Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron loss and significant clinical variability. Blood-based biomarkers offer a scalable approach to stratify patients and support therapeutic development. In a large-scale serum proteomic survival study of ALS, we analyzed 741 patients from independent cohorts in Israel and the UK to identify blood proteins linked to ALS survival. The cohorts were profiled using complementary proteomics technologies, mass spectrometry and the Olink proximity extension assay (PEA), enabling cross-cohort and cross-platform evaluation of concordant protein effects. We identified 19 serum proteins associated with ALS survival across two independent cohorts profiled using orthogonal proteomic platforms. Among these, GSN and IGFBP2 improved survival prediction and patient stratification compared to established clinical covariates and provided additional prognostic information compared to serum neurofilament light chain (NfL). These novel circulating protein biomarker candidates may improve prognostic assessment and support future precision-medicine approaches in ALS. ### Competing Interest Statement The authors have declared no competing interest. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: London - South East REC 16/LO/2136 gave ethical approval for this work. South Central Oxford Research Ethics Committee 08/H0605/85 gave approval for this work. South Central - Hampshire B REC 14/SC/1248 gave approval for this work. South Central - Oxford C REC 17/SC/0277 gave approval for this work. Wales REC 20/WA/0027 gave approval for this work. East Midlands - Leicester Central REC 21/EM/0251 gave approval for this work. Health and Social Care Northern Ireland B REC 15/NI/0096 gave approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data that support the findings of this study are available from the corresponding author upon reasonable request. UK Research and Innovation, MR/Y001095/1, MR/T006927/1 Motor Neurone Disease Association, https://ror.org/02gq0fg61, Lester/2450/795, Thompson/Jan20/952-795, 894-791, 899-792, 913-792 Robert Packard Center for ALS Research, 2006781541

Nerve injury triggers localized translation of axonal mRNAs to respond to injury and nerve regeneration. The core stress granule protein G3BP1 sequesters axonal mRNAs in granules before and after axotomy. G3BP1 granule disassembly can be regulated by post-translational modifications, including phosphorylation of S149 phosphorylation and acetylation of human K376 (mouse K374). Axonal G3BP1 undergoes phosphorylation after axotomy, but acetylation of G3BP1 in axons was unknown. Here we show that rodent G3BP1 undergoes K374 acetylation after axotomy is ELP3-dependent, which enhances axonal protein synthesis, accelerates nerve regeneration, and supports functional recovery. ELP3-depleted neurons exhibit reduced axon growth and increased axonal G3BP1 granules. The proximal axons degenerate rapidly despite maintaining soma connectivity, an effect prevented by expression of acetylmimetic G3BP1.Together, these findings identify G3BP1 acetylation via ELP3 as a critical regulator of both axonal regeneration and neuronal resilience, revealing a post-translational mechanism that links stress granule regulation to neuronal repair and protection.

Neurological disorders, a leading global cause of death and disabilities, encompass conditions affecting both the peripheral and central nervous systems (PNS and CNS, respectively). Limited axon regeneration is a significant challenge in these disorders, and it has been linked to proteins like PTEN. RNA-based therapeutics, particularly siRNAs, hold the potential for silencing these inhibitory pathways, but their clinical application is hindered by poor stability and cellular uptake. Our study addressed this challenge with the development of novel, fully biodegradable dendritic nanoparticles designed specifically for neuron targeting. These nanoparticles were functionalized with the neurotropic binding domain of tetanus toxin, enhancing selective neuronal targeting and cellular internalization. We demonstrated that these targeted dendriplexes not only maintain biocompatibility and efficient siRNA delivery in neuronal cultures but also significantly enhance axonal growth, as shown in microfluidic models. In a groundbreaking PNS-CNS-on-Chip, dendriplexes exhibited effective migration from PNS to CNS neurons, highlighting their potential for targeted therapeutic delivery via a minimally invasive administration. This study pioneers the application of microfluidics to demonstrate the CNS targeting of dendriplexes, paving the way for innovative treatments in the field of nanomedicine.

