ACS Chemical Neuroscience

Serotonin 5-HT4 receptors (5-HT4Rs) have emerged as potential therapeutic targets in neuropsychiatric and neurodegenerative disorders by modulating circuits that shape mood, cognition, and motor function. Ligands for 5-HT4Rs can modify dopamine (DA) and acetylcholine (ACh) transmission but mechanisms and circuits have not been fully resolved. Some 5-HT4R agonists have been suggested to have effects that include inhibition of acetylcholinesterase (AChE), raising speculation that 5-HT4R ligands might modulate ACh and/or DA through this action. Here, we investigated the impact of RS67333, a partial 5-HT4R agonist, on DA and ACh release dynamics in the striatum detected ex vivo in mouse brain slices using fast-scan cyclic voltammetry and genetically encoded ACh sensor GRABACh3.0 respectively. We found that RS67333 significantly modulated electrically evoked DA release in dorsolateral striatum and nucleus accumbens core, effects that were abolished by a nicotinic receptor (nAChR) antagonist. In parallel, RS67333 altered evoked ACh signals by extending extracellular ACh lifetime, and correspondingly, RS67333 was found to inhibit striatal AChE enzymatic activity. By contrast, BIMU8, an alternative 5-HT4R ligand that did not inhibit striatal AChE, had no effect on evoked striatal ACh or DA release. These findings indicate that RS67333 modulates striatal ACh transmission, which shapes downstream regulation of DA release by nAChRs, not through 5-HT4Rs but through AChE inhibition. These findings emphasize the caution due in attributing functions to 5-HT4Rs, but also highlight an alternative pharmacological profile of some purported 5-HT4R ligands as AChE inhibitors of potential utility for treating ACh/DA disorders.

Decreases in brain pH are associated with numerous neurological and neuropsychiatric conditions yet the molecular mechanisms linking decreased brain pH with these disorders are incomplete. The ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the brain and are inhibited by extracellular protons; however, the proton sensitivity of the delta-glutamate receptor subclass of iGluRs is unknown. Using whole-cell patch-clamp recordings of serotonin neurons in mouse brain slices and activating alpha 1-adrenergic receptors to induce delta 1 glutamate receptor (GluD1R) current, we demonstrated that GluD1R current is inhibited by physiological drops in extracellular pH. Unlike other iGluRs, protons inhibited GluD1R current via a voltage-independent decrease in unitary current. Moreover, mice lacking GluD1R showed impaired behavioral responses to inhalation of CO2. Taken together, this study continues to expand on the growing body of evidence positing GluD1R as functional ion channels and suggests that GluD1R facilitate pH sensing in vivo.

Activity of the mesolimbic dopamine system has long been implicated in encoding primary rewards and contributing to the addictive properties of drugs of abuse. Dopamine neurons in the ventral tegmental area (VTADA) of the midbrain typically show patterns of spontaneous burst activity that align with the onset of salient events or rewarding stimuli, resulting in phasic dopamine release in the nucleus accumbens (NAc). Fiber photometry is increasingly being used as an accessible technique to quantify neural activity with high temporal resolution at sensors offering signal specificity in stable recordings over extended periods of time. It has been well established by multiple techniques that opioids increase mesolimbic dopamine activity, likely through disinhibition of VTADA neurons. Here we used fiber photometry to compare sub-second transient events from VTADA neurons with GCaMP6f and dopamine release in the lateral shell of the NAc with dLight1.3b and GRABDA2h in response to morphine treatment. In weekly sessions, one dose of morphine was administered in escalating order (2.5, 5,7.5, and 10 mg/kg, intraperitoneal). Consistent with prior literature, both GCaMP6f in VTADA neurons and dLight1.3b in NAc showed patterns of increased signal following morphine treatment. In contrast, morphine suppressed transient activity at GRABDA2h sensors. Further analyses of whole signal streams from each sensor showed a generalized increase, but reduction in variability of the GRABDA2h signal, consistent with the interpretation of sensor saturation. Such results emphasize the importance of the inclusion of appropriate controls to contextualize the interpretation of biosensor responses, particularly in response to pharmacological treatment. HIGHLIGHTSO_LIMorphine elicited increased signaling in VTADA GCaMP6f and NAc dLight1.3b, consistent with prior literature C_LIO_LIMorphine suppressed NAc GRABDA2h signaling of transient events, suggesting saturation of GRABDA2h sensor C_LIO_LISensor validation with pharmacological challenges is critical for interpretation of data C_LI

