技术资料

Neurotoxicity testing still relies on ethically debated,expensive and time consuming in vivo experiments,which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds,such as picrotoxin (PTX) and endosulfan,and the neurotoxicant methylmercury (MeHg). However,the chemical-induced effects on different parameters for neuronal activity,such as mean spike rate (MSR) and mean burst rate (MBR),may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening. View Publication

Alzheimer's disease (AD) is emerging as a synaptopathology driven by metaplasticity. Indeed,reminiscent of metaplasticity,oligomeric forms of the amyloid-beta$ peptide (oAbeta$) prevent induction of long-term potentiation (LTP) via the prior activation of GluN2B-containing NMDA receptors (NMDARs). However,the downstream Ca2+-dependent signaling molecules that mediate aberrant metaplasticity are unknown. In this study,we show that oAbeta$ promotes the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) via GluN2B-containing NMDARs. Importantly,we find that CaMKII inhibition rescues both the LTP impairment and the dendritic spine loss mediated by oAbeta$. Mechanistically resembling metaplasticity,oAbeta$ prevents subsequent rounds of plasticity from inducing CaMKII T286 autophosphorylation,as well as the associated anchoring and accumulation of synaptic AMPA receptors (AMPARs). Finally,prolonged oAbeta$ treatment-induced CaMKII misactivation leads to dendritic spine loss via the destabilization of surface AMPARs. Thus,our study demonstrates that oAbeta$ engages synaptic metaplasticity via aberrant CaMKII activation. View Publication

Functional evaluation assays using human induced pluripotent stem cell (hiPSC)-derived neurons can predict the convulsion toxicity of new drugs and the neurological effects of antiepileptic drugs. However,differences in responsiveness depending on convulsant type and antiepileptic drugs,and an evaluation index capable of comparing in vitro responses with in vivo responses are not well known. We observed the difference in synchronized burst patterns in the epileptiform activities induced by pentylentetrazole (PTZ) and 4-aminopryridine (4-AP) with different action mechanisms using multi-electrode arrays (MEAs); we also observed that 100 µM of the antiepileptic drug phenytoin suppressed epileptiform activities induced by PTZ,but increased those induced by 4-AP. To compare in vitro results with in vivo convulsive responses,frequency analysis of below 250 Hz,excluding the spike component,was performed. The in vivo convulsive firing enhancement of the high gamma$ wave and beta$ wave component were observed remarkably in in vitro hiPSC-derived neurons with astrocytes in co-culture. MEA measurement of hiPSC-derived neurons in co-culture with astrocytes and our analysis methods,including frequency analysis,appear effective for predicting convulsion toxicity,side effects,and their mechanism of action as well as the comparison of convulsions induced in vivo. View Publication

BACKGROUND Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia,although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling,insulin regulation of the major AD proteins Abeta$ and/or tau,and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS We report herein that wildtype,PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Abeta$42/40 ratio. Unexpectedly,we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux,which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Abeta$42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence,our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD. View Publication

Zika virus (ZIKV) infection is associated with microcephaly in fetuses,but the pathogenesis of ZIKV-related microcephaly is not well understood. Here we show that ZIKV infects the subventricular zone in human fetal brain tissues and that the tissue tropism broadens with the progression of gestation. Our research demonstrates also that intermediate progenitor cells (IPCs) are the main target cells for ZIKV. Post-mitotic committed neurons become susceptible to ZIKV infection as well at later stages of gestation. Furthermore,activation of microglial cells,DNA fragmentation,and apoptosis of infected or uninfected cells could be found in ZIKV-infected brain tissues. Our studies identify IPCs as the main target cells for ZIKV. They also suggest that immune activation after ZIKV infection may play an important role in the pathogenesis of ZIKV-related microcephaly. View Publication

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs,this class of cell is remarkably diverse,comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models,but less attention has been paid to human RGCs. Thus,efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics,confirming the combinatorial expression of molecular markers associated with these subtypes,and also provided insight into more subtype-specific markers. Thus,the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. View Publication

Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain,but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging,in contrast to iPSC differentiation. Here,we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression,impaired axonal mitochondrial morphologies,lower mitochondrial membrane potentials,reduced energy production,and increased oxidized proteins levels. In contrast,the fibroblasts from which iNs were generated show only mild age-dependent changes,consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed,OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging. View Publication

Suprachiasmatic nucleus circadian oscillatory protein (SCOP) (a.k.a. PHLPP1) regulates long-term memory consolidation in the brain. Using a mouse model of controlled cortical impact (CCI) we tested if (1) brain tissue levels of SCOP/PHLPP1 increase after a traumatic brain injury (TBI),and (2) if SCOP/PHLPP1 gene knockout (KO) mice have improved (or worse) neurologic outcomes. Blood chemistry (pH,pCO2,pO2,pSO2,base excess,sodium bicarbonate,and osmolarity) and arterial pressure (MAP) differed in isoflurane anesthetized WT vs. KOs at baseline and up to 1 h post-injury. CCI injury increased cortical/hippocampal SCOP/PHLPP1 levels in WTs 7d and 14d post-injury. Injured KOs had higher brain tissue levels of phosphorylated AKT (pAKT) in cortex (14d post-injury),and higher levels of phosphorylated MEK (pMEK) in hippocampus (7d and 14d post-injury) and in cortex (7d post-injury). Consistent with an important role of SCOP/PHLPP1 on memory function,injured-KOs had near normal performance on the probe trial of the Morris water maze,whereas injured-WTs were impaired. CA1/CA3 hippocampal survival was lower in KOs vs. WTs 24 h post-injury but equivalent by 7d. No difference in 21d cortical lesion volume was detected. SCOP/PHLPP1 overexpression in cultured rat cortical neurons had no effect on 24 h cell death after a mechanical stretch-injury. View Publication

Regulation of AMPA receptor (AMPAR) trafficking is a key modulator of excitatory synaptic transmission; however,intracellular vesicular transport of newly synthesized AMPARs has been little studied due to technical limitations. By combining molecular tools with imaging strategies in cultured rat hippocampal neurons,we found that vesicles containing newly synthesized,GluA1-subunit-containing AMPARs are transported antero- and retrogradely at a mean speed of 1.5 mu$m.s-1. Synaptic activity and variations in intracellular calcium levels bidirectionally modulate GluA1 transport. Chemical long-term potentiation (cLTP) initially induces a halt in GluA1 transport,followed by a sustained increase,while acute glutamate uncaging on synaptic spines arrests vesicular movements. GluA1 phosphomimetic mutants preferentially travel to the dendritic tip,probably to replenish extrasynaptic pools,distal to the soma. Our findings indicate that AMPAR intracellular transport is highly regulated during synaptic plasticity and likely controls AMPAR numbers at the plasma membrane. View Publication