Volume electron microscopy (vEM) has become a powerful tool for 3D ultrastructural analysis of neural circuits, yet its application to human brain organoids remains limited, particularly for connectomic studies. Here, we established a comprehensive and scalable workflow for applying vEM to human cortical organoids, integrating correlative light and electron microscopy, large-area SEM (scanning electron microscopy) mosaic imaging, focused ion beam–SEM (FIB-SEM), and transmission electron microscopy (TEM) validation. By systematically comparing two embedding protocols in use, we demonstrated that the DeFelipe and Fairén (1993); Cano-Astorga et al. (2024a) method provides optimal compatibility with toluidine blue–stained semithin sectioning and enables reliable synapse segmentation and neurite tracing. In contrast, the Deerinck’s protocol (2010) offers enhanced membrane contrast but limits postsynaptic density visualization. Using FIB-SEM imaging of peripheral, neuropil-like regions of cortical organoids, we achieved accurate 3D reconstruction of synapses, neurites and intracellular organelles, enabling quantitative assessment of synaptic apposition surfaces, neurite trajectories, and organelle distribution across defined cellular compartments. Together, our results demonstrate for the first time the feasibility of micro-connectomic reconstruction in human cortical organoids at nanometer resolution. This methodological framework expands the applicability of vEM to organoid systems and provides a robust foundation for future studies of human brain development, disease modeling, and therapeutic evaluation at the synaptic and subcellular level. Workflow for vEM–based micro-connectomic analysis in human cortical organoids. Cortical organoids underwent sample preparation, resin embedding, and semithin/ultrathin sectioning. Large-area SEM mosaic imaging was used to identify regions of interest for targeted FIB-SEM acquisition. Ultrastructural observations were independently validated by TEM, establishing a scalable pipeline for connectome reconstruction and subcellular analysis in human brain organoids. This image was created with BioRender

Major depressive disorder and bipolar disorders are prevalent mental health conditions that significantly impact quality of life and life expectancy. These mood disorders involve major depressive episodes (MDE), which pose a substantial burden for patients and their families. While psychopharmacological therapies are a first-line treatment for MDE, the response is often incomplete. New approaches focused on the human-nature relationship may potentially complement antidepressant treatments, thus reducing psychopharmacological needs. This study aims to evaluate whether green exposure affects depressive symptoms and inflammatory biomarker levels in patients with MDE. This prospective study examined the association between exposure to green environments such as woods, forests, large parks, and gardens for at least 45 minutes twice a week, depressive symptoms, and inflammatory biomarkers in 31 patients with an ongoing MDE. The findings suggest that exposure to greenness, together with the modification of antidepressant therapy, is associated with improved depressive symptoms, lower levels of inflammatory biomarker interleukin-6, and higher concentrations of adiponectin after six weeks of treatment. These results suggest that exposure to green environments may have a favorable impact both on mental health and on inflammatory processes, and thus represent a complementary therapeutic strategy. Such information could be relevant to clinicians and urban planners.

Inspired from the historical debate "nature" versus "nurture", the concept "exposome" recently emerged as a comprehensive framework encompassing the totality of exposures individuals experience, from conception onward, over a complete lifetime, and how they can affect human health. In this narrative review, we present an overview of current knowledge on the exposome, with a particular focus on its impact on brain health across different life stages, highlighting its role during neurodevelopment/childhood, adulthood, physiological aging and neurodegenerative conditions. We explore the three main exposome domains (general external, specific external, internal), underpinning their interconnection. We also provide examples of how specific factors, ranging from chemicals to lifestyle habits, can either positively or negatively influence the CNS structure and function. To this aim, we included articles (mostly between 2005 and 2024) found in open-access databases, selected using the following search terms: "exposome", "central nervous system" and "pregnancy" or "childhood" or "adulthood" or "aging" or "neurodegeneration". Second, we analyzed the relationships between specific exposome domains and each stage of life. Only articles addressing a given risk factor across all life stages were included in the review. In each chapter, we review findings from both human and experimental studies, when available, with an insight on biological mechanisms, in order to link exposure to phenotype. This work highlights the potential of the exposomics' framework to support public health policies and facilitate the prevention and treatment of multifactorial neurological disorders, while underscoring the importance of interdisciplinary efforts to address the multifaceted environmental determinants endangering the CNS health.

