ID: 9300434
Title: Effects of botulinum neurotoxin type A on abducens motoneurons in the cat: ultrastructural and synaptic alterations.
Abstract: The synaptic alterations induced in abducens motoneurons by the injection of 3 ng/kg of botulinum neurotoxin type A into the lateral rectus muscle were studied using ultrastructural and electrophysiological techniques. Motoneurons identified by the retrograde transport of horseradish peroxidase showed a progressive synaptic stripping already noticeable by four days post-injection which increased over the study period. By 35 days post-injection, the normal coverage of motoneurons by synaptic boutons (66.4 +/- 4.0%) significantly decreased to 27.2 +/- 4.0%. Synaptic boutons detached by a widening of the subsynaptic space but remained apposed by synaptic contacts and desmosomes to the motoneuron. Detachment did not affect equally flat and round vesicle-containing boutons. The control motoneuron had almost equal numbers of both types of boutons, but after 35 days post-injection the ratio of round to flat vesicle-containing boutons was 1.20 +/- 0.01. Synaptic boutons impinging on motoneurons showed signs of alterations in membrane turnover, as indicated by an increase in the number of synaptic vesicles and a decrease in the number of coated vesicles and synaptic vesicles near the active zone. Abducens motoneurons had a transient increase in soma size by 15 days that returned to normal at 35 days, but no signs of chromatolysis or organelle degeneration were seen. Accompanying the swelling of motoneurons, a 15-fold increase in the number of spines, very infrequent in controls, was observed. Spines located in the soma and proximal dendritic trunk received synaptic contacts from both flat and round vesicle-containing boutons that could be either partly detached or completely attached to the motoneuron. An increased turnover of the plasmatic membrane of the motoneuron was observed, as indicated by a four-fold increase in the number of somatic coated vesicles. Animals were implanted with bipolar electrodes in the ampulla of both horizontal semicircular canals for evoking contralateral excitatory and ipsilateral inhibitory postsynaptic potentials. Motoneurons were antidromically identified from the lateral rectus muscle. Synaptic potentials of vestibular origin were recorded in abducens motoneurons. In the period between two and six days post-injection, a complete abolition of inhibitory synaptic potentials was observed. By contrast, excitatory synaptic potentials remained, but were reduced by 82%. The imbalance between excitatory and inhibitory inputs to motoneurons induced a progressive increase of firing frequency within a few stimuli applied to the contralateral canal. Between 7 and 15 days post-injection, both excitatory and inhibitory postsynaptic potentials were virtually abolished and remained so up to the longest time checked (105 days). Some motoneurons recorded beyond 60 days post-injection showed signs of recovery of excitatory postsynaptic potentials. During the whole time-span studied, presynaptic wavelets were present, indicating no affecting of the conduction of afferent volleys to the abducens nucleus. Taken together, these data indicate that botulinum neurotoxin at high doses causes profound synaptic alterations in motoneurons responsible for the effects seen in the behavior of motoneurons recorded in alert animals.

ID: 21929507
Title: Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission.
Abstract: Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNTA (botulinum neurotoxin type A) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25 kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNTE (botulinum neurotoxin type E). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNTA or BoNTE to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNTE yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNTA. The BoNTA protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNTE of the BoNTA acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNTA except with high doses, concurring with the lack of evidence for a direct central action when used clinically.

ID: 23110146
Title: The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes.
Abstract: In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

ID: 23300443
Title: Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons.
Abstract: The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.

ID: 25878289
Title: Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a.
Abstract: Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity.

