Area postrema neurons mediate interleukin-6 function in cancer cachexia.

Interleukin-6 (IL-6) has been long considered a key player in cancer cachexia. It is believed that sustained elevation of IL-6 production during cancer progression causes brain dysfunctions, which ultimately result in cachexia. However, how peripheral IL-6 influences the brain remains poorly understood. Here we show that neurons in the area postrema (AP), a circumventricular structure in the hindbrain, is a critical mediator of IL-6 function in cancer cachexia in male mice. We find that circulating IL-6 can rapidly enter the AP and activate neurons in the AP and its associated network. Peripheral tumor, known to increase circulating IL-6, leads to elevated IL-6 in the AP, and causes potentiated excitatory synaptic transmission onto AP neurons and AP network hyperactivity. Remarkably, neutralization of IL-6 in the brain of tumor-bearing mice with an anti-IL-6 antibody attenuates cachexia and the hyperactivity in the AP network, and markedly prolongs lifespan. Furthermore, suppression of Il6ra, the gene encoding IL-6 receptor, specifically in AP neurons with CRISPR/dCas9 interference achieves similar effects. Silencing Gfral-expressing AP neurons also attenuates cancer cachectic phenotypes and AP network hyperactivity. Our study identifies a central mechanism underlying the function of peripheral IL-6, which may serve as a target for treating cancer cachexia.

Human post-mortem organotypic brain slice cultures: a tool to study pathomechanisms and test therapies.

Human brain experimental models recapitulating age- and disease-related characteristics are lacking. There is urgent need for human-specific tools that model the complex molecular and cellular interplay between different cell types to assess underlying disease mechanisms and test therapies. Here we present an adapted ex vivo organotypic slice culture method using human post-mortem brain tissue cultured at an air-liquid interface to also study brain white matter. We assessed whether these human post-mortem brain slices recapitulate the in vivo neuropathology and if they are suitable for pathophysiological, experimental and pre-clinical treatment development purposes, specifically regarding leukodystrophies. Human post-mortem brain tissue and cerebrospinal fluid were obtained from control, psychiatric and leukodystrophy donors. Slices were cultured up to six weeks, in culture medium with or without human cerebrospinal fluid. Human post-mortem organotypic brain slice cultures remained viable for at least six weeks ex vivo and maintained tissue structure and diversity of (neural) cell types. Supplementation with cerebrospinal fluid could improve slice recovery. Patient-derived organotypic slice cultures recapitulated and maintained known in vivo neuropathology. The cultures also showed physiologic multicellular responses to lysolecithin-induced demyelination ex vivo, indicating their suitability to study intrinsic repair mechanisms upon injury. The slice cultures were applicable for various experimental studies, as multi-electrode neuronal recordings. Finally, the cultures showed successful cell-type dependent transduction with gene therapy vectors. These human post-mortem organotypic brain slice cultures represent an adapted ex vivo model suitable for multifaceted studies of brain disease mechanisms, boosting translation from human ex vivo to in vivo. This model also allows for assessing potential treatment options, including gene therapy applications. Human post-mortem brain slice cultures are thus a valuable tool in preclinical research to study the pathomechanisms of a wide variety of brain diseases in living human tissue.

Retracted: The Effects of Hesperidin on Neuronal Apoptosis and Cognitive Impairment in the Sevoflurane Anesthetized Rat are Mediated Through the PI3/Akt/PTEN and Nuclear Factor-κB (NF-κB) Signaling Pathways.

The Editors of Medical Science Monitor wish to inform you that the above manuscript has been retracted from publication due to concerns with the credibility and originality of the study, the manuscript content, and the Figure images. Reference: Haijin Huang, Cuicui Hu, Lin Xu, Xiaoping Zhu, Lili Zhao, Jia Min. The Effects of Hesperidin on Neuronal Apoptosis and Cognitive Impairment in the Sevoflurane Anesthetized Rat are Mediated Through the PI3/Akt/PTEN and Nuclear Factor-kappaB (NF-kappaB) Signaling Pathways. Med Sci Monit, 2020; 26: e920522. DOI: 10.12659/MSM.920522.

sFlt-1 impairs neurite growth and neuronal differentiation in SH-SY5Y cells and human neurons.