Pigment epithelial-derived factor (PEDF) is a multifunctional protein produced predominantly by the retinal pigment epithelium and expressed in many tissues, including the brain, highlighting its participation in crucial processes, such as neuroprotection and angiogenesis. Some neurodevelopmental disorders, such as ASD, are characterized by neurodevelopmental abnormalities, including altered neurite formation, spine formation, and neuronal activities. Many efforts have been made to resolve NDDs, but until now, some symptoms remain untargeted. PEDF is involved in many steps of neurodevelopment. The treatment of PEDF peptide might improve the outcome of NDD symptoms by altering neuronal morphologies. We used PEDF peptides that contain different functional domains to study the effect of administering PEDF peptides on neuronal morphology in a prenatal valproic acid (VPA)-exposed mouse model. We identified that the treatment with PEDF peptides rectified the abnormalities in neurite formation and spine formation in VPA-exposed cortical neurons. In vitro calcium imaging showed abnormalities in the spontaneous activity in VPA-exposed cortical neurons. Treatment of a short PEDF peptide normalized intracellular calcium response to the control level. Accordingly, PEDF peptides have the prospect of serving as potential treatments for patients with neurodevelopmental disorders, such as ASD.

Alpha-Synuclein (-syn) aggregation is central to Parkinson's disease (PD), yet measurements in biofluids are confounded by the coexistence of monomeric and aggregated species. Using Syn-IMAGR, a single-molecule imaging platform with sub-femtomolar sensitivity, we show that purified -syn aggregates undergo dilution-induced disassembly, revealing a concentration-dependent equilibrium. Applied to postmortem brain lysates, Syn-IMAGR distinguishes physiological -syn multimers, which are dimmer and readily dissociate upon dilution, from PD-associated aggregates, which remain detectable and exhibit greater structural resistance to disruption. These results indicate that -syn assemblies occupy distinct stability regimes, with PD-associated aggregates representing a more persistent and less dilution-sensitive structural state. Syn-IMAGR thus provides a quantitative framework for resolving -syn species and for probing their concentration-dependent equilibrium.

Prion diseases are neurodegenerative disorders associated with the structural conversion of the cellular prion protein (PrPc) into its misfolded infectious isoform (PrPSc). Despite substantial efforts, no disease-modifying therapy or cure is currently available. Here, we present an integrated computational-experimental pipeline for the rational design of cyclic peptides targeting PrPc to inhibit its pathogenic conversion. Starting from crystal structures of antibody-bound mouse PrPc, we develop a rational design strategy combined with iterative molecular dynamics simulations and sequence optimization to generate peptides with enhanced binding and structural impact. Three candidates were selected for experimental validation. Our results show that PH1 (49YGPDPSDSYT58, antibody numbering) that binds stably to the &alpha2-&alpha3 interface most effectively reduced PrPSc levels in GT1-7 cells, essentially by inducing allosteric rearrangements that reinforce the intramolecular helical bundle. PL1 (89GQSNTKPYT97) and PL2 (89RQSNTWPYT97) binding the &beta1-&alpha1/&alpha3 junction exerted more modest effects due to the potential competition of the flexible tail to bind at this site. These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.

