, human pluripotent stem cells (HPSCs) have emerged as a promising resource.In the developing brain, cortical diversification is achieved by neural stem cells/neural progenitor cells (NSCs/NPCs) by altering its potency (from multipotent to unipotent) and differentiation capacity (from neurogenesis to gliogenesis). Recent development in tissue reprogramming allows for derivation of NSCs/NPCs from either healthy control subjects manipulated to carry disease mutations or affected individuals carrying specific disease-causing mutations allowing for detailed evaluation of cellular phenotype, pharmacological manipulation, and/or toxicological screening.In this chapter, we will discuss HPSC differentiation into neural stem cells (NSCs) and neurons. We will review the mechanism underlying in vivo neural differentiation and methods which recapitulate this in vitro. We describe a method of deriving NSCs and differentiated mature neurons highlighting key steps of the core protocol. We also provide detailed information of the transcription factor and morphogen map of the developing brain which can be used as a guide to derive region- and lineage-specific NSCs and differentiated neurons.

Derivation of iPSC lines from two patients with familial Alzheimer's disease from India.

The current prevalence of diagnosable dementia in India is 1% of people over 60 years (~3.7 million people), but is estimated to increase significantly, as ~15% world's aged population (>65 years) would be resident here by 2020 (Shah et al., 2016). While several mutations that pose a familial risk have been identified, the ethnic background may influence disease susceptibility, clinical presentation and treatment response. In this study, we report a detailed characterization of two representative HiPSC lines from a well-characterized dementia cohort from India. Availability of these lines, and associated molecular and clinical information, would be useful in the detailed exploration of the genomic contribution(s) to AD.

INDEX-db: The Indian Exome Reference Database (Phase I).

Deep sequencing-based genetic mapping has greatly enhanced the ability to catalog variants with plausible disease association. Confirming how these identified variants contribute to specific disease conditions, across human populations, poses the next challenge. Differential selection pressure may impact the frequency of genetic variations, and thus detection of association with disease conditions, across populations. To understand genotype to phenotype correlations, it thus becomes important to first understand the spectrum of genetic variation within a population by creating a reference map. In this study, we report the development of phase I of a new database of genetic variations called INDian EXome database (INDEX-db), from the Indian population, with an aim to establish a centralized database of integrated information. This could be useful for researchers involved in studying disease mechanisms at clinical, genetic, and cellular levels.

Exome sequencing in families with severe mental illness identifies novel and rare variants in genes implicated in Mendelian neuropsychiatric syndromes.

AIM: Severe mental illnesses (SMI), such as bipolar disorder and schizophrenia, are highly heritable, and have a complex pattern of inheritance. Genome-wide association studies detect a part of the heritability, which can be attributed to common genetic variation. Examination of rare variants with next-generation sequencing may add to the understanding of the genetic architecture of SMI. METHODS: We analyzed 32 ill subjects from eight multiplex families and 33 healthy individuals using whole-exome sequencing. Prioritized variants were selected by a three-step filtering process, which included: deleteriousness by five in silico algorithms; sharing within families by affected individuals; rarity in South Asian sample estimated using the Exome Aggregation Consortium data; and complete absence of these variants in control individuals from the same gene pool. RESULTS: We identified 42 rare, non-synonymous deleterious variants (~5 per pedigree) in this study. None of the variants were shared across families, indicating a 'private' mutational profile. Twenty (47.6%) of the variant harboring genes were previously reported to contribute to the risk of diverse neuropsychiatric syndromes, nine (21.4%) of which were of Mendelian inheritance. These included genes carrying novel deleterious variants, such as the GRM1 gene implicated in spinocerebellar ataxia 44 and the NIPBL gene implicated in Cornelia de Lange syndrome. CONCLUSION: Next-generation sequencing approaches in family-based studies are useful to identify novel and rare variants in genes for complex disorders like SMI. The findings of the study suggest a potential phenotypic burden of rare variants in Mendelian disease genes, indicating pleiotropic effects in the etiology of SMI.

Generation of three spinocerebellar ataxia type-12 patients derived induced pluripotent stem cell lines (IGIBi002-A, IGIBi003-A and IGIBi004-A).

Spinocerebellar ataxia type-12 (SCA12) is a neurological disorder caused due to triplet (CAG) repeat expansion in 5' UTR of PPP2R2B. It is one of the most prominent SCA-subtype in Indian population and till date no patient specific models have been described. Human-induced-pluripotent-stem cell (HiPSC) based disease modelling has become the next generation tool for studying various human pathologies. In the present study we established three SCA12 patient specific iPSC lines. All the generated lines have shown pluripotency markers, normal karyotype, in-vitro three germ layers differentiation potential, vector clearance, SCA12 mutation, parental genomic identity and contamination free culture.