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy which is associated with increased risk of neurodevelopmental disorders in exposed offspring. The pathophysiological mechanisms mediating this relationship are currently unknown, and one potential candidate is the anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which is highly elevated in PE. While sFlt-1 can impair angiogenesis via inhibition of VEGFA signalling, it is unclear whether it can directly affect neuronal development independently of its effects on the vasculature. To test this hypothesis, the current study differentiated the human neural progenitor cell (NPC) line ReNcell® VM into a mixed culture of mature neurons and glia, and exposed them to sFlt-1 during development. Outcomes measured were neurite growth, cytotoxicity, mRNA expression of nestin, MBP, GFAP, and ßIII-tubulin, and neurosphere differentiation. sFlt-1 induced a significant reduction in neurite growth and this effect was timing- and dose-dependent up to 100 ng/ml, with no effect on cytotoxicity. sFlt-1 (100 ng/ml) also reduced ßIII-tubulin mRNA and neuronal differentiation of neurospheres. Undifferentiated NPCs and mature neurons/glia expressed VEGFA and VEGFR-2, required for endogenous autocrine and paracrine VEGFA signalling, while sFlt-1 treatment prevented the neurogenic effects of exogenous VEGFA. Overall, these data provide the first experimental evidence for a direct effect of sFlt-1 on neurite growth and neuronal differentiation in human neurons through inhibition of VEGFA signalling, clarifying our understanding of the potential role of sFlt-1 as a mechanism by which PE can affect neuronal development.

FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control.

Mitochondrial dysfunction and oxidative stress are important mechanisms for secondary injury after traumatic brain injury (TBI), which result in progressive pathophysiological exacerbation. Although the Fibronectin type III domain-containing 5 (FNDC5) was reported to repress oxidative stress by retaining mitochondrial biogenesis and dynamics, its possible role in the secondary injury after TBI remain obscure. In present study, we observed that the level of serum irisin (the cleavage product of FNDC5) significantly correlated with the neurological outcomes of TBI patients. Knockout of FNDC5 increased the lesion volume and exacerbated apoptosis and neurological deficits after TBI in mice, while FNDC5 overexpression yielded a neuroprotective effect. Moreover, FNDC5 deficiency disrupted mitochondrial dynamics and function. Activation of Sirtuin 3 (SIRT3) alleviated FNDC5 deficiency-induced disruption of mitochondrial dynamics and bioenergetics. In neuron-specific SIRT3 knockout mice, FNDC5 failed to attenuate TBI-induced mitochondrial damage and brain injuries. Mechanically, FNDC5 deficiency led to reduced SIRT3 expression via enhanced ubiquitin degradation of transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), which contributed to the hyperacetylation and inactivation of key regulatory proteins of mitochondrial dynamics and function, including OPA1 and SOD2. Finally, engineered RVG29-conjugated nanoparticles were generated to selectively and efficiently deliver irisin to the brain of mice, which yielded a satisfactory curative effect against TBI. In conclusion, FNDC5/irisin exerts a protective role against acute brain injury by promoting SIRT3-dependent mitochondrial quality control and thus represents a potential target for neuroprotection after TBI.

Glioblastoma disrupts cortical network activity at multiple spatial and temporal scales.

The emergence of glioblastoma in cortical tissue initiates early and persistent neural hyperexcitability with signs ranging from mild cognitive impairment to convulsive seizures. The influence of peritumoral synaptic density, expansion dynamics, and spatial contours of excess glutamate upon higher order neuronal network modularity is unknown. We combined cellular and widefield imaging of calcium and glutamate fluorescent reporters in two glioblastoma mouse models with distinct synaptic microenvironments and infiltration profiles. Functional metrics of neural ensembles are dysregulated during tumor invasion depending on the stage of malignant progression and tumor cell proximity. Neural activity is differentially modulated during periods of accelerated and inhibited tumor expansion. Abnormal glutamate accumulation precedes and outpaces the spatial extent of baseline neuronal calcium signaling, indicating these processes are uncoupled in tumor cortex. Distinctive excitability homeostasis patterns and functional connectivity of local and remote neuronal populations support the promise of precision genetic diagnosis and management of this devastating brain disease.

