Transient receptor potential vanilloid 1 (TRPV1) is increasingly recognized as an important regulator of microglial response during neuroinflammation, a key pathological feature of various neurodegenerative diseases such as Alzheimer’s disease. Capsaicin, the primary pungent component in chili peppers, executes its anti-inflammatory properties as a TRPV1 agonist. Although the mechanism of TRPV1 channel activation and signal transduction by capsaicin has been extensively studied, the protein–protein interaction-based mechanisms by which TRPV1 modulates microglial inflammatory responses in the context of neurodegeneration remains unclear. We used a lipopolysaccharide-induced acute neuroinflammation model to examine the effects of TRPV1 activation and pharmacological blockade of TRPV1 channel gating on microglial inflammatory responses. Proximity labeling-based proteomics, bioluminescence resonance energy transfer, and NanoLuc Binary Technology were used to identify and characterize TRPV1-interacting proteins. Signal sequence receptor subunit 4 (SSR4) knockdown and forced interaction between TRPV1 and SSR4 were performed to assess the functional contribution of the TRPV1–SSR4 interaction. Structural modeling, BRET-based domain mapping, and colocalization analyses were further used to examine the molecular and subcellular features of SSR4 recruitment by TRPV1. In the neuroinflammatory model, the TRPV1 agonist capsaicin attenuated pro-inflammatory cytokine production, neuronal apoptosis, microgliosis, and microglial soma enlargement in a TRPV1-dependent manner. Notably, pharmacological blockade of TRPV1 channel gating by capsazepine did not abolish the anti-inflammatory action of capsaicin, suggesting an alternative mechanism of action beyond the canonical ligand-gated Ca2⁺ influx. Screening of TRPV1 prey proteins and validation strategy identified SSR4 as a capsaicin-responsive, TRPV1-associated protein. SSR4 knockdown in microglia significantly diminished the suppression of inflammatory responses caused by TRPV1 activation. Conversely, forced interaction between TRPV1 and SSR4 partially mimicked the anti-inflammatory effects of TRPV1 activation. Further structural and cellular analyses suggested that the transmembrane region of TRPV1 supports SSR4 recruitment. Our findings suggest that capsaicin suppresses microglial inflammation mediated by the TRPV1–SSR4 interaction, providing new insights into the chemical biology basis of TRPV1 interoceptive signaling and potential applications of natural TRPV1 agonists for modifying therapy of neuroinflammation-associated neurodegeneration.
Autophagy-lysosomal dysfunction contributes to lipid droplet (LD) accumulation, which drives pathological progression in Alzheimer's disease (AD). Exercise is reported to restore autophagy-lysosomal function and attenuate cognitive impairment in AD, but its regulation of LD deposition remains unknown. 5×FAD transgenic mice underwent a three-month exercise intervention. Cognitive and emotional behaviors were assessed using the Morris water maze, novel object recognition, and open field test. LD deposition was evaluated by oil red O and BODIPY staining. Proteomic sequencing was performed to identify proteins affected by exercise. Pathological changes were examined by immunofluorescence, western blotting, and transmission electron microscopy. Intracerebroventricular injection of AAV was used to overexpress or knock down microglial TMEM9 to investigate the mechanisms by which physical exercise affects behavior and pathology in AD. In vitro, BV2 cells were cultured and incubated with oligomeric amyloid-β (oAβ), with or without oleic acid to induce LD formation. Chloroquine and rapamycin, which regulate autophagy-lysosomal function, were used to examine the effects of LDs on mitochondrial integrity under glucose deprivation conditions. Lentiviral or plasmid transfection was used to explore the key role of TMEM9 in regulating lipophagy, and the interaction between TMEM9 and ATP6AP1 was detected by co-immunoprecipitation. Clinically, plasma TMEM9 and ATP6AP1 levels in AD patients were measured by ELISA, and receiver operating characteristic (ROC) analysis was performed to evaluate diagnostic performance. Exercise mitigated cerebral LD deposition through enhanced lipophagy, preserving mitochondrial integrity and attenuating cognitive decline. Mechanistically, exercise suppressed microglial TMEM9 expression, restoring LD degradation. Tmem9 downregulation facilitated LKB1 lysosomal translocation, activating AMPK signaling. In vitro, oleic acid-induced LDs preserved mitochondrial membrane potential and increased ATP production in BV2 microglia under glucose deprivation and oAβ exposure. Tmem9 overexpression exacerbated lipophagy impairment and mitochondrial dysfunction, whereas Tmem9 knockdown enhanced lipophagy and cytoprotection. Aβ clearance was negatively regulated by TMEM9. Clinically, AD patients exhibited characteristic Aβ deposition and glucose hypometabolism, with plasma TMEM9 levels strongly correlating with p-tau217 progression. Plasma TMEM9 and the TMEM9/ATP6AP1 ratio emerged as potential AD diagnostic biomarkers (AUC=0.791 and 0.853, respectively). These findings establish exercise-induced TMEM9 inhibition as a novel therapeutic strategy targeting lipidostasis to combat AD progression.
