Abstract Nerve growth factor (NGF) exerts neuroprotective effects in the retina, and accumulating evidence indicates that microglia represent a key cellular target of NGF/TrkA signaling. However, evidence showing that the NGF/TrkA signaling in microglia is required for downstream neuroprotective actions remains unresolved. Here, we directly addressed this question by pharmacologically depleting microglia and assessing the impact on NGF pathway activity and retinal integrity. Adult C57BL/6J mice were treated with the CSF1R inhibitor PLX5622 for three weeks, resulting in a robust (∼77%) depletion of retinal microglia. Microglial ablation induced marked structural and cellular alterations, including significant loss of retinal ganglion cells (RGCs) and thinning of retinal layers, in the absence of any other lesion or insult. Residual microglia exhibited layer-specific phenotypic changes, with a phagocytic profile in the ganglion cell layer and a more ramified morphology in the outer plexiform layer. Strikingly, microglial depletion led to a profound decrease of NGF signaling, with a strong reduction in total and phosphorylated TrkA, and decreased p75NTR levels, in retinal extracts. The amount of TrkA expression is strongly correlated with microglial levels, supporting a primary role of microglia in sustaining NGF signaling in the retina. Together, these findings demonstrate that microglia are required for NGF/TrkA signaling and identify these cells as essential mediators of NGF-dependent neuroprotection in the retina.

Experimental advancements in neuroscience have identified cellular engrams—ensembles of neurons whose activation is necessary and sufficient for memory retrieval. Synaptic plasticity, including long-term potentiation, is fundamental to memory encoding and recall, but the relationship between learning-induced dendritic spine potentiation and neuron-wide activation remains unclear. In this study, we employed a post-synaptic translation-dependent reporter consistent with potentiation (SA-PSDΔVenus) and a neuronal activation reporter (ESARE-dTurquoise) to determine their spatiotemporal correlation in the mouse hippocampal CA1 following contextual fear conditioning (CFC). SA-PSDΔVenus+ spines were enriched in ESARE-dTurquoise+ neurons, with distribution varying across CA1 layers at different phases of memory: SA-PSDΔVenus+ were more frequent in activated neurons in stratum oriens and stratum lacunosum moleculare after CFC (encoding), while recall-activated neurons showed a larger number of SA-PSDΔVenus+ in the stratum radiatum. These findings demonstrate that the relative weight and spatial distribution of potentiated synaptic inputs to hippocampal CA1 pyramidal neurons change between the encoding and retrieval phases of memory.

Epilepsy is a prevalent neurological disorder characterized by recurrent, unprovoked seizures and altered electroencephalographic patterns. This condition is viewed as a malfunctioning of extensive neural networks due to an imbalance of excitatory and inhibitory signals leading neurons to be excessively excitable and to abnormal synchronized electrical activity. Despite the growing number of new antiepileptic drugs, patients suffering from drug-resistant forms of epilepsy do not respond to pharmacological treatment, and the only effective cure remains the neurosurgical resection of the epileptic focus. Nevertheless, several patients fail to achieve seizure freedom after surgical resection. This emphasizes the urgent need for novel human-relevant models to explore the mechanisms underlying drug-refractory forms of epilepsy. While acute and organotypic slices from resected neurological tissue offer a promising method for studying patient-derived brain tissue mechanisms, this technique is limited by its inherently low throughput and challenges in obtaining appropriate control tissue. Recent advances in organoid technology have allowed for the generation of cerebral dorsal/ventral assembloids, which more accurately model the functional connectivity between excitatory and inhibitory neurons and recapitulate key aspects of cortical circuits. This review summarizes current knowledge on the use of human brain organoids and assembloids to model epilepsy, with a particular focus on organoids harboring focal cortical dysplasia-linked mutations. Human brain organoids and assembloids will allow addressing an important question in the field, namely the relative contribution of neurodevelopmental defects vs. those arising at later stages of CNS development. Limitations of this “neuron-only” in vitro model and potential ways to include non-neuronal cells will also be discussed. Finally, we highlight recent advances in employing these new powerful platforms for investigating network dysfunctions underlying FCDs, screening potential antiepileptic drug candidates, and developing personalized therapeutic strategies.