Instability and denervation of the neuromuscular junction (NMJ) are early events in Amyotrophic Lateral Sclerosis (ALS), likely reflecting a progressive decline in the regenerative capacity of motor neurons (MNs) and their environment. To investigate this, we evaluated NMJ regeneration throughout disease progression in SOD1G93A mice following reversible axon terminal degeneration induced by α-Latrotoxin. In parallel, we monitored the expression of CXCR4, a GPCR upregulated during axonal regeneration, and tested whether its pharmacological activation could mitigate ALS- related functional decline. We found that NMJ regenerative capacity is largely preserved during pre- and early symptomatic stages, and remains active in subsets of NMJs even at later stages. CXCR4 is expressed at axon terminals from early disease stages, declining only at end stage. Its expression is conserved across ALS models, including SOD1G93A pigs, hiPSC-derived MN with ALS mutations, and biopsies from sporadic ALS patients. CXCR4 stimulation improved motor function, NMJ innervation, MN survival, and respiratory performance in ALS mice, and axon outgrowth in iPSC-derived MN. These findings identify the NMJ and CXCR4 as viable therapeutic targets in ALS. ### Competing Interest Statement The authors have declared no competing interest.

Sarcopenia is the primary cause of impaired motor performance in the elderly. The current prevailing approach to counteract such condition is increasing the muscle mass through inhibition of the myostatin system: however, this strategy only moderately improves muscular strength, not being able to sustain the innervation of the hypertrophic muscle per se, leading to a progressive worsening of motor performances. Thus, we proposed the administration of ActR-Fc-nLG3, a protein that combines the soluble activin receptor, a strong myostatin inhibitor, with the C-terminal agrin nLG3 domain. This compound has the potential of reinforcing neuro-muscular stability to the hypertrophic muscle. We previously demonstrated an enhancement of motor endurance and ACh receptor aggregation in young mice after ActR-Fc-nLG3 administration. Now we extended these observations by demonstrating that also in aged (2 years-old) mice, long-term administration of ActR-Fc-nLG3 increases in a sustained way both motor endurance and muscle strength, compared with ActR-Fc, a myostatin inhibitor, alone. Histological data demonstrate that the administration of this biological improves neuromuscular stability and fiber innervation maintenance, preventing muscle fiber atrophy and inducing only moderate hypertrophy. Moreover, at the postsynaptic site we observe an increased folding in the soleplate, a likely anatomical substrate for improved neurotransmission efficiency in the NMJ, that may lead to enhanced motor endurance. We suggest that ActR-Fc-nLG3 may become a valid option for treating sarcopenia and possibly other disorders of striatal muscles.

Background One intrastriatal administration of quinolinic acid (QA) in rats induces a lesion with features resembling those observed in Huntington’s disease. Our aim is to evaluate the effects of the cysteinyl leukotriene receptor antagonist montelukast (MLK), which exhibited neuroprotection in different preclinical models of neurodegeneration, on QA-induced neuroinflammation and regional metabolic functions. Methods The right and left striatum of Sprague Dawley and athymic nude rats were injected with QA and vehicle (VEH), respectively. Starting from the day before QA injection, animals were treated with 1 or 10 mg/kg of MLK or VEH for 14 days. At 14 and 30 days post-lesion, animals were monitored with magnetic resonance imaging (MRI) and positron emission tomography (PET) using [ 18 F]-VC701, a translocator protein (TSPO)-specific radiotracer. Striatal neuroinflammatory response was measured post-mortem in rats treated with 1 mg/kg of MLK by immunofluorescence. Rats treated with 10 mg/kg of MLK also underwent a [ 18 F]-FDG PET study at baseline and 4 months after lesion. [ 18 F]-FDG PET data were then used to assess metabolic connectivity between brain regions by applying a covariance analysis method. Results MLK treatment was not able to reduce the QA-induced increase in striatal TSPO PET signal and MRI lesion volume, where we only detected a trend towards reduction in animals treated with 10 mg/kg of MLK. Post-mortem immunofluorescence analysis revealed that MLK attenuated the increase in striatal markers of astrogliosis and activated microglia in the lesioned hemisphere. We also found a significant increase in a marker of anti-inflammatory activity (MannR) and a trend towards reduction in a marker of pro-inflammatory activity (iNOS) in the lesioned striatum of MLK—compared to VEH-treated rats. [ 18 F]-FDG uptake was significantly reduced in the striatum and ipsilesional cortical regions of VEH-treated rats at 4 months after lesion. MLK administration preserved glucose metabolism in these cortical regions, but not in the striatum. Finally, MLK was able to counteract changes in metabolic connectivity and measures of network topology induced by QA, in both lesioned and non-lesioned hemispheres. Conclusions Overall, MLK treatment produced a significant neuroprotective effect by reducing neuroinflammation assessed by immunofluorescence and preserving regional brain metabolism and metabolic connectivity from QA-induced neurotoxicity in cortical and subcortical regions.

