Differentiation Of Granule Cells Research Articles

Similar toxicity potential of glyphosate and glyphosate-based herbicide on cerebellar development after maternal exposure in rats.

Studies on the effects of glyphosate (GlyP) and glyphosate-based herbicides (GBHs) on cerebellar development are extremely limited. This study examined the effects of maternal exposure to GlyP and GBH on rat cerebellar development in male offspring. From day 6 of gestation until day 21 postpartum at weaning, dams were given GlyP at 1.5% or 3.0% in diet or GBH at 1.0% in drinking water (corresponding to 0.36% GlyP). At weaning, GBH exposure was linked to increased numbers of DCX+ migrating granule cells in the cortex and TUNEL+ apoptotic cells in the internal granular layer (IGL), suggesting the disappearance of mismigrated granule cells via apoptosis. GBH also upregulated Nr4a3 and downregulated Cdk5 in the cerebellar vermis, suggesting a causal relation with the impaired granule cell development at this time. GlyP (3.0%) tended to increase in the number of DCX+ migrating granule cells in the IGL and upregulated Nr4a3 at weaning. Both compounds also upregulated genes related to granule cell migration (Astn1, Astn2, Nfia, and/or Nfix) at weaning and in adulthood, which might be an ameliorative response to delayed granule cell migration. Moreover, GBH induced Purkinje cell misalignment at weaning, which could be the result of delayed granule cell migration. In adulthood, GBH was associated with upregulation of the reelin signaling-related genes Reln, Dab1, and Efnb1, suggesting a compensatory response to Purkinje cell misalignment. GlyP induced the same gene expression changes. These results suggest that GBH reversibly disrupts cerebellar development, primarily by targeting granule cell migration and differentiation, whereas GlyP exhibited similar toxic potential as GBH.

Activation of TrkB signaling mitigates cerebellar anomalies caused by Rbm4-Bdnf deficiency

A molecular and functional link between neurotrophin signaling and cerebellar foliation is lacking. Here we show that constitutive knockout of two homologous genes encoding the RNA binding protein RBM4 results in foliation defects at cerebellar lobules VI–VII and delayed motor learning in mice. Moreover, the features of Rbm4 double knockout (dKO), including impaired differentiation of cerebellar granule cells and dendritic arborization of Purkinje cells, are reminiscent of neurotrophin deficiency. Loss of RBM4 indeed reduced brain-derived neurotrophic factor (BDNF). RBM4 promoted the expression of BDNF and full-length TrkB, implicating RBM4 in efficient BDNF-TrkB signaling. Finally, prenatal supplementation with 7,8-dihydroxyflavone, a TrkB agonist, restored granule cell differentiation, Purkinje cell dendritic complexity and foliation—the intercrural fissure in particular—in the neonatal cerebellum of Rbm4dKO mice, which also showed improved motor learning in adulthood. This study provides evidence that prenatal activation of TrkB signaling ameliorates cerebellar malformation caused by BDNF deficiency.

Reduced Cerebellar BDNF Availability Affects Postnatal Differentiation and Maturation of Granule Cells in a Mouse Model of Cholesterol Dyshomeostasis

Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder due to mutations in the NPC1 gene resulting in the accumulation of cholesterol within the endosomal/lysosomal compartments. The prominent feature of the disorder is the progressive Purkinje cell degeneration leading to ataxia.In a mouse model of NPC1 disease, we have previously demonstrated that impaired Sonic hedgehog signaling causes defective proliferation of granule cells (GCs) and abnormal cerebellar morphogenesis. Studies conducted on cortical and hippocampal neurons indicate a functional interaction between Sonic hedgehog and brain-derived neurotrophic factor (BDNF) expression, leading us to hypothesize that BDNF signaling may be altered in Npc1 mutant mice, contributing to the onset of cerebellar alterations present in NPC1 disease before the appearance of signs of ataxia.We characterized the expression/localization patterns of the BDNF and its receptor, tropomyosin-related kinase B (TrkB), in the early postnatal and young adult cerebellum of the Npc1nmf164 mutant mouse strain.In Npc1nmf164 mice, our results show (i) a reduced expression of cerebellar BDNF and pTrkB in the first 2 weeks postpartum, phases in which most GCs complete the proliferative/migrative program and begin differentiation; (ii) an altered subcellular localization of the pTrkB receptor in GCs, both in vivo and in vitro; (iii) reduced chemotactic response to BDNF in GCs cultured in vitro, associated with impaired internalization of the activated TrkB receptor; (iv) an overall increase in dendritic branching in mature GCs, resulting in impaired differentiation of the cerebellar glomeruli, the major synaptic complex between GCs and mossy fibers.

