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Hom ozygous variants in the peptidyl-tRNA hydrolase 2 gene (PTRH2) cause infantile-onset multisystem neurologic, endocrine, and pancreatic disease. The objective is to delineate the mechanisms underlying the core cerebellar phenotype in this disease. For this, we generated constitutive (Ptrh2LoxPxhCMVCre, Ptrh2−/− mice) and Purkinje cell (PC) specific (Ptrh2LoxPxPcp2Cre, Ptrh2ΔPCmice) Ptrh2 mutant mouse models and investigated the effect of the loss of Ptrh2 on cerebellar development. We show that Ptrh2−/− knockout mice had severe postnatal runting and lethality by postnatal day 14. Ptrh2ΔPC PC specific knockout mice survived until adult age; however, they showed progressive cerebellar atrophy and functional cerebellar deficits with abnormal gait and ataxia. PCs of Ptrh2ΔPC mice had reduced cell size and density, stunted dendrites, and lower levels of ribosomal protein S6, a readout of the mammalian target of rapamycin pathway. By adulthood, there was a marked loss of PCs. Thus, we identify a cell autonomous requirement for PTRH2 in PC maturation and survival. Loss of PTRH2 in PCs leads to downregulation of the mTOR pathway and PC atrophy. This suggests a molecular mechanism underlying the ataxia and cerebellar atrophy seen in patients with PTRH2 mutations leading to infantile-onset multisystem neurologic, endocrine, and pancreatic disease.

Cerebellar granule neuron progenitors (CGNPs) give rise to the cerebellar granule neurons in the developing cerebellum. Generation of large number of these neurons is made possible by the high proliferation rate of CGNPs in the external granule layer (EGL) in the dorsal cerebellum. Here, we show that upregulation of β-catenin can maintain murine CGNPs in a state of proliferation. Further, we show that β-catenin mRNA and protein levels can be regulated by the mitogen Sonic hedgehog (Shh). Shh signaling led to an increase in the level of the transcription factor N-myc. N-myc was found to bind the β-catenin promoter, and the increase in β-catenin mRNA and protein levels could be prevented by blocking N-myc upregulation downstream of Shh signaling. Furthermore, blocking Wingless-type MMTV integration site (Wnt) signaling by Wnt signaling pathway inhibitor Dickkopf 1 (Dkk-1) in the presence of Shh did not prevent the upregulation of β-catenin. We propose that in culture, Shh signaling regulates β-catenin expression through N-myc and results in increased CGNP proliferation.

Objectives In the premature newborn, perinatal inflammation mediated by microglia contributes significantly to neurodevelopmental injuries including white matter injury (WMI). Brain inflammation alters development through neuroinflammatory processes mediated by activation of homeostatic microglia toward a pro-inflammatory and neurotoxic phenotype. Investigating immune regulators of microglial activation is crucial to find effective strategies to prevent and treat WMI. Methods Ex vivo microglial cultures and a mouse model of WMI induced by perinatal inflammation (interleukin-1-beta, IL-1β; postnatal day 1-5) were used to uncover and elucidate the role of microRNA-146b-5p in microglial activation and WMI. Results A specific reduction in vivo in microglia of Dicer, a protein required for microRNAs maturation, reduces pro-inflammatory activation of microglia and prevents hypomyelination in our model of WMI. Microglial miRNome analysis in the WMI model identified miRNA-146b-5p as a candidate modulator of microglial activation. Ex vivo microglial cell culture treated with the pro-inflammatory stimulus lipopolysaccharide (LPS) led to overexpression of immunomodulatory miRNA-146b-5p but its drastic reduction in the microglial extracellular vesicles (EVs). To increase miR-146b-5p expression, we employed a 3DNA nanocarrier to deliver synthetic miR-146b-5p specifically to microglia. Enhancing microglial miR-146b-5p overexpression significantly decreased LPS-induced activation, down-regulated IRAK1 and restored miRNA-146b-5p levels in EVs. In our WMI model, 3DNA miR-146b-5p treatment significantly prevented microglial activation, hypomyelination and cognitive defect induced by perinatal inflammation. Interpretations These findings support that miR-146b-5p is a major regulator of microglia phenotype and could be targeted to reduce the incidence and the severity of perinatal brain injuries and their long-term consequences. This article is protected by copyright. All rights reserved.

Groundwater is the main source of drinking water for a significant portion of the human population. In many agricultural areas, diffuse pollution such as high levels of total dissolved salts including nitrate, puts the quality of this resource at risk. However, the effect of exposure to these water contaminants on brain development is currently poorly understood. Here we characterised water from a borewell located in an intensely cultivated area (agricultural) or water from a borewell located in a nearby pristine forest. The agricultural borewell water was rich in nitrates with high total dissolved salts. We then studied the consequence of drinking the agricultural water on mouse brain development. For this, the agricultural borewell water or forest water was given to mice for 6 weeks before and during pregnancy and lactation. The brains of the offspring born to these dams were analysed at postnatal day (P)5 and P21 and compared using immunohistochemistry for changes in glial cells, neurons, myelin, and cell death across many brain regions. Brains from offspring born to dams who had been given agricultural water (versus forest control water) were significantly smaller, and at P21 had a significant degeneration of neurons and increased numbers of microglia in the motor cortex, had fewer white matter astrocytes and an increase in cell death, particularly in the dentate gyrus. This study shows that brain development is sensitive to water composition. It points to the importance of assessing neurodevelopmental delays when considering the effect of water contaminated with agricultural run offs on human health. MAIN FINDING: Pregnant and lactating mice were given borewell water from intensely cultivated land. Offspring brains reveal degeneration of neurons and a loss of astrocytes, increase in microglial cells and cell death, pointing to neurodevelopmental problems.