Sex- and cycle-dependent changes in spine density and size in hippocampal CA2 neurons.

Sex hormones affect structural and functional plasticity in the rodent hippocampus. However, hormone levels not only differ between males and females, but also fluctuate across the female estrous cycle. While sex- and cycle-dependent differences in dendritic spine density and morphology have been found in the rodent CA1 region, but not in the CA3 or the dentate gyrus, comparable structural data on CA2, i.e. the hippocampal region involved in social recognition memory, is so far lacking. In this study, we, therefore, used wildtype male and female mice in diestrus or proestrus to analyze spines on dendritic segments from identified CA2 neurons. In basal stratum oriens, we found no differences in spine density, but a significant shift towards larger spine head areas in male mice compared to females. Conversely, in apical stratum radiatum diestrus females had a significantly higher spine density, and females in either cycle stage had a significant shift towards larger spine head areas as compared to males, with diestrus females showing the larger shift. Our results provide further evidence for the sexual dimorphism of hippocampal area CA2, and underscore the importance of considering not only the sex, but also the stage of the estrous cycle when interpreting morphological data.

Single-nucleus analysis reveals microenvironment-specific neuron and glial cell enrichment in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complicated neurodegenerative disease. Neuron-glial cell interactions are an important but not fully understood process in the progression of AD. We used bioinformatic methods to analyze single-nucleus RNA sequencing (snRNA-seq) data to investigate the cellular and molecular biological processes of AD. METHOD: snRNA-seq data were downloaded from Gene Expression Omnibus (GEO) datasets and reprocessed to identify 240,804 single nuclei from healthy controls and patients with AD. The cellular composition of AD was further explored using Uniform Manifold Approximation and Projection (UMAP). Enrichment analysis for the functions of the DEGs was conducted and cell development trajectory analyses were used to reveal underlying cell fate decisions. iTALK was performed to identify ligand-receptor pairs among various cell types in the pathological ecological microenvironment of AD. RESULTS: Six cell types and multiple subclusters were identified based on the snRNA-seq data. A subcluster of neuron and glial cells co-expressing lncRNA-SNHG14, myocardin-related transcription factor A (MRTFA), and MRTFB was found to be more abundant in the AD group. This subcluster was enriched in mitogen-activated protein kinase (MAPK)-, immune-, and apoptosis-related pathways. Through molecular docking, we found that lncRNA-SNHG14 may bind MRTFA and MRTFB, resulting in an interaction between neurons and glial cells. CONCLUSIONS: The findings of this study describe a regulatory relationship between lncRNA-SNHG14, MRTFA, and MRTFB in the six main cell types of AD. This relationship may contribute to microenvironment remodeling in AD and provide a theoretical basis for a more in-depth analysis of AD.

Apolipoprotein E2 Expression Alters Endosomal Pathways in a Mouse Model With Increased Brain Exosome Levels During Aging.

The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging.

Plp1-expresssing perineuronal DRG cells facilitate colonic and somatic chronic mechanical pain involving Piezo2 upregulation in DRG neurons.

Satellite glial cells (SGCs) of dorsal root ganglia (DRGs) are activated in a variety of chronic pain conditions; however, their mediation roles in pain remain elusive. Here, we take advantage of proteolipid protein (PLP)/creERT-driven recombination in the periphery mainly occurring in SGCs of DRGs to assess the role of SGCs in the regulation of chronic mechanical hypersensitivity and pain-like responses in two organs, the distal colon and hindpaw, to test generality. We show that PLP/creERT-driven hM3Dq activation increases, and PLP/creERT-driven TrkB.T1 deletion attenuates, colon and hindpaw chronic mechanical hypersensitivity, positively associating with calcitonin gene-related peptide (CGRP) expression in DRGs and phospho-cAMP response element-binding protein (CREB) expression in the dorsal horn of the spinal cord. Activation of Plp1+ DRG cells also increases the number of small DRG neurons expressing Piezo2 and acquiring mechanosensitivity and leads to peripheral organ neurogenic inflammation. These findings unravel a role and mechanism of Plp1+ cells, mainly SGCs, in the facilitation of chronic mechanical pain and suggest therapeutic targets for pain mitigation.