Neuronal plasticity, essential for learning and memory, involves structural changes triggered by neurotrophic factors such as brain-derived neurotrophic factor (BDNF). BDNF activates its receptor, TrkB, to induce local and long-distance signaling, promoting dendritic branching. While BDNF activation of the mechanistic target of rapamycin (mTOR) pathway is well-documented in dendritic spines, its role in axons remains unclear. Using compartmentalized cultures of embryonic cortical neurons from mice, this study demonstrates that axonal BDNF triggers mTOR-dependent phosphorylation of translation regulators (4E-BP1 and S6), driving local protein synthesis. Fluorescent labeling, immunostaining, and radial microfluidic chambers were employed to isolate axonal compartments and assess local translation. Axonal BDNF increased Rab5 protein translation in a TrkB- and mTOR-dependent manner, highlighting the importance of localized protein synthesis for axonal trafficking. Furthermore, axonal protein synthesis was required for retrograde transport of internalized cargoes and CREB activation in the nucleus, linking local translation to long- distance signaling. These findings highlight the crucial role of axonal mTOR activation in regulating local proteostasis and neuronal function, offering new insights into axonal trafficking mechanisms and their implications for neuroplasticity. This work advances our understanding of how mTOR activation in axons and localized translation induced by BDNF are essential for axonal transport and efficient signaling in cortical neurons. ### Competing Interest Statement The authors have declared no competing interest.

Amyotrophic lateral sclerosis (ALS) is characterized by neuromuscular junction (NMJ) disruption and neurodegeneration. Recent findings highlight a pivotal role for TAR DNA-binding protein 43 (TDP-43) in forming axonal pathological condensates and facilitating NMJ disruption through inhibition of local protein synthesis. However, the mechanisms that drive local TDP-43 accumulation remain unknown. Here we identify that the TDP-43 axonal accumulation in peripheral nerves of SOD1 patients and mice stems from its aberrant local synthesis. This is a non-cell-autonomous process driven by muscle-derived miR-126a-5p extracellular vesicles (EVs). Inhibiting muscle secretion of miR-126a-5p prompts presynaptic TDP-43 synthesis and accumulation, which disrupts axonal translation and causes NMJ degeneration. Introducing miR-126 to SOD1G93A mice, primary co-cultures and human induced pluripotent stem cell (iPSC)-derived co-cultures with ALS mutations exhibits neuroprotective effects and delays motor decline. These findings identify a transcellular communication axis between muscles and motor neurons that regulates axonal local synthesis and NMJ maintenance, offering insights into ALS onset and progression.

Gene therapy using small interfering RNA (siRNA) holds promise for treating neurological disorders by silencing specific genes, like the phosphatase and tensin homolog (PTEN) gene, which restricts axonal growth. Effective siRNA delivery to neurons, however, poses challenges due to premature nucleic acid degradation and unspecific delivery. Chitosan-based delivery systems, noted for their biocompatibility, face limitations such as low transfection efficiency and lack of neurotropism. Building on the previous successes with neuron-targeted DNA delivery using chitosan, a novel approach for siRNA delivery aimed at PTEN downregulation is proposed. This involves using thiolated trimethyl chitosan (TMCSH)-based siRNA nanoparticles functionalized with the neurotropic C-terminal fragment of the tetanus neurotoxin heavy chain (HC) for efficient delivery to peripheral and central neurons. These polyplexes demonstrate suitable physicochemical properties, biocompatibility, and no adverse effects on neuronal electrophysiology. Diverse neuronal models, including 3D ex vivo cultures and microfluidics, confirm the polyplexes' efficiency and neurospecificity. HC-functionalization significantly enhances neuronal binding, and live cell imaging reveals fivefold faster retrograde transport along axons. Furthermore, siRNA delivery targeting PTEN promoted axonal outgrowth in embryonic cortical neurons. In conclusion, the proposed polyplexes represent a promising platform for neuronal siRNA delivery, offering potential for clinical translation and therapeutic applications.

Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited.This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.

>Neurons are highly polarized cells with axons reaching over a meter long in adult humans.To survive and maintain their proper function,neurons depend on specific mechanisms that regulate spatiotemporal signaling and metabolic events,which need to be carried out at the right place,time,and intensity.Such mechanisms include axonal transport,local synthesis,and liquid-liquid phase separations.Alterations and malfunctions in these processes are correlated to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).ALS is a fast-progressing fatal motor neuron disease pathologically characterized by muscle atrophy,neuromuscular junction degeneration,and eventually,motor neuron death(Rothstein,2009).While the precise mechanisms underlying ALS pathogenesis remain elusive,TAR DNA binding protein 43 (TDP-43) mislocalization and aggregation play a key role in the disease process (Buratti and Baralle,2009;Lee et al.,2012;Piol et al.,2023).Concurrently,disruptions in axonal transport,and alterations in local synthesis,which are vital for neuronal function,have also been observed in ALS.Understanding the bidirectional interplay between TDP-43aggregation and axonal transport/local synthesis events holds promise for elucidating ALS pathology and advancing therapeutic strategies.