Globally, more than 3.5 billion people are at risk of dengue infection and an estimated 390 million cases are detected annually. The pandemic has brought with it the simultaneous circulation of multiple serotypes and in its aftermath, endemic dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Nearly 3 million children were hospitalized with this syndrome in the past 3 decades, mainly in South-East Asia. Recent outbreaks in the Pacific Islands, China, India, Sri Lanka, Cuba and Venezuela indicate that the high intensity and rapid spread of dengue transmission which is closely connected with urbanization factors, increase with temperature and precipitation and changes in spatial patterns.Since the mid-1990s, India has been experiencing frequent dengue epidemics. The overall cost of dengue in India in 2016 was about US$5.71 billion. Over the years, dengue has transitioned into an urban pandemic, largely as an epidemic starting in urban centres and gradually expanding to surrounding peri-urban and rural areas. Urban areas are characterized by relatively higher temperature (Urban Heat Island), and higher concentration of population, and are more favorable for the transmission of Aedes aegypti mosquito. The vector thrives in areas with standing water, including puddles, water tanks, containers and old tires. Lack of reliable sanitation and regular garbage collection and water storage practices contribute to the breeding and spread of the mosquitoes.Delhi is hyperendemic with a frequent occurrence of concurrent infections with multiple DENV serotypes. The first severe outbreak took place in the state during 1996 with 10253 cases and 253 deaths. Since then, outbreaks in the city have been reported during 2006, 2010, 2013 and 2015, indicating the decreasing gap between the outbreaks. Dengue cases in the city show an increasing trend in the past decade with 2015, the most recent incidence of outbreak reporting 15857 cases and 60 deaths as reported under the National Vector Borne Disease Surveillance Programme (NVBDSP). A lag period of two months has been observed between the peaks of rainfall and dengue cases in Delhi indicating a lack of sufficient and timely preventive and public health efforts by the local health administration alongside other systemic challenges to be responsible for the outbreaks. However, since 2015, the reported dengue-related cases and deaths are showing steady decline with 80% reduction in reported cases.The municipality and other urban local bodies are taking several measures for dengue control and prevention. Several fever clinics have been opened up in the government hospitals, dispensaries and mohalla (community) clinics. The private hospitals have also been permitted to increase the bed strength by 20% during the season only for fever patients. To coordinate the efforts of the Delhi government and the civic bodies, a dengue control cell is also created. In addition, an intensive anti dengue campaign has been launched to increase awareness and public participation is contributing to improved disease control. Several studies have emphasized on the need for epidemiological and entomological surveillance to monitor trends in dengue distribution, seasonal patterns and circulating serotypes to guide dengue control activities with efforts currently being made to ensure early warning signals for timely detection of outbreaks.

Zika virus (ZV) infects neural stem cells (NSCs) and causes quiescence in NSCs, reducing the pool of brain cells, leading to microcephaly. Despite conscientious efforts, the molecular mechanisms for ZV-mediated effects on NSCs lack clarity. This study aimed to explore the underlying mechanisms for ZV-mediated induction of quiescence in the primary cultures of human fetal neural stem cells (fNSCs). We demonstrate that expression of ZV envelope (E) protein displays maximum quiescence in human fNSCs by accumulating cells in the G0/G1 phase of the cell cycle as compared to other non-structural proteins, viz. NS2A, NS4A and NS4B. E protein induces immature differentiation by induction of pro-neuronal genes in proliferating fNSCs, induces apoptosis in differentiating fNSCs 3 days post differentiation, and disrupts migration of cells from differentiating neurospheres. In utero electroporation of mouse brain with E protein shows drastic downregulation of proliferating cells in ventricular and subventricular zone regions. Global microRNA sequencing suggests that E protein modulates miRNA circuitry. Among differentially expressed miRNAs, we found 14 upregulated and 11 downregulated miRNAs. Mir-204-3p and mir-1273g-3p directly regulate NOTCH2 and PAX3 expression, respectively, by binding to their 3'UTR. Bioinformatic analysis using GO analysis for the targets of differentially expressed miRNAs revealed enrichment of cell cycle and developmental processes. Furthermore, WNT, CCKR, PDGF, EGF, p53, and NOTCH signaling pathways were among the top enriched pathways. Thus, our study provides evidence for the involvement of ZV E protein and novel insights into the molecular mechanism through identification of miRNA circuitry. Art work depicting the effect of Zika virus E protein on human fetal neural stem cells.