Abstract 3567: Cancer-epigenetic mouse models for medulloblastoma

Abstract Medulloblastoma (MB) is the most common childhood brain tumors (up to 20% of all childhood brain tumors) and occurs mainly in children (<15 years old. MB is located in the cerebellum in the brain and can spread to the spine or to other parts of brain. Because MB growth is very fast, MB is categorized as a type of high-grade malignant primary tumors. MB development is often associated with the dysregulation of signaling pathways, such as the wingless (WNT) and sonic hedgehog (SHH). Methylated histone H3 lysine 4 (H3K4) is a major methylation mark. Trimethyl H3K4 (H3K4me3) occupies as much as 75% of all human gene promoters. We previously reported that the H3K4 methyltransferase KMT2D (also called MLL4, MLL2, and ALR; a transcriptional coactivator, is required for neuronal differentiation of human neuron-lineage NT2/D1 stem cells. Interestingly, our analysis of several databases has shown that Kmt2d is the most frequently mutated epigenetic modifier (8%‒10%) in medulloblastoma (MB). Recently, we showed that brain-specific knockout of Kmt2d alone induces spontaneous MB in mice. Kmt2d loss highly upregulated several oncogenic signaling programs and downregulated tumor suppressive genes. In this study, we sought to determine whether Kmt2d loss cooperates with another oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1) is one of the most frequently mutated genes in MB, and KMT2D is known to be a shared driver gene mutation in MB. For these reasons, we examined the effect of Kmt2d loss on of Ptch+/−-driven MB genesis. A heterozygous loss of Kmt2d increased the incidence and progression of Ptch+/−-driven MB genesis. We have shown that Kmt2d loss reduces epigenomic signals for super-enhancers/enhancers and broad H3K4me3 in 4-month-old cerebella. In addition, we determine the effect of heterozygous Kmt2d loss on cell proliferation by comparing the Nes-Cre Kmt2dfl/+ Ptch+/- (KP) cerebella to Ptch+/- cerebella at 2- & 4-month-old time points using IHC staining of Ki-67 (a proliferation marker). MB’s from KP mice showed two different mechanisms by activating oncogenic cellular signaling kinases (e.g., phospho [p]-AKT and p-p38) levels as well as MYCN signaling pathway. Our transcriptomic analysis revealed several tumor suppressive and transcriptional corepressor genes to be downregulated such as Ncor2 and Dnmt3a. We found Ncor2 depletion in Ptch-/+ neurospheres shows upregulation of similar oncogenic genes as in KP mice tumors. Kmt2d loss reduced cerebellar granule cell differentiation. Our analysis for oncogenic kinases may provide a basis for a novel MB treatment plan involving the use of cellular kinase inhibitors. As KMT2D is among the most often mutated genes in human MBs, this mouse model may be useful for future preclinical therapeutic experiments for the treatment of MB patients. KMT2D in medulloblastoma genesis using mouse models will provide epigenetic and mechanistic insights into how heterozygous KMT2D loss promotes medulloblastoma. Citation Format: Shilpa S. Dhar, Ali S. Rizvi, Lauren Reed, Calena Brown, Min Gyu Lee. Cancer-epigenetic mouse models for medulloblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3567.

Microglia modulate proliferation, neurite generation and differentiation of human neural progenitor cells.

