Altered transcriptomes, cell type proportions, and dendritic spine morphology in hippocampus of suicide deaths.

Suicide is a condition resulting from complex environmental and genetic risks that affect millions of people globally. Both structural and functional studies identified the hippocampus as one of the vulnerable brain regions contributing to suicide risk. Here, we have identified the hippocampal transcriptomes, gene ontology, cell type proportions, dendritic spine morphology, and transcriptomic signature in iPSC-derived neuronal precursor cells (NPCs) and neurons in postmortem brain tissue from suicide deaths. The hippocampal tissue transcriptomic data revealed that NPAS4 gene expression was downregulated while ALDH1A2, NAAA, and MLXIPL gene expressions were upregulated in tissue from suicide deaths. The gene ontology identified 29 significant pathways including NPAS4-associated gene ontology terms "excitatory post-synaptic potential", "regulation of postsynaptic membrane potential" and "long-term memory" indicating alteration of glutamatergic synapses in the hippocampus of suicide deaths. The cell type deconvolution identified decreased excitatory neuron proportion and an increased inhibitory neuron proportion providing evidence of excitation/inhibition imbalance in the hippocampus of suicide deaths. In addition, suicide deaths had increased dendric spine density, due to an increase of thin (relatively unstable) dendritic spines, compared to controls. The transcriptomes of iPSC-derived hippocampal-like NPCs and neurons revealed 31 and 33 differentially expressed genes in NPC and neurons, respectively, of suicide deaths. The suicide-associated differentially expressed genes in NPCs were RELN, CRH, EMX2, OXTR, PARM1 and IFITM2 which overlapped with previously published results. The previously-known suicide-associated differentially expressed genes in differentiated neurons were COL1A1, THBS1, IFITM2, AQP1, and NLRP2. Together, these findings would help better understand the hippocampal neurobiology of suicide for identifying therapeutic targets to prevent suicide.

PIEZO1-HaloTag hiPSCs: Bridging Molecular, Cellular and Tissue Imaging.

PIEZO1 channels play a critical role in numerous physiological processes by transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of endogenous PIEZO1 activity and localization in regulating mechanotransduction. To enable physiologically and clinically relevant human-based studies, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with super-resolution imaging, our chemogenetic approach allows precise visualization of PIEZO1 in various cell types. Further, the PIEZO1-HaloTag hiPSC technology allows non-invasive monitoring of channel activity via Ca2+-sensitive HaloTag ligands, with temporal resolution approaching that of patch clamp electrophysiology. Using lightsheet imaging of hiPSC-derived neural organoids, we also achieve molecular scale PIEZO1 imaging in three-dimensional tissue samples. Our advances offer a novel platform for studying PIEZO1 mechanotransduction in human cells and tissues, with potential for elucidating disease mechanisms and development of targeted therapeutics.

Piezo1 regulates cholesterol biosynthesis to influence neural stem cell fate during brain development.

Mechanical forces and tissue mechanics influence the morphology of the developing brain, but the underlying molecular mechanisms have been elusive. Here, we examine the role of mechanotransduction in brain development by focusing on Piezo1, a mechanically activated ion channel. We find that Piezo1 deletion results in a thinner neuroepithelial layer, disrupts pseudostratification, and reduces neurogenesis in E10.5 mouse embryos. Proliferation and differentiation of Piezo1 knockout (KO) mouse neural stem cells (NSCs) isolated from E10.5 embryos are reduced in vitro compared to littermate WT NSCs. Transcriptome analysis of E10.5 Piezo1 KO brains reveals downregulation of the cholesterol biosynthesis superpathway, in which 16 genes, including Hmgcr, the gene encoding the rate-limiting enzyme of the cholesterol biosynthesis pathway, are downregulated by 1.5-fold or more. Consistent with this finding, membrane lipid composition is altered, and the cholesterol levels are reduced in Piezo1 KO NSCs. Cholesterol supplementation of Piezo1 KO NSCs partially rescues the phenotype in vitro. These findings demonstrate a role for Piezo1 in the neurodevelopmental process that modulates the quantity, quality, and organization of cells by influencing cellular cholesterol metabolism. Our study establishes a direct link in NSCs between PIEZO1, intracellular cholesterol levels, and neural development.

The roles of serotonin in cell adhesion and migration, and cytoskeletal remodeling.