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by mutations in the MECP2 gene. Although recent therapeutic advances, such as the approval of Trofinetide, offer partial relief, no comprehensive curative treatment is currently available. Among the emerging strategies, nerve growth factor (NGF) has gained attention due to its neurotrophic and immunomodulatory properties. This review, in addition to discussing the key features of RTT and the role of growth factors, also highlights recent evidence supporting NGF-based strategies for RTT, focusing on two independent studies that tested intranasal administration of NGF-like molecules in Mecp2-mutant mice. Both recombinant human NGF (rhNGF) and a modified, “painless” variant (hNGFp) improved behavioral (cognitive and motor) symptoms. While rhNGF primarily restored mitochondrial function, hNGFp restored neuroinflammatory responses through microglial regulation. Despite differences in molecular mechanisms and dosages, both molecules demonstrated efficacy without adverse effects, especially when administered intranasally, preventively, and over longer periods. These findings suggest that NGF may act through dual mechanisms, by supporting energy homeostasis and regulating immune responses. The use of intranasal delivery further enhances translational potential by overcoming blood–brain barrier limitations. Together, these studies provide a strong rationale for pursuing NGF-based therapies in RTT and encourage further investigations to optimize dosing, timing, and safety in preclinical and clinical settings.

Brain development and plasticity depend on specific microRNA (miRNA) expression patterns across cell types and subcellular compartments. Nevertheless, comprehensive profiling of localized brain miRNAs is still limited by challenges in isolating individual cell types or compartments and in detection sensitivity. To overcome these limitations, we advanced HIV-1 Gag’s ability to bind host miRNAs within Virus-like Particles to develop Synthetic Nano-Particles for Precise endogenous miRNA loading and export (SNaP). Our data establish SNaP’s modularity and portability to clinically relevant neural cells, with particle yields matching benchmark packaging cells. The integration of SNaP with a cell-specific promoter enabled lineage-restricted miRNA export, while incorporating a dendritic localization signal improved the specificity of post-synaptic miRNA recovery over traditional synaptosomes. Additional engineering with a miRNA-binding module synergistically increased synaptic miRNA packaging in a sequence-independent manner. Collectively, this work positions SNaP as a technological advancement supporting the high-resolution, spatially resolved profiling of miRNAs, adaptable to diverse polarized or heterogeneous culture systems.

Abstract The mutational landscape of neural innervation axes in cancer has garnered increasing attention due to their significant influence on tumorigenesis, metastasis, and treatment resistance. This study provides an overview of the alterations of neural signaling pathways based on multi-omics data analysis. In particular, we conducted a comprehensive analysis of mutations in key genes associated with neurotrophin signaling, norepinephrine and cholinergic pathways, alongside paracrine and synaptogenic factors. Our findings reveal that 65% of patients in a pancancer cohort exhibit at least one somatic alteration in these pathways, with neurotrophin genes being the most frequently altered. Notably, alterations in these genes (e.g., NGF) correlate with poor patient survival across various cancer types, suggesting their oncogenic potential. We also identified significant co-occurrence of mutations and differential expression patterns from both bulk and single-cell RNAseq data, indicating complex interactions among these pathways that contribute to tumor innervation and neuroplasticity. This study underscores the necessity of understanding the crosstalk between neural signaling and cancer biology to improve patient stratification based on mutational profiles and identify potential therapeutic targets. Further research utilizing advanced genomic techniques is essential to elucidate the mechanistic roles of these pathways in tumor innervation.

Neurodegenerative diseases, like Alzheimer's disease (AD), are characterized by the accumulation of tau aggregates, leading to neuronal dysfunction and cognitive decline. This study explores the development of dual-acting compounds combining sigma-1 receptor (σ1R) agonists and histone deacetylase inhibitors (HDACi) to target these pathological mechanisms. Compounds 2d and 3a demonstrated high affinity for σ1R and significantly reduced tau aggregation and phosphorylation in vitro, notably at the AT8 epitope. These dual-acting compounds destabilized tau aggregates, increased tau solubility, and showed favorable pharmacokinetic properties, with compound 2d exhibiting enhanced chemical stability and longer half-life than 3a. In vivo, both compounds confirmed a σ1R agonist profile by reversing the effect of the σ1R antagonist BD-1063. This dual-action approach, acting on both HDAC and σ1R pathways, holds significant potential for treating tauopathies. While further optimization and clinical evaluation are needed, these findings provide a strong foundation for the continued development of multimodal therapies for neurodegenerative diseases treatment.