Abstract Background Alzheimer’s disease (AD) and related dementias (ADRDs) remain incurable, partly due to their complex and poorly understood pathogenic mechanisms. Loss of vacuolar protein sorting-associated protein 35 (VPS35), a core component of the retromer complex, has been implicated in AD and ADRDs. While Vps35 deficiency is known to increase amyloidogenesis by regulating its cargo proteins, such as APP and BACE1, the precise mechanism by which VPS35 dysfunction leads to cognitive impairment remains elusive. While neuronal VPS35 regulates trafficking and functions of multiple synaptic membrane proteins, such as postsynaptic glutamatergic and GABAnergic receptors and pre-synaptic vesicle protein SV2A, and Levetiracetam (LEV), an antiepileptic drug that improves cognition, interacts with the SV2A, exactly how LEV enhances cognition is still a matter of debate. Methods Given the abundant expression of VPS35 in pyramidal excitatory neurons, we generated a conditional knockout mouse model with Vps35 deletion from postnatal glutamatergic excitatory neurons ( Vps35 CaMKIIα ) and characterized its phenotypes using a combination of behavioral assessments, and electrophysiological, biochemical, and immunohiotological analyses in the hippocampal CA1 region. A targeted pharmacological screen was conducted to evaluate the therapeutic potential of LEV. Results Vps35 deletion did not disrupt the gross brain development but led to progressive cognitive decline beginning at three months of age. Electrophysiological analyses revealed earlier-onset hippocampal CA1 hyperexcitability, marked by depolarized resting membrane potentials and reduced Kir 2.1 expression. However, restoring Kir 2.1 to suppress hyperexcitability failed to rescue cognitive deficits. Instead, cognitive impairment was associated with weakened GABAergic transmission and disruption of the excitatory/inhibitory balance in the Schaffer collateral–CA1 circuit. Notably, LEV treatment diminished these deficits in mutant mice. Intriguingly, LEV increased the levels of retromer component, VPS26A, and multiple pre- and postsynaptic GABAergic proteins, in addition to SV2A, all of which were reduced in Vps35 -deficient CA1 neurons. Conclusions These findings identify VPS35 in excitatory neurons as a critical factor for inhibitory synapse maintenance and network homeostasis. This study also highlights LEV as a potential therapeutic strategy for ADRDs through the preservation of GABAergic synapses.
Abstract The discovery of the glymphatic system and meningeal lymphatic vessels has transformed our understanding of brain fluid homeostasis and central–peripheral communication, highlighting the critical role of cerebrospinal fluid in brain clearance. While substantial progress has been made in defining the structural components and physiological regulators of brain clearance, major uncertainties remain regarding (1) how these systems interact as an integrated physiological network, (2) how they fail in disease, and (3) whether they can be therapeutically manipulated. In this review, we first summarize the anatomical and physiological organization of the glymphatic–lymphatic axis, with particular emphasis on its regulatory mechanisms in light of rapid advances in this field. We then discuss how the glymphatic–lymphatic axis functionally integrates with peripheral immune and metabolic systems and describe how impairment of these clearance pathways is associated with the accumulation of pathological proteins, neuroinflammation, and vascular dysfunction and may contribute to the progression of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Finally, we discuss emerging therapeutic strategies and biomarkers targeting the glymphatic–lymphatic axis. By redefining the central–peripheral crosstalk, the glymphatic–lymphatic axis provides a unifying framework for understanding brain clearance, neurodegeneration, and future therapeutic interventions.