Spinal muscular atrophy (SMA) is a severe autosomal recessive neuromuscular disease affecting children and young adults, caused by mutations of the survival motor neuron 1 gene (SMN1). SMA is characterized by the degeneration of spinal alpha motor neurons (αMNs), associated with muscle paralysis and atrophy, as well as other peripheral alterations. Both growth hormone-releasing hormone (GHRH) and its potent agonistic analog, MR-409, exert protective effects on muscle atrophy, cardiomyopathies, ischemic stroke, and inflammation. In this study, we aimed to assess the protective role of MR-409 in SMNΔ7 mice, a widely used model of SMA. Daily subcutaneous treatment with MR-409 (1 or 2 mg/kg), from postnatal day 2 (P2) to euthanization (P12), increased body weight and improved motor behavior in SMA mice, particularly at the highest dose tested. In addition, MR-409 reduced atrophy and ameliorated trophism in quadriceps and gastrocnemius muscles, as determined by an increase in fiber size, as well as upregulation of myogenic genes and inhibition of proteolytic pathways. MR-409 also promoted the maturation of neuromuscular junctions, by reducing multi-innervated endplates and increasing those mono-innervated. Finally, treatment with MR-409 delayed αMN death and blunted neuroinflammation in the spinal cord of SMA mice. In conclusion, the present study demonstrates that MR-409 has protective effects in SMNΔ7 mice, suggesting that GHRH agonists are promising agents for the treatment of SMA, possibly in combination with SMN-dependent strategies.

Background Huntington’s disease (HD) is a motor and cognitive neurodegenerative disorder due to prominent loss of striatal medium spiny neurons (MSNs). Cell replacement using human embryonic stem cells (hESCs) derivatives may offer new therapeutic opportunities to replace degenerated neurons and repair damaged circuits. Methods With the aim to develop effective cell replacement for HD, we assessed the long-term therapeutic value of hESC-derived striatal progenitors by grafting the cells into the striatum of a preclinical model of HD [i.e., adult immunodeficient rats in which the striatum was lesioned by monolateral injection of quinolinic acid (QA)]. We examined the survival, maturation, self-organization and integration of the graft as well as its impact on lesion-dependent motor alterations up to 6 months post-graft. Moreover, we tested whether exposing a cohort of QA-lesioned animals to environmental enrichment (EE) could improve graft integration and function. Results Human striatal progenitors survived up to 6 months after transplantation and showed morphological and neurochemical features typical of human MSNs. Donor-derived interneurons were also detected. Grafts wired in both local and long-range striatal circuits, formed domains suggestive of distinct ganglionic eminence territories and displayed emerging striosome features. Moreover, over time grafts improved complex motor performances affected by QA. EE selectively increased cell differentiation into MSN phenotype and promoted host-to-graft connectivity. However, when combined to the graft, the EE paradigm used in this study was insufficient to produce an additive effect on task execution. Conclusions The data support the long-term therapeutic potential of ESC-derived human striatal progenitor grafts for the replacement of degenerated striatal neurons in HD and suggest that EE can effectively accelerate the maturation and promote the integration of human striatal cells.

Background Huntington Disease (HD) is a monogenetic dominant neurodegenerative disorder that progressively affects motor and cognitive functions as a consequence of the prominent loss of medium spiny projection neurons (MSNs) in the corpus striatum of the basal ganglia1. Human embryonic stem cells (hESCs) may provide novel therapeutic chances to replace deteriorating neurons and restore damaged circuits through cell replacement. Aims Here, we evaluated the long-term therapeutic potential of hESC-derived striatal progenitors transplanted into the striatum of a quinolinic acid (QA)-based rat model of Huntington’s Disease (HD) with the goal of developing effective cell replacement for HD. Methods To this aim, hESC were differentiated according to an established in vitro differentiation protocol (Delli Carri, Development; 2013) and day 20 progenitors transplanted in the striatum of adult athymic nude rats. The motor performance of the animals was then monitored up to six months of housing in standard and enriched environment (EE) and histological analysis performed at end of observation. Results Transplanted human striatal progenitors displayed morphological and neurochemical characteristics typical of human medium spiny neurons (MSNs), or interneurons, and they continued to function for up to 6 months following transplantation. Grafts generated domains that resembled discrete ganglionic eminence areas and were wired in both local and long-range striatal circuits and were able to ameliorate QA-affected motor capabilities. Moreover, exposure to EE facilitated host-to-graft connection, increased cell differentiation into the MSN phenotype, and improved task performances. Conclusions These findings indicate that human striatal progenitor grafts have the ability to mitigate HD signs in the long term, and EE can efficiently hasten the maturation and support the integration of human striatal neurons.