Misfolded proteins are tightly associated with various neurodegenerative diseases, and removing these misfolded proteins is one of the actively pursued approaches for seeking therapeutics for these diseases. In this study, we demonstrated that molecularly produced light (molecular light) from ADLumin-5, a self-photosensitizing chemiluminescence compound, could induce photo-oxidation and photodegradation of misfolded proteins, including beta-amyloid, tau, alpha-synucleins, and TDP-43 proteins in vitro. We validated the oxidation and degradation via LC-MS, MADLI-MS, and western blotting. Using beta-amyloid as a showcase, we demonstrated that, upon photo-oxidation and photodegradation, the toxicities of this misfolded protein were significantly reduced. To investigate the therapeutic effects of ADLumin-5 in vivo, we used the 5xFAD mouse model for longitudinal treatment for 4 months. In vivo molecular imaging results indicated that ADLumin-5 could reduce the accumulation of beta-amyloid proteins. Our study presents a novel approach to seek therapeutics for neurodegenerative disease via molecular light-induced degradation of misfolded proteins. In addition, because ADLumin-5 is dual-functional--enabling both photodegradation and in vivo imaging of misfolded protein changes--it can be considered a photo-theranostic agent for neurodegenerative diseases, representing a novel approach to drug discovery for neurodegenerative diseases.

Traumatic brain injury (TBI) has long-term consequences that include chronic traumatic encephalopathy (CTE) and an elevated risk for Alzheimer Disease (AD). These dementias ultimately manifest as tauopathies but may begin with acute neuronal dysfunction including post-traumatic seizures. Provocative evidence suggests that these prodromal seizures are a viable target to mitigate the later onset of dementias, and anti-epileptic drugs (AED) that increase the threshold of action potentials have indeed been shown to mitigate later tauopathies[1, 2]. Here, we test whether AEDs and other compounds that modulate synaptic transmission, applied immediately after TBI, can also act as prophylactics that block subsequent CTE-like tau aggregation and neurodegeneration in a larval zebrafish model. Levetiracetam (LEV) is an AED that modulates synaptic vesicle release. Application of LEV immediately following TBI abrogated TBI-induced tau tau aggregation (IC50 = 3.168 x10-3 mM) and cell death in the larval zebrafish TBI model. We next considered a polypharmacy approach involving mGluR2, because mGluR2 positively allosteric modulators (PAMs) such as JNJ-42153605 have previously been able to improve LEVs action in reducing some recalcitrant forms of seizure in a mouse model. We found that JNJ-42153605 was itself effective at blocking TBI-induced tau aggregation (IC50 = 8.691 x10-5 mM). Moreover, a subeffective dose of JNJ-42153605 (10-5 mM) was able to substantially improve the efficacy of LEV (~16-fold) in its prophylactic actions. Thus, LEV and JNJ-42153605 applied briefly after TBI offer a potent polypharmacy approach, at least in our preclinical animal model, to tackle the later tau aggregation and neurodegeneration that follows from TBI neurotrauma. These results warrant further investigation, including testing into mammalian TBI models (with longer disease course).

The aggregation of -synuclein (Syn) is a molecular hallmark of Parkinson's disease (PD) and other synucleinopathies. Understanding the molecular mechanisms that determine the aggregation of this protein may thus facilitate the development of disease-modifying therapies. While Syn is most commonly expressed as a 140-residue protein (Syn-140), recent evidence suggests an involvement of alternatively spliced Syn isoforms in disease onset and progression. Here, we report and characterise the interaction between Syn-140 and the aggregation-prone Syn-112 variant, one of the most abundant Syn splice isoforms. We found that amounts as low as 1% of Syn-112 accelerate the nucleation and aggregation of Syn-140. To further investigate this phenomenon, we employed MALDI-MS and NMR spectroscopy, confirming that Syn-140 and Syn-112 monomers interact strongly with one another. Furthermore, to assess the association of Syn-112 with disease pathology, we performed immunohistochemical staining combined with confocal microscopy on PD brain samples. Thereby, we found an increase in the number as well as the area of Syn-112 immunoreactive aggregates compared to healthy controls. These results illustrate how low-abundance Syn splice isoforms can modulate the aggregation landscape of Syn-140 and in turn contribute to the molecular heterogeneity of synucleinopathies.