ID: 37037273
Title: Botulinum neurotoxin A modulates the axonal release of pathological tau in hippocampal neurons.
Abstract: Pathological tau aggregates propagate across functionally connected neuronal networks in human neurodegenerative pathologies, such as Alzheimer's disease. However, the mechanism underlying this process is poorly understood. Several studies have showed that tau release is dependent on neuronal activity and that pathological tau is found in the extracellular space in free form, as well as in the lumen of extracellular vesicles. We recently showed that metabotropic glutamate receptor activity and SNAP25 integrity modulate the release of pathological tau from human and mouse synaptosomes. Here, we have leveraged botulinum neurotoxins (BoNTs), which impair neurotransmitter release by cleaving specific synaptic SNARE proteins, to dissect molecular mechanisms related to tau release at synapses. In particular, we have tested the effect of botulinum neurotoxin A (BoNT/A) on the synaptic release of tau in primary mouse neurons. Hippocampal neurons were grown in microfluidic chambers and transduced with lentiviruses expressing human tau (hTau). We found that neuronal stimulation significantly increases the release of mutant hTau, whereas wild-type hTau is unaffected. Importantly, BoNT/A blocks mutant hTau release, indicating that this process is controlled by SNAP25, a component of the SNARE complex, in intact neurons. These results suggest that BoNTs are potent tools to study the spreading of pathological proteins in neurodegenerative diseases and could play a central role in identifying novel molecular targets for the development of therapeutic interventions to treat tauopathies.

ID: 37394036
Title: Intercellular transmission of pathogenic proteins in ALS: Exploring the pathogenic wave.
Abstract: In patients with amyotrophic lateral sclerosis (ALS), disease symptoms and pathology typically spread in a predictable spatiotemporal pattern beginning at a focal site of onset and progressing along defined neuroanatomical tracts. Like other neurodegenerative diseases, ALS is characterized by the presence of protein aggregates in postmortem patient tissue. Cytoplasmic, ubiquitin-positive aggregates of TDP-43 are observed in approximately 97% of sporadic and familial ALS patients, while SOD1 inclusions are likely specific to cases of SOD1-ALS. Additionally, the most common subtype of familial ALS, caused by a hexanucleotide repeat expansion in the first intron of the C9orf72 gene (C9-ALS), is further characterized by the presence of aggregated dipeptide repeat proteins (DPRs). As we will describe, cell-to-cell propagation of these pathological proteins tightly correlates with the contiguous spread of disease. While TDP-43 and SOD1 are capable of seeding protein misfolding and aggregation in a prion-like manner, C9orf72 DPRs appear to induce (and transmit) a 'disease state' more generally. Multiple mechanisms of intercellular transport have been described for all of these proteins, including anterograde and retrograde axonal transport, extracellular vesicle secretion, and macropinocytosis. In addition to neuron-to-neuron transmission, transmission of pathological proteins occurs between neurons and glia. Given that the spread of ALS disease pathology corresponds with the spread of symptoms in patients, the various mechanisms by which ALS-associated protein aggregates propagate through the central nervous system should be closely examined.

ID: 38110531
Title: Entry and exit of extracellular vesicles to and from the blood circulation.
Abstract: Extracellular vesicles (EVs) are biological nanoparticles that promote intercellular communication by delivering bioactive cargo over short and long distances. Short-distance communication takes place in the interstitium, whereas long-distance communication is thought to require transport through the blood circulation to reach distal sites. Extracellular vesicle therapeutics are frequently injected systemically, and diagnostic approaches often rely on the detection of organ-derived EVs in the blood. However, the mechanisms by which EVs enter and exit the circulation are poorly understood. Here, the lymphatic system and transport across the endothelial barrier through paracellular and transcellular routes are discussed as potential pathways for EV entry to and exit from the blood circulatory system.

ID: 39196607
Title: Botulinum toxin intoxication requires retrograde transport and membrane translocation at the ER in RenVM neurons.
Abstract: Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to the cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localized Golgi in a retromer-dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by the toxin, revealing a new pathway mediating BoNT/A cellular toxicity.