Microglia, the primary immune cells of the brain, significantly influence the fate of neurons after neural damage. Depending on the local environment, they exhibit a wide range of phenotypes, including patrolling (naïve), proinflammatory, and anti-inflammatory characteristics, which greatly affects neurotoxicity. Despite the fact that neural progenitor cells (NPCs) and hippocampal neurons represent cell populations, which play pivotal role in neural regeneration, interaction between microglia and these cell types is poorly studied. In the present work, we investigated how microglial cells affect the proliferation and neurite outgrowth of human stem cell-derived NPCs, and how microglia stimulation with proinflammatory or anti-inflammatory agents modulates this interaction. We found that naïve microglia slightly diminish NPC proliferation and have no effect on neurite outgrowth. In contrast, proinflammatory stimulated microglia promote both proliferation and neurite generation, whereas microglia stimulated with anti-inflammatory cytokines augment neurite outgrowth leaving NPC proliferation unaffected. We also studied how microglia influence neurite development and differentiation of hippocampal dentate gyrus granule cells differentiated from NPCs. We found that proinflammatory stimulated microglia inhibit axonal development but facilitate dendrite generation in these differentiating neurons. Our results elucidate a fine-tuned modulatory effect of microglial cells on cell types crucial for neural regeneration, opening perspectives for novel regenerative therapeutic interventions.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

Reelin Is Required for Maintenance of Granule Cell Lamination in the Healthy and Epileptic Hippocampus.

One characteristic feature of mesial temporal lobe epilepsy is granule cell dispersion (GCD), a pathological widening of the granule cell layer in the dentate gyrus. The loss of the extracellular matrix protein Reelin, an important positional cue for neurons, correlates with GCD formation in MTLE patients and in rodent epilepsy models. Here, we used organotypic hippocampal slice cultures (OHSC) from transgenic mice expressing enhanced green fluorescent protein (eGFP) in differentiated granule cells (GCs) to monitor GCD formation dynamically by live cell video microscopy and to investigate the role of Reelin in this process. We present evidence that following treatment with the glutamate receptor agonist kainate (KA), eGFP-positive GCs migrated mainly toward the hilar region. In the hilus, Reelin-producing neurons were rapidly lost following KA treatment as shown in a detailed time series. Addition of recombinant Reelin fragments to the medium effectively prevented the KA-triggered movement of eGFP-positive GCs. Placement of Reelin-coated beads into the hilus of KA-treated cultures stopped the migration of GCs in a distance-dependent manner. In addition, quantitative Western blot analysis revealed that KA treatment affects the Reelin signal transduction pathway by increasing intracellular adaptor protein Disabled-1 synthesis and reducing the phosphorylation of cofilin, a downstream target of the Reelin pathway. Both events were normalized by addition of recombinant Reelin fragments. Finally, following neutralization of Reelin in healthy OHSC by incubation with the function-blocking CR-50 Reelin antibody, GCs started to migrate without any direction preference. Together, our findings demonstrate that normotopic position of Reelin is essential for the maintenance of GC lamination in the dentate gyrus and that GCD is the result of a local Reelin deficiency.

Notch Signaling between Cerebellar Granule Cell Progenitors.

Cerebellar granule cells (GCs) are cells which comprise over 50% of the neurons in the entire nervous system. GCs enable the cerebellum to properly regulate motor coordination, learning, and consolidation, in addition to cognition, emotion and language. During GC development, maternal GC progenitors (GCPs) divide to produce not only postmitotic GCs but also sister GCPs. However, the molecular machinery for regulating the proportional production of distinct sister cell types from seemingly uniform GCPs is not yet fully understood. Here we report that Notch signaling creates a distinction between GCPs and leads to their proportional differentiation in mice. Among Notch-related molecules, Notch1, Notch2, Jag1, and Hes1 are prominently expressed in GCPs. In vivo monitoring of Hes1-promoter activities showed the presence of two types of GCPs, Notch-signaling ON and OFF, in the external granule layer (EGL). Single-cell RNA sequencing (scRNA-seq) and in silico analyses indicate that ON-GCPs have more proliferative and immature properties, while OFF-GCPs have opposite characteristics. Overexpression as well as knock-down (KD) experiments using in vivo electroporation showed that NOTCH2 and HES1 are involved cell-autonomously to suppress GCP differentiation by inhibiting NEUROD1 expression. In contrast, JAG1-expressing cells non-autonomously upregulated Notch signaling activities via NOTCH2-HES1 in surrounding GCPs, eventually suppressing their differentiation. These findings suggest that Notch signaling results in the proportional generation of two types of cells, immature and differentiating GCPs, which contributes to the well-organized differentiation of GCs.

