Ramesh Menon

Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32(+) medium-sized spiny neurons

Medium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington’s disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGFβ inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1+/GSX2+ telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1+/FOXP2+/CTIP2+/calbindin+/DARPP-32+ MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32+ neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.

iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF

The possibility of generating neural stem/precursor cells (NPCs) from induced pluripotent stem cells (iPSCs) has opened a new avenue of research that might nurture bench-to-bedside translation of cell transplantation protocols in central nervous system myelin disorders. Here we show that mouse iPSC-derived NPCs (miPSC-NPCs)-when intrathecally transplanted after disease onset-ameliorate clinical and pathological features of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Transplanted miPSC-NPCs exert the neuroprotective effect not through cell replacement, but through the secretion of leukaemia inhibitory factor that promotes survival, differentiation and the remyelination capacity of both endogenous oligodendrocyte precursors and mature oligodendrocytes. The early preservation of tissue integrity limits blood-brain barrier damage and central nervous system infiltration of blood-borne encephalitogenic leukocytes, ultimately responsible for demyelination and axonal damage. While proposing a novel mechanism of action, our results further expand the therapeutic potential of NPCs derived from iPSCs in myelin disorders.

MiR-30e and miR-181d control Radial Glia cell proliferation via HtrA1 modulation

The precise mechanisms by which microRNAs (miRNAs) contribute to the dynamic regulation of gene expression during the forebrain development are still partly elusive. Here we show that the depletion of miRNAs in the cerebral cortex and hippocampus, via genetic inactivation of Dicer after the onset of forebrain neurogenesis, profoundly impairs the morphological and proliferative characteristics of neural stem and progenitor cells. The cytoarchitecture and self-renewal potential of radial glial (RG) cells located within the cerebral cortex and the hippocampus were profoundly altered, thus causing a significant derangement of both the post natal dorsal sub-ventricular zone and the dentate gyrus. This effect was attributed to the High-temperature requirement A serine peptidase 1 (HtrA1) gene product whose overexpression in the developing forebrain recapitulated some of the aspects of the Dicer−/− phenotype. MiR-30e and miR-181d were identified as posttranscriptional negative regulators of HtrA1 by binding to its 3′ untranslated region. In vivo overexpression of miR-30e and miR-181d in Dicer−/− forebrain rescued RG proliferation defects.

Gender-based blood transcriptomes and interactomes in multiple sclerosis: involvement of SP1 dependent gene transcription.

In this study we investigated the contribution of gender to global gene expression in peripheral blood mononuclear cells from multiple sclerosis (MS) patients and healthy controls. We observed that, in contrast to the conventional approach, gender-based case-control comparisons resulted in genelists with significantly reduced heterogeneity in human populations. In addition, MS was characterized by significant changes both in the quantity and in the quality of the sex-specific genes. Application of stringent statistics defined gender-based signatures which classified a second independent MS population with high precision. The global unsupervised cluster analyses for 60 subjects showed that 29/31 female and 27/29 male samples were properly identified. Notably, MS was associated in women and in men with distinct gene signatures which however shared several molecular functions, biological processes and interactors. Issues regarding epigenetic control of gene expression appeared as the main common theme for disease, with a central role for the functional modules related to histone deacetylase, NF-kappaB and androgen receptor signaling. Moreover, in silico analyses predicted that the differential expression in MS women and men were depending on the transcription factor SP1. Specific targeting of this pathway by the bis-anthracycline WP631 impaired T cell responses in vitro and in vivo, and reduced the incidence and the severity of experimental autoimmune encephalomyelitis, indicating that SP1 dependent gene transcription sustains neuroinflammation. Thus, the gender-based approach with its reduced heterogeneity and the systems biology tools with the identification of the molecular and functional networks successfully uncovered the differences but also the commonalities associated to multiple sclerosis in women and men. In conclusion, we propose gender-based systems biology as a novel tool to gain fundamental information on disease-associated functional processes.

Molecular and functional definition of the developing human striatum

The complexity of the human brain derives from the intricate interplay of molecular instructions during development. Here we systematically investigated gene expression changes in the prenatal human striatum and cerebral cortex during development from post-conception weeks 2 to 20. We identified tissue-specific gene coexpression networks, differentially expressed genes and a minimal set of bimodal genes, including those encoding transcription factors, that distinguished striatal from neocortical identities. Unexpected differences from mouse striatal development were discovered. We monitored 36 determinants at the protein level, revealing regional domains of expression and their refinement, during striatal development. We electrophysiologically profiled human striatal neurons differentiated in vitro and determined their refined molecular and functional properties. These results provide a resource and opportunity to gain global understanding of how transcriptional and functional processes converge to specify human striatal and neocortical neurons during development.