A distinctive feature of neocortical development is the highly coordinated production of different progenitor cell subtypes, which are critical for ensuring adequate neurogenic outcome and the development of normal neocortical size. To further understand the mechanisms that underlie neocortical growth, we focused our studies on the microcephaly gene Mcph1, and we report here that Mcph1 (1) exerts its functions in rapidly dividing apical radial glial cells (aRGCs) during mouse neocortical development stages that precede indirect neurogenesis; (2) is expressed at mitochondria; and (3) controls the proper proliferation and survival of RGCs, potentially through crosstalk with cellular metabolic pathways involving the stimulation of mitochondrial activity via VDAC1/GRP75 and AKT/HK2/VDAC1 and glutaminolysis via ATF4/PCK2. We currently report the description of a MCPH-gene implication in the interplay between bioenergetic pathways and neocortical growth, thus pointing to alterations of cellular metabolic pathways, in particular glutaminolysis, as a possible cause of microcephalic pathogenesis.

Genetic anomalies have a role in autism spectrum disorders (ASD). Each genetic factor is responsible for a small fraction of cases. Environment factors, like preterm delivery, have an important role in ASD. Preterm infants have a 10-fold higher risk of developing ASD. Preterm birth is often associated with maternal/fetal inflammation, leading to a fetal/neonatal inflammatory syndrome. There are demonstrated experimental links between fetal inflammation and the later development of behavioral symptoms consistent with ASD. Preterm infants have deficits in connectivity. Most ASD genes encode synaptic proteins, suggesting that ASD are connectivity pathologies. Microglia are essential for normal synaptogenesis. Microglia are diverted from homeostatic functions towards inflammatory phenotypes during perinatal inflammation, impairing synaptogenesis. Preterm infants with ASD have a different phenotype from term born peers. Our original hypothesis is that exposure to inflammation in preterm infants, combined with at risk genetic background, deregulates brain development leading to ASD.

An inherent asymmetry exists between the two centrosomes of a dividing cell. One centrosome is structurally more mature (mother centrosome) than the other (daughter centrosome). Post division, one daughter cell inherits the mother centrosome while the other daughter cell inherits the daughter centrosome. Remarkably, the kind of centrosome inherited is associated with cell fate in several developmental contexts such as in radial glial progenitors in the developing mouse cortex, Drosophila neuroblast divisions and in Drosophila male germline stem cells. However, the role of centrosome inheritance in granule neuron progenitors in the developing cerebellum has not been investigated. Here, we show that mother and daughter centrosomes do exist in these progenitors, and the amount of pericentriolar material (PCM) each centrosome possesses is different. However, we failed to observe any correlation between the fate adopted by the daughter cell and the nature of centrosome it inherited.

Abstract Cell division and differentiation are two fundamental physiological processes that need to be tightly balanced to achieve harmonious development of an organ or a tissue without jeopardizing its homeostasis. The role played by the centriolar protein STIL is highly illustrative of this balance at different stages of life as deregulation of the human STIL gene expression has been associated with either insufficient brain development (primary microcephaly) or cancer, two conditions resulting from perturbations in cell cycle and chromosomal segregation. This review describes the recent advances on STIL functions in the control of centriole duplication and mitotic spindle integrity, and discusses how pathological perturbations of its finely tuned expression result in chromosomal instability in both embryonic and postnatal situations, highlighting the concept that common key factors are involved in developmental steps and tissue homeostasis.

"The goal of this study was to examine the effect of maternal iron deficiency on the developing hippocampus in order to define a developmental window for this effect, and to see whether iron deficiency causes changes in glucocorticoid levels. The study was carried out using pre-natal, post-natal, and pre+post-natal iron deficiency paradigm. Iron deficient pregnant dams and their pups displayed elevated corticosterone which, in turn, differentially affected glucocorticoid receptor (GR) expression in the CA1 and the dentate gyrus. Brain Derived Neurotrophic Factor (BDNF) was reduced in the hippocampi of pups following elevated corticosterone levels. Reduced neurogenesis at P7 was seen in pups born to iron deficient mothers, and these pups had reduced numbers of hippocampal pyramidal and granule cells as adults. Hippocampal subdivision volumes also were altered. The structural and molecular defects in the pups were correlated with radial arm maze performance; reference memory function was especially affected. Pups from dams that were iron deficient throughout pregnancy and lactation displayed the complete spectrum of defects, while pups from dams that were iron deficient only during pregnancy or during lactation displayed subsets of defects. These findings show that maternal iron deficiency is associated with altered levels of corticosterone and GR expression, and with spatial memory deficits in their pups."

Maternal malnutrition affects every aspect of fetal development. The present study asked the question whether a low-protein diet of the mother could result in motor deficits in the offspring. Further, to examine whether cerebellar pathology was correlated with motor deficits, several parameters of the postnatal development of the cerebellum were assayed. This is especially important because the development of the cerebellum is unique in that the time scale of development is protracted compared with that of the cortex or hippocampus. The most important result of the study is that animals born to protein-deficient mothers showed significant delays in motor development as assessed by rotarod and gait analysis. These animals also showed reduced cell proliferation and reduced thickness in the external granular layer. There was a reduction in the number of calbindin-positive Purkinje cells (PC) and granular cells in the internal granular layer. However, glial fibrillary acidic protein-positive population including Bergmann glia remained unaffected. We therefore conclude that the development of the granular cell layer and the PC is specifically prone to the effects of protein malnutrition potentially due to their protracted developmental period from approximately embryonic day 11 to 13 until about the third postnatal week.