Sevoflurane-Induced Neuroapoptosis in Rat Dentate Gyrus Is Activated by Autophagy Through NF-κB Signaling on the Late-Stage Progenitor Granule Cells.

ObjectiveThe mechanisms by which exposure of the late-stage progenitor cells to the anesthesia sevoflurane alters their differentiation are not known. We seek to query whether the effects of sevoflurane on late-stage progenitor cells might be regulated by apoptosis and/or autophagy.MethodsTo address the short-term impact of sevoflurane exposure on granule cell differentiation, we used 5-bromo-2-deoxyuridine (BrdU) to identify the labeled late-stage progenitor granule cells. Male or female rats were exposed to 3% sevoflurane for 4 h when the labeled granule cells were 2 weeks old. Differentiation of the BrdU-labeled granule cells was quantified 4 and 7 days after exposure by double immunofluorescence. The expression of apoptosis and autophagy in hippocampal dentate gyrus (DG) was determined by western blot and immunofluorescence. Western blot for the expression of NF-κB was used to evaluate the mechanism. Morris water maze (MWM) test was performed to detect cognitive function in the rats on postnatal 28–33 days.ResultsExposure to sevoflurane decreased the differentiation of the BrdU-labeled late-stage progenitor granule cells, but increased the expression of caspase-3, autophagy, and phosphorylated-P65 in the hippocampus of juvenile rats and resulted in cognitive deficiency. These damaging effects of sevoflurane could be mitigated by inhibitors of autophagy, apoptosis, and NF-κB. The increased apoptosis could be alleviated by pretreatment with the autophagy inhibitor 3-MA, and the increased autophagy and apoptosis could be reduced by pretreatment with NF-κB inhibitor BAY 11-7085.ConclusionThese findings suggest that a single, prolonged sevoflurane exposure could impair the differentiation of late-stage progenitor granule cells in hippocampal DG and cause cognitive deficits possibly via apoptosis activated by autophagy through NF-κB signaling. Our results do not preclude the possibility that the affected differentiation and functional deficits may be caused by depletion of the progenitors pool.

Abstract 1083: Heterozygous knockout of the histone modifier MLL4 promotes Ptch+/−-driven medulloblastoma

Abstract Medulloblastoma (MB) is the most common primary childhood brain tumor. MB development frequently results from the dysregulation of sonic hedgehog and wingless pathways. In contrast to our understanding of these pathways, the pathogenic roles of aberrant epigenetic modifiers in MB remain little known. MLL4 is one of the most commonly mutated genes in MB. In our recent study, we showed that brain-specific knockout of the gene encoding the histone H3 lysine 4 methyltransferase MLL4 (also called COMPASS-like methyltransferase, KMT2D, MLL2, and ALR) induced spontaneous MBs. Mll4 loss highly upregulated several oncogenic signaling programs, including expression of several oncogenic Ras activators (e.g., Rasgrp1, Rasgrf1, and Rasgef1a). In contrast, Mll4 loss downregulated expression of tumor-suppressive genes, such as Dnmt3a. In this study, we sought to determine whether Mll4 loss cooperates with other oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1) is one of the most frequently mutated genes in MB, and its loss induces MB in mouse models. Thus, we examined the effect of Mll4 loss on of Ptch+/−-driven MB genesis. A heterozygous loss of Mll4 increased the incidence and progression of Ptch+/−-driven MB genesis. MBs were confirmed by H & E staining, and immunohistochemical staining using anti-Ki-67 showed that cell proliferation was markedly increased. Calbindin signals were undetectable in Nestin-Cre Mll4f/+ Ptch+/−, and NeuN levels were decreased in Nestin-Cre Mll4f/+ Ptch+/− cerebellum compared to Ptch+/− cerebellum, suggesting the impairment of Purkinje and granule cells. Mll4 loss reduced cerebellar granule cell differentiation. These findings indicate that a haploinsufficiency of MLL4 promotes Ptch+/−-driven MB genesis. This mouse model may be useful for future preclinical therapeutic experiments for the treatment of MB patients with MLL4 mutations. Citation Format: Shilpa S. Dhar, Dongyu Zhao, Tao Lin, Roy V. Sillitoe, Kaifu Chen, Min Gyu Lee. Heterozygous knockout of the histone modifier MLL4 promotes Ptch+/−-driven medulloblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1083.

