技术资料

BACKGROUND: Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo,where activity of inputs can be differentially regulated,but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here,we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. METHODOLOGY/PRINCIPAL FINDINGS: GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons,both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures,synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. CONCLUSIONS/SIGNIFICANCE: The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde 'reward' signal generated by WT neurons,although in this paradigm there was no 'punishment' signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system. View Publication

A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention,small molecule protein kinase inhibitors present a promising therapeutic strategy. Here,we report a robust cell-based phenotypic assay,utilizing primary rat hippocampal neurons,for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium-throughput screening,as indicated by its Z'-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened,revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches toward the development of drugs for treating SCI and related neurological pathologies. View Publication

The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells,called induced pluripotent stem cells (iPSCs),can be an unlimited source of specialized cell types for the body. Thus,autologous somatic cell replacement therapy becomes possible,as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited,and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons. View Publication

Induced pluripotent stem cells (iPSC) and their differentiated derivatives offer a unique source of human primary cells for toxicity screens. Here,we report on the comparative cytotoxicity of 80 compounds (neurotoxicants,developmental neurotoxicants,and environmental compounds) in iPSC as well as isogenic iPSC-derived neural stem cells (NSC),neurons,and astrocytes. All compounds were tested over a 24-h period at 10 and 100$\$,in duplicate,with cytotoxicity measured using the MTT assay. Of the 80 compounds tested,50 induced significant cytotoxicity in at least one cell type; per cell type,32,38,46,and 41 induced significant cytotoxicity in iPSC,NSC,neurons,and astrocytes,respectively. Four compounds (valinomycin,3,3',5,5'-tetrabromobisphenol,deltamethrin,and triphenyl phosphate) were cytotoxic in all four cell types. Retesting these compounds at 1,10,and 100$\$ using the same exposure protocol yielded consistent results as compared with the primary screen. Using rotenone,we extended the testing to seven additional iPSC lines of both genders; no substantial difference in the extent of cytotoxicity was detected among the cell lines. Finally,the cytotoxicity assay was simplified by measuring luciferase activity using lineage-specific luciferase reporter iPSC lines which were generated from the parental iPSC line. This article is part of a Special Issue entitled SI: PSC and the brain. View Publication

To address existing limitations in live neuron imaging,we have developed NeuO,a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications. View Publication

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected,and the functional consequences,have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51),but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD),a model of global brain ischemia,as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity,the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore,GDNF protected hippocampal neurons when present before,during or after OGD,and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF. View Publication

AbstractBack to TopbackslashnNeurons produced from stem cells have emerged as a tool to identify new therapeutic targets for neurological diseases such as amyotrophic lateral sclerosis (ALS). However,it remains unclear to what extent these new mechanistic insights will translate to animal models,an important step in the validation of new targets. Previously,we found that glia from mice carrying the SOD1G93A mutation,a model of ALS,were toxic to stem cell–derived human motor neurons. We use pharmacological and genetic approaches to demonstrate that the prostanoid receptor DP1 mediates this glial toxicity. Furthermore,we validate the importance of this mechanism for neural degeneration in vivo. Genetic ablation of DP1 in SOD1G93A mice extended life span,decreased microglial activation,and reduced motor neuron loss. Our findings suggest that blocking DP1 may be a therapeutic strategy in ALS and demonstrate that discoveries from stem cell models of disease can be corroborated in vivo. View Publication

OBJECTIVE Charcot-Marie-Tooth (CMT) disease is a group of inherited peripheral neuropathies associated with mutations or copy number variations in over 70 genes encoding proteins with fundamental roles in the development and function of Schwann cells and peripheral axons. Here,we used iPSC-derived cells to identify common pathophysiological mechanisms in axonal CMT. METHODS iPSC lines from patients with two distinct forms of axonal CMT (CMT2A and CMT2E) were differentiated into spinal cord motor neurons and used to study axonal structure and function and electrophysiological properties in vitro. RESULTS iPSC-derived motor neurons exhibited gene and protein expression,ultrastructural and electrophysiological features of mature primary spinal cord motor neurons. Cytoskeletal abnormalities were found in neurons from a CMT2E (NEFL) patient and corroborated by a mouse model of the same NEFL point mutation. Abnormalities in mitochondrial trafficking were found in neurons derived from this patient,but were only mildly present in neurons from a CMT2A (MFN2) patient. Novel electrophysiological abnormalities,including reduced action potential threshold and abnormal channel current properties were observed in motor neurons derived from both of these patients. INTERPRETATION Human iPSC-derived motor neurons from axonal CMT patients replicated key pathophysiological features observed in other models of MFN2 and NEFL mutations,including abnormal cytoskeletal and mitochondrial dynamics. Electrophysiological abnormalities found in axonal CMT iPSC-derived human motor neurons suggest that these cells are hyperexcitable and have altered sodium and calcium channel kinetics. These findings may provide a new therapeutic target for this group of heterogeneous inherited neuropathies. View Publication

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized,the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV,an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013),productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation,even during early stages of infection,leading to progenitor depletion,disruption of the VZ,impaired neurogenesis,and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly. View Publication

BACKGROUND Fragile X syndrome (FXS),a common cause of intellectual disability and autism,results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to<200 repeats. Such expanded alleles,known as full mutation (FM) alleles,are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP,a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. METHODS We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing,and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. RESULTS We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs),some silenced alleles contract and when the repeat number drops below ˜400,DNA methylation erodes,even when the repeat number remains<200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore,there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. CONCLUSIONS Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo,(ii) large unmethylated alleles may be deleterious in stem cells,(iii) methylation can occur on alleles with<400 repeats very early in embryogenesis,and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with<200 repeats. View Publication

Dendritic protein synthesis plays a critical role in several forms of synaptic plasticity,including BDNF (brain-derived neurotrophic factor)-mediated long-term synaptic potentiation (LTP). Dendritic transcripts are typically transported in a repressed state as components of large ribonucleoprotein complexes,and then translated upon stimulation at,or in the vicinity,of activated synapses. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a trans-acting factor involved in dendritic mRNA trafficking,but how the distribution of the protein in dendrites is regulated has not been characterized. Here we found that a fraction of hnRNP A2/B1 is present at the synapse under resting conditions in cultured hippocampal neurons. Accordingly,this ribonucleoprotein was detected in free mRNP,monosomal,and polyribosomal fractions obtained from synaptoneurosomes. Neuronal activity and BDNF treatment increased hnRNP A2/B1 protein levels in the cell body and dendritic compartments,and induced the delivery of this protein to synaptic sites. The activity-dependent accumulation of hnRNP A2/B1 at the synapse required,at least in part,the activation of TrkB receptors,presumably by BDNF. This neurotrophin also upregulated the hnRNP A2/B1 mRNA in the soma but was without effect on the abundance of neuritic hnRNP A2/B1 transcripts. These results show that the distribution of hnRNP A2/B1 is regulated by BDNF and by neuronal activity,an effect that may have a role in BDNF-induced synaptic plasticity events. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-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 $\$-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. View Publication