Human Neuronal Cultures Research Articles

Upregulation of miR-370 and miR-543 is associated with reduced expression of heat shock protein 40 in spinocerebellar ataxia type 3

Molecular chaperones are important regulators of protein folding and proteasomal removal of misfolded proteins. In spinocerebellar ataxia type 3 (SCA3), the co-chaperone DnaJ homology subfamily B member 1 (DNAJB1 or heat shock protein 40) is recruited to protein aggregates formed by the disease-causing mutant polyglutamine (polyQ) protein ataxin-3 (ATXN3). Over-expression of DNAJB1 reduces polyQ protein toxicity. Here, we identified two miRNAs, miR-370 and miR-543, that function in posttranscriptional regulation of DNAJB1 expression. MiRNAs are small endogenously produced RNAs controlling mRNA stability and play a role in polyQ disease pathogenesis. In human neuronal cultures derived from SCA3 patient-specific induced pluripotent stem cell (iPSC) lines, miR-370 and miR-543 levels are upregulated, while DNAJB1 expression is concurrently reduced. These findings suggest that downregulation of DNAJB1 by these two miRNAs is an early event that could contribute to SCA3 pathogenesis. Inhibition of these two miRNAs in turn could stabilize DNAJB1 and thereby be beneficial in SCA3 disease.

NEURONAL APOE MODULATES A SPORADIC ALZHEIMER’S DISEASE PHENOTYPE IN PATIENT-DERIVED INDUCED NEURONS

The APOE-ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (sAD) and may be enriched in other clinically relevant populations. While APOE is predominantly expressed by astrocytes in the central nervous system, neuronal expression of APOE is of increasing interest in age-related cognitive impairment, neurological injury, and neurodegeneration. The human and mouse APOE promoter sequences are dissimilar, and contain distinct regions that control expression in a species specific, cell-type specific manner. APOE mRNA and protein is detectable in primary human neuronal cultures in a manner suggestive of de novo protein synthesis. However, neuronal expression of APOE has not been linked to neurotoxicity in a human system. We reprogrammed induced pluripotent stem cells (iPSCs) from three unrelated sAD patients with the ε3/ε4 genotype, used CRISPR/Cas9 gene editing to generate ε3/ε3 isogenic lines, and differentiated them into pure cultures of glutamatergic neurons with no detectable glia. APOE is synthesized and secreted by human induced neurons. Genetic correction of ε4 to ε3 reduces tau phosphorylation and phosphoactivation of a specific kinase. Moreover, ε4 is associated with altered amyloid secretion, and exacerbates the neurotoxic effects of a calcium ionophore. APOE is expressed in human glia-free neuronal culture system, and APOE isoform alters sAD-associated molecular pathways. More detailed mechanistic studies are required to better understand this phenomenon. However, the net effect is that endogenous expression of APOE-ε4 weakens neurons and contributes to the sAD cellular phenotype independently of, but likely cooperatively with, glial APOE.

Modeling Alzheimer’s disease brains in vitro

Alzheimer’s disease (AD) research is hampered by a lack of models that recapitulate all key disease features. Park et al. introduce a microfluidic device containing a 3D culture of human neurons, astrocytes, and microglia that develop AD-like pathology, revealing a potentially important inflammatory mechanism of neurodegeneration.

Aromatase/Seladin-1 Interactions in Human Neuronal Cell Culture, the Hippocampus of Healthy Rats and Transgenic Alzheimer’s Disease Mice

Background/Aims: Decreasing levels of aromatase and seladin-1 could be one of the molecular mechanisms of Alzheimer’s disease (AD). Aromatase is an enzyme that catalyzes estrogen biosynthesis from androgen precursors, and seladin-1 is an enzyme that converts desmosterol to cholesterol, which is the precursor of all hormones. Verifying the potential relationship between these proteins and accordingly determining new therapeutic targets constitute the aims of this study. Methods: Changes in protein levels were compared in vitro in aromatase and seladin-1 inhibitor-administered human neuroblastoma (SH-SY5Y) cells in vivo in intracerebroventricular (icv) aromatase or seladin-1 inhibitor-administered rats, as well as in transgenic AD mice in which the genes encoding these proteins were knocked out. Results and Conclusions: In the cell cultures, we observed that seladin-1 protein levels increased after aromatase enzyme inhibition. The hippocampal aromatase protein levels decreased following chronic seladin-1 inhibition in icv inhibitor-administered rats; however, the aromatase levels in the dentate gyrus of seladin-1 knockout (SelKO) AD male mice increased. These findings indicate a partial relationship between these proteins and their roles in AD pathology.

