rapid-detection-neurodevelopmental-phenotypes-human-neural-precursor

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Rutgers Robert Wood Johnson Medical School

Numerous studies have focused on the regulation of leptin signalling and the functions of leptin in energy homoeostasis; however, little is known about how leptin secretion is regulated. In the present study we studied leptin storage and secretion regulation in 3T3-L1 and primary adipocytes. Leptin is stored in membrane-bound vesicles that are localized predominantly in the ER (endoplasmic reticulum) and close to the plasma membrane of both 3T3-L1 and primary adipocytes. Insulin increases leptin secretion as early as 15 min without affecting the leptin mRNA level. Interestingly, treatment with the protein synthesis inhibitor cycloheximide and the ER-Golgi trafficking blocker Brefeldin A inhibit both basal and ISLS (insulin-stimulated leptin secretion), suggesting that insulin stimulates leptin secretion by up-regulating leptin synthesis and that leptin-containing vesicles go through the ER-Golgi route. The PI3K (phosphoinositide 3-kinase)/Akt, but not MAPK (mitogen-activated protein kinase), pathway is involved in ISLS in vitro and in vivo. Although Ca2+ triggers synaptic vesicle and secretory granule exocytosis, Ca2+ influx alone is not sufficient to induce leptin secretion. Remarkably, Ca2+ is required for ISLS possibly due to its involvement in insulin-stimulated Akt phosphorylation. We conclude that insulin stimulates leptin release through the PI3K/Akt pathway and that Ca2+ is required for robust Akt phosphorylation and leptin secretion.

Insulin-stimulated leptin secretion requires calcium and PI3K/Akt activation.

Glucagon-like peptide-1 (GLP-1) and its analogs act as appetite suppressants and have been proven to be clinically efficacious in reducing body weight in obese individuals. Central GLP-1 is expressed in a small population of brainstem cells located in the nucleus tractus solitarius (NTS), which project to a wide range of brain areas. However, it remains unclear how endogenous GLP-1 released in the brain contributes to appetite regulation. Using chemogenetic tools, we discovered that central GLP-1 acts on the midbrain ventral tegmental area (VTA) and suppresses high-fat food intake. We used integrated pathway tracing and synaptic physiology to further demonstrate that activation of GLP-1 receptors specifically reduces the excitatory synaptic strength of dopamine (DA) neurons within the VTA that project to the nucleus accumbens (NAc) medial shell. These data suggest that GLP-1 released from NTS neurons can reduce highly palatable food intake by suppressing mesolimbic DA signaling.

Endogenous Glucagon-like Peptide-1 Suppresses High-Fat Food Intake by Reducing Synaptic Drive onto Mesolimbic Dopamine Neurons.

Nanotechnology-based approaches offer the chemical control required to develop precision tools suitable for applications in neuroscience. We report a novel approach employing hybrid upconversion nanomaterials, combined with the photoresponsive ion channel channelrhodopsin-2 (ChR2), to achieve near-infrared light (NIR)-mediated optogenetic control of neuronal activity. Current optogenetic methodologies rely on using visible light (e.g. 470 nm blue light), which tends to exhibit high scattering and low tissue penetration, to activate ChR2. In contrast, our approach enables the use of 980 nm NIR light, which addresses the short-comings of visible light as an excitation source. This was facilitated by embedding upconversion nanomaterials, which can convert NIR light to blue luminescence, into polymeric scaffolds. These hybrid nanomaterial scaffolds allowed for NIR-mediated neuronal stimulation, with comparable efficiency as that of 470 nm blue light. Our platform was optimized for NIR-mediated optogenetic control by balancing multiple physicochemical properties of the nanomaterial (e.g. size, morphology, structure, emission spectra, concentration), thus providing an early demonstration of rationally-designing nanomaterial-based strategies for advanced neural applications.

Hybrid upconversion nanomaterials for optogenetic neuronal control.

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼ 3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼ 38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance.

Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds.

Direct conversion of somatic cells into neurons holds great promise for regenerative medicine. However, neuronal conversion is relatively inefficient in human cells compared to mouse cells. It has been unclear what might be the key barriers to reprogramming in human cells. We recently elucidated an RNA program mediated by the polypyrimidine tract binding protein PTB to convert mouse embryonic fibroblasts (MEFs) into functional neurons. In human adult fibroblasts (HAFs), however, we unexpectedly found that invoking the documented PTB-REST-miR-124 loop generates only immature neurons. We now report that the functionality requires sequential inactivation of PTB and the PTB paralog nPTB in HAFs. Inactivation of nPTB triggers another self-enforcing loop essential for neuronal maturation, which comprises nPTB, the transcription factor BRN2, and miR-9. These findings suggest that two separate gatekeepers control neuronal conversion and maturation and consecutively overcoming these gatekeepers enables deterministic reprogramming of HAFs into functional neurons.

Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells.

Alcohol abuse produces an enormous impact on health, society, and the economy. Currently, there are very limited therapies available, largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse.

Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells.