ID: 41540479
Title: Running exercise mitigates amyloidosis in 5xFAD mice by improving the structure and function of the meningeal lymphatic system.
Abstract: BACKGROUND: Alzheimer’s disease (AD) progression is closely linked to the accumulation of amyloid-[Formula: see text] (A[Formula: see text]), with impaired clearance mechanisms playing a key role. The meningeal lymphatic (mLym) system, which drains cerebrospinal fluid (CSF) and waste from the brain to peripheral lymph nodes, has emerged as a critical pathway for A[Formula: see text] removal. While physical exercise is known to improve cognitive function and reduce AD risk, its effect on the mLym system and downstream AD pathology have not been fully elucidated. METHODS: Three-month-old 5xFAD mice underwent a 3-month wheel-running exercise regimen. The function of the mLym system was assessed before and after exercise using high-frequency ultrasound imaging with nanoparticle tracers to monitor CSF drainage to deep cervical lymph nodes. The study evaluated changes in mLym vessel structure, A[Formula: see text] deposition, and cognitive performance. Additionally, the effects of serum and extracellular vesicles (EVs) from exercised rats on the expression of lymphatic vessel-related genes (LYVE-1, VEGFR3, and VEGF-C) were examined in lymphatic endothelial and microglial cell lines. RESULTS: Compared to 3-month-old 5xFAD mice and age-matched wild-type controls, 6-month-old 5xFAD mice displayed progressive decline in mLym function, reduced vessel integrity, and increased amyloid plaque burden, accompanied by impaired learning and memory. These changes were associated with decreased expression of LYVE-1 and VEGFR3 in the meninges and VEGF-C in the brain. Exercise intervention reversed these deficits, restoring mLym function and vessel structure, enhancing A[Formula: see text] clearance, and improving cognitive performance. Surgical ligation of mLym vessels accelerated amyloid accumulation and removed the exercise-induced benefits, underscoring the system’s importance in A[Formula: see text] removal. In vitro, A[Formula: see text] oligomers suppressed VEGFR3 and VEGF-C expression, while serum and EVs from exercised rats counteracted this effect. Proteomic analysis of EVs from exercised animals revealed upregulation of CD9, suggesting a link to VEGFR3 signaling. CONCLUSIONS: This study demonstrates that A[Formula: see text] oligomers impair mLym function, exacerbating amyloid pathology. Exercise preserves the structure and function of the mLym system, promoting A[Formula: see text] clearance and mitigating AD progression. These findings highlight the therapeutic potential of targeting the meningeal lymphatic system to slow or prevent AD.

ID: 41989572
Title: Botulinum toxin from foodborne hazard to aesthetic and biomedical tool: mechanisms, applications, detection strategies, and future perspectives.
Abstract: As powerful biological toxins, botulinum neurotoxins (BoNTs), which are mainly generated by the anaerobic bacterium Clostridium botulinum, are at the same time useful therapeutic and cosmetic agents. This review gives an extensive review of the microbiological etiology, pathophysiology, clinical use, detection strategies, and emerging challenges regarding BoNT. On a molecular scale, BoNTs are endopeptidases, which are zinc-dependent and which inhibit the fusion of synaptic vesicles by cleaving SNARE proteins, thus preventing the release of acetylcholine and causing reversible neuromuscular paralysis. This property is the basis of their medical use in treating a wide range of neuromuscular and glandular conditions, such as dystonia, spasticity, chronic migraine, and hyperhidrosis, and aesthetic surgeries using compounds like Botox and its derivatives. Although they are clinically successful, their therapeutic use is associated with possible adverse effects, including local muscle weakness as well as uncommon systemic complications, especially in the case of high dosage or incorrect administration. The review also compares existing analysis techniques to detect BoNT, such as immunological assays, molecular diagnostics, biosensor technologies, and mass spectrometry with their benefits and drawbacks in sensitivity, specificity, and toxin functional evaluation. Also , neutralization of the body formation, resistance to treatment, variability of toxin preparations, and regulatory difficulties are addressed. Future opportunities focus on the creation of non-animal testing models, advanced biosensing technology and individualized therapeutic methods to increase safety, diagnostic accuracy, and prolonged clinical performance.