Glioblastoma (GB) is the most aggressive primary brain tumor, characterized by poor prognosis, profound immunosuppression, and limited response to current therapies. To overcome these barriers, we developed CTX-CNF1, a brain-penetrant recombinant protein that integrates direct glioma targeting with myeloid cell–mediated immune reprogramming. CTX-CNF1 was generated by fusing Chlorotoxin (CTX), which enables selective delivery across the blood–brain barrier, with Cytotoxic Necrotizing Factor 1 (CNF1), a bacterial protein with antitumor and immunostimulatory properties. Therapeutic efficacy was evaluated in immunocompetent orthotopic GB mouse models using survival analysis, MRI, motor assessment, and immunophenotyping. Systemic CTX-CNF1 administration was well tolerated and significantly prolonged survival, reduced tumor burden, and preserved motor function. A substantial proportion of treated animals achieved complete tumor regression and resisted tumor re-challenge, consistent with the induction of durable antitumor immunity. Mechanistically, CTX-CNF1 promoted a pro-inflammatory reprogramming of tumor-associated myeloid cells and enhanced CD8⁺ T-cell recruitment and cytotoxicity, thereby counteracting GB-associated immunosuppression. Therapeutic efficacy was confirmed across distinct glioma models. In combination settings, CTX-CNF1 selectively enhanced the activity of immune checkpoint blockade in a context-dependent manner. CTX-CNF1 represents a dual-action therapeutic strategy that combines tumor targeting with immune modulation and effective brain delivery. By coupling direct antitumor effects with remodeling of the tumor immune microenvironment, CTX-CNF1 supports the development of combinatorial approaches to overcome therapeutic resistance in GB and other immune-cold tumors.

IntroductionRecent research revealed that Tau plays critical roles in various neuronal functions. We previously demonstrated that destabilization and nuclear delocalization of Tau alter the expression of glutamatergic genes, mediating early neuronal damage.MethodsIn this study, we discovered that changes in Tau availability are linked to global alterations in gene expression that affect multiple neuronal pathways. Comparison with the human temporal region showed that the Tau-dependent modulation of gene expression closely resembles the intermediate stages of Alzheimer’s disease (AD) that precede the definitive pathological condition.ResultsFurthermore, we identified the chromatin remodeling pathway as being significantly affected by Tau in both our cellular model and AD brains, with reductions in heterochromatin markers. Our findings indicate that Tau is able to globally affect the neuronal transcriptome and that its subcellular unbalance changes gene expression in the intermediate stages of AD development. In addition, we found that the chromatin architecture is affected by Tau during the progression of AD.DiscussionThese results provide new insights into the molecular mechanisms underlying early stages of AD development and highlight the central role of Tau and the contribution of nuclear Tau in this process.

Nerve growth factor (NGF) binding to the receptor tyrosine kinase, TrkA, drives neurotrophic signaling essential for neuronal development and survival. This interaction simultaneously drives peripheral pain, making this pathway an attractive but complicated therapeutic target for chronic pain. By integrating single-molecule microscopy, structural and electrophysiology analyses, with a human NGF variant, NGF painless , which retains neurotrophic effects but abolishes pain, we delineate the molecular mechanisms that bias TrkA signaling towards neurotrophic functions without triggering nociception. We show that, unlike wild-type NGF, NGF painless fails to sensitize TRPV1 channels to capsaicin, thus disengaging TrkA from the nociceptive pathway. We further show that this selective loss of nociceptive TrkA signaling by NGF painless results from its reduced ability to activate PLCγ1 and trigger calcium release compared to NGF, while still preserving the ERK and AKT signaling essential for neurotrophic functions. This biased signaling arises from reduced electrostatic complementarity at the TrkA:NGF painless complex interface, which shortens the lifetime of this functional complex on native membranes. Mutations in TrkA that restore the electrostatic complementarity at the TrkA:NGF painless interface eliminate biased signaling. This mechanistic understanding of TrkA binding by NGF painless , and how it differs from NGF, will spur the development of two therapeutic classes of molecules - one that selectively suppresses nociceptive signaling while preserving neurotrophic functions in chronic pain, and another that enhances neurotrophic activity without evoking peripheral pain in neurodegenerative conditions.

Brain development and plasticity depend on specific microRNA (miRNA) expression patterns across cell types and subcellular compartments. Nevertheless, comprehensive profiling of localized brain miRNAs is still limited by challenges in isolating individual cell types or compartments and in detection sensitivity. To overcome these limitations, we advanced HIV-1 Gag’s ability to bind host miRNAs within Virus-like Particles to develop Synthetic Nano-Particles for Precise miRNA loading and export (SNaP). Our data establish SNaP’s modularity and portability to clinically-relevant neural cells, with particle yields matching benchmark packaging cells. SNaP integration with a cell-specific promoter enabled lineage-restricted miRNA export, while incorporating a Dendritic Localization Signal improved the specificity of postsynaptic miRNA recovery over traditional synaptosomes. Additional engineering with a miRNA-binding module synergistically boosted synaptic miRNA packaging in a sequence-independent manner. Collectively, our work positions SNaP as a technological advancement supporting the high-resolution, spatially resolved profiling of non-coding RNAs, adaptable to diverse polarized or heterogeneous tissues.