Developing two reference control samples for the Indian population.

Human induced Pluripotent Stem Cells (HiPSCs) have immense potential in research and therapeutics. Under the aegis of Department of Biotechnology funded national program entitled, "The Accelerator program for Discovery in Brain Disorders using Stem Cells (ADBS)" we have established a HiPSC biorepository (https://www.ncbs.res.in/adbs/bio-repository) with an objective to study severe mental illness. The repository comprises of HiPSC lines derived from healthy control donors and individuals with life time diagnosis of severe mental illness from dense families. In the current report we submit information regarding two population control reference lines (male = 1; female = 1) from this biorepository.

Discovery biology of neuropsychiatric syndromes (DBNS): a center for integrating clinical medicine and basic science.

BACKGROUND: There is emerging evidence that there are shared genetic, environmental and developmental risk factors in psychiatry, that cut across traditional diagnostic boundaries. With this background, the Discovery biology of neuropsychiatric syndromes (DBNS) proposes to recruit patients from five different syndromes (schizophrenia, bipolar disorder, obsessive compulsive disorder, Alzheimer's dementia and substance use disorders), identify those with multiple affected relatives, and invite these families to participate in this study. The families will be assessed: 1) To compare neuro-endophenotype measures between patients, first degree relatives (FDR) and healthy controls., 2) To identify cellular phenotypes which differentiate the groups., 3) To examine the longitudinal course of neuro-endophenotype measures., 4) To identify measures which correlate with outcome, and 5) To create a unified digital database and biorepository. METHODS: The identification of the index participants will occur at well-established specialty clinics. The selected individuals will have a strong family history (with at least another affected FDR) of mental illness. We will also recruit healthy controls without family history of such illness. All recruited individuals (N = 4500) will undergo brief clinical assessments and a blood sample will be drawn for isolation of DNA and peripheral blood mononuclear cells (PBMCs). From among this set, a subset of 1500 individuals (300 families and 300 controls) will be assessed on several additional assessments [detailed clinical assessments, endophenotype measures (neuroimaging- structural and functional, neuropsychology, psychophysics-electroencephalography, functional near infrared spectroscopy, eye movement tracking)], with the intention of conducting repeated measurements every alternate year. PBMCs from this set will be used to generate lymphoblastoid cell lines, and a subset of these would be converted to induced pluripotent stem cell lines and also undergo whole exome sequencing. DISCUSSION: We hope to identify unique and overlapping brain endophenotypes for major psychiatric syndromes. In a proportion of subjects, we expect these neuro-endophenotypes to progress over time and to predict treatment outcome. Similarly, cellular assays could differentiate cell lines derived from such groups. The repository of biomaterials as well as digital datasets of clinical parameters, will serve as a valuable resource for the broader scientific community who wish to address research questions in the area.

Mutation burden profile in familial Alzheimer's disease cases from India.

This study attempts to identify coding risk variants in genes previously implicated in Alzheimer's disease (AD) pathways, through whole-exome sequencing of subjects (N = 17) with AD, with a positive family history of dementia (familial AD). We attempted to evaluate the mutation burden in genes encoding amyloid precursor protein metabolism and previously linked to risk of dementias. Novel variants were identified in genes involved in amyloid precursor protein metabolism such as PSEN1 (chr 14:73653575, W161C, tgg > tgT), PLAT (chr 8:42039530,G272R), and SORL1 (chr11:121414373,G601D). The mutation burden assessment of dementia-related genes for all 17 cases revealed 45 variants, which were either shared across subjects, or were present in just the 1 patient. The study shows that the clinical characteristics, and genetic correlates, obtained in this sample are broadly comparable to the other studies that have investigated familial forms of AD. Our study identifies rare deleterious genetic variations, in the coding region of genes involved in amyloid signaling, and other dementia-associated pathways.

Effect of CLU and PICALM polymorphisms on AD risk: A study from south India.