Axon degeneration and Neuromuscular Junction (NMJ) disruption are key pathologies in the fatal neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Despite accumulating evidence that axons and NMJs are impacted at a very early stage of the disease, current knowledge about the mechanisms leading to their degeneration remains elusive. Cytoplasmic mislocalization and accumulation of the protein TDP-43 are considered key pathological hallmarks of ALS, as they occur in ~ 97% of ALS patients, both sporadic and familial. Recent studies have identified pathological accumulation of TDP-43 in intramuscular nerves of muscle biopsies collected from pre-diagnosed, early symptomatic ALS patients. These findings suggest a gain of function for TDP-43 in axons, which might facilitate early NMJ disruption. In this review, we dissect the process leading to axonal TDP-43 accumulation and phosphorylation, discuss the known and hypothesized roles TDP-43 plays in healthy axons, and review possible mechanisms that connect TDP-43 pathology to the axon and NMJ degeneration in ALS.

Supplementary Table from An Exercise-Induced Metabolic Shield in Distant Organs Blocks Cancer Progression and Metastatic Dissemination

Rapid responses to changes within subcellular compartments of highly polarized cells, such as neuron axons, depend on local translation and post-transcriptional regulation. The mechanism by which microRNAs (miRNAs) regulate this process is not fully understood. Here, using live cell imaging and RNA sequencing analysis, we demonstrated how miRNAs can differentially control hundreds of transcripts at the subcellular level. We demonstrated that the seed match length of the miRNA target-sequence regulates both mRNA stability and protein translation rates. While longer seed matches have an increased inhibitory effect, transcriptome analysis did not reveal differences in seed match length between axonal and somata mRNAs of motor neurons. However, mRNA variants with longer 3′UTR are enriched in axons and contain multiple repeats of specific miRNA target sequences. Finally, we demonstrated that the long 3′UTR mRNA variant of the motor protein Kif5b is enriched explicitly in motor neuron axons and contains multiple sequence repeats for binding miR-129-5p. This subsequently results in the differential post-transcriptional regulation of kif5b and its synthesis in axons. Thus, we suggest that the number of miRNA binding sites at the 3′UTR of the mRNA, rather than the miRNA seed match length, regulates the axonal transcriptome.

Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.

When digging in the ground during egg laying the female locust extends her abdomen to 2-3 times of its original length. How the abdominal nervous system accommodates such extreme elongation remains unknown. We characterized and quantified the system's biomechanical response using controlled ex vivo elongation and force measurements. The microstructure of the nerves was studied using histology and high-resolution confocal microscopy. Although the nervous system of sexually mature females demonstrated fully reversible hyper-extensibility of up to 275%, the elongation observed in premature females and males was much more limited. The unique extension dynamics of the different groups were captured by their very different force-displacement curves. Confocal microscopy suggested that elongation is not owing to undulations of the nervous system structure. Thus, the exceptional resistance to deformation and rupture presents the female locust abdominal nervous system as a valuable model for understanding the functionality and pathology related to nerve extension and reversible elongation.

Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.

Nuclear-encoded mitochondrial protein mRNAs have been found to be localized and locally translated within neuronal processes. However, the mechanism of transport for those mRNAs to distal locations is not fully understood. Here, we describe axonal co-transport of Cox7c with mitochondria. Fractionation analysis and single-molecule fluorescence in situ hybridization (smFISH) assay revealed that endogenous mRNA encoding Cox7c was preferentially associated with mitochondria in a mouse neuronal cell line and within mouse primary motor neuron axons, whereas other mRNAs that do not encode mitochondrial protein were much less associated. Live-cell imaging of MS2-tagged Cox7c mRNA further confirmed the preferential colocalization and co-transport of Cox7c mRNA with mitochondria in motor neuron axons. Intriguingly, the coding region, rather than the 3' untranslated region (UTR), was the key domain for the co-transport. Our results reveal that Cox7c mRNA can be transported with mitochondria along significant distances and that its coding region is a major recognition feature. This is consistent with the idea that mitochondria can play a vital role in spatial regulation of the axonal transcriptome at distant neuronal sites.

The molecular communication mechanisms within the Motor Neurons (MN) distant axon and its soma, as well as between MN and their neighboring cells and extracellular environment are of keen interest for our understanding of neurodevelopment and neurodegenerative diseases. One tool that has significantly improved our ability to study such processes with high spatiotemporal resolution is microfluidic devices. Here we describe a step-by-step guide to the neuromuscular co-culturing procedure and demonstrate how to track trophic factors transmission from muscle-to-neuron and their transport along the axons.