Current efforts to improve muscle performance are focused on muscle trophism via inhibition of the myostatin pathway: however they have been unsuccessful in the clinic to date. In this study, a novel protein has been created by combining the soluble activin receptor, a strong myostatin inhibitor, to the C-terminal agrin nLG3 domain (ActR-Fc-nLG3) involved in the development and maintenance of neuromuscular junctions. Both domains are connected via the constant region of an Igg1 monoclonal antibody. Surprisingly, young male mice treated with ActR-Fc-nLG3 showed a remarkably increased endurance in the rotarod test, significantly longer than the single domain compounds ActR-Fc and Fc-nLG3 treated animals. This increase in endurance was accompanied by only a moderate increase in body weights and wet muscle weights of ActR-Fc-nLG3 treated animals and were lower than expected. The myostatin inhibitor ActR-Fc induced, as expected, a highly significant increase in body and muscle weights compared to control animals and ActR-Fc-nLG3 treated animals. Moreover, the prolonged endurance effect was not observed when ActR-Fc and Fc-nLG3 were dosed simultaneously as a mixture and the body and muscle weights of these animals were very similar to ActR-Fc treated animals, indicating that both domains need to be on one molecule. Muscle morphology induced by ActR-Fc-nLG3 did not appear to be changed however, close examination of the neuromuscular junction showed significantly increased acetylcholine receptor surface area for ActR-Fc-nLG3 treated animals compared to controls. This result is consistent with published observations that endurance training in rats increased acetylcholine receptor quantity at neuromuscular junctions and provide evidence that improving nerve-muscle interaction could be an important factor for sustaining long term muscle activity.

Onuf’s nucleus is a small group of neurons located in the ventral horns of the sacral spinal cord. The motor neurons (MNs) of Onuf’s nucleus innervate striated voluntary muscles of the pelvic floor and are histologically and biochemically comparable to the other somatic spinal MNs. However, curiously, these neurons also show some autonomic-like features as, for instance, they receive a strong peptidergic innervation. The review provides an overview of the histological, biochemical, metabolic, and gene expression peculiarities of Onuf’s nucleus. Moreover, it describes the aging-related pathologies as well as several traumatic and neurodegenerative disorders in which its neurons are involved: indeed, Onuf’s nucleus is affected in Parkinson’s disease (PD) and Shy-Drager Syndrome (SDS), whereas it is spared in Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), Duchenne Muscular Dystrophy (DMD). We summarize here the milestone studies that have contributed to clarifying the nature of Onuf’s neurons and in understanding what makes them either vulnerable or resistant to damage. Altogether, these works can offer the possibility to develop new therapeutic strategies for counteracting neurodegeneration.

Huntington disease (HD) is an inherited late-onset neurological disorder characterized by progressive neuronal loss and disruption of cortical and basal ganglia circuits. Cell replacement using human embryonic stem cells may offer the opportunity to repair the damaged circuits and significantly ameliorate disease conditions. Here, we showed that in-vitro-differentiated human striatal progenitors undergo maturation and integrate into host circuits upon intra-striatal transplantation in a rat model of HD. By combining graft-specific immunohistochemistry, rabies virus-mediated synaptic tracing, and ex vivo electrophysiology, we showed that grafts can extend projections to the appropriate target structures, including the globus pallidus, the subthalamic nucleus, and the substantia nigra, and receive synaptic contact from both host and graft cells with 6.6 ± 1.6 inputs cell per transplanted neuron. We have also shown that transplants elicited a significant improvement in sensory-motor tasks up to 2 months post-transplant further supporting the therapeutic potential of this approach.