CRH neurons in the hypothalamic paraventricular nucleus (PVN) play a central role in regulating the hypothalamus-pituitary-adrenal (HPA) axis and are directly influenced by 17β-estradiol (E2). Although compelling evidence has suggested the existence of membrane-associated estrogen receptors (mERs) in hypothalamic and other central nervous system neurons, it remains unknown whether E2 impacts CRH neuronal excitability through this mechanism. The purpose of the current study is to examine the existence and function of mER signaling in PVN CRH neurons. Whole-cell recordings were made from CRH neurons identified by Alexa Fluor 594 labeling and post hoc immunostaining in ovariectomized female mice. E2 (100nM) rapidly suppressed the M-current (a voltage-dependent K(+) current) and potentiated glutamatergic excitatory postsynaptic currents. The putative Gq-coupled mER (Gq-mER) characterized in hypothalamic proopiomelanocortin neurons initiates a phospholipase C-protein kinase C-protein kinase A pathway; therefore, we examined the involvement of this pathway using selective inhibitors. Indeed, the ER antagonist ICI 182780 and inhibitors of Gq-phospholipase C-protein kinase C-protein kinase A blocked E2's actions, suggesting dependence on the Gq-mER. Furthermore, STX, a selective ligand for the Gq-mER, mimicked E2's actions. Finally, to examine the in vivo effect of Gq-mER activation, E2 or STX injection increased c-fos expression in CRH neurons in the PVN, suggesting CRH neuronal activation. This corresponded to an increase in plasma corticosterone. We conclude that the Gq-mER plays a critical role in the rapid regulation of CRH neuronal activity and the HPA axis. Our findings provide a potential underlying mechanism for E2's involvement in the pathophysiology of HPA-associated mood disorders.

Gq Protein-Coupled Membrane-Initiated Estrogen Signaling Rapidly Excites Corticotropin-Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus in Female Mice.

Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of nicotine in both DA and glutamatergic neurons. Glutamatergic N398 neurons responded to lower nicotine doses (0.1 μM) with greater frequency and amplitude but they also exhibited rapid desensitization, consistent with previous analyses of N398-associated nicotinic receptor function. This study offers a proof-of-principle for utilizing human neurons to study gene variants contribution to addiction.

Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele.

Neuroinflammation, a common neuropathologic feature of neurodegenerative disorders including Parkinson disease (PD), is frequently exacerbated by microglial activation. The extracellular protein α-synuclein (ASYN), whose aggregation is characteristic of PD, remains a key therapeutic target, but the control of synuclein trafficking and aggregation within microglia has been challenging. First, we established that microglial internalization of monomeric ASYN was mediated by scavenger receptors (SR), CD36 and SRA1, and was rapidly accompanied by the formation of ASYN oligomers. Next, we designed a nanotechnology approach to regulate SR-mediated intracellular ASYN trafficking within microglia. We synthesized mucic acid-derivatized sugar-based amphiphilic molecules (AM) with optimal stereochemistry, rigidity, and charge for enhanced dual binding affinity to SRs and fabricated serum-stable nanoparticles via flash nanoprecipitation comprising hydrophobe cores and amphiphile shells. Treatment of microglia with AM nanoparticles decreased monomeric ASYN internalization and intracellular ASYN oligomer formation. We then engineered composite deactivating NPs with dual character, namely shell-based SR-binding amphiphiles, and core-based antioxidant poly (ferrulic acid), to investigate concerted inhibition of oxidative activation. In ASYN-challenged microglia treated with NPs, we observed decreased ASYN-mediated acute microglial activation and diminished microglial neurotoxicity caused by exposure to aggregated ASYN. When the composite NPs were administered in vivo within the substantia nigra of fibrillar ASYN-challenged wild type mice, there was marked attenuation of activated microglia. Overall, SR-targeting AM nanotechnology represents a novel paradigm in alleviating microglial activation in the context of synucleinopathies like PD and other neurodegenerative diseases.

Polymer brain-nanotherapeutics for multipronged inhibition of microglial α-synuclein aggregation, activation, and neurotoxicity.

Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene-mutation-cell-behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPaseare associated with various tumors, and ∼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and ∼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three general features of BRaf signaling. This study establishes the first efficient procedure that permits large-scale functional analysis of human disease-linked mutations essential for precision medicine.

BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method.

Calcium (Ca1 and Ca2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C termini, which associate with calmodulin (CaM), a universal calcium sensor. Ca3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCa3) and human Ca3.1, Ca3.2, and Ca3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I-II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high-nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM inH-N HSQC NMR spectra. Intact Ca-binding sites on CaM and an intact gating brake sequence (first 39 amino acids of the I-II linker) were required in Ca3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Ca3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM.

Article	Year
Insulin-stimulated leptin secretion requires calcium and PI3K/Akt activation. The Biochemical journal\| PubMed ID: 24405299	2014
Endogenous Glucagon-like Peptide-1 Suppresses High-Fat Food Intake by Reducing Synaptic Drive onto Mesolimbic Dopamine Neurons. Cell reports\| PubMed ID: 26212334	2015
Hybrid upconversion nanomaterials for optogenetic neuronal control. Nanoscale\| PubMed ID: 26415758	2015
Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds. Nature communications\| PubMed ID: 26983594	2016
Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells. Nature neuroscience\| PubMed ID: 27110916	2016
Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells. Molecular brain\| PubMed ID: 27160314	2016
Gq Protein-Coupled Membrane-Initiated Estrogen Signaling Rapidly Excites Corticotropin-Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus in Female Mice. Endocrinology\| PubMed ID: 27387482	2016
Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele. Scientific reports\| PubMed ID: 27698409	2016
Polymer brain-nanotherapeutics for multipronged inhibition of microglial α-synuclein aggregation, activation, and neurotoxicity. Biomaterials\| PubMed ID: 27736702	2016
BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method. Genes & development\| PubMed ID: 28404629	2017
Calmodulin regulates Ca3 T-type channels at their gating brake. The Journal of biological chemistry\| PubMed ID: 28972185	2017

Zhiping Pang

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