Effects of Phosphatidylserine Source of Docosahexaenoic Acid on Cerebellar Development in Preterm Pigs.

Preterm birth, a major contributor to infant mortality and morbidity, impairs development of the cerebellum, the brain region involved in cognitive processing and motor function. Previously, we showed that at term-equivalent age, preterm pigs that received formula supplemented with docosahexaenoic acid (DHA) esterified to phosphatidylserine (PS) had cerebellar weights similar to those of newborn term pigs and were heavier than control preterm pigs. However, whether PS-DHA promotes the development of specific cerebellar cell populations or enhances key developmental processes remains unknown. Here we investigated the effects of the PS-DHA on development of the cerebellum in preterm pigs delivered via caesarean section and reared for ten days on a milk replacer with either PS-DHA (experimental group) or sunflower oil (control group). Upon necropsy, key cerebellar populations were analyzed using immunohistochemistry. Consumption of PS-DHA was associated with the expansion of undifferentiated granule cell precursors and increased proliferation in the external granule cell layer (EGL). Preterm pigs that received PS-DHA also had significantly fewer apoptotic cells in the internal granule cell layer (IGL) that contains differentiated granule neurons. PS-DHA did not affect the number of differentiating granule cells in the inner EGL, thickness of the inner EGL, density of Purkinje cells, or Bergmann glial fibers, or diameter of Purkinje cells. Thus, PS-DHA may support cerebellar development in preterm subjects by enhancing proliferation of granule cells, a process specifically inhibited by preterm birth, and increasing the survival of granule cells in the IGL. These findings suggest that PS-DHA is a promising candidate for clinical studies directed at enhancing brain development.

Early Ethanol Exposure Inhibits the Differentiation of Hippocampal Dentate Gyrus Granule Cells in a Mouse Model of Fetal Alcohol Spectrum Disorders.

Alcohol consumption during pregnancy may damage the developing central nervous system of the fetus and lead to brain structural and functional deficits in the children, known as fetal alcohol spectrum disorders. The underlying mechanisms have not been fully elucidated. Previously, using a third trimester-equivalent mouse model, ethanol (EtOH)-induced behavioral deficits (including spatial learning and memory dysfunction) in the mice were detected on postnatal day (PD) 35. The hippocampal formation is critically involved in spatial learning/memory and contains 2 major neuron populations: the pyramidal cells in the hippocampus proper and the dentate gyrus granule cells (DGGCs) in the dentate gyrus (DG). In rodents, while the pyramidal cells are almost exclusively generated prenatally, the DG granule neurons are majorly generated during the first 2weeks postnatally, which coincides with the period of EtOH exposure in our mouse model. Therefore, in the current study the effects of EtOH exposure on the development of the DGGCs were examined. C57BL/6 mice were treated with 4g/kg of EtOH by intubation on PD 4 to 10 to mimic alcohol exposure to the fetus during the third trimester in humans, and the development of DGGCs was examined by immunohistochemistry and quantified on PD 35. EtOH exposure does not affect the number of total or newly generated DGGCs, but reduces the number of mature DGGCs on PD 35 in both male and female mice. The ratio of immature DGGCs over total DGGCs was increased, and the ratio of mature DGGCs over total DGGCs was decreased by EtOH exposure. In addition, no sex-dependent effects of EtOH treatment were detected. Our data indicate that EtOH exposure in mice during PD 4 to 10 does not affect the generation/proliferation but inhibits the differentiation of the DGGCs on PD 35.

Epileptiform activity induced by kainate application results in decreased Reelin expression and motility of differentiated granule cells

Epileptiform activity induced by kainate application results in decreased Reelin expression and motility of differentiated granule cells

Rho family GTPases, Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.