Genetically defined nucleus incertus neurons differ in connectivity and function.

The nucleus incertus (NI), a conserved hindbrain structure implicated in the stress response, arousal, and memory, is a major site for production of the neuropeptide relaxin-3. On the basis of goosecoid homeobox 2 (gsc2) expression, we identified a neuronal cluster that lies adjacent to relaxin 3a (rln3a) neurons in the zebrafish analogue of the NI. To delineate the characteristics of the gsc2 and rln3a NI neurons, we used CRISPR/Cas9 targeted integration to drive gene expression specifically in each neuronal group, and found that they differ in their efferent and afferent connectivity, spontaneous activity, and functional properties. gsc2 and rln3a NI neurons have widely divergent projection patterns and innervate distinct subregions of the midbrain interpeduncular nucleus (IPN). Whereas gsc2 neurons are activated more robustly by electric shock, rln3a neurons exhibit spontaneous fluctuations in calcium signaling and regulate locomotor activity. Our findings define heterogeneous neurons in the NI and provide new tools to probe its diverse functions.

Defective ferritinophagy and imbalanced iron metabolism in PBDE-47-triggered neuronal ferroptosis and salvage by Canolol.

The brominated flame retardant 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) is a ubiquitous environmental pollutant that causes neurotoxicity. However, incomplete understanding of the underlying mechanisms has hampered the development of effective intervention strategies. Oxidative stress and related cell death are the modes of action for PBDE-47 neurotoxicity, which are also the characteristics of ferroptosis. Nonetheless, the role of ferroptosis in PBDE-47-induced neurotoxicity remains unclear. In the present study, we found that PBDE-47 triggered ferroptosis in neuron-like PC12 cells, as evidenced by intracellular iron overload, lipid peroxidation, and mitochondrial damage. This was confirmed by ferroptosis inhibitors including the lipid reactive oxygen species scavenger ferrostatin-1 and iron chelator deferoxamine mesylate. Mechanistically, PBDE-47 impaired ferritinophagy by disrupting nuclear receptor coactivator 4-mediated lysosomal degradation of the iron storage protein ferritin. Moreover, PBDE-47 disturbed iron metabolism by increasing cellular iron import via upregulation of transferrin receptor 1 and decreasing cellular iron export via downregulation of ferroportin 1 (FPN1). Intriguingly, rescuing lysosomal function by overexpressing cathepsin B (CatB) mitigated PBDE-47-induced ferroptosis by partially restoring dysfunctional ferritinophagy and enhancing iron excretion via the upregulation of FPN1. However, FPN1 knockdown reversed the beneficial effects of CatB overexpression on the PBDE-47-induced iron overload. Finally, network pharmacology integrated with experimental validation revealed that Canolol, the main phenolic compound in canola oil, protected against PBDE-47-evoked iron overload, resulting in ferroptosis by restoring defective ferritinophagy and improving abnormal iron metabolism via lowering iron uptake and facilitating iron excretion. Overall, these data suggest that ferroptosis is a novel mechanism of PBDE-47-induced neuronal death and that manipulation of ferritinophagy and iron metabolism via Canolol represents a promising therapeutic strategy.

Induction of indoleamine 2,3-dioxygenase 1 expression in neurons of the central nervous system through inhibition of histone deacetylases blocks the progression of experimental autoimmune encephalomyelitis.