The c-Jun NH2-terminal protein kinase (JNK) is a Janus-faced kinase, which, in the nervous system, plays important roles in a broad range of physiological and pathological processes. Three genes, encoding for 10 JNK isoforms, have been identified: jnk1, jnk2, and jnk3. In the developing spinal cord, JNK proteins control neuronal polarity, axon growth/pathfinding, and programmed cell death; in adulthood they can drive degeneration and regeneration, after pathological insults. Indeed, recent studies have highlighted a role for JNK in motor neuron (MN) diseases, such as amyotrophic lateral sclerosis and spinal muscular atrophy. In this review we discuss how JNK-dependent signaling regulates apparently contradictory functions in the spinal cord, in both the developmental and adult stages. In addition, we examine the evidence that the specific targeting of JNK signaling pathway may represent a promising therapeutic strategy for the treatment of MN diseases.

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields.

May 6, 2019April 9, 2019Free AccessA novel Biological (FOX-115) improves endurance of motor performances in old mice (P2.4-033)Ruggero G. Fariello, Roberta Schellino, Marina Boido, Jan Willem Vrijbloed, and Alessandro VercelliAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.P2.4-033 Letters to the Editor

Spinal muscular atrophy (SMA) is a severe neurodegenerative disorder that occurs in early childhood. The disease is caused by the deletion/mutation of the survival motor neuron 1 (SMN1) gene resulting in progressive skeletal muscle atrophy and paralysis, due to the degeneration of spinal motor neurons (MNs). Currently, the cellular and molecular mechanisms underlying MN death are only partly known, although recently it has been shown that the c-Jun NH2-terminal kinase (JNK)-signaling pathway might be involved in the SMA pathogenesis. After confirming the activation of JNK in our SMA mouse model (SMN2+/+; SMNΔ7+/+; Smn−/−), we tested a specific JNK-inhibitor peptide (D-JNKI1) on these mice, by chronic administration from postnatal day 1 to 10, and histologically analyzed the spinal cord and quadriceps muscle at age P12. We observed that D-JNKI1 administration delayed MN death and decreased inflammation in spinal cord. Moreover, the inhibition of JNK pathway improved the trophism of SMA muscular fibers and the size of the neuromuscular junctions (NMJs), leading to an ameliorated innervation of the muscles that resulted in improved motor performances and hind-limb muscular tone. Finally, D-JNKI1 treatment slightly, but significantly increased lifespan in SMA mice. Thus, our results identify JNK as a promising target to reduce MN cell death and progressive skeletal muscle atrophy, providing insight into the role of JNK-pathway for developing alternative pharmacological strategies for the treatment of SMA.

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, character- ized by motor impairment, muscle atrophy and premature death following motor neuron (MN) degeneration, due to the lack of SMN (survival motor neuron) protein. Currently, the cellular and molecular mechanisms underlying MN death are only partly known [1], although recently it has been shown that the JNK-signalling pathway might be involved in the SMA pathogen- esis. After confirming the activation of JNK in our SMA mouse model (SMN2+/+; SMN∆7+/+; Smn-/-), we tested on these mice a synthetic JNK-inhibitor peptide (D-JNKI), by chronic admin- istration from postnatal day 1 (P1) to P10; then, at age P12, we analyzed their spinal cords and quadriceps muscles. We observed that D-JNKI administration delayed MN death and decreasedneuroinflammation in the spinal cord. Moreover, by inhibiting JNK pathway, the muscular fib- ers and the neuromuscular junctions appeared respectively more trophic and mature. The his- tological/molecular results positively correlated with improved motor performances and hind- limb muscular tone. Finally, the treatment slightly, but significantly increased lifespan in SMA mice. Overall, our results identify JNK as a promising target to reduce MN cell death and pro- gressive skeletal muscle atrophy, providing insight into the role of JNK-pathway for developing alternative pharmacological strategies for the treatment of SMA.This work was supported by grants from CRT Foundation, Girotondo/ONLUS and SMArathon-ONLUS foundations.

The guidance protein Semaphorin7A (Sema7A) is required for the proper development of the immune and nervous systems. Despite strong expression in the mature brain, the role of Sema7A in the adult remains poorly defined. Here we show that Sema7A utilizes different cell surface receptors to control the proliferation and differentiation of neural progenitors in the adult hippocampal dentate gyrus (DG), one of the select regions of the mature brain where neurogenesis occurs. PlexinC1 is selectively expressed in early neural progenitors in the adult mouse DG and mediates the inhibitory effects of Sema7A on progenitor proliferation. Subsequently, during differentiation of adult-born DG granule cells, Sema7A promotes dendrite growth, complexity and spine development through β1-subunit-containing integrin receptors. Our data identify Sema7A as a key regulator of adult hippocampal neurogenesis, providing an example of how differential receptor usage spatiotemporally controls and diversifies the effects of guidance cues in the adult brain.