Alzheimer's Disease Blood Biomarkers Associated With Neuroinflammation as Therapeutic Targets for Early Personalized Intervention.

The definitive diagnosis of Alzheimer's Disease (AD) without the need for neuropathological confirmation remains a challenge in AD research today, despite efforts to uncover the molecular and biological underpinnings of the disease process. Furthermore, the potential for therapeutic intervention is limited upon the onset of symptoms, providing motivation for studying and treating the AD precursor mild cognitive impairment (MCI), the prodromal stage of AD instead. Applying machine learning classification to transcriptomic data of MCI, AD, and cognitively normal (CN) control patients, we identified differentially expressed genes that serve as biomarkers for the characterization and classification of subjects into MCI or AD groups. Predictive models employing these biomarker genes exhibited good classification performances for CN, MCI, and AD, significantly above random chance. The PI3K-Akt, IL-17, JAK-STAT, TNF, and Ras signaling pathways were also enriched in these biomarker genes, indicating their diagnostic potential and pathophysiological roles in MCI and AD. These findings could aid in the recognition of MCI and AD risk in clinical settings, allow for the tracking of disease progression over time in individuals as part of a therapeutic approach, and provide possible personalized drug targets for early intervention of MCI and AD.

WNK3 Maintains the GABAergic Inhibitory Tone, Synaptic Excitation and Neuronal Excitability via Regulation of KCC2 Cotransporter in Mature Neurons.

The activation of chloride (Cl-)permeable gamma (γ)-aminobutyric acid type A(GABAA) receptors induces synaptic inhibition in mature and excitation in immature neurons. This developmental "switch" in GABA function controlled by its polarity depends on the postnatal decrease in intraneuronal Cl- concentration mediated by KCC2, a member of cation-chloride cotransporters (CCCs). The serine-threonine kinase WNK3 (With No Lysine [K]), is a potent regulator of all CCCs and is expressed in neurons. Here, we characterized the functions of WNK3 and its role in GABAergic signaling in cultured embryonic day 18 (E18) hippocampal neurons. We observed a decrease in WNK3 expression as neurons mature. Knocking down of WNK3 significantly hyperpolarized EGABA in mature neurons (DIV13-15) but had no effect on immature neurons (DIV6-8). This hyperpolarized EGABA in WNK3-deficient neurons was not due to the total expression of NKCC1 and KCC2, that remained unchanged. However, there was a reduction in phosphorylated KCC2 at the membrane, suggesting an increase in KCC2 chloride export activity. Furthermore, hyperpolarized EGABA observed in WNK3-deficient neurons can be reversed by the KCC2 inhibitor, VU024055, thus indicating that WNK3 acts through KCC2 to influence EGABA . Notably, WNK3 knockdown resulted in morphological changes in mature but not immature neurons. Electrophysiological characterization of WNK3-deficient mature neurons revealed reduced capacitances but increased intrinsic excitability and synaptic excitation. Hence, our study demonstrates that WNK3 maintains the "adult" GABAergic inhibitory tone in neurons and plays a role in the morphological development of neurons and excitability.

Rare Functional Variants Associated with Antidepressant Remission in Mexican-Americans: Short title: Antidepressant remission and pharmacogenetics in Mexican-Americans.

INTRODUCTION: Rare genetic functional variants can contribute to 30-40% of functional variability in genes relevant to drug action. Therefore, we investigated the role of rare functional variants in antidepressant response. METHOD: Mexican-American individuals meeting the Diagnostic and Statistical Manual-IV criteria for major depressive disorder (MDD) participated in a prospective randomized, double-blind study with desipramine or fluoxetine. The rare variant analysis was performed using whole-exome genotyping data. Network and pathway analyses were carried out with the list of significant genes. RESULTS: The Kernel-Based Adaptive Cluster method identified functional rare variants in 35 genes significantly associated with treatment remission (False discovery rate, FDR <0.01). Pathway analysis of these genes supports the involvement of the following gene ontology processes: olfactory/sensory transduction, regulation of response to cytokine stimulus, and meiotic cell cycleprocess. LIMITATIONS: Our study did not have a placebo arm. We were not able to use antidepressant blood level as a covariate. Our study is based on a small sample size of only 65 Mexican-American individuals. Further studies using larger cohorts are warranted. CONCLUSION: Our data identified several rare functional variants in antidepressant drug response in MDD patients. These have the potential to serve as genetic markers for predicting drug response. TRIAL REGISTRATION: ClinicalTrials.gov NCT00265291.

Publisher Correction: Integrated analysis of a compendium of RNA-Seq datasets for splicing factors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrated analysis of a compendium of RNA-Seq datasets for splicing factors.