BACKGROUND: A wide array of histone deacetylase (HDAC) inhibitors and aryl hydrocarbon receptor (AHR) agonists commonly arrest experimental autoimmune encephalomyelitis (EAE). However, it is not known whether HDAC inhibition is linked to the AHR signaling pathway in EAE. METHODS: We investigated how the pan-HDAC inhibitor SB939 (pracinostat) exerted immunoregulatory action in the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE mouse model by evaluating changes in of signal transducer and activator of transcription 3 (STAT3) acetylation and the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and AHR in inflamed spinal cords during EAE evolution. We proved the involvement of IDO1 and the AHR in SB939-mediated immunosuppression using Ido1-/- and Ahr-/- mice. RESULTS: Administration with SB939 halted EAE progression, which depended upon IDO1 expression in neurons of the central nervous system (CNS). Our in vitro and in vivo studies demonstrated that SB939 sustained the interleukin-6-induced acetylation of STAT3, resulting in the stable transcriptional activation of Ido1. The therapeutic effect of SB939 also required the AHR, which is expressed mainly in CD4+ T cells and macrophages in CNS disease lesions. Finally, SB939 was shown to markedly reduce the proliferation of CD4+ T cells in inflamed neuronal tissues but not in the spleen or draining lymph nodes. CONCLUSIONS: Overall, our results suggest that IDO1 tryptophan metabolites produced by neuronal cells may act on AHR in pathogenic CD4+ T cells in a paracrine fashion in the CNS and that the specific induction of IDO1 expression in neurons at disease-afflicted sites can be considered a therapeutic approach to block the progression of multiple sclerosis without affecting systemic immunity.

Priming of hippocampal microglia by IFN-γ/STAT1 pathway impairs social memory in mice.

Social behavior is inextricably linked to the immune system. Although IFN-γ is known to be involved in social behavior, yet whether and how it encodes social memory remains unclear. In the current study, we injected with IFN-γ into the lateral ventricle of male C57BL/6J mice, and three-chamber social test was used to examine the effects of IFN-γ on their social preference and social memory. The morphology of microglia in the hippocampus, prelimbic cortex and amygdala was examined using immunohistochemistry, and the phenotype of microglia were examined using immunohistochemistry and enzyme-linked immunosorbent assays. The IFN-γ-injected mice were treated with lipopolysaccharide, and effects of IFN-γ on behavior and microglial responses were evaluated. STAT1 pathway and microglia-neuron interactions were examined in vivo or in vitro using western blotting and immunohistochemistry. Finally, we use STAT1 inhibitor or minocycline to evaluated the role of STAT1 in mediating the microglial priming and effects of primed microglia in IFN-γ-induced social dysfunction. We demonstrated that 500 ng of IFN-γ injection results in significant decrease in social index and social novelty recognition index, and induces microglial priming in hippocampus, characterized by enlarged cell bodies, shortened branches, increased expression of CD68, CD86, CD74, CD11b, CD11c, CD47, IL-33, IL-1ß, IL-6 and iNOS, and decreased expression of MCR1, Arg-1, IGF-1 and BDNF. This microglia subpopulation is more sensitive to LPS challenge, which characterized by more significant morphological changes and inflammatory responses, as well as induced increased sickness behaviors in mice. IFN-γ upregulated pSTAT1 and STAT1 and promoted the nuclear translocation of STAT1 in the hippocampal microglia and in the primary microglia. Giving minocycline or STAT1 inhibitor fludarabin blocked the priming of hippocampal microglia induced by IFN-γ, ameliorated the dysfunction in hippocampal microglia-neuron interactions and synapse pruning by microglia, thereby improving social memory deficits in IFN-γ injected mice. IFN-γ initiates STAT1 pathway to induce priming of hippocampal microglia, thereby disrupts hippocampal microglia-neuron interactions and neural circuit link to social memory. Blocking STAT1 pathway or inhibiting microglial priming may be strategies to reduce the effects of IFN-γ on social behavior.

Oridonin exerts anticonvulsant profile and neuroprotective activity in epileptic mice by inhibiting NLRP3-mediated pyroptosis.