Cyclin dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2) has gained attention in the last years following the discovery, in 2005, that recessive mutations cause primary autosomal recessive microcephaly. This disease is seen as an isolated developmental defect of the brain, particularly of the cerebral cortex, and was thus historically also referred to as microcephalia vera. Unraveling the pathomechanisms leading to this human disease is fascinating scientists because it can convey insight into basic mechanisms of physiologic brain development (particularly of cortex formation). It also finds itself in the spotlight because of its implication in trends in mammalian evolution with a massive increase in the size of the cerebral cortex in primates. Here, we provide a timely overview of the current knowledge on the function of CDK5RAP2 and mechanisms that might lead to disease in humans when the function of this protein is disturbed.

Successful axon targeting during development is critically dependent on directionality of axon extension and requires coordination between the extrinsic cues that provide spatial information to the axon and the intrinsic responses that regulate structural specification of the axon during neuronal polarization. How these responses are coordinated is unclear but are known to involve aligning the centrosome with the base of the emerging axon. We have used a novelin vitromicropatterning assay that spatially segregates the extrinsic cues used by polarizing cerebellar granule cells to orient axon extension and used it to investigate the signaling mechanisms responsible for coordinating centrosome positioning with intrinsic responses. The results show that, when laminin and/or vitronectin are used as spatially restricted cues in association with substrate-associated sonic hedgehog, they are sufficient to induce cell cycle arrest, that laminin and vitronectin then induce integrin-mediated signaling that upregulates phosphoinositide-3 kinase and PKC function to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3) that is associated with the centrosome, that this PIP3 can interact with PKC-phosphorylated growth-associated protein GAP-43, and that PKC-phosphorylated GAP-43 in turn is required for positioning Par6, Cdc42, and IQGAP1, all intrinsic response components, in proximity to the centrosome, such that, in the absence of GAP-43, they are mislocalized and microtubules are not oriented appropriately. We conclude from these results that GAP-43 plays an important role in coordinating extrinsic signaling and intrinsic responses in polarizing cerebellar granule neurons.

Brain lesions induced in newborn mice by the glutamatergic agonists ibotenate (acting on NMDA and metabotropic receptors) or S-willardiine (acting on AMPA-kainate receptors) mimic some aspects of periventricular white matter lesions and neocortical grey matter damage observed in human neonates at risk for developing cerebral palsy. The neonatal mouse brain can be sensitized to excitotoxic damage by IL-1beta exposure similar to that observed in the human situation. Positive modulators of AMPA receptors have received increasing attention as potential neuroprotective agents in a number of neurodegenerative disorders of the adult. However whether they can also act as a neuroprotectant in neonatal brain damage has yet to be defined. Therefore the present study uses a well-defined rodent model of neonatal excitotoxic brain lesions to assess the neuroprotective effects of S18986, a positive allosteric modulator of AMPA receptors, as well as its mechanisms of action. In this model, S18986 provided a dose-dependent and long-lasting protection of developing white matter and cortical grey matter against an excitotoxic insult and also when this was combined with a sensitizing inflammatory insult. Neuroprotective effects of S18986 in cortical grey matter involved decreased necrotic and apoptotic cell death. S18986-induced neuroprotection against NMDA receptor-mediated brain lesions was blocked by inhibitors of ERK and PI3 kinase-Akt pathways. S18986 effects were abolished by a neutralizing anti-BDNF antibody and real time PCR confirmed the stimulation by S18986 of BDNF production in the neonatal brain. The present study provides strong experimental support for the role of S18986 as a candidate molecule for therapy in cases of excitotoxic perinatal brain lesions and identifies BDNF as a key mediator of this S18986-mediated neuroprotection.

The ability to study the characteristics of serotonin release from human serotonergic neurons is valuable both in terms of understanding disease pathology and in trying to understand how drugs that affect the serotonergic system alter neurotransmitter release. There is, however, no good in vitro system to model human serotonergic neurons. Although human embryonic stem (hES) cells offer an attractive model system, the derivation of serotonergic neurons from these cells has remained at a low efficiency. To address this problem, Nestin positive precursors from HUES7 hES cell line were first generated. These Nestin positive cells when terminally differentiated gave rise to 20% MAP-2 positive neurons. A high percentage (40%) of these neurons could be converted to serotonergic neurons. These serotonergic neurons expressed both serotonin and the neuron-specific tryptophan hydroxylase enzyme. In addition, they expressed several of the transcription factors that have been associated with serotonergic differentiation including Mash1 and Pet1. Finally, during the process of neuronal differentiation, the serotonin content, the localization of serotonin vesicles, and their ability to release serotonin following depolarization was characterized using a live cell serotonin imaging technique based on three-photon microscopy. Thus, for the first time, we demonstrate the feasibility of characterizing the development and function of human serotonergic neurons in vitro.

