技术资料

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here,we describe a new recessive neurodevelopmental syndrome with global developmental delay,seizures and intellectual disability. Using linkage analysis and exome sequencing,we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation,p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically,the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo,CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43,the ortholog of human CAMK2A. In vitro,neurons derived from patient iPSCs displayed profound synaptic defects. Together,our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders. View Publication

Heterozygous loss-of-function mutations in GRIN2B,a subunit of the NMDA receptor,cause intellectual disability and language impairment. We developed clonal models of GRIN2B deletion and loss-of-function mutations in a region coding for the glutamate binding domain in human cells and generated neurons from a patient harboring a missense mutation in the same domain. Transcriptome analysis revealed extensive increases in genes associated with cell proliferation and decreases in genes associated with neuron differentiation,a result supported by extensive protein analyses. Using electrophysiology and calcium imaging,we demonstrate that NMDA receptors are present on neural progenitor cells and that human mutations in GRIN2B can impair calcium influx and membrane depolarization even in a presumed undifferentiated cell state,highlighting an important role for non-synaptic NMDA receptors. It may be this function,in part,which underlies the neurological disease observed in patients with GRIN2B mutations. View Publication

We have developed models of HIV latency using microglia derived from adult human patient brain cortex and transformed with the SV40 T large and hTERT antigens. Latent clones infected by HIV reporter viruses display high levels of spontaneous HIV reactivation in culture. BrainPhys,a medium highly representative of the CNS extracellular environment,containing low glucose and 1{\%} FBS,reduced,but did not prevent,HIV reactivation. We hypothesized that spontaneous HIV reactivation in culture was due to the expression of pro-inflammatory genes,such as TNF-alpha$,taking place in the absence of the natural inhibitory signals from astrocytes and neurons. Indeed,expression and secretion of TNF-alpha$ is strongly reduced in HIV-latently infected microglia compared to the subset of cells that have undergone spontaneous HIV reactivation. Whereas inhibitors of NF-kappa$B or of macrophage activation only had a short-term silencing effect,addition of dexamethasone (DEXA),a glucocorticoid receptor (GR) agonist and mediator of anti-inflammation,silenced the HIV provirus in a long-term,and shRNA-mediated knock-down of GR activated HIV. DEXA also decreased secretion of a number of cytokines,including TNF-alpha$. Chromatin immunoprecipitation analysis revealed that DEXA strongly increased GR occupancy at the HIV promoter,and reduced histone 3 acetylated levels. Moreover,TNF-alpha$ expression inhibitors in combination with DEXA induced further HIV silencing and increased the histone 3 lysine 27 tri-methylated epigenetic mark of repression at the HIV promoter region. We conclude that GR is a critical repressor of HIV transcription in microglia,and a novel potential pharmacological target to restrict HIV expression in the CNS. View Publication

We introduce a novel all-optical assay for functional studies of biological neural networks in vitro. We created a novel optogenetic construct named OptoCaMP which is a combination of a channelrhodopsin variant (CheRiff) and a red genetically encoded calcium indicator (GECI) (jRCaMP1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. Additionally,we have developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity,in both stimulated and non-stimulated neurons,thus allowing the investigation of the spread of excitation through an interconnected network. Finally,we demonstrate the sensitivity of this assay to the change of neural network connectivity. View Publication

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study,we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42),indicating an increase in transient mPTP opening. Subsequently,the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42,during which time global cellular reactive oxygen species increased,mitochondrial membrane potential decreased,cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism,then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ. View Publication

BACKGROUND Transplanting autologous patient-derived enteric neuronal stem/progenitor cells (ENSCs) is an innovative approach to replacing missing enteric neurons in patients with Hirschsprung disease (HSCR). Using autologous cells eliminates immunologic and ethical concerns raised by other cell sources. However,whether postnatal aganglionic bowel is permissive for transplanted ENSCs and whether ENSCs from HSCR patients can be successfully isolated,cultured,and transplanted in vivo remains unknown. METHODS ENSCs isolated from the ganglionic intestine of Ednrb(-/-) mice (HSCR-ENSCs) were characterized immunohistochemically and evaluated for their capacity to proliferate and differentiate in vitro. Fluorescently labeled ENSCs were co-cultured ex vivo with aganglionic Ednrb(-/-) colon. For in vivo transplantation,HSCR-ENSCs were labeled with lentivirus expressing green fluorescent protein (GFP) and implanted into aganglionic embryonic chick gut in ovo and postnatal aganglionic Ednrb(-/-) rectum in vivo. KEY RESULTS HSCR-ENSCs maintain normal capacity self-renewal and neuronal differentiation. Moreover,the Ednrb(-/-) aganglionic environment is permissive to engraftment by wild-type ENSCs ex vivo and supports migratrion and neuroglial differentiation of these cells following transplantation in vivo. Lentiviral GFP-labeled HSCR-ENSCs populated embryonic chick hindgut and postnatal colon of Ednrb(-/-) HSCR,with cells populating the intermuscular layer and forming enteric neurons and glia. CONCLUSIONS & INFERENCES ENSCs can be isolated and cultured from mice with HSCR,and transplanted into the aganglionic bowel of HSCR littermates to generate enteric neuronal networks. These results in an isogenic model establish the potential of using autologous-derived stem cells to treat HSCR and other intestinal neuropathies. View Publication

Cell therapy offers an innovative approach for treating enteric neuropathies. Postnatal gut-derived enteric neural stem/progenitor cells (ENSCs) represent a potential autologous source,but have a limited capacity for proliferation and neuronal differentiation. Since serotonin (5-HT) promotes enteric neuronal growth during embryonic development,we hypothesized that serotonin receptor agonism would augment growth of neurons from transplanted ENSCs. Postnatal ENSCs were isolated from 2 to 4 week-old mouse colon and cultured with 5-HT4 receptor agonist (RS67506)-loaded liposomal nanoparticles. ENSCs were co-cultured with mouse colon explants in the presence of RS67506-loaded (n = 3) or empty nanoparticles (n = 3). ENSCs were also transplanted into mouse rectum in vivo with RS67506-loaded (n = 8) or blank nanoparticles (n = 4) confined in a thermosensitive hydrogel,Pluronic F-127. Neuronal density and proliferation were analyzed immunohistochemically. Cultured ENSCs gave rise to significantly more neurons in the presence of RS67506-loaded nanoparticles. Similarly,colon explants had significantly increased neuronal density when RS67506-loaded nanoparticles were present. Finally,following in vivo cell delivery,co-transplantation of ENSCs with 5-HT4 receptor agonist-loaded nanoparticles led to significantly increased neuronal density and proliferation. We conclude that optimization of postnatal ENSCs can support their use in cell-based therapies for neurointestinal diseases. View Publication