Diabetic neuropathic pain is a debilitating complication of diabetes for which disease-modifying therapies remain limited. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in neuroprotection and metabolic regulation, but its role in diabetic neuropathic pain remains unclear. Here, we found that patients with diabetic neuropathic pain had reduced serum PACAP-38 levels and elevated pro-inflammatory cytokines compared with patients with diabetes mellitus alone. In streptozotocin-induced diabetic neuropathic pain (DNP) mice, PACAP partially improved body weight recovery but did not markedly reduce fasting hyperglycemia, glucose tolerance or pancreatic islet injury. Importantly, PACAP improved gait performance, attenuated mechanical, thermal, and cold hypersensitivity, and preserved intraepidermal nerve fibers. Transcriptomic and validation analyses showed that diabetic neuropathic pain was associated with endothelial impairment, hypoxia-related signaling, mitochondrial alterations, and redox imbalance in dorsal root ganglia. PACAP treatment partially modulated DNP-related transcriptional changes, including those linked to mitochondrial metabolism, oxidative stress-related processes, and neuronal function. Mechanistically, PACAP increased ERK phosphorylation and PGC1α expression, enhanced mitochondrial biogenesis-related responses, preserved mitochondrial ultrastructure, and improved mitochondrial membrane potential. ERK inhibition with U0126 attenuated PACAP-mediated mitochondrial protection and analgesic effects. These findings suggest that PACAP alleviates DNP through an ERK-PGC1α-associated mechanism that contributes to the restoration of mitochondrial homeostasis in sensory neurons, rather than by substantially correcting systemic hyperglycemia.
Neonicotinoid pesticides are widely used systemic insecticides with increasing evidence of neurotoxicity. However, their potential contribution to Alzheimer's disease (AD)-related neuroinflammation remains poorly understood. In this study, seven representative neonicotinoid pesticides were systematically evaluated by integrating physicochemical characterization, target prediction, pathway enrichment analysis, human AD snRNA-seq, molecular docking, molecular dynamics simulations, and experimental validation. Among the seven compounds, dinotefuran was prioritized based on its favorable blood-brain barrier permeability and enrichment in inflammation-related pathways. Integration of target prediction with AD transcriptomic analyses identified CHUK and NFKB1 as key candidate targets associated with NF-κB signaling. snRNA-seq analysis further demonstrated preferential enrichment of NF-κB signaling activity in microglia from AD brains. Molecular docking and molecular dynamics simulations supported potential interactions between dinotefuran and IKKα (CHUK) as well as NF-κB1 (p50). In Aβ-treated BV2 microglia, dinotefuran further enhanced activation of the CHUK/NFKB1-mediated NF-κB signaling pathway, accompanied by increased expression of IKKα, phosphorylation of IκBα and p65, enhanced NF-κB1 activation, and elevated secretion of IL-1β, TNF-α, IL-6, IFN-α, and IFN-β. These pro-inflammatory effects were markedly attenuated by the NF-κB inhibitor BAY11-7082. Collectively, our findings suggest that CHUK/NFKB1-associated NF-κB signaling may contribute to dinotefuran-enhanced inflammatory responses in Aβ-treated BV2 microglia, providing preliminary mechanistic insights into the potential involvement of neonicotinoid pesticides in AD-related neuroinflammation.