Cell-Biological Requirements for the Generation of Dentate Gyrus Granule Neurons.

The dentate gyrus (DG) receives highly processed information from the associative cortices functionally integrated in the trisynaptic hippocampal circuit, which contributes to the formation of new episodic memories and the spontaneous exploration of novel environments. Remarkably, the DG is the only brain region currently known to have high rates of neurogenesis in adults (Andersen et al., 1966, 1971). The DG is involved in several neurodegenerative disorders, including clinical dementia, schizophrenia, depression, bipolar disorder and temporal lobe epilepsy. The principal neurons of the DG are the granule cells. DG granule cells generated in culture would be an ideal model to investigate their normal development and the causes of the pathologies in which they are involved and as well as possible therapies. Essential to establish such in vitro models is the precise definition of the most important cell-biological requirements for the differentiation of DG granule cells. This requires a deeper understanding of the precise molecular and functional attributes of the DG granule cells in vivo as well as the DG cells derived in vitro. In this review we outline the neuroanatomical, molecular and cell-biological components of the granule cell differentiation pathway, including some growth- and transcription factors essential for their development. We summarize the functional characteristics of DG granule neurons, including the electrophysiological features of immature and mature granule cells and the axonal pathfinding characteristics of DG neurons. Additionally, we discuss landmark studies on the generation of dorsal telencephalic precursors from pluripotent stem cells (PSCs) as well as DG neuron differentiation in culture. Finally, we provide an outlook and comment critical aspects.

A Peptidomic Approach to Characterize Peptides Involved in Cerebellar Cortex Development Leads to the Identification of the Neurotrophic Effects of Nociceptin

The cerebellum is a brain structure involved in motor and cognitive functions. The development of the cerebellar cortex (the external part of the cerebellum) is under the control of numerous factors. Among these factors, neuropeptides including PACAP or somatostatin modulate the survival, migration and/or differentiation of cerebellar granule cells. Interestingly, such peptides contributing to cerebellar ontogenesis usually exhibit a specific transient expression profile with a low abundance at birth, a high expression level during the developmental processes, which take place within the first two postnatal weeks in rodents, and a gradual decline toward adulthood. Thus, to identify new peptides transiently expressed in the cerebellum during development, rat cerebella were sampled from birth to adulthood, and analyzed by a semi-quantitative peptidomic approach. A total of 33 peptides were found to be expressed in the cerebellum. Among these 33 peptides, 8 had a clear differential expression pattern during development, 4 of them i.e. cerebellin 2, nociceptin, somatostatin and VGF [353-372], exhibiting a high expression level during the first two postnatal weeks followed by a significative decrease at adulthood. A focus by a genomic approach on nociceptin, confirmed that its precursor mRNA is transiently expressed during the first week of life in granule neurons within the internal granule cell layer of the cerebellum, and showed that the nociceptin receptor is also actively expressed between P8 and P16 by the same neurons. Finally, functional studies revealed a new role for nociceptin, acting as a neurotrophic peptide able to promote the survival and differentiation of developing cerebellar granule neurons.

Preterm birth disrupts cerebellar development by affecting granule cell proliferation program and Bergmann glia

Preterm birth is a leading cause of long-term motor and cognitive deficits. Clinical studies suggest that some of these deficits result from disruption of cerebellar development, but the mechanisms that mediate cerebellar abnormalities in preterm infants are largely unknown. Furthermore, it remains unclear whether preterm birth and precocious exposure to the ex-utero environment directly disrupt cerebellar development or indirectly by increasing the probability of cerebellar injury, including that resulting from clinical interventions and protocols associated with the care of preterm infants. In this study, we analyzed the cerebellum of preterm pigs delivered via c-section at 91% term and raised for 10 days, until term-equivalent age. The pigs did not receive any treatments known or suspected to affect cerebellar development and had no evidence of brain damage. Term pigs sacrificed at birth were used as controls. Immunohistochemical analysis revealed that preterm birth did not affect either size or numbers of Purkinje cells or molecular layer interneurons at term-equivalent age. The number of granule cell precursors and Bergmann glial fibers, however, were reduced in preterm pigs. Preterm pigs had reduced proliferation but not differentiation of granule cells. qRT-PCR analysis of laser capture microdissected external granule cell layer showed that preterm pigs had a reduced expression of Ccnd1 (Cyclin D1), Ccnb1 (Cyclin B1), granule cell master regulatory transcription factor Atoh1, and signaling molecule Jag1. In vitro rescue experiments identified Jag1 as a central granule cell gene affected by preterm birth. Thus, preterm birth and precocious exposure to the ex-utero environment disrupt cerebellum by modulating expression of key cerebellar developmental genes, predominantly affecting development of granule precursors and Bergmann glia.