OBJECTIVES: To study the association of apolipoprotein E (APOE), Clusterin (CLU) and phosphatidylinositol binding clathrin assembly protein (PICALM) polymorphisms in Alzheimer's disease (AD) subjects compared to cognitively normal control subjects in an Indian population. METHODS: The study subjects included persons with AD (N=243) and age group matched healthy controls (N=164). All the AD subjects were evaluated using a standard protocol. DNA was isolated from whole blood. APOE (rs7412, rs429358), CLU (rs11136000) and PICALM (rs3851179) were genotyped. General linear model was used to test the association between the individual risk genotypes and AD. RESULTS: The presence of APOE ε4 was associated with AD after adjusting for age and gender (p<0.0001). There was no association observed with AD at both rs11136000 CLU (p=0.25) and rs3851179 PICALM (p=0.54). CONCLUSION: Our results confirmed a significant association of APOE ε4 carrier status with AD. No association was observed for CLU and PICALM with AD. This might be due to a different genetic background. There are no previous reports of these polymorphisms in an Indian cohort. Future Indian AD studies should investigate additional SNPs in a larger sample size in these genes.

Genotoxic Effects of Culture Media on Human Pluripotent Stem Cells.

Culture conditions play an important role in regulating the genomic integrity of Human Pluripotent Stem Cells (HPSCs). We report that HPSCs cultured in Essential 8 (E8) and mTeSR, two widely used media for feeder-free culturing of HPSCs, had many fold higher levels of ROS and higher mitochondrial potential than cells cultured in Knockout Serum Replacement containing media (KSR). HPSCs also exhibited increased levels of 8-hydroxyguanosine, phospho-histone-H2a.X and p53, as well as increased sensitivity to γ-irradiation in these two media. HPSCs in E8 and mTeSR had increased incidence of changes in their DNA sequence, indicating genotoxic stress, in addition to changes in nucleolar morphology and number. Addition of antioxidants to E8 and mTeSR provided only partial rescue. Our results suggest that it is essential to determine cellular ROS levels in addition to currently used criteria i.e. pluripotency markers, differentiation into all three germ layers and normal karyotype through multiple passages, in designing culture media.

Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications.

We have recently described manufacturing of human induced pluripotent stem cells (iPSC) master cell banks (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Reports, 5(4), 647-659, 2015). In this manuscript, we describe the detailed characterization of the two iPSC clones generated using this process, including whole genome sequencing (WGS), microarray, and comparative genomic hybridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibration material and with a reporter subclone and lines made by a similar process from different donors. We believe that iPSCs are likely to be used to make multiple clinical products. We further believe that the lines used as input material will be used at different sites and, given their immortal status, will be used for many years or even decades. Therefore, it will be important to develop assays to monitor the state of the cells and their drift in culture. We suggest that a detailed characterization of the initial status of the cells, a comparison with some calibration material and the development of reporter sublcones will help determine which set of tests will be most useful in monitoring the cells and establishing criteria for discarding a line.

Cellular models to study bipolar disorder: A systematic review.

BACKGROUND: There is an emerging interest in the use of cellular models to study psychiatric disorders. We have systematically reviewed the application of cellular models to understand the biological basis of bipolar disorder (BD). METHOD: Published scientific literature in MEDLINE, PsychINFO and SCOPUS databases were identified with the following search strategy: [(Lymphoblastoid OR Lymphoblast OR Fibroblast OR Pluripotent OR Olfactory epithelium OR Olfactory mucosa) AND (Bipolar disorder OR Lithium OR Valproate OR Mania)]. Studies were included if they had used cell cultures derived from BD patients. RESULTS: There were 65 articles on lymphoblastoid cell lines, 14 articles on fibroblasts, 4 articles on olfactory neuronal epithelium (ONE) and 2 articles on neurons reprogrammed from induced pluripotent stem cell lines (IPSC). Several parameters have been studied, and the most replicated findings are abnormalities in calcium signaling, endoplasmic reticulum (ER) stress response, mitochondrial oxidative pathway, membrane ion channels, circadian system and apoptosis related genes. These, although present in basal state, seem to be accentuated in the presence of cellular stressors (e.g. oxidative stress--rotenone; ER stress--thapsigargin), and are often reversed with in-vitro lithium. CONCLUSION: Cellular modeling has proven useful in BD, and potential pathways, especially in cellular resilience related mechanisms have been identified. These findings show consistency with other study designs (genome-wide association, brain-imaging, and post-mortem brain expression). ONE cells and IPSC reprogrammed neurons represent the next generation of cell models in BD. Future studies should focus on family-based study designs and combine cell models with deep sequencing and genetic manipulations.

A method to identify and isolate pluripotent human stem cells and mouse epiblast stem cells using lipid body-associated retinyl ester fluorescence.

We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450-500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies.

Apolipoprotein E4 and brain white matter integrity in Alzheimer's disease: tract-based spatial statistics study under 3-Tesla MRI.