Traumatic brain injury (TBI) induces rapid neuroinflammatory responses not only in the brain but also in anatomically and immunologically connected central nervous system (CNS) compartments, including the retina. In our study, we investigated retinal microglial activation, retinal ganglion cell (RGC) calcium dynamics, and caspase-3 activation in adult mice subjected to severe traumatic brain injury using the Marmarou impact-acceleration model at 24 and 48 h post-injury. Carrying out Ca{superscript 2}-imaging, immunohistochemistry, and ex vivo time-lapse microscopy, we found robust microglial activation in both the superficial and deep retinal layers following TBI, accompanied by increased microglial motility. RGCs exhibited a transient surge in degeneration-induced spontaneous activity at 24 h, followed by a marked reduction below control levels at 48 h, consistent with early degenerative changes. Activated caspase-3 levels were significantly elevated in both microglia and other retinal cell types at both time points, indicating ongoing apoptotic effects. Together, these findings demonstrate that TBI rapidly triggers inflammatory and apoptotic mechanisms in the retina, which are detectable within the first 48 hours. Our results highlight the retina as a sensitive indicator of early CNS pathology after traumatic injury and underscore the potential of retinal analysis for monitoring TBI-induced neurodegeneration for future clinical implementation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/734783v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@5bc694org.highwire.dtl.DTLVardef@14a4ce4org.highwire.dtl.DTLVardef@fe2d32org.highwire.dtl.DTLVardef@149419d_HPS_FORMAT_FIGEXP M_FIG C_FIG

CaMKII is a multifunctional kinase essential for synaptic plasticity and memory formation. While its canonical role involves enzymatic phosphorylation, recent evidence suggests CaMKII also functions through liquid-liquid phase separation (LLPS) with substrate proteins, including GluN2B-containing NMDA receptors. However, the physiological significance remains unclear. Here, we generated CaMKII subtype knock-in (KI) mice carrying a point mutation (I205K) in the hydrophobic pocket, a key interface required for LLPS. These mice exhibited a complete loss of structural long-term potentiation (sLTP) despite normal spine morphology, marked hyperactivity and profound deficits in aversive memory formation. Atomoxetine, an approved attention-deficit/hyperactivity disorder (ADHD) treatment, ameliorated the hyperactive phenotype. Notably, we identified a patient carrying the I205N variant presenting with ADHD and mild intellectual disability, mirroring the behavioral features observed in I205K KI mice. Additional neurodevelopmental disorder-associated variants within the same hydrophobic pocket similarly disrupted LLPS in vitro. Molecular dynamics simulations revealed these variants destabilize the CaMKII-GluN2B interaction through distinct mechanisms that perturb the dynamic stability of the binding interface. These findings establish CaMKII-mediated phase separation as critical for linking synaptic molecular assembly to cognitive function and provide a unifying molecular basis for synaptic disorganization, hyperactivity, and memory deficits associated with neurodevelopmental disorders. SignificanceCaMKII is essential for synaptic plasticity and memory, yet the contribution of its non-catalytic functions, including liquid-liquid phase separation (LLPS), to brain function remains unclear. Here, we show that disrupting CaMKII LLPS with its synaptic partners impairs synaptic localization, abolishes LTP, and causes profound memory deficits in vivo. A knock-in mouse carrying the I205K mutation showed hyperactivity and impaired learning. This recapitulate the clinical phenotypes of a human patients with the I205N variant, which is associated with ADHD and intellectual disability. Together, these findings establish LLPS as a critical mechanism underlying CaMKII function in the brain and provide a disease-relevant framework for understanding synaptic dysfunction in neurodevelopmental disorders.