Positive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor modulators include benzamide compounds that allosterically modulate AMPA glutamate receptors. These small molecules that cross the blood-brain barrier have been shown to act as a neuroprotectant by increasing the levels of endogenous brain-derived neurotrophic factor (BDNF). Positive AMPA receptor modulators have also been shown to increase the levels of growth-associated protein-43 (GAP-43). GAP-43 plays a major role in many aspects of neuronal function in vertebrates. The goal of this study was to determine whether GAP-43 was important in mediating the actions of positive AMPA receptor modulator (S18986) and BDNF. Using cortical cultures from GAP-43 knockout and control mice, we show that (1) GAP-43 is upregulated in response to S18986 and BDNF in control cultures; (2) this upregulation of GAP-43 is essential for mediating the neuroprotective effects of S18986 and BDNF; (3) administration of S18986 and BDNF leads to an increase in the expression of the glutamate transporters GLT-1 and GLAST that are key to limiting excitotoxic cell death and this increase in GLT-1 and GLAST expression is completely blocked in the absence of GAP-43. Taken together this study concludes that GAP-43 is an important mediator of the neurotrophic effects of S18986 and BDNF on neuronal survival and plasticity, and is essential for the success of positive AMPA receptor modulator-BDNF-based neurotrophin therapy.

The management of biomedical waste is a crucial issue in health and environmental management. Rules in India were promulgated in 1998, originally with a deadline of December 2000 and extended to December 2002; however, the actual situation remains far from satisfactory. A study conducted in 2001 by CEE, New Delhi; indicated an implementation deficit. To gauge the present situation, a survey was undertaken during 2005-2006. A systematic analysis of current biomedical waste management practices in smaller nursing homes and hospitals in Delhi was carried out. A total of 53 nursing homes, with bed strengths ranging from 20 to over 200, were included. The survey results show that there is a marked improvement in the segregation practices of biomedical waste in small private hospitals and nursing homes. The majority of nursing homes and hospitals were found to be using a service provider for the collection, management, and disposal of healthcare wastes. Data was collected through a questionnaire and field visits. This paper discusses the relevant data indicative of current practices of healthcare waste management in the nursing homes and small healthcare facilities in Delhi.

In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.

The capacity of pluripotent embryonic stem cells (ES cells) to proliferate and differentiate makes them promising tools in the field of cell therapy. In spite of the controversy surrounding the numerous ethical questions raised by this technology, it has been shown to have therapeutic potential for heart, lung, liver, bone and connective tissue regeneration. In addition, a very attractive aspect of this technology is its potential for the treatment of cerebral pathology. A number of studies using ES cell transplants report the differentiation of ES cells in the brain or spinal cord of rodents, and the improvement of locomotor and/or cognitive deficits caused by brain injury. This review offers a synthesis of recent advances in the field of both human and rodent stem cell manipulation to select populations of neurons, astrocytes and oligodendrocytes. In parallel, this review emphasizes the striking similarities that exist between genetically programmed embryonic development of the nervous system and the differentiation of ES cells in vitro.

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

Objective Transplanting exogenous neuronal progenitors to replace damaged neurons in the adult brain following injury or neurodegenerative disorders and achieve functional amelioration is a realistic goal. However, studies so far have rarely taken into consideration the preexisting inflammation triggered by the disease process that could hamper the effectiveness of transplanted cells. Here, we examined the fate and long-term consequences of human cerebellar granule neuron precursors (GNP) transplanted into the cerebellum of Harlequin mice, an adult model of progressive cerebellar degeneration with early-onset microgliosis. Methods Human embryonic stem cell-derived progenitors expressing Atoh1, a transcription factor key to GNP specification, were generated in vitro and stereotaxically transplanted into the cerebellum of preataxic Harlequin mice. The histological and functional impact of these transplants was followed using immunolabeling and Rotarod analysis. Results Although transplanted GNPs did not survive beyond a few weeks, they triggered the proliferation of endogenous nestin-positive precursors in the leptomeninges that crossed the molecular layer and differentiated into mature neurons. These phenomena were accompanied by the preservation of the granule and Purkinje cell layers and delayed ataxic changes. In vitro neurosphere generation confirmed the enhanced neurogenic potential of the cerebellar leptomeninges of Harlequin mice transplanted with exogenous GNPs. Interpretation The cerebellar leptomeninges of adult mice contain an endogenous neurogenic niche that can be stimulated to yield mature neurons from an as-yet unidentified population of progenitors. The transplantation of human GNPs not only stimulates this neurogenesis, but, despite the potentially hostile environment, leads to neuroprotection and functional amelioration.