ID: 42102607
Title: BoNT-A amelliorates itch sensitization in atopic dermatitis mice by inhibiting histamine release.
Abstract: In this study, we thoroughly investigated the potential mechanisms underlying the therapeutic effects of Botulinum Toxin Type A (BoNT-A) on ameliorating pruritic behavior and allodynia in a murine model of atopic dermatitis (AD). Recognizing the pivotal role of mast cells in AD pathogenesis, we hypothesized that BoNT-A inhibits mast cell degranulation via the cleavage of SNAP-25. Using P815 cells and primary mast cells, we confirmed the expression of SNAP-25 and SV2, and through siRNA-mediated knockdown, we validated that SNAP-25 is a pivotal mediator of mast cell degranulation and the specific target required for BoNT-A' s inhibitory action. In our in vivo experiments utilizing MC903-induced AD mice, we observed that BoNT-A administration significantly reduced scratching bouts and alleviated pruritus allodynia while decreasing mast cell recruitment in both epidermal and dermal layers. Furthermore, to elucidate the involvement of the central nervous system, we constructed a BoNT-A/FITC complex and demonstrated that the toxin undergoes retrograde transport from peripheral nerves to the spinal cord dorsal horn. Therefore, it appears to exert an antipruritic effect by downregulating the expression of pruritus-related ion channels and neuropeptides, specifically TRPV1 and CGRP. Consequently, our findings establish that BoNT-A exerts its therapeutic effects through a dual mechanism: peripherally by inhibiting mast cell degranulation via the SNAP-25/SV2 pathway, and centrally by modulating neurogenic inflammation within the spinal cord, thus providing a novel perspective for the treatment of allergic diseases.

ID: 42275483
Title: Intranasal Delivery of Bacterial Extracellular Vesicles Enables RNA Cargo Entry Into the Brain.
Abstract: Extracellular vesicles (EVs) released by bacteria are potent mediators of host-microbe interactions. They modulate immune responses, deliver functional molecules and influence disease progression. However, whether bacterial EVs can access the brain and functionally affect host cells remains unclear. In this study, we engineered Escherichia coli-derived EVs by electroporating Cre recombinase mRNA (Ec EVCre) and assessed their transport and functional delivery following intranasal administration. Using mT/mG reporter mice, we observed EV uptake in the olfactory epithelium and recombination-driven GFP expression in a subset of neurons in the olfactory bulb, providing proof-of-concept for the functional delivery of bacterial EV-associated mRNA into the brain. Single-cell RNA sequencing and imaging analyses of the olfactory regions revealed neuronal and immune cell subsets as key EV targets. Microfluidic biochip chamber assays with cultured sensory neurons demonstrated that EVs undergo retrograde axonal transport from neurite terminals to the soma via signalling endosomes. Pharmacological inhibition significantly impaired EV uptake, supporting the involvement of endocytic pathways. In addition to neuronal entry, we discovered that phagocytic cells, including neutrophils and macrophages, can engulf EVCre in the nasal mucosa and migrate into the brain, providing an alternative immune-mediated route for vesicle delivery. Together, these findings indicate that bacterial EVs exploit both neuronal and phagocytic pathways to deliver functional RNA cargo into the brain, providing novel insights into microbial access to the central nervous system and its implications for neuroimmune interactions.

ID: 42342866
Title: Bioenergetic failure in diabetic peripheral neuropathy: from glucotoxicity to multidimensional metabolic imbalance.
Abstract: Diabetic peripheral neuropathy (DPN), particularly distal symmetric polyneuropathy, characterized by length‑dependent axonal damage, is a common chronic complication of type 2 diabetes mellitus. The pathogenesis of DPN is complicated, yet one thing is clear: long axons require a great deal of energy. When axonal transport is compromised by impaired energy metabolism, neuronal somata fall into an energy deficit that triggers neurodegeneration. While earlier work centered on hyperglycemia-induced cytotoxicity, recent studies have increasingly implicated dysregulation of glucose, lipid, and amino acid metabolism as key contributors to DPN. In this review, we integrate the anatomical organization of peripheral nerves, bioenergetic pathways, and axon-Schwann cell interactions to establish a framework for understanding how glucose, lipid, and amino acid dysregulation converge to induce bioenergetic failure in DPN. Based on these mechanisms, we further discuss novel strategies aimed at restoring metabolic homeostasis in neurons and Schwann cells. Importantly, correcting a single metabolic pathway is unlikely to halt or reverse DPN. Instead, restoring global energy homeostasis to rebalance axonal energy supply and demand may be essential for preserving peripheral nerve function.

