Neurons In Early Stages Research Articles

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

Defined, Simplified, Scalable, and Clinically Compatible Hydrogel-Based Production of Human Brain Organoids

Human brain organoids present a new paradigm for modeling human brain organogenesis, providing unprecedented insight to the molecular and cellular processes of brain development and maturation. Other potential applications include in vitro models of disease and tissue trauma, as well as three-dimensional (3D) clinically relevant tissues for pharmaceuticals development and cell or tissue replacement. A key requirement for this emerging technology in both research and medicine is the simple, scalable, and reproducible generation of organoids using reliable, economical, and high-throughput culture platforms. Here we describe such a platform using a defined, clinically compliant, and readily available hydrogel generated from gelatin methacrylate (GelMA). We demonstrate the efficient production of organoids on GelMA from human induced pluripotent stem cells (iPSCs), with scalable production attained using 3D printed GelMA-based multiwell arrays. The differentiation of iPSCs was systematic, rapid, and direct to enable iPSCs to form organoids in their original position following seeding on GelMA, thereby avoiding further cell and organoid disruption. Early neural precursors formed by day 5, neural rosettes and early-stage neurons by day 14, and organoids with cellular and regional heterogeneity, including mature and electrophysiologically active neurons, by day 28. The optimised method provides a simplified and well-defined platform for both research and translation of iPSCs and derivative brain organoids, enabling reliable 3D in vitro modelling and experimentation, as well as the provision of clinically relevant cells and tissues for future therapeutics.

Identification of inflammasome signaling proteins in neurons and microglia in early and intermediate stages of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that destroys memory and cognitive function. Inflammasome activation has been suggested to play a critical role in the neuroinflammatory response in AD progression, but the cell-type expression of inflammasome proteins in the brain has not been fully characterized. In this study, we used samples from the hippocampus formation, the subiculum, and the entorhinal cortex brain from 17 donors with low-level AD pathology and 17 intermediate AD donors to assess the expression of inflammasome proteins. We performed analysis of hippocampal thickness, β-amyloid plaques, and hyperphosphorylated tau to ascertain the cellular pathological changes that occur between low and intermediate AD pathology. Next, we determined changes in the cells that express the inflammasome sensor proteins NOD-like receptor proteins (NLRP) 1 and 3, and caspase-1. In addition, we stained section with IC100, a humanized monoclonal antibody directed against the inflammasome adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and a commercially available anti-ASC antibody. Our results indicate that hippocampal cortical thickness did not significantly change between low and intermediate AD pathology, but there was an increase in pTau and β-amyloid clusters in intermediate AD cases. NLRP3 was identified mainly in microglial populations, whereas NLRP1 was seen in neuronal cytoplasmic regions. There was a significant increase of ASC in neurons labeled by IC100, whereas microglia in the hippocampus and subiculum were labeled with the commercial anti-ASC antibody. Caspase-1 was present in the parenchyma in the CA regions where amyloid and pTau were identified. Together, our results indicate increased inflammasome protein expression in the early pathological stages of AD, that IC100 identifies neurons in early stages of AD and that ASC expression correlates with Aβ and pTau in postmortem AD brains.

A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

SummaryThe maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicular glutamate transporter 1 [VGlut1]) and inhibitory (GABA) markers, while late-stage cultures (day 40) expressed solely VGlut1, indicating a purely excitatory phenotype without containing glial cells. This maturation process was further validated utilizing patch clamp and MEA analysis. Particularly, induced long-term potentiation (LTP) successfully persisted for 1 h in day 40 cultures, but only achieved LTP in the presence of the GABAA receptor antagonist picrotoxin in day 21 cultures. This system was also applied to epilepsy modeling utilizing bicuculline and its correction utilizing the anti-epileptic drug valproic acid.

Balance alterations and reduction of pedunculopontine cholinergic neurons in early stages of parkinsonism in middle-aged rats.

The purpose of the present study was to investigate balance alterations and the possible role of the cholinergic neurons in the pedunculopontine nucleus (PPN) in the early stages of a progressive animal model of Parkinson's disease (PD). Twenty-eight middle-aged (8-9months) male Wistar rats received 4 or 10 subcutaneous vehicle (control, CTL) or reserpine (RES) injections (0.1mg/kg). The animals were submitted to different behavioral tests. Forty-eight hours after the 4th injection, half of the animals of each group (n=7) were perfused and submitted to immunohistochemical analysis for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT). The remaining animals (n=7 per group) were killed 48h after the 10th injection. RES group presented motor deficits in the catalepsy and open field tests starting at days 12 and 20 of treatment, respectively (only for the animals that received 10 injections). On the other hand, dynamic and static balance changes were observed at earlier stages of RES treatment, starting at days 6 and 4, respectively. At this point of the treatment, there was no decrease in the number of TH immunoreactivity neurons in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and dorsal striatum (DS). However, a decrease was observed in SNpc and dorsal striatum of animals that received 10 injections. In contrast, there was a decrease in the number of ChAT immunoreactive cells in PPN concomitantly to the balance alterations at the early stages of treatment (after 4 RES injections). Thus, by mimicking the progressiveness of PD, the reserpine model made it possible to identify static and dynamic balance impairments prior to the motor alterations in the catalepsy and open field tests. In addition, changes in balance were accompanied by a reduction in the number of ChAT immunoreactive cells in NPP in the early stages of treatment.