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS.

SummaryHuman induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, “Optopatch,” pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

Generation of three-dimensional human neuronal cultures: application to modeling CNS viral infections

BackgroundA variety of neurological disorders including neurodegenerative diseases and infection by neurotropic viruses can cause structural and functional changes in the central nervous system (CNS), resulting in long-term neurological sequelae. An improved understanding of the pathogenesis of these disorders is important for developing efficacious interventions. Human induced pluripotent stem cells (hiPSCs) offer an extraordinary window for modeling pathogen-CNS interactions, and other cellular interactions, in three-dimensional (3D) neuronal cultures that can recapitulate several aspects of in vivo brain tissue.MethodsHerein, we describe a prototype of scaffold-free hiPSC-based adherent 3D (A-3D) human neuronal cultures in 96-well plates. To test their suitability for drug screening, A-3D neuronal cultures were infected with herpes simplex virus type 1 (HSV-1) with or without acyclovir.ResultsThe half maximal inhibitory concentration (IC50) of acyclovir was 3.14 μM and 3.12 μM determined using flow cytometry and the CX7 High Content Screening platform, respectively.ConclusionsOur A-3D neuronal cultures provide an unprecedented opportunity for high-content drug screening programs to treat human CNS infections.

Graphene foam as a biocompatible scaffold for culturing human neurons.

In this study, we explore the use of electrically active graphene foam as a scaffold for the culture of human-derived neurons. Human embryonic stem cell (hESC)-derived cortical neurons fated as either glutamatergic or GABAergic neuronal phenotypes were cultured on graphene foam. We show that graphene foam is biocompatible for the culture of human neurons, capable of supporting cell viability and differentiation of hESC-derived cortical neurons. Based on the findings, we propose that graphene foam represents a suitable scaffold for engineering neuronal tissue and warrants further investigation as a model for understanding neuronal maturation, function and circuit formation.

Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures

Long-term neural differentiation of human pluripotent stem cells (hPSCs) is associated with enhanced neuronal maturation, which is a necessity for creation of representative in vitro models. It also induces neurogenic-to-gliogenic fate switch, increasing proportion of endogenous astrocytes formed from the common neural progenitors. However, the significance of prolonged differentiation on the neural cell type composition and functional development of hPSC-derived neuronal cells has not been well characterized. Here, we studied two hPSC lines, both of which initially showed good neuronal differentiation capacity. However, the propensity for endogenous astrogenesis and maturation state after extended differentiation varied. Live cell calcium imaging revealed that prolonged differentiation facilitated maturation of GABAergic signaling. According to extracellular recordings with microelectrode array (MEA), neuronal activity was limited to fewer areas of the culture, which expressed more frequent burst activity. Efficient maturation after prolonged differentiation also promoted organization of spontaneous activity by burst compaction. These results suggest that although prolonged neural differentiation can be challenging, it has beneficial effect on functional maturation, which can also improve transition to different neural in vitro models and applications.

Differential susceptibility of striatal, hippocampal and cortical neurons to Caspase-6.