Serotonin is well known as a neurotransmitter. Its roles in neuronal processes such as learning, memory or cognition are well established, and also in disorders such as depression, schizophrenia, bipolar disorder, and dementia. However, its effects on adhesion and cytoskeletal remodelling which are strongly affected by 5-HT receptors, are not as well studied with some exceptions for e.g. platelet aggregation. Neuronal function is strongly dependent on cell-cell contacts and adhesion-related processes. Therefore the role played by serotonin in psychiatric illness, as well as in the positive and negative effects of neuropsychiatric drugs through cell-related adhesion can be of great significance. In this review, we explore the role of serotonin in some of these aspects based on recent findings.

Serotonin 2A (5-HT2A) receptor affects cell-matrix adhesion and the formation and maintenance of stress fibers in HEK293 cells.

5-HT2A, a G-protein coupled receptor, is widely expressed in the human body, including in the gastrointestinal tract, platelets and the nervous system. It mediates various functions, for e.g. learning, memory, mood regulation, platelet aggregation and vasoconstriction, but its involvement in cell-adhesion remains largely unknown. Here we report a novel role for 5-HT2A in cell-matrix adhesion.In HEK293 cells, which are loosely adherent, expression and stimulation of human or rat 5-HT2A receptor by agonists such as serotonin or 2,5-dimethoxy-4-iodoamphetamine (DOI) led to a significant increase in adhesion, while inhibition of 5-HT2A by antipsychotics, such as risperidone, olanzapine or chlorpromazine prevented it. 5-HT2A activation gave rise to stress fibers in these cells and was also required for their maintenance. Mechanistically, the 5-HT2A-mediated adhesion was mediated by downstream PKC and Rho signaling. Since 5-HT2A is associated with many disorders such as dementia, depression and schizophrenia, its role in cell-matrix adhesion could have implications for neural circuits.

Dopamine requires unique residues to signal via the serotonin 2A receptor.

Serotonin is an important neurotransmitter and neuromodulator. Disruption of the serotonergic system has been implicated in various psychiatric disorders such as schizophrenia and bipolar disorder. Most of the drugs targeting these neurotransmitter systems are classified primarily as agonists or inverse agonists/antagonists, with their described function being limited to activating the canonical signaling pathway(s), or inhibiting the pathway(s) respectively. Previous work with the human 5-HT2A has shown the receptor to be activated by dopamine, also an endogenous ligand. Dopamine is the cognate ligand of the dopaminergic system, which significantly overlaps with the serotonergic system in the brain. The two systems innervate many of the same brain areas, and the central serotonergic systems also regulate dopamine functions. Our aim was to investigate the downstream signaling set up by the receptor on being activated by dopamine. We show that dopamine is a functionally selective ligand at 5-HT2A and have examined dopamine as a ligand with respect to some receptor-dependent phenotypes. Our results show that dopamine acts as an agonist at the human serotonin 2A receptor and brings about its activation and internalization. Using in vitro assays, we have established differences in the signaling pathways set up by dopamine as compared to serotonin. Using site-specific mutagenesis we have identified residues important for this functional selectivity, shown by dopamine at this receptor. Our identification of specific residues important in the functional selectivity of dopamine at 5-HT2A could have far reaching implications for the field of GPCR signaling and drug-design. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea.

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a noncanonical enzyme, phenylalanine hydroxylase. Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescues the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

5-HT2A deletion protects against Clozapine-induced hyperglycemia.

Clozapine is an antipsychotic known for its superior efficacy in treating drug-resistant Schizophrenia. However, Clozapine induces various side effects such as hyperglycemia, agranulocytosis, weight gain etc. The mechanisms of these Clozapine-induced side effects have remained largely elusive though an important role is ascribed to 5-HT2A (Serotonin receptor subtype-2A). In this pilot study, we report for the first time that the 5-HT2A 'global' knockout mice (Htr2a-/-) are resistant to the Clozapine-induced hyperglycemia. Importantly though, the Htr2a-/- mice exhibit near normal basal glucose metabolism in the glucose tolerance tests. Collectively, the Htr2a-/- mice provide an important tool to study the Clozapine-induced hyperglycemia.

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.

Does retinoic acid reverse cell cycle dysregulation in Alzheimer's disease lymphocytes?