Microglia, the resident immune cells of the brain, play a crucial role in sculpting neuronal circuits during development, and their dysfunction is increasingly implicated in neurodevelopmental disorders such as Down syndrome (DS). Here, we reveal a previously unrecognized pathological mechanism whereby microglia contribute to synaptic and neuronal activity deficits in DS: a selective disruption of microglia–interneuron interactions. Using primary neuron–microglia co-cultures from Ts65Dn mice, we show that while trisomy in neurons drives excitatory synaptic deficits and major microglial morphological changes, microglial trisomy disrupts the regulation of inhibitory synapses in a cell-autonomous manner. To investigate these pathological interactions in vivo, we developed a novel spatial distribution analysis tool that, combined with chemogenetic approaches targeting parvalbumin (PV) interneurons in Ts65Dn mice, allowed us to reveal a disrupted microglia–PV interneuron crosstalk characterized by reduced physical association and impaired microglial responsiveness to PV activity. Finally, by targeting microglia via P2Y12 receptor inhibition, we restored cortical connectivity, rescued PV interneuron function, and improved cognitive performance in Ts65Dn mice. Overall, these findings establish microglia–interneuron dysregulation as a key driver of neuronal activity and synaptic dysfunction in the Ts65Dn mouse model of DS and identify the microglia as a promising therapeutic target to counter circuit dysfunction and cognitive deficits.

Retinitis pigmentosa (RP) is a family of genetically heterogeneous diseases still without a cure. Despite the causative genetic mutation typically not expressed in cone photoreceptors, these cells inevitably degenerate following the primary death of rods, causing blindness. The reasons for the “bystander” degeneration of cones are presently unknown but decrement of survival factors, oxidative stress, and inflammation all play a role. Targeting these generalized biological processes represents a strategy to develop mutation-agnostic therapies for saving vision in large populations of RP individuals. A classical method to support neuronal survival is by employing neurotrophic factors, such as NGF. This study uses painless human NGF (hNGFp), a TrkA receptor-biased variant of the native molecule with lower affinity for nociceptors and limited activity as a pain inducer; the molecule has identical neurotrophic power of the native form but a reduced affinity for the p75NTR receptors, known to trigger apoptosis. hNGFp has a recognized activity on brain microglial cells, which are induced to a phenotype switch from a highly activated to a more homeostatic configuration. hNGFp was administered to RP-like mice in vivo with the aim of decreasing retinal inflammation and also providing retinal neuroprotection. However, the ability of this treatment to counteract the bystander degeneration of cones remained limited.

IntroductionFrontotemporal dementia (FTD) is an extremely heterogeneous and complex neurodegenerative disease, exhibiting different phenotypes, genetic backgrounds, and pathological states. Due to these characteristics, and to the fact that clinical symptoms overlap with those of other neurodegenerative diseases or psychiatric disorders, the diagnosis based only on the clinical evaluation is very difficult. The currently used biomarkers help in the clinical diagnosis, but are insufficient and do not cover all the clinical needs.MethodsBy the means of a new immunoassay, we have measured and analyzed the proNGF levels in 43 cerebrospinal fluids (CSF) from FTD patients, and compared the results to those obtained in CSF from 84 Alzheimer’s disease (AD), 15 subjective memory complaints (SMC) and 13 control subjects.ResultsA statistically significant difference between proNGF levels in FTD compared to AD, SMC and controls subjects was found. The statistical models reveal that proNGF determination increases the accuracy of FTD diagnosis, if added to the clinically validated CSF biomarkers.DiscussionThese results suggest that proNGF could be included in a panel of biomarkers to improve the FTD diagnosis.

Background and PurposeRetinal ganglion cells (RGCs) are the output stage of retinal information processing, via their axons forming the optic nerve (ON). ON damage leads to axonal degeneration and death of RGCs, and results in vision impairment. Nerve growth factor (NGF) signalling is crucial for RGC operations and visual functions. Here, we investigate a new neuroprotective mechanism of a novel therapeutic candidate, a p75-less, TrkA-biased NGF agonist (hNGFp) in rat RGC degeneration, in comparison with wild type human NGF (hNGFwt).Experimental ApproachBoth neonate and adult rats, whether subjected or not to ON lesion, were treated with intravitreal injections or eye drops containing either hNGFp or hNGFwt. Different doses of the drugs were administered at days 1, 4 or 7 after injury for a maximum of 10 days, when immunofluorescence, electrophysiology, cellular morphology, cytokine array and behaviour studies were carried out. Pharmacokinetic evaluation was performed on rabbits treated with hNGFp ocular drops.ResultshNGFp exerted a potent RGC neuroprotection by acting on microglia cells, and outperformed hNGFwt in rescuing RGC degeneration and reducing inflammatory molecules. Delayed use of hNGFp after ON lesion resulted in better outcomes compared with treatment with hNGFwt. Moreover, hNGFp-based ocular drops were less algogenic than hNGFwt. Pharmacokinetic measurements revealed that biologically relevant quantities of hNGFp were found in the rabbit retina.Conclusions and ImplicationsOur data point to microglia as a new cell target through which NGF-induced TrkA signalling exerts neuroprotection of the RGC, emphasizing hNGFp as a powerful treatment to tackle retinal degeneration.