ID: 42350670
Title: Metabolic state determines the brain and direct islet effects of liraglutide on enhanced insulin secretion.
Abstract: Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist for type 2 diabetes and obesity management, shows variable patient responses. We investigated the metabolic state-dependent mechanisms underlying this heterogeneity and how liraglutide's mode of action shifts across stages of metabolic dysfunction. We employed human pancreatic islets from donors across metabolic states (normoglycaemic [HbA1c <42 mmol/l (<6.0%)], glucose intolerance [HbA1c 42-47 mmol/l (6.0-6.4%)] and type 2 diabetes [HbA1c ≥48 mmol/l (≥6.5%)]) using dynamic perifusion and static incubation techniques to assess glucose-stimulated insulin secretion. GLP-1R mRNA levels were measured in 112 donor islets stratified by HbA1c. Mechanistic investigations used tanycyte-specific GLP-1R knockdown (GLP-1RTanycyteKD) mice and botulinum toxin B-expressing (iBot) mice to distinguish between central and peripheral pathways. Oral glucose tolerance tests, pyruvate tolerance tests and positron emission tomography were performed to assess in vivo metabolic effects. Liraglutide (25 nmol/l) enhanced glucose-stimulated insulin secretion specifically in donors with glucose intolerance (n=7, p=0.021), with no effect in normoglycaemic islets (n=7), despite preserved GLP-1 (7-36) responsiveness. In type 2 diabetes islets, GLP-1R mRNA levels progressively decreased with rising HbA1c (p=0.015, normoglycaemic [n=48] vs type 2 diabetes [n=10]). In chow-fed mice, liraglutide's insulin-stimulating effects required tanycyte-mediated hypothalamic access, as demonstrated by abolished responses in GLP-1RTanycyteKD mice. However, during metabolic dysfunction (a 12-week high-fat diet), direct islet responsiveness was restored independent of tanycyte function. Advanced metabolic disease (a 27-week high-fat diet) maintained islet responsiveness ex vivo while losing in vivo insulin enhancement, revealing insulin-independent glucose-lowering mechanisms involving hepatic gluconeogenesis suppression and enhanced peripheral glucose uptake. Liraglutide operates through complementary, metabolic state-dependent pathways: tanycyte-mediated brain actions predominate in healthy conditions, direct islet effects emerge during glucose intolerance and insulin-independent mechanisms maintain efficacy across metabolic states. This mechanistic framework enables potential patient stratification in type 2 diabetes therapy, suggesting that matching liraglutide's predominant mechanism to individual metabolic profiles could optimise treatment outcomes.

ID: 42401592
Title: Munc18-1 is crucial for photoreceptor function, survival and regulation of syntaxin-3 localization and expression.
Abstract: The function of the SNARE complex regulator, Munc18-1, in photoreceptor cells is unknown. Here, we found that removing Munc18-1 from photoreceptors results in major degeneration starting at P14. In the absence of Munc18-1, before major photoreceptor degeneration, functional and synaptic impairments were present, indicating a critical function of Munc18-1. Furthermore, Munc18-1 played a critical role in expression and localization of syntaxin-3. The syntaxin-3 protein level is dramatically reduced in the soma and plasma membrane of photoreceptors without Munc18-1. At the photoreceptor synapses, the colocalization of syntaxin-3 and its SNARE partner, SNAP-25, was reduced, potentially suggesting an altered syntaxin-3 synaptic localization. In the Munc18-1-deficient photoreceptors, immature synapses and outer segment lesions were found. Taken together, these findings provide evidence that Munc18-1 is important for maintaining sufficient syntaxin-3 expression in the cell body and synapses of photoreceptors. The lack of Munc18-1, combined with poor syntaxin-3 expression, contributes to photoreceptor functional impairment and degeneration.