Aberrant re-entry of neurons into cell cycle appears to be an early event in Alzheimer's disease (AD) and targeting this dysregulation may have therapeutic potential. We have examined whether cell cycle dysregulation in AD can be detected using patient and control derived B-lymphocytes. Cell cycle analysis using flow cytometry demonstrated that cell cycle dysregulation occurs in AD lymphocytes, with a significant difference in the distribution of cells in G0/G1, S and G2/M phases of cell cycle as compared to control lymphocytes. Using global gene expression analysis by RNA sequencing and cell cycle analysis, we examined the role of Retinoic Acid (RA), a candidate molecule predicted to be of therapeutic potential in cell cycle dysregulation associated with AD. CCND1, CCNE2, E2F transcription factors which are known to be dysregulated in AD were among the 32 genes that showed differential expression in response to RA treatment thus suggesting a protective role of RA. However, the cell cycle analysis demonstrated that RA did not reverse the cellular phenotype in AD lymphocytes. This suggests that though RA might have a protective role by influencing the expression of cell cycle genes, it might not be able to arrest abnormal re-entry into cell cycle.

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.

Identifying the In Vivo Cellular Correlates of Antipsychotic Drugs.

GPCRs such as 5-HT2A and D2 are implicated in the therapeutic and the side effects of antipsychotics. However, the pattern of brain activity that leads to the behavioral effects of antipsychotics is poorly understood. To address this question, we used the transgenic 'FosTRAP' mice (Mus musculus), where a fluorescent reporter marks the cells responsive to the stimulus of interest. Here, the stimulus was an administration of various antipsychotic drugs. In case of typical antipsychotics such as Haloperidol, the c-fos active cells were predominantly found in the striatum, whereas in case of the atypical antipsychotics (Clozapine and Olanzapine), c-fos-induced cells were more numerous in the cortical regions, e.g., orbital cortex, piriform cortex. Curiously, we also observed ependymal cells to be a novel cellular target of atypical antipsychotics. 5-HT2A is considered to be a major target for atypical antipsychotics. Therefore, we bred 'FosTRAP' mice with 5-HT2A knock-out (KO) mice and tested their response to the prototype of atypical antipsychotics, Clozapine. Interestingly, the absence of 5-HT2A did not significantly affect the number of c-fos-induced cells in the cortical regions. However, the ependymal cells showed a dramatically reduced response to Clozapine in the absence of 5-HT2A. In summary, the TRAP system has allowed us to identify various region-specific activity induced by antipsychotics and novel cellular targets of the antipsychotics. These results serve as a "proof of principle" study that can be extended to explore the biochemical and physiological changes brought about by antipsychotics and specifically identify antipsychotic-responsive cells in the live tissue.

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.

Sedative effect of Clozapine is a function of 5-HT2A and environmental novelty.

Antipsychotic drugs are the mainstay in the treatment of schizophrenia and bipolar disorder. However, antipsychotics often exhibit sedation or activity suppression among many other side effects, and the factors that influence them remain poorly understood. We now show, using a 5-HT2A knockout (Htr2a-/-) mouse, that environmental circumstances can affect suppression of activity induced by the atypical antipsychotic- Clozapine. We observed that Htr2a-/- mice were more resistant to Clozapine-induced suppression of activity (CISA) and this behaviour was dependent on the environment being 'novel'. In their 'home' environment, at identical doses the mice exhibited CISA. Interestingly, the effect of genotype and environmental novelty on CISA could not be extended to the other antipsychotics that were tested, i.e. Haloperidol and Risperidone. Haloperidol-induced activity suppression was independent of context and genotype. Whereas context affected Risperidone-induced activity suppression only in the Htr2a+/+ mice. Furthermore, we observed that caffeine, a stimulant, elicited resistance to CISA similar to that seen in the 'novel' context. Our study establishes a previously unknown interaction between the environmental context, 5-HT2A and CISA and emphasises the role of non-pharmacological factors such as environment on the effects of the drug, which seem antipsychotic-specific. Our findings should advance the understanding of the side effects of individual antipsychotics and the role of environment to overcome side effects such as sedation.

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.

Fluorescence anisotropy uncovers changes in protein packing with inclusion growth in a cellular model of polyglutamine aggregation.