GAP-43 is expressed in proliferating neuroblasts in vivo and in vitro, but its role during early neurogenesis has not been investigated. Here we show that neuroectodermal differentiation stimulated by retinoic acid (RA) in the embryonal carcinoma (EC) line P19 is accompanied by upregulation of GAP-43 expression in neuroepithelial precursor cells. In contrast, when upregulation of GAP-43 expression was prevented in 3 independent P19 lines because of a targeted insertion into the gene, generation of neuroepithelial precursors was inhibited. Consequently, neuronal number was significantly decreased, neuronal morphology was abnormal and fewer than 20% of all neurons were able to initiate neuritogenesis. Extracellular matrix (ECM) was unable to rescue initiation of neuritogenesis in the mutant cells, however those neurites that were extended responded normally to ECM-stimulated neurite outgrowth-promoting signals. These data suggest that GAP-43 function is required for commitment to a neuronal phenotype as well as initiation of neurite extension. However, stimulation of neurite outgrowth by ECM in P19s occurs independently of GAP-43.

Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed “T-shaped” axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

There is increasing agreement that greenhouse gas emissions generated by human activity are responsible for global climate change (International Panel on Climate Change, 2007). There has been an observed 0.5°C rise in average global temperatures since the mid-1970s, a change that the international panel attributes partly to these anthropogenic emissions. It is also believed that these changes will have, in many parts of the world, adverse health effects ranging from heat-stroke and injury from extreme weather events to hunger and infectious diseases.

International audience; Autosomal recessive primary microcephaly (MCPH) is a genetic disorder that causes a reduction of cortical outgrowth without severe interference with cortical patterning. It is associated with mutations in a number of genes encoding protein involved in mitotic spindle formation and centrosomal activities or cell cycle control. We have shown previously that blocking vasoactive intestinal peptide (VIP) during gestation in mice by using a VIP antagonist (VA) results in microcephaly. Here, we have shown that the cortical abnormalities caused by prenatal VA administration mimic the phenotype described in MCPH patients and that VIP blockade during neurogenesis specifically disrupts Mcph1 signaling. VA administration reduced neuroepithelial progenitor proliferation by increasing cell cycle length and promoting cell cycle exit and premature neuronal differentiation. Quantitative RT-PCR and Western blot showed that VA downregulated Mcph1. Inhibition of Mcph1 expression led to downregulation of Chk1 and reduction of Chk1 kinase activity. The inhibition of Mcph1 and Chk1 affected the expression of a specific subset of cell cycle–controlling genes and turned off neural stem cell proliferation in neurospheres. Furthermore, in vitro silencing of either Mcph1 or Chk1 in neurospheres mimicked VA-induced inhibition of cell proliferation. These results demonstrate that VIP blockade induces microcephaly through Mcph1 signaling and suggest that VIP/Mcph1/Chk1 signaling is key for normal cortical development.

The regulation of cell proliferation in the external granular layer (EGL) of the developing cerebellum is important for its normal patterning. An important signal that regulates EGL cell proliferation is Sonic hedgehog (Shh). Shh is secreted by the Purkinje cells (PC) and has a mitogenic effect on the granule cell precursors of the EGL. Deregulation of Shh signaling has been associated with abnormal development, and been implicated in medulloblastomas, which are tumors that arise from the cerebellum. Given the importance of the Shh pathway in cerebellum development and disease, there has been no systematic study of its expression pattern during human cerebellum development. In this study, we describe the expression pattern of Shh, its receptor patched, smoothened, and its effectors that belong to the Gli family of transcription factors, during normal human cerebellum development from 10 weeks of gestational age, and in medulloblastomas that represents a case of abnormal cell proliferation in the cerebellum. This expression pattern is compared to equivalent stages in the normal development of cerebellum in mouse, as well as in tumors. Important differences between human and mouse that reflect differences in the normal developmental program between the 2 species are observed. First, in humans there appears to be a stage of Shh signaling within the EGL, when the PC are not yet the source of Shh. Second, unlike in the postnatal mouse cerebellum, expression of Shh in the PC in the postnatal human cerebellum is downregulated. Finally, medulloblastomas in the human but not in patched heterozygote mouse express Shh. These results highlight cross-species differences in the regulation of the Shh signaling pathway.

A rapid growth in human cerebellar development occurs in the third trimester, which is impeded by preterm delivery. The goal of this study was to characterize the impact of preterm delivery on the developmental program of the human cerebellum. Still born infants, which meant that all development up to that age had taken place in-utero, were age paired with preterm delivery infants, who had survived in an ex-utero environment, which meant that their development had also taken place outside the uterus. The two groups were assessed on quantitative measures that included molecular markers of granule neuron, purkinje neuron and bergmann glia differentiation, as well as the expression of the sonic hedgehog signaling pathway, that is important for cerebellar growth. We report that premature birth and development in an ex-utero environment leads to a significant decrease in the thickness and an increase in the packing density of the cells within the external granular layer and the inner granular layer well, as a reduction in the density of bergmann glial fibres. In addition, this also leads to a reduced expression of sonic hedgehog in the purkinje layer. We conclude that the developmental program of the cerebellum is specifically modified by events that follow preterm delivery.

