Scientific Resources

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

Store-operated calcium entry (SOCE) is a Calcium (Ca2+) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons,the presence and functions of SOCE are still in question. Here,we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase,which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs),being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions,but the underlying mechanism and,specifically,the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons,we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD,pointing out a key function of calcium and SOCE in synaptic plasticity. View Publication

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However,methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately,these methods entail numerous challenges,including poor efficiency,variable cell type heterogeneity,and lengthy,expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol,these lines can be inducibly differentiated into either cortical (i3 Neurons) or lower motor neurons (i3 LMN) in a rapid,efficient,and scalable manner (Wang et al.,2017). In this manuscript,we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i3 Neurons or i3 LMNs,and we present neuronal culture conditions for various experimental applications. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

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 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

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature,post-mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti-depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin-2 (NEUROG2) to generate a homogenous population of post-mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline,the vast majority of cells are post-mitotic,and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f,we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2-responsive genes were determined by RNA-Seq. FGF2-regulated genes previously identified in non-neuronal cell types were up-regulated (EGR1,ETV4,SPRY4,and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron-specific genes were also identified that may mediate FGF2-dependent increases in synaptic efficacy including NRXN3,SYT2,and GALR1. Since several of these genes have been implicated in MDD previously,these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD. View Publication

Hypoxic stressors contribute to neuronal death in many brain diseases. Astrocyte processes surround most neurons and are therefore anatomically well-positioned to shield them from hypoxic injury. Excitatory amino acid transporters (EAATs),represent the sole mechanism of active reuptake of glutamate into the astrocytes and neurons and are essential to dampen neuronal excitation following glutamate release at synapses. Glutamate clearance impairment from any factors is bound to result in an increase in hypoxic neuronal injury. The brain energy metabolism under hypoxic conditions depends on monocarboxylate transporters (MCTs) that are expressed by neurons and glia. Previous co-immunoprecipitation experiments revealed that MCT4 directly modulate EAAT1 in astrocytes. The reduction in both surface proteins may act synergistically to induce neuronal hyperexcitability and excitotoxicity. Therefore we hypothesized that astrocytes would respond to hypoxic conditions by enhancing their expression of MCT4 and EAAT1,which,in turn,would enable them to better support neurons to survive lethal hypoxia injury. An oxygen deprivation (OD) protocol was used in primary cultures of neurons,astrocytes,and astrocytes-neurons derived from rat hippocampus,with or without MCT4-targeted short hairpin RNA (shRNA) transfection. Cell survival,expression of MCT4,EAAT1,glial fibrillary acidic protein and neuronal nuclear antigen were evaluated. OD resulted in significant cell death in neuronal cultures and up-regulation of MCT4,EAAT1 expression respectively in primary cell cultures,but no injury in neuron-astrocyte co-cultures and astrocyte cultures. However,neuronal cell death in co-cultures was increased exposure to shRNA-MCT4 prior to OD. These findings demonstrate that the MCT4-mediated expression of EAAT1 is involved in the resistance to hypoxia injury in astrocyte-neuron co-cultures. View Publication

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly,which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However,mechanisms ofNPCs maintenance remain unclear. Here,we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC),which includes Nde1,Aurora A,andOFD1,recruited to the ciliary base for timely cilium disassembly. In contrast,mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment,long cilia,retarded cilium disassembly,and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus,our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control. View Publication

NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex,GluN1 is combined primarily with GluN2A and GluN2B,which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B,the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components,in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis,revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions. View Publication

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells,which is partially associated with the induction or repression of genes that influence the ischemic response. However,the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults,we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD),an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD,total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes,whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD,confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins,such as those encoding for PICK1,GRIP1,TARPγ3,calsyntenin-2/3,SAPAP2 and SNAP-25,were down-regulated after OGD. Additionally,OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors,but increased the mRNA expression of the GluN3A subunit,thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together,our results present the expression profile elicited by in vitro ischemia in hippocampal neurons,and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins,suggesting that the synaptic proteome may change after ischemia. View Publication