In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications.

Abstract Rett syndrome is a rare genetic neurodevelopmental disease, affecting 1 in over 10 000 females born worldwide, caused by de novo mutations in the X-chromosome-located methyl-CpG-binding protein 2 (MeCP2) gene. Despite the great effort put forth by the scientific community, a therapy for this devastating disease is still needed. Here, we tested the therapeutic effects of a painless mutein of the nerve growth factor (NGF), called human NGF painless (hNGFp), via a non-invasive intranasal delivery in female MeCP2+/− mice. Of note, previous work had demonstrated a broad biodistribution of hNGFp in the mouse brain by the nasal delivery route. We report that (i) the long-term lifelong treatment of MeCP2+/− mice with hNGFp, starting at 2 months of age, increased the chance of survival while also greatly improving behavioural parameters. Furthermore, when we assessed the phenotypic changes brought forth by (ii) a short-term 1-month-long hNGFp-treatment, starting at 3 months of age (right after the initial presentation of symptoms), we observed the rescue of a well known neuronal target population of NGF, cholinergic neurons in the medial septum. Moreover, we reveal a deficit in microglial morphology in MeCP2+/− mice, completely reversed in treated animals. This effect on microglia is in line with reports showing microglia to be a TrkA-dependent non-neuronal target cell population of NGF in the brain. To understand the immunomodulatory activity of hNGFp, we analysed the cytokine profile after hNGFp treatment in MeCP2+/− mice, to discover that the treatment recovered the altered expression of key neuroimmune-communication molecules, such as fractalkine. The overall conclusion is that hNGFp delivered intranasally can ameliorate symptoms in the MeCP2+/− model of Rett syndrome, by exerting strong neuroprotection with a dual mechanism of action: directly on target neurons and indirectly via microglia.

Cognitive frailty (CF) is a heterogeneous syndrome that is becoming one of the most serious health problems as the world’s population age is increasing. Elucidating its biological mechanisms as well as prevention and treatments is becoming increasingly significant, particularly in view of the associated health costs. We presented the study protocol of a research project funded by the Italian Ministry of Health (grant number RF-2016-02363298) aiming to investigate the cognitive and neuropsychological effects of a 5-week treatment with therapy based on the regenerative properties of ozone (O3) in a cohort of subjects stratified according to CF scores. We also studied the potential effects of O3 on blood-based biomarkers indicative of specific biological systems that may be altered in CF. Seventy-five older persons were recruited and randomly assigned to receive the active treatment (150 cc of oxygen-O2-O3 mixture at the concentration of 30 µg of O3 per cc of O2), O2, or the placebo (air) for 5 weeks. The main endpoints were the change in the scores of clinical scales from baseline (T0) to weeks 3 (T3), 9 (T9), and 15 (T15) after treatment and the change in biomarker levels resulting from transcriptomics, proteomics, and metabolomic patterns at the same times. The positive results from this study could have important clinical implications.

Neurotrophin receptors of the Trk family are involved in the regulation of brain development and neuroplasticity, and therefore can serve as targets for anti-cancer and stroke-recovery drugs, antidepressants, and many others. The structures of Trk protein domains in various states upon activation need to be elucidated to allow rational drug design. However, little is known about the conformations of the transmembrane and juxtamembrane domains of Trk receptors. In the present study, we employ NMR spectroscopy to solve the structure of the TrkB dimeric transmembrane domain in the lipid environment. We verify the structure using mutagenesis and confirm that the conformation corresponds to the active state of the receptor. Subsequent study of TrkB interaction with the antidepressant drug fluoxetine, and the antipsychotic drug chlorpromazine, provides a clear self-consistent model, describing the mechanism by which fluoxetine activates the receptor by binding to its transmembrane domain. Neurotrophin receptor TrkB regulates neuronal growth and neuroplasticity. Here, the authors present the NMR structure of the intramembrane region of TrkB activated by antidepressant drugs, yielding insights into receptor function.