It is well established that activating the mGluR2 metabotropic glutamate receptor (mGluR2), which is the main presynaptic autoreceptor for glutamate in the brain, attenuates the behavioral and electrophysiological actions of LSD and other psychedelics. However, the mechanisms responsible for these actions are controversial. The two competing mechanistic hypotheses have been proposed to explain this phenomenon are: (1) direct actions mediated by mGluR2/5-HT2A heterodimers, and (2) inhibition of 5-HT2A-mediated excitation of pyramidal neurons via presynaptic inhibition of glutamate release by mGluR2 receptors. Consistent with prior reports, we show mGluR2 agonist pretreatment attenuates the head twitch response induced by the psychedelic drug 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in these mice. We next employed multiple orthogonal in vivo and in vitro approaches to explore the potential for direct physical interactions between mGluR2 and 5-HT2A receptors. We next engineered mice to express mGluR2-mCherry-CT and 5-HT2A-eGFP-CT tagged receptors and found no evidence for receptor colocalization or oligomerization under basal or 5-HT2A agonist-exposed conditions in vitro or in vivo. Radioligand binding and kinetic analyses revealed no evidence for mGluR2-mediated modulation of 5-HT2A ligand binding in vitro or in vivo. Collectively, our findings support models in which mGluR2 signaling modulates the activity of Gq-coupled 5-HT2A receptors in layer V pyramidal neurons, rather than models positing the requirement of mGluR2/5-HT2A multimers.

The inhibition of A{beta}42 ({beta}-amyloid) fibril formation is a key therapeutic strategy in Alzheimer's disease research. Paddlewheel diruthenium complexes have shown promising activity against A{beta}42 aggregation and preformed fibril disaggregation, yet their molecular mode of action remains poorly understood. In this work, we perform atomistic simulations to explore how charge modulation influences the interactions of three analogous paddlewheel diruthenium complexes, the parent neutral complex [Ru2Cl(D-p-FPhF)(O2CCH3)3], and its anionic [Ru2Cl2(D-p-FPhF)(O2CCH3)3]- and cationic [Ru2(D-p-FPhF)(O2CCH3)3]+ counterparts (D-p-FPhF- is the N,N' -bis(4-fluorophenyl)formamidinato ligand) with A{beta}42. Our results indicate that electrostatic tuning governs binding affinity and the extent of interaction across the A{beta}42 fibril surface. As the complexes' charge changes from -1 to +1, the interaction pattern shifts from localized contacts to widespread, multi-site engagement encompassing key charged, aromatic, and hydrophobic regions of A{beta}42. This enhanced binding correlates with longer-lived, thermodynamically stable interactions at the fibril interface, which effectively lower the free energy penalty for fibril disassembly. Overall, our findings propose a mechanism in which charge-dependent activation through ligand exchange enhances fibril recognition and promotes disruptive binding modes, demonstrating the potential of charge-tunable diruthenium complexes as therapeutic modulators of A{beta}42 fibril stability.