ID: 42422258
Title: Neuromuscular-related interventions for post-stroke dysphagia: a comprehensive narrative review.
Abstract: To systematically review the efficacy, mechanisms, and application characteristics of neuromuscular-related interventions for post-stroke dysphagia (PSD), and to examine strategy selection across different swallowing stages to inform individualized rehabilitation strategies. PubMed, Web of Science, Embase, and MEDLINE were systematically searched for studies published between 1995 and 2025. Titles and abstracts were screened, and the full texts of eligible studies were retrieved for further analysis. Interventions related to neural and muscular regulation were categorized and synthesized into four main groups: exercise training and behavioral interventions; peripheral neuromuscular stimulation; central nervous system modulation techniques; and other adjunctive interventions. A total of 293 publications were included, of which 56.63% were randomized controlled trials. The interventions were summarized into four major categories comprising more than 10 techniques. Exercise training and behavioral interventions (e.g., oral motor exercises) enhanced swallowing muscle strength and coordination. Peripheral neuromuscular stimulation (e.g., neuromuscular electrical stimulation and acupuncture) enhanced or modulated swallowing function by directly stimulating relevant nerves or muscles. Central nervous system modulation techniques (e.g., transcranial magnetic stimulation and transcranial direct current stimulation) influenced swallowing-related neural networks indirectly by regulating cortical excitability. Other adjunctive interventions included botulinum toxin injection, which directly targeted the cricopharyngeal muscle. Further analysis examined the selection of key rehabilitation techniques across different clinical stages of PSD, integrating central and peripheral neuromodulation approaches. It explored the potential implications of soft-tissue surgery and meridian-muscle theory for PSD management to inform individualized clinical decision-making. Neuromuscular interventions were found to be widely used in PSD management, particularly transcranial magnetic stimulation, acupuncture, and neuromuscular electrical stimulation. Future strategies should integrate pathology, clinical manifestations, and lesion localization to develop central lesion-oriented multimodal therapies that combine peripheral nerve and muscle interventions, potentially improving clinical outcomes.

ID: 42427545
Title: Dystrophic changes of nigrostriatal axons harboring a Synj1 Parkinson mutation suggest catastrophic failure of endocytic mechanisms.
Abstract: Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

ID: 42458952
Title: Syntaxin-4 at the Crossroads of Synaptic Plasticity and Neurodegeneration: From Molecular Mechanisms to Biomarker and Therapeutic Potential.
Abstract: Syntaxin-4 (Stx4), a member of the Qa-SNARE protein family, is a pivotal regulator of membrane trafficking. Stx4 is predominantly localized at the postsynaptic membrane of neurons and in glial cells, where it is essential for synaptic plasticity and the maintenance of neuronal homeostasis. Stx4 facilitates the activity-dependent exocytosis of glutamatergic receptors, thereby governing the balance between Long-Term Potentiation (LTP) and Long-Term Depression (LTD). Emerging evidence suggests that dysregulation of Stx4 is intricately linked to the pathogenesis of neurodegenerative diseases. In Alzheimer's disease, Stx4 is involved in synaptic dysfunction and cognitive impairment through its interactions with amyloid-β (Aβ) and tau pathologies, affecting receptor recruitment at dendritic spines. In Parkinson's Disease (PD), Stx4 contributes to α-synuclein proteostasis, dopaminergic signaling, neuroinflammation, and the maintenance of blood-brain barrier integrity. While its presence in cerebrospinal fluid highlights its potential as a candidate biomarker for these diseases, further validation is required to establish clinical utility. This review summarizes current mechanistic insights into Stx4's multifaceted roles in the neurodegenerative disorders and explores the therapeutic prospects of targeting Stx4-mediated pathways due to its translational potential.