AKAP-mediated feedback control of cAMP gradients in developing hippocampal neurons.

Cyclic AMP (cAMP) and protein kinase A (PKA), classical examples of spatially compartmentalized signaling molecules, are critical axon determinants that regulate neuronal polarity and axon formation, yet little is known about micro-compartmentalization of cAMP and PKA signaling and its role in developing neurons. Here, we revealed that cAMP forms a gradient in developing hippocampal neurons, with higher cAMP levels in more distal regions of the axon compared to other regions of the cell. Interestingly, this cAMP gradient changed according to the developmental stage and depended on proper anchoring of PKA by A-kinase anchoring proteins (AKAPs). Disrupting PKA anchoring to AKAPs increased the cAMP gradient in early-stage neurons and led to enhanced axon elongation. Our results provide new evidence for a local negative feedback loop, assembled by AKAPs, for the precise control of a growth-stage-dependent cAMP gradient to ensure proper axon growth.

Neural Quadratic Discriminant Analysis: Nonlinear Decoding with V1-Like Computation.

Linear-nonlinear (LN) models and their extensions have proven successful in describing transformations from stimuli to spiking responses of neurons in early stages of sensory hierarchies. Neural responses at later stages are highly nonlinear and have generally been better characterized in terms of their decoding performance on prespecified tasks. Here we develop a biologically plausible decoding model for classification tasks, that we refer to as neural quadratic discriminant analysis (nQDA). Specifically, we reformulate an optimal quadratic classifier as an LN-LN computation, analogous to "subunit" encoding models that have been used to describe responses in retina and primary visual cortex. We propose a physiological mechanism by which the parameters of the nQDA classifier could be optimized, using a supervised variant of a Hebbian learning rule. As an example of its applicability, we show that nQDA provides a better account than many comparable alternatives for the transformation between neural representations in two high-level brain areas recorded as monkeys performed a visual delayed-match-to-sample task.

Building Bridges for Spinal Cord Repair

For nearly a century, neuroscientists have sought to restore neurological function across spinal cord lesions. Lu etal. now present significant progress toward this goal, showing in rats that transplanted neural stem cells establish a functional bridge across completely transected spinal cords.

Transcriptional signatures mediated by acetylation overlap with early-stage Alzheimer’s disease

The mechanisms by which environmental influences lead to the development of complex neurodegenerative diseases are largely unknown. It is known, however, that epigenetic mechanisms can mediate alterations in transcription due to environmental influences. In order to identify genes susceptible to regulation in the adult cortex by one type of epigenetic mechanism, histone, and protein acetylation, we treated mice with the histone deacetylase inhibitor Trichostatin A (TSA). After 1week of treatment with TSA, RNA was extracted from the brain cortices of mice and gene expression differences were analyzed by microarray profiling. The altered genes were then compared with genes differentially expressed in microarray studies of disease by database and literature searches. Genes regulated by TSA were found to significantly overlap with differentially expressed genes in the Alzheimer's disease (AD) brain. Several TSA-regulated genes involved in chromatin remodeling and epigenetic reprogramming including histone cluster 1, H4h (Hist1H4h), methionine adenosyltransferase II, alpha (Mat2a), and 5-methyltetrahydrofolate homocysteine reductase (Mtrr) overlapped with genes altered in early-stage AD in gray matter. We also show that the expression of hemoglobin, which has been shown to be altered in neurons in the AD brain, is regulated by TSA treatment. This analysis suggests involvement of epigenetic mechanisms in neurons in early stages of AD.