Autosomal recessive primary microcephaly (MCPH), historically referred to as Microcephalia vera , is a genetically and clinically heterogeneous disease. Patients with MCPH typically exhibit congenital microcephaly as well as mental retardation, but usually no further neurological findings or malformations. Their microcephaly with grossly preserved macroscopic organization of the brain is a consequence of a reduced brain volume, which is evident particularly within the cerebral cortex and thus results to a large part from a reduction of grey matter. Some patients with MCPH further provide evidence of neuronal heterotopias, polymicrogyria or cortical dysplasia suggesting an associated neuronal migration defect. Genetic causes of MCPH subtypes 1–7 include mutations in genes encoding microcephalin, cyclin-dependent kinase 5 regulatory associated protein 2 (CDK5RAP2), abnormal spindle-like, microcephaly associated protein (ASPM), centromeric protein J (CENPJ), and SCL/TAL1-interrupting locus (STIL) as well as linkage to the two loci 19q13.1–13.2 and 15q15–q21. Here, we provide a timely overview of current knowledge on mechanisms leading to microcephaly in humans with MCPH and abnormalities in cell division/cell survival in corresponding animal models. Understanding the pathomechanisms leading to MCPH is of high importance not only for our understanding of physiologic brain development (particularly of cortex formation), but also for that of trends in mammalian evolution with a massive increase in size of the cerebral cortex in primates, of microcephalies of other etiologies including environmentally induced microcephalies, and of cancer formation.

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves.

Health is linked to amenities like clean drinking water, sewerage systems, poverty, literacy levels and infrastructure. Higher government spending on health care can result in lower incidence of diseases especially communicable ailments. According to ‘Healthcare in India’ published by the Foundation for Research in Community Health, among the leading causes of deaths in India, communicable diseases especially infectious, parasitic, gastrointestinal and respiratory diseases make up 43.3%. Tuberculosis (TB) accounts for a loss of approximately 11 million disability adjusted life years (DALYs). It remains one of the leading causes of disease and death in India in spite of the National TB program (NTP) being in place for over 30 years. Similarly, there are an estimated 4–5 million people infected with HIV in India today. Nearly one in every 150 adults in India is infected. Prevalence of HIV infection has been on the rise in practically all states and all population groups in India. HIV is also spreading rapidly in rural India as in urban areas. New health problems such as drug resistant forms of several communicable diseases have emerged, including malaria, filariasis and multi-drug resistant TB. Hepatitis B and C are other major health problems with an estimated 350 million carriers in the world today. Of these 43 million are in India and 200,000 people die every year due to these deadly diseases. Many of the communicable diseases are spread through the environment, through particulate matter or aerosols in the air, through water and food. They are also transmitted through untreated waste – biomedical and others, which often act as carriers or vehicles for microorganisms and cause injury to the people handling these wastes thus creating a portal of entry for these pathogens. The environment is also the reservoir for many unwanted chemicals, which are genotoxins and these

Growth-associated protein 43 (GAP-43) is required for development of a functional cerebral cortex in vertebrates; however, its role in cerebellar development is not well understood. Recently, we showed that absence of GAP-43 caused defects in proliferation, differentiation, and polarization of cerebellar granule cells. In this paper, we show that absence of GAP-43 causes defects in cerebellar patterning that reflect both cell-autonomous and non-autonomous functions. Cell-autonomous effects of GAP-43 impact precursor proliferation and axon targeting: In its absence, (1) proliferation of granule cell precursors in response to sonic hedgehog and fibroblast growth factor is inhibited, (2) proliferation of neuroepithelial precursors is inhibited, and (3) targeting of climbing fibers to the central lobe is disrupted. Cell non-autonomous effects of GAP-43 impact differentiated Purkinje cells in which GAP-43 has been downregulated: In its absence, both maturation and mediolateral patterning of Purkinje cells are inhibited. Both cell-autonomous and non-autonomous functions of GAP-43 involve its phosphorylation by protein kinase C. GAP-43 is phosphorylated in granule cell precursors in response to sonic hedgehog in vitro, and phosphorylated GAP-43 is also found in proliferating neuroepithelium and climbing fibers. Phosphorylated GAP-43 is specifically enriched in the presynaptic terminals of parallel and climbing fibers that innervate Purkinje cell bodies and dendrites. The cell-autonomous and non-autonomous effects of GAP-43 converge on the central lobe. The multiple effects of GAP-43 on cerebellar development suggest that it is a critical downstream transducer of signaling mechanisms that integrate generation of cerebellar structure with functional parcellation at the central lobe.

Several studies using animal models have suggested that the effects of nutritional insult on the developing brain are long-lasting and lead to permanent deficits in learning and behavior. Malnutrition can refer to the availability of all the nutrients but in insufficient quantities or it may imply that one or more of essential nutrients is either missing or is present, but in the wrong proportions in the diet. The hypothesis addressed in this study is that different domains of cognitive functioning can be affected by malnutrition and this can be related to the type of nutritional deficiency that the brain has been exposed to during development. To study the effect of nutritional deprivation during brain development, a paradigm of maternal malnutrition during the period of gestation and lactation was used and its effects were studied on the F1 offspring using Swiss albino mice. Three different types of malnutrition were used, that involve, caloric restriction, inadequate amount of protein in the diet and condition of low iron content. Our results show that the domain of spatial memory affected in the F1 generation depended on the kind of malnutrition that the mother was subjected to. Further our study shows that although hippocampal volume was reduced in all F1 pups, hippocampal subregions of the F1 animals were differentially vulnerable depending on type of malnutrition that the mother was subjected to. These results highlight the importance of qualifying the kind of malnutrition that is suffered by the mother during the period of gestation and lactation as it has consequences for the cognitive domain affected in the offspring. Awareness of this should inform prevention strategies in trying to reverse the effects of adverse maternal nutrition during critical periods in brain development.