A vast amount of public RNA-sequencing datasets have been generated and used widely to study transcriptome mechanisms. These data offer precious opportunity for advancing biological research in transcriptome studies such as alternative splicing. We report the first large-scale integrated analysis of RNA-Seq data of splicing factors for systematically identifying key factors in diseases and biological processes. We analyzed 1,321 RNA-Seq libraries of various mouse tissues and cell lines, comprising more than 6.6 TB sequences from 75 independent studies that experimentally manipulated 56 splicing factors. Using these data, RNA splicing signatures and gene expression signatures were computed, and signature comparison analysis identified a list of key splicing factors in Rett syndrome and cold-induced thermogenesis. We show that cold-induced RNA-binding proteins rescue the neurite outgrowth defects in Rett syndrome using neuronal morphology analysis, and we also reveal that SRSF1 and PTBP1 are required for energy expenditure in adipocytes using metabolic flux analysis. Our study provides an integrated analysis for identifying key factors in diseases and biological processes and highlights the importance of public data resources for identifying hypotheses for experimental testing.

MeCP2 Dysfunction in Rett Syndrome and Neuropsychiatric Disorders.

Elucidating the functions of a particular gene is paramount to the understanding of how its dysfunction contributes to disease. This is especially important when the gene is implicated in multiple different disorders. One such gene is methyl-CpG-binding protein 2 (MECP2), which has been most prominently associated with the neurodevelopmental disorder Rett syndrome, as well as major neuropsychiatric disorders such as autism and schizophrenia. Being initially identified as a transcriptional regulator that modulates gene expression and subsequently also shown to be involved in other molecular events, dysfunction of the MeCP2 protein has the potential to affect many cellular processes. In this chapter, we will briefly review the functions of the MeCP2 protein and how its mutations are implicated in Rett syndrome and other neuropsychiatric disorders. We will further discuss about the mouse models that have been generated to specifically dissect the function of MeCP2 in different cell types and brain regions. It is envisioned that such thorough and targeted examination of MeCP2 functions can aid in enlightening the role that it plays in normal and dysfunctional physiological systems.

Behavioral Characterization of MeCP2 Dysfunction-Associated Rett Syndrome and Neuropsychiatric Disorders.

The methyl-CpG-binding protein 2 (MECP2) gene has been implicated in multiple neuropsychiatric disorders such as autism and schizophrenia and, most notably, Rett syndrome (RTT). Mouse models of MeCP2 dysfunction that have been developed are thus important not only for examining the protein's contribution to RTT, but also for elucidating the etiologies of other MECP2-associated neuropsychiatric disorders. In this chapter, we present protocols for three behavioral assays for characterizing major functional domains of MeCP2 dysfunction-the open field test for measuring general locomotor activity and anxiety-like behavior, the three-chambered Crawley box test for assessing social preference and social novelty, and the rotarod assay for testing locomotor coordination. It is hoped that these information facilitate systematic characterization of mouse models that may aid in elucidating the role of MeCP2 in neurological disorders, as well as assessing the effects of putative mechanistic and therapeutic interventions.

Choline Rescues Behavioural Deficits in a Mouse Model of Rett Syndrome by Modulating Neuronal Plasticity.

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls, with 95% of RTT cases resulting from mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Choline, a dietary micronutrient found in most foods, has been shown to be important for brain development and function. However, the exact effects and mechanisms are still unknown. We found that 13 mg/day (1.7 × required daily intake) of postnatal choline treatment to Mecp2-conditional knockout mice rescued not only deficits in motor coordination, but also their anxiety-like behaviour and reduced social preference. Cortical neurons in the brains of Mecp2-conditional knockout mice supplemented with choline showed enhanced neuronal morphology and increased density of dendritic spines. Modelling RTT in vitro by knocking down the expression of the MeCP2 protein with shRNA, we found that choline supplementation to MeCP2-knockdown neurons increased their soma sizes and the complexity of their dendritic arbors. Rescue of the morphological defects could lead to enhanced neurotransmission, as suggested by an observed trend of increased expression of synaptic proteins and restored miniature excitatory postsynaptic current frequency in choline-supplemented MeCP2-knockdown neurons. Through the use of specific inhibitors targeting each of the known physiological pathways of choline, synthesis of phosphatidylcholine from choline was found to be essential in bringing about the changes seen in the choline-supplemented MeCP2-knockdown neurons. Taken together, these data reveal a role of choline in modulating neuronal plasticity, possibly leading to behavioural changes, and hence, a potential for using choline to treat RTT.

Choline Ameliorates Disease Phenotypes in Human iPSC Models of Rett Syndrome.

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2 (R306C) and MECP2 (1155Δ32)) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons, the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input, evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase, an important enzyme in cholinergic signaling, and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models, thereby illustrating the potential for using choline as a nutraceutical to treat RTT.

An optogenetic approach for assessing formation of neuronal connections in a co-culture system.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.