Endoplasmic Reticulum Stress in Motor Neurons of the Spinal Cord in Sporadic Amyotrophic Lateral Sclerosis

The accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) lumen causes a cellular stress response termed the unfolded protein response. Although ER stress has been implicated in various neurodegenerative diseases, the morphological features of aggregated proteins in ER lumina that may cause neurodegeneration have not been well characterized. We examined anterior horn neurons using immunohistochemistry and electron microscopy in 12 sporadic amyotrophic lateral sclerosis (ALS) patients and 12 controls. Approximately 2.6% of both normal-appearing and degenerated motor neurons in ALS cases were immunostained for the ER chaperone protein glucose-regulated protein 78, and approximately 0.1% of these neurons was glucose-regulated protein 78 positive in controls (p = 0.0004). Amyotrophic lateral sclerosis cases also tended to have glucose-regulated protein 78-positive motor neurons more frequently than control cases (p = 0.08). By electron microscopy,neurons in ALS patients showed accumulations of amorphous and granular material suggestive of misfolded or unfolded proteins in dilated predominantly normal-appearing ER. There were also wavy membranous structures extending from the ER membranes that lacked membrane-bound ribosomes, electron-dense material resembling Bunina bodies, Hirano bodies, honeycomb-like structures, and membrane-particle complexes associated with the ER in these neurons. Control sample neurons demonstrated none of these features. These ER alterations suggest that the unfolded protein response is activated in motor neurons in ALS patients and provide the first morphological evidence that ER stress may be involved in the neurodegeneration of motor neurons in early stages of sporadic ALS.

Ca 2+ and acidosis synergistically lead to the dysfunction of cortical GABAergic neurons during ischemia

Cell death in cerebral ischemia is presumably initiated by neural excitotoxicity resulted from the dysfunction of inhibitory neurons in early stage. Molecular processes underlying the ischemic injury of inhibitory neurons remain to be elusive, which we investigated by biochemical manipulations with cellular imaging and patch clamp at GFP-labeled GABAergic cells in cortical slices. Ischemia induces Ca 2+ elevation, acidosis and dysfunction in GABAergic cells. An elevation of cytoplasmic Ca 2+ or H + impairs the encoding of action potentials in these neurons. The effects of Ca 2+ and H + are additive in nature and occlude ischemic outcomes. Ischemia impairs spike production through prolonging spike refractory periods and raising threshold potentials. Therefore, calcium toxicity and acidosis during ischemia synergistically impair the dynamics of sodium channels and function of cortical GABAergic neurons, which lead to neural excitotoxicity. Our results also suggest that the cocktail therapeutics is needed to prevent neuronal death from ischemia.

A threshold explains modulation of neural responses to opposite-contrast stereograms.

Disparity-sensitive neurons respond to contrast-inverted stereograms (aRDS) that do not evoke depth percepts. This is in conflict with the idea that such neurons are the direct correlate of depth perception. However, the output of neurons responding to aRDS may be further processed: neurons at later processing stages show weaker responses to aRDS than early stage neurons. Here, we show that such a response hierarchy emerges in a three-layered neural network. A numerical analysis demonstrates that threshold operations can largely explain the network's behavior as well as the electrophysiological data. An extension of the energy neuron model for disparity-sensitive neurons predicts increased responses to aRDS for an identifiable sub-class of cells and can thus be tested in electrophysiological experiments.

Shape and contour detection

Detectability of contours may be affected by long-range interactions between neurons in early stages of visual cortex. Specifically, neurons with receptive fields arrayed along the length of a contour may facilitate each other in a position- and orientation-dependent manner. Accordingly, the overall geometry of a contour should significantly influence both the strength of these long-range interactions and the contour’s detectability. Psychophysical experiments measuring the detectability of sampled, curvilinear contours hidden by randomly-oriented and -positioned noise elements revealed two main findings. First, changes in direction of curvature degraded contour detectability. Second, the effect of changes in magnitude of curvature were predicted by the average of local curvature along the length of the contour. While the first result emphasizes the importance of uniform direction of curvature, the second result rules out penalties for deviation from circularity.

Fate of presynaptic afferents to Purkinje cells in the adult nervous mutant mouse: A model to study presynaptic stabilization

The hemispheric cerebellar cortex from 174- to 420-day-old nervous mice has been studied with Golgi, Cajal-reduced silver and electron microscopic techniques. In all mice, the existence of a continuous process of Purkinje cell death, indicated by the presence of few of these neurons in early stages of degeneration, has been established. The morphology of such degenerating cells, as well as the removal of the necrotic debris and the consecutive isolation of presynaptic fibers by glia are described. In the molecular layer, and in spite of the important Purkinje cell loss, numerous varicosities belonging to parallel fibers persist. They can be either synapsing on interneurons or concentrated in small clusters of 2–10 boutons covered by astrocytic processes. The synaptic investment of stellate and basket cells, as well as the length and the branching pattern of their dendritic fields are not significantly changed, besides the increase in afferent terminals deprived of their postsynaptic targets. Basket formations, as numerous as in control cerebellum, are forming a discontinuous row at the superficial third of the granular layer. Over 90% of these baskets are devoid of Purkinje cells, however, their ultrastructural features mimic those of the same terminals in control mice, with the exception that few of them can either establish heterologous synapses with granule cell dendrites, or develop gap junctions between them. The significance of the persistence of normal looking presynaptic elements long time after deprvation of their postsynaptic targets is discussed in relation to the role that function can play in the stabilization of synaptic contacts.