Active cysteinyl protease Caspase-6 is associated with early Alzheimer and Huntington diseases. Higher entorhinal cortex and hippocampal Caspase-6 levels correlate with lower cognitive performance in aged humans. Caspase-6 induces axonal degeneration in human primary neuron cultures and causes inflammation and neurodegeneration in mouse hippocampus, and age-dependent memory impairment. To assess whether Caspase-6 causes damage to another neuronal system, a transgenic knock-in mouse overexpressing a self-activated form of Caspase-6 five-fold in the striatum, the area affected in Huntington disease, and 2.5-fold in the hippocampus and cortex, was generated. Detection of Tubulin cleaved by Caspase-6 confirmed Caspase-6 activity. The Caspase-6 expressing mice and control littermates were subjected to behavioral tests to assess Huntington disease-relevant psychiatric, motor, and cognitive deficits. Depression was excluded with the forced swim and sucrose consumption tests. Motor deficits were absent in the nesting, clasping, rotarod, vertical pole, gait, and open field analyzes. However, Caspase-6 mice developed age-dependent episodic and spatial memory deficits identified by novel object recognition, Barnes maze and Morris water maze assays. Neuron numbers were maintained in the striatum, hippocampus, and cortex. Microglia and astrocytes were increased in the hippocampal stratum lacunosum molecular and in the cortex, but not in the striatum. Synaptic mRNA profiling identified two differentially expressed genes in transgenic hippocampus, but none in striatum. Caspase-6 impaired synaptic transmission and induced neurodegeneration in hippocampal CA1 neurons, but not in striatal medium spiny neurons. These data revealed that active Caspase-6 in the striatal medium spiny neurons failed to induce inflammation, neurodegeneration or behavioral abnormalities, whereas active Caspase-6 in the cortex and hippocampus impaired episodic and spatial memories, and induced inflammation, neuronal dysfunction, and neurodegeneration. The results indicate age and neuronal subtype-dependent Caspase-6 toxicity and highlight the importance of targeting the correct neuronal subtype to identify underlying molecular mechanisms of neurodegenerative diseases.

Targeting Human Astrocytes' Calcium-sensing Receptors for Treatment of Alzheimer's Disease.

Understanding the pathophysiology of Alzheimer's disease (AD) in the principal human neural cells is necessary for finding therapeutics for this illness. To help do this, we have been using freshly cultured functionally normal cerebral cortical adult human astrocytes (NAHAs) and postnatal neurons. The findings show that amyloid-β oligomers (Aβ-os) binding to calcium-sensing receptors (CaSRs) on NAHAs and neuron surfaces trigger signals capable of driving AD pathogenesis. This Aβ•CaSR signalling shifts the amyloid precursor protein (APP) from its α-secretase shedding producing neurotrophic/neuroprotective soluble (s)APPα to its β-secretase cleaving engendering AD-driving Aβ42/Aβ42-os peptides. Aβ•CaSR signalling in NAHAs also drives the release of toxic hyper-phosphorylated Tau proteins in exosomes, and of nitric oxide, and VEGF-A. These several harmful agents comprise the neuron-killing machinery, driving the very slowly spreading AD neurocontagion. VEGF-A over-secretion from Aβ-exposed blood vessel-attached astrocytes induces a functional magnetic resonance imaging- detectable hippocampal neoangiogenesis which indicates approaching AD in amnestic minor cognitive impairment (aMCI) patients. Most important in AD's regard, selective allosteric CaSR antagonists (calcylitics) added to Aβ42/Aβ42-os-exposed NAHAs (or to human neuron cultures) rescue the extracellular shedding of neurotrophic/ neuroprotective sAPPα and suppress all the neurotoxic effects of Aβ•CaSR signalling even when multiple microglial cytokines are also present. Therefore, since the multipotent calcilytics would be reasonably safe and inexpensive drugs for humans, it is worthwhile testing them as AD therapeutics in clinical trials especially in persons in the earliest detectable stages of AD neuropathology progression such as aMCI.

Functional Comparison of Cultures of IPSC-Derived Human Neurons and Rodent Neurons

Functional Comparison of Cultures of IPSC-Derived Human Neurons and Rodent Neurons

Assessing electrical activity of human neuronal cell culture networks grown on 3D fibrous scaffolds using different techniques: A comparative study

Assessing electrical activity of human neuronal cell culture networks grown on 3D fibrous scaffolds using different techniques: A comparative study

Impairment of Thiamine Transport at the GUT-BBB-AXIS Contributes to Wernicke's Encephalopathy.