ID: 42466399
Title: Neuronal Mitochondrial Dysfunction Drives Astrocytic Mitochondrial Transfer after TBI: Reveals the Therapeutic Potential of astrocytic EV-Mito.
Abstract: Mitochondria are dynamic organelles essential for neuronal survival and synaptic function, and their dysfunction is a key consequence of excitotoxicity following traumatic brain injury (TBI). While intercellular mitochondrial transfer and exogenous mitochondrial transplantation have emerged as mechanisms to restore cellular bioenergetics, its in vivo relevance in the central nervous system remains incompletely understood. Here, we used astrocyte and neuron-specific mitochondrial reporters (GFP or Dendra2) in mice to assess cell-type-specific mitochondrial morphology, bioenergetics, and transfer 24hrs after TBI. Neurons exhibited marked mitochondrial dysfunction, including altered morphology and reduced bioenergetic capacity across somatic, synaptic, and non-neuronal fractions. In contrast, astrocytic mitochondria showed morphological changes but preserved bioenergetic function. Concomitantly, astrocyte-to-neuron mitochondrial transfer was significantly increased following injury, although transfer to synapses remained limited. Single-cell RNA sequencing of astrocytes revealed upregulation of genes involved in extracellular vesicle (EV) biogenesis and mitochondrial translation following injury compared to controls. In vitro co-culture studies confirmed that astrocytes transfer mitochondria to neurons via EVs containing mitochondria (EV-mito). Isolated EV-mito from astrocyte-conditioned media improves neuronal mitochondrial function under NMDA (N-methyl-D-aspartate) induced excitotoxic conditions. Together, these findings demonstrate that neuronal mitochondrial dysfunction drives astrocyte-mediated mitochondrial transfer as an adaptive neuroprotective response after TBI. This process preserves neuronal bioenergetics in the soma and neurites but not at synapses, highlighting both its therapeutic potential and spatial limitations.

ID: 42503395
Title: Engineered extracellular vesicles derived from sweet potato loaded with siPOLD1 for targeted therapy of glioma.
Abstract: Glioma is the most common malignant tumor of the central nervous system, with high malignancy and poor prognosis, necessitating the development of novel targeted therapies. DNA polymerase delta catalytic subunit 1 (POLD1) is implicated in multiple cancers, but its role in glioma remains unclear. Plant-derived extracellular vesicles (PDEVs) have emerged as biocompatible, targetable nanocarriers with promising applications in cancer therapy. This study aims to elucidate the oncogenic function of POLD1 in glioma and develop a PDEVs -based delivery system for targeted therapy, with the goal of improving the current therapeutic landscape for glioma. POLD1 expression and prognostic significance were analyzed using clinical samples and databases. In vitro, CCK-8, Transwell, and flow cytometry assays evaluated the impact of POLD1 knockdown on glioma cell proliferation, invasion, migration, cell cycle, and apoptosis. In vivo tumorigenesis and survival were assessed in mouse models. Sweet potato-derived nano-vesicles (SPDELNVs) were isolated and characterized. An engineered A2-SPDELNVs-siPOLD1 system was developed via surface modification and siRNA loading, and its targeting efficiency and therapeutic efficacy were evaluated both in vitro and in vivo. POLD1 was upregulated in glioma tissues and correlated with poor prognosis. Its knockdown suppressed proliferation, invasion, and migration, induced cell cycle arrest, and promoted apoptosis in vitro. In vivo, POLD1 targeting inhibited tumor growth and prolonged survival. SPDELNVs showed intrinsic anti-glioma activity and efficient cellular uptake. The engineered A2-SPDELNVs-siPOLD1 effectively delivered siRNA, silenced POLD1, and significantly inhibited tumor progression both in vitro and in vivo, with enhanced survival. Our findings uncover the oncogenic role of POLD1 in glioma and validate it as a promising therapeutic target. Furthermore, we establish a novel, plant-based A2-SPDELNVs-siPOLD1 delivery platform with effective BBB penetration and tumor targeting, offering a promising strategy for the treatment of glioma.

