AB5622 Sigma-AldrichAnti-Microtubule-Associated Protein 2 (MAP2) Antibody

Direct conversion of mammalian fibroblasts into induced neuronal (iN) cells has been attained by forced expression of pro-neural transcriptional factors, or by combining defined factors with either microRNAs or small molecules. Here, we show that neuronal cells can be converted from postnatal human fibroblasts into cell populations with neuronal purities of up to greater than 80% using a combination of six chemical compounds. The chemical compound-induced neuronal cells (CiNCs) express neuron-specific proteins and functional neuron markers. The efficiency of CiNCs is unaffected by either the donor's age or cellular senescence (passage number). We propose this chemical direct converting strategy as a potential approach for highly efficient generation of neuronal cells from human fibroblasts for such uses as in neural disease modeling and regenerative medicine.

Levodopa-induced dyskinesias (LIDs) are major complications in the pharmacological management of Parkinson's disease (PD). Abnormal glutamatergic transmission in the striatum is considered a key factor in the development of LIDs. This work aims at: (i) characterizing N-methyl-D-aspartate (NMDA) receptor GluN2A/GluN2B subunit ratio as a common synaptic trait in rat and primate models of LIDs as well as in dyskinetic PD patients; and (ii) validating the potential therapeutic effect of a cell-permeable peptide (CPP) interfering with GluN2A synaptic localization on the dyskinetic behavior of these experimental models of LIDs. Here we demonstrate an altered ratio of synaptic GluN2A/GluN2B-containing NMDA receptors in the striatum of levodopa-treated dyskinetic rats and monkeys as well as in post-mortem tissue from dyskinetic PD patients. The modulation of synaptic NMDA receptor composition by a cell-permeable peptide interfering with GluN2A subunit interaction with the scaffolding protein postsynaptic density protein 95 (PSD-95) leads to a reduction in the dyskinetic motor behavior in the two animal models of LIDs. Our results indicate that targeting synaptic NMDA receptor subunit composition may represent an intriguing therapeutic approach aimed at ameliorating levodopa motor side effects.

Spatial water maze (WM) overtraining induces hippocampal mossy fiber (MF) expansion, and it has been suggested that spatial pattern separation depends on the MF pathway. We hypothesized that WM experience inducing MF expansion in rats would improve spatial pattern separation in the hippocampal network. We first tested this by using the the delayed non-matching to place task (DNMP), in animals that had been previously trained on the water maze (WM) and found that these animals, as well as animals treated as swim controls (SC), performed better than home cage control animals the DNMP task. The "catFISH" imaging method provided neurophysiological evidence that hippocampal pattern separation improved in animals treated as SC, and this improvement was even clearer in animals that experienced the WM training. Moreover, these behavioral treatments also enhance network reliability and improve partial pattern separation in CA1 and pattern completion in CA3. By measuring the area occupied by synaptophysin staining in both the stratum oriens and the stratun lucidum of the distal CA3, we found evidence of structural synaptic plasticity that likely includes MF expansion. Finally, the measures of hippocampal network coding obtained with catFISH correlate significantly with the increased density of synaptophysin staining, strongly suggesting that structural synaptic plasticity in the hippocampus induced by the WM and SC experience is related to the improvement of spatial information processing in the hippocampus.

Due to their capacity to self-replicate or produce specific differentiated cell types, neural stem cells (NSCs) and bone marrow derived-mesenchymal stem cells (BMSCs) are potential sources for cell transplantation therapies, particularly for neural injury. However, the interaction between NSCs and BMSCs during differentiation has not yet been defined. The interaction is believed to improve the effectiveness and efficiency of cell therapy. In the present study, human NSCs and BMSCs were cultured and the Transwell co-culture system was used to observe the interplay between NSCs and BMSCs during differentiation. The results revealed that NSCs promoted BMSCs to differentiate into neurons and NSCs; whereas, BMSCs did not affect the differentiation of NSCs. Simultaneously, co-culture increased the concentration of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which are secreted by NSCs and BMSCs. The present findings suggest that co-culture of NSCs and BMSCs can promote the differentiation and this process may be modulated by BDNF and NGF.

Gaucher disease (GD) is caused by insufficient activity of acid β-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD, induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs, NPCs, and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition, GD2 neurons showed increased α-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons, but intriguingly, those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes, sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings, providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge, this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease.

Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking.

Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

Exosomes are nano-sized vesicles of endocytic origin released into the extracellular space upon fusion of multivesicular bodies with the plasma membrane. Exosomes represent a novel mechanism of cell-cell communication allowing direct transfer of proteins, lipids and RNAs. In the nervous system, both glial and neuronal cells secrete exosomes in a way regulated by glutamate. It has been hypothesized that exosomes can be used for interneuronal communication implying that neuronal exosomes should bind to other neurons with some kind of specificity. Here, dissociated hippocampal cells were used to compare the specificity of binding of exosomes secreted by neuroblastoma cells to that of exosomes secreted by cortical neurons. We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells. In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons. Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication.

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types.

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.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.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.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.