Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neuron function. Although ophthalmic deficits are not considered a classic symptom of ALS, recent studies suggest that changes in retinal cells, similar to those in the spinal cord motor neurons, have been observed in postmortem human tissues and animal models.Methods: In this study, we examined by immunofluorescence analysis the retinal cell layers of sporadic ALS patients in post-mortem retinal slices. We evaluated the presence of cytoplasmic TDP-43 and SQSTM1/p62 aggregates, activation of the apoptotic pathway, and microglia and astrocytes reactivity.Results: We found in the retinal ganglion cell layer of ALS patients the increase of mislocalized TDP-43, SQSTM1/p62 aggregates, activation of cleaved caspase-3, and microglia density, suggesting that retinal changes can be used as an additional diagnostic tool for ALS.Discussion: The retina is considered part of the central nervous system, and neurodegenerative changes in the brain may be accompanied by structural and possibly functional changes in the neuroretina and ocular vasculature. Therefore, using in vivo retinal biomarkers as an additional diagnostic tool for ALS may provide an opportunity to longitudinally monitor individuals and therapies over time in a noninvasive and cost-effective manner.

Maintaining the excitability of neurons and circuits is fundamental for healthy brain functions. The global compensatory increase in excitatory synaptic strength, in response to decreased activity, is one of the main homeostatic mechanisms responsible for such regulation. This type of plasticity has been extensively characterized in rodents in vivo and in vitro, but few data exist on human neurons maturation. We have generated an in vitro cortical model system, based on differentiated human-induced pluripotent stem cells, chronically treated with tetrodotoxin, to investigate homeostatic plasticity at different developmental stages. Our findings highlight the presence of homeostatic plasticity in human cortical networks and show that the changes in synaptic strength are due to both pre- and post-synaptic mechanisms. Pre-synaptic plasticity involves the potentiation of neurotransmitter release machinery, associated to an increase in synaptic vesicle proteins expression. At the post-synaptic level, we report an increase in the expression of post-synaptic density proteins, involved in glutamatergic receptor anchoring. These results extend our understanding of neuronal homeostasis and reveal the developmental regulation of its expression in human cortical networks. Since induced pluripotent stem cell-derived neurons can be obtained from patients with neurodevelopmental and neurodegenerative diseases, our platform offers a versatile model for assessing human neural plasticity under physiological and pathological conditions.

INTRODUCTION: The intronic MAPT mutation IVS10+16 is linked to familial frontotemporal dementia, causing hyperphosphorylation and accumulation of tau protein, resulting in synaptic and neuronal loss and neuroinflammation in patients. This mutation disrupts MAPT gene splicing, increasing exon 10 inclusion and leading to an imbalance of 3R and 4R Tau isoforms.METHODS: We generated patterned cortical organoids from isogenic control and mutant human induced pluripotent stem cell (iPSC) lines. Nanostring gene expression analysis, immunofluorescence, and calcium imaging recordings were used to study the impact of the MAPT IVS10+16 mutation on neuronal development and function.RESULTS: Tau mutant cortical organoids showed altered mitochondrial function and gene expression related to neuronal development, with synaptic markers and neuronal activity reduction. Bezafibrate treatment restored mitochondrial content and rescued synaptic functionality and tau physiology.DISCUSSION: These findings suggest that targeting mitochondrial function with bezafibrate could potentially reverse tau‐induced neurodevelopmental deficits, highlighting its therapeutic potential for tauopathies like frontotemporal dementia.Highlights: The IVS 10+16 MAPT mutation significantly disrupts cortical differentiation and synaptic maturation, evidenced by downregulated genes associated with synapses and neuronal development. Tau‐mutant cortical organoids exhibit mitochondrial dysfunction, with fewer and smaller mitochondria alongside tau hyperphosphorylation and aggregation, which further contribute to neuronal damage and disease progression. Treatment with bezafibrate effectively normalizes mitochondrial parameters, enhances neuronal integrity and synaptic maturation, and restores network functionality, showcasing its promise as a therapeutic strategy for tauopathies. The 3D in vitro disease model used in this study proves valuable for studying tauopathies and testing new drugs, effectively mimicking key aspects of tau‐related neurodegeneration.

It has been recently demonstrated that the exposure of naive neuronal cells to light—at the basis of optogenetic techniques and calcium imaging measurements—may alter neuronal firing. Indeed, understanding the effect of light on nongenetically modified neurons is crucial for a correct interpretation of calcium imaging and optogenetic experiments. Here we investigated the effect of continuous visible LED light exposure (490 nm, 0.18 − 1.3 m W / m m 2 ) on spontaneous activity of primary neuronal networks derived from the early postnatal mouse cortex. We demonstrated, by calcium imaging and patch clamp experiments, that illumination higher than 1.0 m W / m m 2 causes an enhancement of network activity in cortical cultures. We investigated the possible origin of the phenomena by blocking the transient receptor potential vanilloid 4 (TRPV4) channel, demonstrating a complex connection between this temperature-dependent channel and the measured effect. The results presented here shed light on an exogenous artifact, potentially present in all calcium imaging experiments, that should be taken into account in the analysis of fluorescence data.

The comprehension of neuronal network functioning, from most basic mechanisms of signal transmission to complex patterns of memory and decision making, is at the basis of the modern research in experimental and computational neurophysiology. While mechanistic knowledge of neurons and synapses structure increased, the study of functional and effective networks is more complex, involving emergent phenomena, nonlinear responses, collective waves, correlation and causal interactions. Refined data analysis may help in inferring functional/effective interactions and connectivity from neuronal activity. The Transfer Entropy (TE) technique is, among other things, well suited to predict structural interactions between neurons, and to infer both effective and structural connectivity in small- and large-scale networks. To efficiently disentangle the excitatory and inhibitory neural activities, in the article we present a revised version of TE, split in two contributions and characterized by a suited delay time. The method is tested on in silico small neuronal networks, built to simulate the calcium activity as measured via calcium imaging in two-dimensional neuronal cultures. The inhibitory connections are well characterized, still preserving a high accuracy for excitatory connections prediction. The method could be applied to study effective and structural interactions in systems of excitable cells, both in physiological and in pathological conditions.

Human induced pluripotent stem cell (iPSC)-derived neural organoids have emerged as valuable models for investigating human neurodevelopment and neurological disorders. However, their complex three-dimensional architecture poses significant challenges for conventional histological approaches, which rely on physical sectioning and inevitably disrupt spatial relationships within the tissue. Volumetric imaging of intact organoids typically requires light-sheet fluorescence microscopy, a technology not widely accessible to standard cell biology laboratories. Here, we show that solvent-based tissue clearing, using the iDISCO+ and Visikol^® HISTO protocols, combined with conventional laser-scanning and spinning-disk confocal microscopy, platforms already available in most imaging facilities, is sufficient to resolve neuroepithelial rosette-like structures, neuronal networks, and astroglial components within intact human cortical and retinal organoids, while preserving immunofluorescent labeling and tissue architecture. The workflow was also compatible with commonly used immunofluorescence markers. Although light-sheet fluorescence microscopy remains advantageous for large-scale whole-sample imaging, our results show that cleared human organoids within the size range analyzed here can be effectively visualized using accessible confocal systems. This study provides a practical strategy for three-dimensional imaging of intact human neural organoids, facilitating spatial analysis of developmental organization and disease-relevant phenotypes in laboratories without dedicated light-sheet microscopy infrastructure.