INTRODUCTION: Apolipoprotein E4 (ApoE ε4) polymorphism is a known genetic risk factor for Alzheimer's disease (AD). OBJECTIVES: To evaluate the role of ApoE ε4 on white matter structural integrity in AD. METHODS: Subjects were 32 patients with probable AD (ApoE ε4-positive: n = 15) and 18 matched controls (ApoE ε4-positive: n = 6). All subjects were right-handed, evaluated using standard scales and genotyped at the ApoE locus. Diffusion tensor imaging was performed with a 3-tesla MRI scanner and analyzed using the tract-based spatial statistics method. RESULTS: AD patients had significantly lower fractional anisotropy (FA) in bilateral temporoparietal, limbic and parahippocampal regions in comparison to healthy comparison subjects. ApoE ε4 carriers among both AD and healthy comparison subjects showed lower FA in limbic and medial temporal regions. CONCLUSIONS: There is a modest association between ApoE ε4 carrier status and reduction in white matter tract integrity at medial temporal and limbic regions in both healthy and AD subjects.

Genetic polymorphism in the serotonin transporter promoter region and ecological success in macaques.

A well-characterised sequence length polymorphism in the serotonin transporter promoter region (5-HTTLPR) influences individual behavioural traits and cognitive abilities in humans and rhesus macaques. Macaques have been classified into four continuous grades on the basis of their behavioural attributes, ranging from highly hierarchical and nepotistic species to the most egalitarian and tolerant ones. A comparative study of several species that spanned these grades revealed only rhesus macaques to be polymorphic at the 5-HTTLPR and concluded that the polymorphism was responsible for their despotic and aggressive behaviour (Wendland et al., Behav Genet 36:163-172, 2006). We studied wild populations of three other species and found that the egalitarian and tolerant bonnet and Arunachal macaques are also polymorphic while liontailed macaques, although belonging to the same group, are monomorphic. We thus reject a role for this particular polymorphism in interspecific behavioural variability and show that polymorphic species enjoy greater ecological success possibly due to their higher intraspecific variability in individual behavioural traits.

North and South Indian populations share a common ancestral origin of Friedreich's ataxia but vary in age of GAA repeat expansion.

Friedreich's ataxia (FRDA) is caused by expansion of GAA repeats in the frataxin (FXN) gene on chromosome 9q13-q21.1. We analysed the origin of FRDA in 21 North Indian (NI) and eight South Indian (SI) families using five single nucleotide polymorphisms (SNPs) and a microsatellite marker spanning the GAA repeats. The NI and SI families were derived from Indo-European and Dravidian linguistic backgrounds respectively. The frequency of large normal (LNs) alleles of the GAA repeat correlate with the overall lower prevalence of FRDA in India compared to the European population. All of the expanded alleles in the Indian population share a common core haplotype suggesting a founder effect. The expanded alleles in the NI population demonstrate more similarity to those of Europeans in terms of age of GAA repeat expansion (15975 +/- 2850 years) and association of LNs with expanded alleles. FRDA seems to have been introduced recently in the South Indian population since the average estimated age of the mutation in SI is 5425 +/- 1750 years and unlike NI some of the haplotypes of LNs are not associated with the expanded alleles.

Phylogenetic analysis and selection pressures of 5-HT receptors in human and non-human primates: receptor of an ancient neurotransmitter.

Neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) an ancient neurotransmitter, involved in several neurophysiological and behavioral functions, acts by interacting with multiple receptors (5-HT(1)-5-HT(7)). Alterations in serotonergic signalling have also been implicated in various psychiatric disorders. The availability of the genome data of non-human primates permits comparative analysis of human 5-HT receptors with sequences of non-human primates to understand evolutionary divergence. We compared and analyzed serotonergic receptor sequences from human and non-human primates. Phylogenetic analysis by Maximum Likelihood (ML) method classified human and primate 5-HT receptors into six unique clusters. There was considerable conservation of 5-HT receptor sequences between human and non-human primates; however, a greater diversity at the sub-group level was observed. Compared to the other subgroups, larger multiplicity and expansion was seen within the 5-HT(4) receptor subtype in both human and non-human primates. Analysis of non-synonymous and synonymous substitution ratios (Ka/Ks ratio) using the Nei-Gojobori method suggests that 5-HT receptor sequences have undergone negative (purifying) selection over the course of evolution in human, chimpanzee and rhesus monkey. Abnormal human and non-human primate psychopathalogy and behavior, in the context of these variations is discussed. Analysis of these 5-HT receptors in other species will help understand the molecular evolution of 5-HT receptors, and its possible influence on complex behaviors, and psychiatric disorders.