In the central nervous system (CNS), generation of phenotypic diversity within the neuronal lineage is precisely regulated in a spatial and temporal fashion. Neural basic helix-loop-helix (bHLH) transcription factors are cell intrinsic factors that control commitment to neuronal lineage and play an important role in neuronal cell type specification. The ability to differentiate human embryonic stem (hES) cells into neurons provides a good model system to address human neuronal specification. Previous studies have shown neurogenin-2 (Ngn2) to be involved in the development of mesencephalic dopaminergic neurons. Toward the goal of correlating neuronal phenotype with early gene expression pattern, we have characterized the expression of Ngn2 during hES cell differentiation. Our results show that treatment of embryoid bodies (EBs) with retinoic acid (RA) leads to the greatest proportion of tyrosine hydroxylase (TH)-positive cells followed by vasoactive intestinal peptide (VIP)-treated EBs as compared to untreated EBs. This increase in the proportion of TH-positive neurons was correlated with the unique morphology of RA-treated aggregates and the spatial delocalization of the expression of Ngn2 within the EB. Neurospheres derived from RA-treated EBs contained many nestin-positive cells within regions that expressed Ngn2. We show that the extent of nestin-positive cells that arise from the region of Ngn2 expression is correlated with the appearance of TH-positive neurons. Our results show for the first time the expression of Ngn2 during the differentiation of hES cells.

Growth-associated protein-43 (GAP-43) is a major growth cone protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of homozygote GAP-43 (−/−) mice failed to form the anterior commissure (AC), hippocampal commissure (HC), and corpus callosum (CC)in vivo. Instead, although midline fusion was normal, selective fasciculation between commissural axons was inhibited, and TAG-1-labeled axons tangled bilaterally into Probst's bundles. Moreover, their growth cones had significantly smaller lamellas and reduced levels of F-actinin vitro. Likewise, 100% of GAP-43 (+/−) mice with one disrupted allele also showed defects in HC and CC, whereas the AC was unaffected. Individual GAP-43 (+/−) mice could be assigned to two groups based on the amount that PKC phosphorylation of GAP-43 was reduced in neocortical neurons. In mice with ∼1% phosphorylation, the HC and CC were absent, whereas in mice with ∼10% phosphorylation, the HC and CC were smaller. Both results suggest that PKC-mediated signaling in commissural axons may be defective. However, although Probst's bundles formed consistently at the location of the glial wedge, both GAP-43 (−/−) and GAP-43 (+/+) cortical axons were still repulsed by Slit-2in vitro, precluding failure of this deflective signal from the glial wedge as the source of the phenotype. Nonetheless, the data show that a functional threshold of GAP-43 is required for commissure formation and suggests that failure to regulate F-actin in commissural growth cones may be related to inhibited PKC phosphorylation of GAP-43.

GAP-43 is first expressed in proliferating neuroblasts and is required for maturation of neurons. When GAP-43 is not expressed in differentiating embryonal carcinoma P19 cells, reduced numbers of neurons were generated. Here we show that neuronal differentiation is initially disrupted at the onset of cell-cycle arrest in aggregated, proliferating neuronal precursors. The ratio of nestin:beta-tubulin-labeled progeny generated at this stage suggests that the differentiation is asymmetric. Apoptosis of immature neurons subsequently produced was also significantly induced. In vivo, too, proliferation of neuroblasts was significantly reduced in cortex of GAP-43(-/-) mice at E14.5. These data demonstrate that when GAP-43 is not expressed in proliferating neuroblasts, neural differentiation is not initiated appropriately, inducing apoptosis. Moreover, the concurrent inhibition of Ca2+-dependent adhesion between differentiating P19 cells in aggregates implicates GAP-43 in CAM-mediated signaling during neurogenesis, as has been previously shown in growth cones.

There is strong evidence that growth-associated protein (GAP-43), a protein found only in the nervous system, regulates the response of neurons to axonal guidance signals. However, its role in complex spatial patterning in cerebral cortex has not been explored. We show that mice lacking GAP-43 expression (−/−) fail to establish the ordered whisker representation (barrel array) normally found in layer IV of rodent primary somatosensory cortex. Thalamocortical afferents to −/− cortex form irregular patches in layer IV within a poorly defined cortical field, which varies between hemispheres, rather than the stereotypic, whisker-specific, segregated map seen in normal animals. Furthermore, many thalamocortical afferents project abnormally to widely separated cortical targets. Taken together, our findings indicate a loss of identifiable whisker territories in the GAP-43 −/− mouse cortex. Here, we present a disrupted somatotopic map phenotype in cortex, in clear contrast to the blurring of boundaries within an ordered whisker map in other barrelless mutants. Our results indicate that GAP-43 expression is critical for the normal establishment of ordered topography in barrel cortex.