BACKGROUND: Epilepsy is a chronic disabling disease poorly controlled by available antiseizure medications. Oridonin, a bioactive alkaloid with anti-inflammatory properties and neuroprotective effects, can inhibit the increased excitability of neurons caused by glutamate accumulation at the cellular level. However, whether oridonin affects neuronal excitability and whether it has antiepileptic potential has not been reported in animal models or clinical studies. METHOD: Pentylenetetrazol was injected into mice to create a model of chronic epilepsy. Seizure severity was assessed using the Racine scale, and the duration and latency of seizures were observed. Abnormal neuronal discharge was detected using electroencephalography, and neuronal excitability was assessed using calcium imaging. Damage to hippocampal neurons was evaluated using Hematoxylin-Eosin and Nissl staining. The expression of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and other pyroptosis-related proteins was determined using western blotting and immunofluorescence. A neuronal pyroptosis model was established using the supernatant of BV2 cells treated with lipopolysaccharide and adenosine triphosphate to stimulate hippocampal neurons. RESULTS: Oridonin (1 and 5 mg/kg) reduced neuronal damage, increased the latency of seizures, and shortened the duration of fully kindled seizures in chronic epilepsy model mice. Oridonin decreased abnormal discharge during epileptic episodes and suppressed increased neuronal excitability. In vitro experiments showed that oridonin alleviated pyroptosis in hippocampal HT22 neurons. CONCLUSION: Oridonin exerts neuroprotective effects by inhibiting pyroptosis through the NLRP3/caspase-1 pathway in chronic epilepsy model mice. It also reduces pyroptosis in hippocampal neurons in vitro, suggesting its potential as a therapy for epilepsy.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Functional Analysis of NMDAR Subunit Components in Postsynaptic Currents of Identified Cells and Synapses in Brain Slices.

Epilepsy is one of the most represented neurological diseases worldwide. However, in many cases, the precise molecular mechanisms of epileptogenesis and ictiogenesis are unknown. Because of their important role in synaptic function and neuronal excitability, NMDA receptors are implicated in various epileptogenic mechanisms. Most of these are subunit specific and require a precise analysis of the subunit composition of the NMDARs implicated. Here, we describe an express electrophysiological method to analyze the contribution of NMDAR subunits to spontaneous postsynaptic activity in identified cells in brain slices using patch clamp whole cell recordings.

Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Analysis of Functional NMDA Receptors in Astrocytes.

Neuronal N-methyl-D-aspartate (NMDA) receptors are well known for their pivotal role in memory formation. Originally, they were thought to be exclusive to neurons. However, numerous studies revealed their functional expression also on various types of glial cells in the nervous system. Here, the methodology on how to study the physiology of NMDA receptors selectively on astrocytes will be described in detail. Astrocytes are the main class of neuroglia that control transmitter and ion homeostasis, which link cerebral blood flow and neuronal energy demands, but also affect synaptic transmission directly.

Construction of human 3D striato-nigral assembloids to recapitulate medium spiny neuronal projection defects in Huntington's disease.

The striato-nigral (Str-SN) circuit is composed of medium spiny neuronal projections that are mainly sent from the striatum to the midbrain substantial nigra (SN), which is essential for regulating motor behaviors. Dysfunction of the Str-SN circuitry may cause a series of motor disabilities that are associated with neurodegenerative disorders, such as Huntington's disease (HD). Although the etiology of HD is known as abnormally expanded CAG repeats of the huntingtin gene, treatment of HD remains tremendously challenging. One possible reason is the lack of effective HD model that resembles Str-SN circuitry deficits for pharmacological studies. Here, we first differentiated striatum-like organoids from human pluripotent stem cells (hPSCs), containing functional medium spiny neurons (MSNs). We then generated 3D Str-SN assembloids by assembling striatum-like organoids with midbrain SN-like organoids. With AAV-hSYN-GFP-mediated viral tracing, extensive MSN projections from the striatum to the SN are established, which formed synaptic connection with GABAergic neurons in SN organoids and showed the optically evoked inhibitory postsynaptic currents and electronic field potentials by labeling the striatum-like organoids with optogenetic virus. Furthermore, these Str-SN assembloids exhibited enhanced calcium activity compared to that of individual striatal organoids. Importantly, we further demonstrated the reciprocal projection defects in HD iPSC-derived assembloids, which could be ameliorated by treatment of brain-derived neurotrophic factor. Taken together, these findings suggest that Str-SN assembloids could be used for identifying MSN projection defects and could be applied as potential drug test platforms for HD.