The aggregation of polyglutamine-rich proteins is closely linked with numerous neurodegenerative disorders. In pathological and cellular models, the appearance of protein-rich inclusions in cells acts as a read out of protein aggregation. The precise organization of aggregated protein in these inclusions and their mode of growth are still poorly understood. Here, fluorescence anisotropy-based measurements have been used to probe protein packing across inclusions of varying brightness, formed by an monomeric enhanced green fluorescent protein (mEGFP)-tagged polyglutamine model peptide in cells. High-resolution, confocal-based steady-state anisotropy measurements report a large depolarization, consistent with extensive homo-Förster (fluorescence) resonance energy transfer (FRET) between the sequestered mEGFP-tagged protein molecules. An inverse correlation of fluorescence anisotropy with intensity is seen across inclusions, which becomes emphasized when the observed fluorescence anisotropy values of inclusions are corrected for the fluorescence contribution of the diffusible protein, present within and around smaller inclusions. Homo-FRET becomes enhanced as inclusion size increases. This enhancement is confirmed by two-photon excitation-based time-resolved fluorescence anisotropy decay measurements, which also suggest that the mEGFP-tagged protein molecules are arranged in multiple ways within inclusions. Bright inclusions display faster FRET rates with a larger number of mEGFP moieties participating in homo-FRET than faint inclusions do. These results are consistent with a model in which the protein is more closely packed in the brighter inclusions. In such a possible mechanism, the higher packing density of protein molecules in brighter inclusions would suggest that inclusion growth could involve an intermolecular compaction event within the inclusion, as more monomers and aggregates are recruited into the growing inclusion.

Functional selectivity in serotonin receptor 2A (5-HT2A) endocytosis, recycling, and phosphorylation.

G protein-coupled receptor (GPCR) signaling is modulated by endocytosis and endosomal sorting of receptors between degradation and recycling. Differential regulation of these processes by endogenous ligands and synthetic drugs is a poorly understood area of GPCR signaling. Here, we describe remarkable diversity in the regulation of trafficking of GPCR induced by multiple ligands. We show that the serotonin receptor 2A (5-HT(2A)), a prototypical GPCR in the study of functional selectivity at a signaling receptor, is functionally selective in endocytosis and recycling in response to five ligands tested: endogenous agonists serotonin (5-HT) and dopamine (DA), synthetic agonist 1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (DOI), antagonist ketanserin, and inverse agonist and antipsychotic drug clozapine. Only four ligands (5-HT, DA, DOI, and clozapine) bring about receptor endocytosis. As we have earlier described with 5-HT and DA, there is ligand-specific requirement for protein kinase C (PKC) in endocytosis. We now show 5-HT(2A) phosphorylation by PKC is necessary for 5-HT-mediated and DOI-mediated receptor endocytosis, but DA-mediated and clozapine-mediated internalization is not affected if PKC is inhibited. Internalized receptors are recycled to the cell surface, but there is variability in the time course of recycling. 5-HT- and DA-internalized receptors are recycled in 2.5 hours while agonist DOI and antagonist clozapine bring about recycling in 7.5 hours. Recycling in response to those ligands that require PKC activation to effect receptor endocytosis is dependent on receptor dephosphorylation by protein phosphatase 2A (PP2A). Thus, internalization and phosphorylation/dephosphorylation cycles may play a significant role in the regulation of 5-HT(2A) by functionally and therapeutically important ligands.

Differences in the C-terminus contribute to variations in trafficking between rat and human 5-HT(2A) receptor isoforms: identification of a primate-specific tripeptide ASK motif that confers GRK-2 and beta arrestin-2 interactions.

Internalization and recycling of G-protein coupled receptors are important cellular processes regulating receptor function. These are receptor-subtype and cell type-specific. Although important, trafficking variations between receptor isoforms of different species has received limited attention. We report here, differences in internalization and recycling between rat and human serotonin 2A receptor (5-HT(2A)R) isoforms expressed in human embryonic kidney 293 cells in response to serotonin. Although the human and rat 5-HT(2A)Rs differ by only a few amino acids, the human receptor takes longer to recycle to the cell surface after internalization, with the additional involvement of beta arrestin-2 and G-protein receptor kinase 2. The interaction of beta arrestin-2 with the human receptor causes the delay in recycling and is dependent on a primate-specific ASK motif present in the C-terminus of the receptor. Conversion of this motif to NCT, the corresponding sequence present in the rat isoform, results in the human isoform trafficking like the rat receptor. Replacing the serine 457 with alanine in the ASK motif of human isoform resulted in faster recycling, although with continued arrestin-dependent internalization. This study establishes significant differences between the two isoforms with important implications in our understanding of the human 5-HT(2A)R functions; and indicates that extrapolating results from non-human receptor isoforms to human subtypes is not without caveats.