ID: 42504872
Title: Unconventional Protein Secretion in the Central Nervous System: Mechanisms and Roles in Physiology and Disease.
Abstract: Extracellular secretion of neurotransmitters, proteins, and peptides by cells of the central nervous system underpins neurological function and homeostasis. Decades of elegant research have illuminated the molecular mechanisms and machinery that support the release of neurotransmitters via synaptic vesicle exocytosis, as well as the secretion of signal-peptide bearing proteins through the endoplasmic reticulum (ER)-Golgi based secretory pathways. However, it is now increasingly appreciated that signal-peptide lacking "leaderless" proteins can also be secreted via ER-Golgi-independent mechanisms collectively termed unconventional protein secretion (UcPS). In this review, we highlight the physiological and pathological consequences of UcPS in the central nervous system. UcPS supports the secretion of aggregation-prone proteins such as α-synuclein and mutant huntingtin, pro-inflammatory mediators including interleukin-1β and high mobility group box protein 1, and neuroprotective or angiogenic factors such as fibroblast growth factor 2. Furthermore, several retroelement-derived proteins, encoded by ancient genomic elements with structural homology to retroviruses, are also secreted via unconventional pathways, and are thought to regulate essential CNS processes such as synaptic plasticity. These diverse cargoes underscore the functional range of UcPS in neuronal and glial biology. We summarize current understanding of the major UcPS pathways used by CNS cells. These mechanisms include plasma-membrane pore-mediated release facilitated by proteins such as gasdermin-D, as well as vesicular routes in which UcPS cargoes enter organelles of the autophagic and endolysosomal systems that subsequently fuse with the plasma membrane to enable extracellular release. Finally, we discuss key unresolved questionRecent evidence from HeLa cells suggests regarding the regulation of UcPS, including the molecular features that target select leaderless cargoes toward UcPS, how the balance between conventional secretion and UcPS shifts under cellular stress, and the current understanding of the diverse molecular machinery that mediates the vesicular form of UcPS.

ID: 42506696
Title: Effects of Botulinum Toxin Type A in Essential Blepharospasm: Evidence from a Clinical and Neurophysiological Pilot Study.
Abstract: Essential blepharospasm (BEB) is a focal dystonia characterized by abnormal brainstem excitability and impaired inhibitory control within trigeminal-facial circuits. Botulinum toxin type A (BoNT-A) is the established first-line treatment, primarily acting at the neuromuscular junction. However, whether BoNT-A also modulates central brainstem circuits in BEB patients remains unclear, and dedicated neurophysiological studies have yielded conflicting results. To investigate whether BoNT-A modulates brainstem interneuronal excitability in BEB using the blink reflex recovery cycle and to correlate clinical outcomes with neurophysiological results. Thirteen patients with BEB underwent neurophysiological and clinical evaluation before (T0) and one month after (T1) BoNT-A treatment. The blink reflex recovery cycle was assessed at interstimulus intervals (ISIs) of 200, 300, 500, and 1000 ms. Clinical severity was assessed using the BSPSS, JRS, and BDS scales. The R2 amplitude ratio showed a statistically significant decrease after treatment across all ISIs (all p ≤ 0.001), indicating reduced brainstem interneuronal excitability. All clinical scales demonstrated statistically significant improvement after treatment (BSPSS, JRS, BDS; all p < 0.001). This pilot study provides preliminary evidence that BoNT-A treatment may reduce brainstem interneuronal excitability in BEB patients, as evidenced by a substantial and consistent decrease in R2 amplitude ratios of the blink reflex recovery cycle. These findings are consistent with a central modulatory effect of BoNT-A beyond its established peripheral action. Larger controlled studies are warranted to confirm these results.

ID: 42543397
Title: Autonomous intranasal delivery systems for central nervous system therapeutics.
Abstract: Intranasal delivery provides a rapid, non-invasive route to the central nervous system, bypassing the blood-brain barrier and first-pass metabolism. However, its therapeutic potential remains constrained by the nasal cavity's complex anatomy, the restricted surface area and permeability of the olfactory epithelium, and short drug residence times. Recent advances in nanotechnology and synthetic biology have enabled the development of autonomous and programmable delivery systems that can target the olfactory epithelium, enhance brain entry and sustain therapeutic release. This review highlights current strategies for engineering intranasal drug delivery vectors that can replicate or extend cellular functions to enable autonomous nose-to-brain drug delivery. These vectors include: synthetic nanoparticles that mimic essential cellular activities and allow for modular surface modification; extracellular vesicles that naturally carry therapeutic cargo and exhibit parent-cell-derived tropism; and living therapeutics, such as engineered microbes, viruses or stem cells, that respond dynamically to host environments and can be genetically programmed for precise payload production. Emphasis is placed on the modular design of functional components, host-responsive interactions tailored to anatomical and physiological cues, and the integration of programmable functions that collectively drive delivery autonomy and therapeutic efficacy. Together, these advances position intranasal delivery as a versatile platform for treating neurological disorders, offering a foundation for future translational development.