Wernicke's encephalopathy, a common neurological disease, is caused by thiamine (vitamin B1) deficiency. Neuropathy resulting from thiamine deficiency is a hallmark of Wernicke-Korsakoff syndrome in chronic alcohol users. The underlying mechanisms of this deficiency and progression of neuropathy remain to be understood. To uncover the unknown mechanisms of thiamine deficiency in alcohol abuse, we used chronic alcohol consumption or thiamine deficiency diet ingestion in animal models. Observations from animal models were validated in primary human neuronal culture for neurodegenerative process. We employed radio-labeled bio-distribution of thiamine, qualitative and quantitative analyses of the various biomarkers and neurodegenerative process. In the present studies, we established that disruption of thiamine transport across the intestinal gut blood-brain barrier axis as the cause of thiamine deficiency in the brain for neurodegeneration. We found that reduction in thiamine transport across these interfaces was the cause of reduction in the synthesis of thiamine pyrophosphate (TPP), an active cofactor for pyruvate dehydrogenase E1α (PDHE1α). Our findings revealed that decrease in the levels of PDHE1α cofactors switched on the activation of PD kinase (PDK) in the brain, thereby triggering the neuronal phosphorylation of PDHE1α (p-PDHE1α). Dysfunctional phosphorylated PDHE1α causes the reduction of mitochondrial aerobic respiration that led to neurodegeneration. We concluded that impairment of thiamine transport across the gut-BBB-axis that led to insufficient TPP synthesis was critical to Wernicke-neuropathy, which could be effectively prevented by stabilizing the thiamine transporters.

Is trehalose an autophagic inducer? Unraveling the roles of non-reducing disaccharides on autophagic flux and alpha-synuclein aggregation

Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.

Retracted: Anti-ribosomal-phosphoprotein autoantibodies penetrate to neuronal cells via neuronal growth associated protein, affecting neuronal cells in vitro.

OBJECTIVE Anti-ribosomal-phosphoprotein antibodies (anti-Ribos.P Abs) are detected in 10-45% of NPSLE patients. Intracerebroventricular administration of anti-ribosomal-P Abs induces depression-like behaviour in mice. We aimed to discern the mechanism by which anti-Ribos.P Abs induce behavioural changes in mice. METHODS Anti-Ribos.P Abs were exposed to human and rat neuronal cell cultures, as well as to human umbilical vein endothelial cell cultures for a control. The cellular localization of anti-Ribo.P Abs was found by an immunofluorescent technique using a confocal microscope. Identification of the target molecules was undertaken using a cDNA library. Immunohistochemistry and an inhibition assay were carried out to confirm the identity of the target molecules. Neuronal cell proliferation was measured by bromodeoxyuridine, and Akt and Erk expression by immunoblot. RESULTS Human anti-Ribos.P Abs penetrated into human neuronal cells and rat hippocampal cell cultures in vitro, but not to endothelial cells as examined. Screening a high-content human cDNA-library with anti-Ribos.P Abs identified neuronal growth-associated protein (GAP43) as a target for anti-Ribos.P Abs. Ex vivo anti-Ribos.P Abs bind to mouse brain sections of hippocampus, dentate and amygdala. Anti-Ribos.P Abs brain-binding was prevented by GAP43 protein. Interestingly, GAP43 inhibited in a dose-dependent manner the anti-Ribos.P Abs binding to recombinant-ribosomal-P0, indicating mimicry between the ribosomal-P0 protein and GAP43. Furthermore, anti-Ribos.P Abs reduced neuronal cell proliferation activity in vitro (P < 0.001), whereas GAP43 decreased this inhibitory activity by a factor of 7.6. The last was related to Akt and Erk dephosphorylation. CONCLUSION Anti-Ribos.P Abs penetrate neuronal cells in vitro by targeting GAP43. Anti -Ribos.P Abs inhibit neuronal-cell proliferation via inhibition of Akt and Erk. Our data contribute to deciphering the mechanism for anti-Ribos.P Abs' pathogenic activity in NPSLE.

Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation.

Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy.IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.

Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1\u03b2

BackgroundThe cause of neurodegeneration in progressive forms of multiple sclerosis is unknown. We investigated the impact of specific neuroinflammatory markers on human neurons to identify potential therapeutic targets for neuroprotection against chronic inflammation.MethodsSurface immunocytochemistry directly visualized protease-activated receptor-1 (PAR1) and interleukin-1 (IL-1) receptors on neurons in human postmortem cortex in patients with and without neuroinflammatory lesions. Viability of cultured neurons was determined after exposure to cerebrospinal fluid from patients with progressive multiple sclerosis or purified granzyme B and IL-1β. Inhibitors of PAR1 activation and of PAR1-associated second messenger signaling were used to elucidate a mechanism of neurotoxicity.ResultsImmunohistochemistry of human post-mortem brain tissue demonstrated cells expressing higher amounts of PAR1 near and within subcortical lesions in patients with multiple sclerosis compared to control tissue. Human cerebrospinal fluid samples containing granzyme B and IL-1β were toxic to human neuronal cultures. Granzyme B was neurotoxic through activation of PAR1 and subsequently the phospholipase Cβ-IP3 second messenger system. Inhibition of PAR1 or IP3 prevented granzyme B toxicity. IL-1β enhanced granzyme B-mediated neurotoxicity by increasing PAR1 expression.ConclusionsNeurons within the inflamed central nervous system are imperiled because they express more PAR1 and are exposed to a neurotoxic combination of both granzyme B and IL-1β. The effects of these inflammatory mediators may be a contributing factor in the progressive brain atrophy associated with neuroinflammatory diseases. Knowledge of how exposure to IL-1β and granzyme B act synergistically to cause neuronal death yields potential novel neuroprotective treatments for neuroinflammatory diseases.

Pharmacologically increasing microtubule acetylation corrects stress-exacerbated effects of organophosphates on neurons.

Many veterans of the 1990-1991 Gulf War contracted Gulf War Illness (GWI), a multisymptom disease that primarily affects the nervous system. Here, we treated cultures of human or rat neurons with diisopropyl fluorophosphate (DFP), an analog of sarin, one of the organophosphate (OP) toxicants to which the military veterans were exposed. All observed cellular defects produced by DFP were exacerbated by pretreatment with corticosterone or cortisol, which, in rat and human neurons, respectively, serves in our experiments to mimic the physical stress endured by soldiers during the war. To best mimic the disease, DFP was used below the level needed to inhibit acetylcholinesterase. We observed a diminution in the ratio of acetylated to total tubulin that was correctable by treatment with tubacin, a drug that inhibits HDAC6, the tubulin deacetylase. The reduction in microtubule acetylation was coupled with deficits in microtubule dynamics, which were correctable by HDAC6 inhibition. Deficits in mitochondrial transport and dopamine release were also improved by tubacin. Thus, various negative effects of the toxicant/stress exposures were at least partially correctable by restoring microtubule acetylation to a more normal status. Such an approach may have therapeutic benefit for individuals suffering from GWI or other neurological disorders linked to OP exposure.

Function of Metallothionein-3 in Neuronal Cells: Do Metal Ions Alter Expression Levels of MT3?

A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression analysis identified significant variations in the mRNA levels of genes associated with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3.

Molecular Profiling of Human Induced Pluripotent Stem Cell-Derived Hypothalamic Neurones Provides Developmental Insights into Genetic Loci for Body Weight Regulation.

Recent data suggest that common genetic risks for metabolic disorders such as obesity may be human-specific and exert effects via the central nervous system. To overcome the limitation of human tissue access for study, we have generated induced human pluripotent stem cell (hiPSC)-derived neuronal cultures that recapture many features of hypothalamic neurones within the arcuate nucleus. In the present study, we have comprehensively characterised this model across development, benchmarked these neurones to invivo events, and demonstrate a link between obesity risk variants and hypothalamic development. The dynamic transcriptome across neuronal maturation was examined using microarray and RNA sequencing methods at nine time points. K-means clustering of the longitudinal data was conducted to identify co-regulation and microRNA control of biological processes. The transcriptomes were compared with those of 103 samples from 13 brain regions reported in the Genotype-Tissue Expression database (GTEx) using principal components analysis. Genes with proximity to body mass index (BMI)-associated genetic variants were mapped to the developmentally expressed genesets, and enrichment significance was assessed with Fisher's exact test. The human neuronal cultures have a transcriptional and physiological profile of neuropeptide Y/agouti-related peptide arcuate nucleus neurones. The neuronal transcriptomes were highly correlated with adult hypothalamus compared to any other brain region from the GTEx. Also, approximately 25% of the transcripts showed substantial changes in expression across neuronal development and potential co-regulation of biological processes that mirror neuronal development invivo. These developmentally expressed genes were significantly enriched for genes in proximity to BMI-associated variants. We confirmed the utility of this invitro human model for studying the development of key hypothalamic neurones involved in energy balance and show that genes at loci associated with body weight regulation may share a pattern of developmental regulation. These data support the need to investigate early development to elucidate the human-specific central nervous system pathophysiology underlying obesity susceptibility.