Shared Molecular and Functional Frameworks among Five Complex Human Disorders: A Comparative Study on Interactomes Linked to Susceptibility Genes

Background Genome-wide association studies (gwas) are invaluable in revealing the common variants predisposing to complex human diseases. Yet, until now, the large volumes of data generated from such analyses have not been explored extensively enough to identify the molecular and functional framework hosting the susceptibility genes. Methodology/Principal Findings We investigated the relationships among five neurodegenerative and/or autoimmune complex human diseases (Parkinsons disease-Park, Alzheimers disease-Alz, multiple sclerosis-MS, rheumatoid arthritis-RA and Type 1 diabetes-T1D) by characterising the interactomes linked to their gwas-genes. An initial study on the MS interactome indicated that several genes predisposing to the other autoimmune or neurodegenerative disorders may come into contact with it, suggesting that susceptibility to distinct diseases may converge towards common molecular and biological networks. In order to test this hypothesis, we performed pathway enrichment analyses on each disease interactome independently. Several issues related to immune function and growth factor signalling pathways appeared in all autoimmune diseases, and, surprisingly, in Alzheimers disease. Furthermore, the paired analyses of disease interactomes revealed significant molecular and functional relatedness among autoimmune diseases, and, unexpectedly, between T1D and Alz. Conclusions/Significance The systems biology approach highlighted several known pathogenic processes, indicating that changes in these functions might be driven or sustained by the framework linked to genetic susceptibility. Moreover, the comparative analyses among the five genetic interactomes revealed unexpected genetic relationships, which await further biological validation. Overall, this study outlines the potential of systems biology to uncover links between genetics and pathogenesis of complex human disorders.

Activated macrophages release microvesicles containing polarized M1 or M2 mRNAs

MVs are known vehicles of horizontal communication among cells, currently under scrutiny as powerful biomarkers in several pathological processes. The potential advantage of MVs relies on the assumption that their content reflects processes ongoing in pathologically relevant cell types. We have described that MVs of myeloid origin in the CSF are a marker of microglia/macrophage activation. Myeloid cells have different activation types, resulting in diverse functional phenotypes. Knowledge on the activation type of myeloid cells during disease would be of paramount importance for the understanding of ongoing pathogenic processes. We show here that macrophages activated in vitro in different ways all release increased amounts of MVs compared with NS cells. Moreover, we show that macrophage‐derived MVs contain a repertoire of mRNAs that is not the result of casual sampling from the parental cells, as it is characterized by distinct mRNA enrichments and species. Nevertheless, mRNA content of MVs clearly allows identification in vivo of the activated phenotype of the cell of origin, indicating carryover of functional macrophage traits. We propose that detection of mRNAs in myeloid MVs permits identification of myeloid cell activation type during disease, allowing for further stratification of pathological processes.

Transcript profiling of different types of multiple sclerosis lesions yields FGF1 as a promoter of remyelination

Chronic demyelination is a pathological hallmark of multiple sclerosis (MS). Only a minority of MS lesions remyelinates completely. Enhancing remyelination is, therefore, a major aim of future MS therapies. Here we took a novel approach to identify factors that may inhibit or support endogenous remyelination in MS. We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays. In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter. Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core. In functional experiments, FGF1 accelerated developmental myelination in dissociated mixed cultures and promoted remyelination in slice cultures, whereas it decelerated differentiation of purified primary oligodendrocytes, suggesting that promotion of remyelination by FGF1 is based on an indirect mechanism. The analysis of human astrocyte responses to FGF1 by genome wide expression profiling showed that FGF1 induced the expression of the chemokine CXCL8 and leukemia inhibitory factor, two factors implicated in recruitment of oligodendrocytes and promotion of remyelination. Together, this study presents a transcript profiling of remyelinated MS lesions and identified FGF1 as a promoter of remyelination. Modulation of FGF family members might improve myelin repair in MS.

Human neurotrophin receptor p75NTR defines differentiation-oriented skeletal muscle precursor cells: implications for muscle regeneration.

Satellite cells are resident stem cells of adult skeletal muscle that have roles in tissue repair. Although several efforts have led to the functional characterization of distinct myogenic populations in animal models, the translation of these findings to humans has been limited. Here, we analyzed the expression and function of the neurotrophin receptor p75NTR in human skeletal muscle precursor cells. We combined histological investigations of muscle biopsies with molecular and cellular analyses of primary muscle precursor cells. p75NTR is expressed by most satellite cells in vivo and is a marker for regenerating fibers in inflamed and dystrophic muscle. p75NTR mRNA and protein are also detectable in primary myoblasts, and these levels increase transiently when cell differentiation is triggered. Transcriptome analyses of p75NTRhigh versus p75NTRlow muscle cells showed that p75NTR is the prototype marker for a precursor cell population that has a broad transcriptional repertoire associated with muscle development and maturation. Several in vitro experiments, including receptor blockade and gene silencing in myoblasts, proved that p75NTR specifically regulates myogenesis and dystrophin expression. Taken together, the results indicate that p75NTR is a novel marker of human differentiation-prone muscle precursor cells that is involved in myogenesis in vivo and in vitro.

Dysregulation of MS risk genes and pathways at distinct stages of disease

Objective: To perform systematic transcriptomic analysis of multiple sclerosis (MS) risk genes in peripheral blood mononuclear cells (PBMCs) of subjects with distinct MS stages and describe the pathways characterized by dysregulated gene expressions. Methods: We monitored gene expression levels in PBMCs from 3 independent cohorts for a total of 297 cases (including clinically isolated syndromes (CIS), relapsing-remitting MS, primary and secondary progressive MS) and 96 healthy controls by distinct microarray platforms and quantitative PCR. Differential expression and pathway analyses for distinct MS stages were defined and validated by literature mining. Results: Genes located in the vicinity of MS risk variants displayed altered expression in peripheral blood at distinct stages of MS compared with the healthy population. The frequency of dysregulation was significantly higher than expected in CIS and progressive forms of MS. Pathway analysis for each MS stage–specific gene list showed that dysregulated genes contributed to pathogenic processes with scientific evidence in MS. Conclusions: Systematic gene expression analysis in PBMCs highlighted selective dysregulation of MS susceptibility genes playing a role in novel and well-known pathogenic pathways.