Migraine, most notably migraine with aura, elevates the risk of ischaemic stroke, though the underlying causal molecular connections have not been fully elucidated. This study sought to uncover migraine-associated causal genes that confer susceptibility to ischaemic stroke and to dissect their cell-type-specific functional mechanisms operating within the neurovascular unit. We combined publicly available human transcriptomic resources, genetics-driven causal inference, machine-learning-assisted biomarker screening, mouse-derived single-cell and spatial transcriptomic atlases, immune-cell deconvolution, and in-silico molecular docking. Twenty migraine-related genes were detected with causal associations to ischaemic-stroke risk. We prioritised three consensus hub genes: CD79A, PKN2, and PRKDC. In stroke patients, CD79A was significantly downregulated, while PKN2 and PRKDC exhibited elevated expression; their reliable diagnostic capacity was further confirmed in an independent external cohort. Single-cell and spatial transcriptomic profiling mapped these hub genes to endothelial, myeloid and glial cell populations residing within a dynamic inflammatory-vascular pathological niche in mouse ischaemic brain lesions. Mechanistic analyses pointed to perturbed mitochondrial energy homeostasis and dysregulated adaptive immune responses as downstream consequences modulated by these hub genes. Molecular-docking simulations demonstrated high-affinity interactions between Tamibarotene, Dirithromycin and the three encoded hub proteins, highlighting these compounds as promising candidates for drug repurposing. Collectively, our multi-omics analysis pinpoints CD79A, PKN2 and PRKDC as migraine-derived causal targets for ischaemic stroke, delivers cell- and spatial-resolved mechanistic perspectives on neurovascular-unit injury, and puts forward lead compounds awaiting further pre-clinical validation.
Glymphatic system (GS) dysfunction contributes to impaired solute clearance, neuroinflammation, and secondary injury after traumatic brain injury (TBI). We investigated whether temporal interference stimulation (TIS) targeting the lateral hypothalamus (LH) improves neurological outcomes and GS function after TBI and explored the underlying mechanism. Finite element modeling was used to optimize the electrode montage and stimulation parameters for LH-targeted TIS. Treatment effects were assessed by behavioral testing, histopathology, glymphatic tracer imaging, and neurovascular evaluation. LH activation, Orexin A (OXA)/orexin receptor 2 (OX2R)-CaMKII signaling, and aquaporin 4 (AQP4) polarization were examined using immunofluorescence, transcriptomic profiling, molecular analyses, and pharmacological pathway inhibition. Mechanical stretch injury and astrocyte-neuron or astrocyte-endothelial co-culture models were used for further mechanistic validation. LH-targeted TIS increased LH neuronal activation with spatial specificity and no detectable short-term adverse effects, improved neurological and cognitive performance, and reduced neuronal injury, cerebral edema, and neuroinflammation after TBI. TIS restored GS clearance, enhanced OXA/OX2R-CaMKII signaling, strengthened dystrophin-associated AQP4 anchoring, re-established perivascular AQP4 polarization, and modulated astrocyte reactivity-associated markers. OX2R blockade markedly attenuated the effects of TIS on GS function, neuroinflammation, and behavioral outcomes. In vitro, OXA reduced C3 expression, increased S100A10 expression, and attenuated neuronal and endothelial injury in astrocyte co-cultures; these effects were diminished by AQP4 overexpression. Collectively, LH-targeted TIS improves GS function after TBI, at least partly through OXA/OX2R-CaMKII-associated regulation of dystrophin-associated AQP4 organization and polarization, thereby supporting astrocyte, neurovascular, and synaptic homeostasis and improving neurological outcomes.
The indications for acute reperfusion therapies in ischemic stroke have recently expanded to include patients with large ischemic cores, highlighting an urgent need for targeted neurotherapeutics that maximize tissue salvage. Preclinical studies suggest that early reperfusion, even after extensive ischemic injury, can mitigate infarct expansion and activate endogenous regenerative pathways, though the precise mechanisms driving these therapeutic benefits remain incompletely understood. In this study, we investigated the cellular and molecular mechanisms underlying successful reperfusion to identify novel avenues for immunotherapy. Using transient (t-MCAO) and permanent (p-MCAO) middle cerebral artery occlusion mouse models, we comprehensively analyzed central and peripheral immune cell dynamics. Our analysis revealed that successful reperfusion significantly elevates regulatory T cell (Treg) proportions compared to non-reperfused controls. Across experimental groups, higher interleukin-10 (IL-10) levels generally accompanied greater Treg abundance and better motor performance. Targeted intra-arterial delivery of IL-10 at the exact time of reperfusion directly attenuated cerebral injury and improved neurological outcomes. These findings suggest that amplifying anti-inflammatory signaling post-reperfusion may provide neuroprotection, positioning intra-arterial IL-10 as a potential adjunctive neurotherapeutic strategy to enhance the efficacy of mechanical thrombectomy.