Differential Expression of Piccolo Splice Isoforms During Cerebellar Development

Piccolo is a multi‐domain protein with restricted expression to the presynaptic active zone, the site of synaptic vesicle fusion and neurotransmitter release. Alternative splicing of the Piccolo gene (PCLO) results in two primary splice isoforms: isoform‐1 (I‐1) with two C2 domains (C2A and C2B) and isoform‐2 (I‐2) with only a single C2A motif. In addition, an exon‐skipping event generates Piccolo isoforms that alter the C2A domain resulting in exclusion of a 27‐nucleotide exon located in the C2A domain (e.g. short variant). The result is a lack of 9 amino acids in the domain resulting in increased calcium and phospholipid binding affinity versus the longer C2A domain (e.g. long variant) that includes the 9‐peptide region. Consistent with the importance of the Ca2+‐binding property of C2 domain‐containing proteins in synaptic function, genome‐wide association studies have identified variations in or near the C2A domain of human PCLO that predispose individuals to affective disorders. The most compelling evidence for the importance of the C2A domain comes from recent identification of the genetic cause of pontocerebellar hypoplasia type III (PCH3): a neurological disorder that results in severe underdevelopment of the cerebellum. Analysis of genomic DNA from two affected individuals with PCH3 identified a homozygous nonsense variant in PCLO that introduces a STOP codon in the 3′ coding region including the C2A domain. The purpose of this study was to examine the temporal expression of Piccolo splice variants during critical periods of mouse cerebellar and cortical development. We performed real‐time quantitative RT‐PCR gene expression analysis of Piccolo splice isoforms (e.g. I‐1, I‐2, long and short) from RNA isolated from cerebellum at key stages of cerebellar granule cell differentiation (P0, P5, P15, adult) and also from the cortex at the same ages. Our analysis demonstrated that expression levels of the I‐1 and I‐2 splice variants were relatively consistent between cerebellum and cortex at all examined developmental time points. In contrast, we showed that during initial stages of cerebellar development (P0 and P5), the more calcium‐sensitive short variant had significantly increased expression levels relative to the long variant. Our results indicate that increased expression of the short variant may play a role in neuronal proliferation during initial cerebellar development when considering the augmented calcium permeability of immature neurons during this time period.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential 3' Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types.

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3'UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3'UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3'UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.

Liver X receptor β regulates the development of the dentate gyrus and autistic-like behavior in the mouse

The dentate gyrus (DG) of the hippocampus is a laminated brain region in which neurogenesis begins during early embryonic development and continues until adulthood. Recent studies have implicated that defects in the neurogenesis of the DG seem to be involved in the genesis of autism spectrum disorders (ASD)-like behaviors. Liver X receptor β (LXRβ) has recently emerged as an important transcription factor involved in the development of laminated CNS structures, but little is known about its role in the development of the DG. Here, we show that deletion of the LXRβ in mice causes hypoplasia in the DG, including abnormalities in the formation of progenitor cells and granule cell differentiation. We also found that expression of Notch1, a central mediator of progenitor cell self-renewal, is reduced in LXRβ-null mice. In addition, LXRβ deletion in mice results in autistic-like behaviors, including abnormal social interaction and repetitive behavior. These data reveal a central role for LXRβ in orchestrating the timely differentiation of neural progenitor cells within the DG, thereby providing a likely explanation for its association with the genesis of autism-related behaviors in LXRβ-deficient mice.