Vitamin D3 (VD) deficiency is a global health concern, and its supplementation has been shown to alleviate inflammation and oxidative stress across numerous neurological disorders. However, the beneficial effect of this common nutraceutical in schizophrenia (SCZ) remains inadequately explored. The present study investigated the presupplementation effects of VD on positive and cognitive symptoms in a MK-801induced mouse model of SCZ. MK-801, a non-competitive NMDA receptor antagonist, is a widely used drug that mimics some of the psychotic symptoms associated with SCZ. The repeated administration of a single dose of MK-801 (0.5mg/kg; intraperitoneally) for two weeks produced hyperlocomotion, anxiety- like behavior, and working memory deficits in MK-801-induced SCZ-like mice. These behavioral abnormalities were significantly attenuated in VS5 mice (SCZ mice presupplemented with 500 IU/kg/day of VD). At the molecular level, VD rescued gene expression of major NMDA receptor subunits (NR1, NR2A, NR2B), 7 nicotinic acetylcholine receptors (7nAChRs), and neurotrophin factors (NGF and BDNF). A restoration of PSD-95 protein expression, accompanied by downregulation of calcineurin, was also observed in the prefrontal cortex (PFC) of VS5 mice, suggesting protective effects of VD on synaptic communication and function in SCZ. In vitro studies showed that calcitriol (1 M) treatment of HEK-293 T cells transfected with 7nAChRs potentiated the single-channel current amplitude and demonstrated a direct modulatory effect of this nutraceutical on 7nAChRs expression and function. In silico JASPAR analysis further identified putative Vitamin D response elements (VDREs) within the promoter regions of various target genes, supporting the genomic action of VD. Additionally, VD deficiency was observed in Indian SCZ patients, highlighting its potential clinical relevance. Together with our previous findings (Manjari et al., 2022, 2023), the present study also demonstrates anti-inflammatory, anti-cholinesterase, neurotrophic, and synaptic-enhancing effects of VD, deepening our understanding of the multifaceted neuroprotective effects of the "D3" neurosteroid in neuropsychiatric disorders such as SCZ. HighlightsO_LIVD presupplementation improves the behavioral deficits in MK-801 induced SCZ mice. C_LIO_LINutraceutical intervention normalizes the gene expression of major NMDARs subunits namely, NR1, NR2A, NR2B, in the PFC of SCZ mice. C_LIO_LIVD mediates a restoration in the expression and function of 7nAChRs in SCZ mice. C_LIO_LIVD exhibits neuroprotective, neurotrophic, synaptoprotective, anti-inflammatory and anti-acetylcholinesterase effects, highlighting its therapeutic potential in SCZ. C_LI

Synucleinopathies are a group of neurodegenerative disorders characterized by the accumulation of aggregated -synuclein (-syn), including Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. These diseases are marked by locomotor and non-motor impairments, as well as mitochondrial dysfunction and the loss of dopaminergic (DA) neurons. We have developed several anti--syn single-domain antibodies (sdAbs) and demonstrated the diagnostic imaging potential of two of them and the acute therapeutic benefit of one in clearing -syn in a mouse model. However, whether these sdAbs can suppress -syn-mediated neuronal loss and locomotor impairment in vivo remains unclear. We evaluated the therapeutic potential of five anti--syn sdAbs to clear pathological -syn in mouse neuronal culture and then demonstrated their in vivo efficacy in a Drosophila model of synucleinopathy. The sdAbs differed in their efficacy to lower levels of phospho-serine 129 -syn, prevent loss of DA neurons, alleviate mitochondrial dysfunction, improve motor function, and prolong survival in synucleinopathy flies. The most effective sdAb, 2H1, has not been reported before. It binds strongly to the aggregation prone region of -syn and robustly improves all these disease parameters. Additionally, that sdAb is associated with -syn in the fly neurons, as shown through proximity dependent turboID biotinylation assays. The sdAb-turboID also biotinylated -syn-associated proteins involved in synapse/vesicle trafficking pathways, pinpointing the location of their intracellular interaction. Our findings provide an insight into the therapeutic mechanism of action of these sdAbs and strongly support their clinical development.

HLA-DR-restricted T-cell reactivity to amyloid {beta} (A{beta}) has been associated with Alzheimers disease (AD), but structural evidence for HLA presentation of A{beta}-derived peptides remains elusive. We present the crystal structure of the A{beta}1-15 fragment bound to HLA-DR1, providing, to the best of our knowledge, the first experimental structure of an Alzheimers A{beta} peptide bound to an HLA molecule. The molecular architecture of this complex defines a peptide:MHC interaction dictated by engagement of A{beta}1-15 peptide central core with further involvement of N- and C-terminal peptide flanks. The structure reveals that DR{beta}1 Arg70, a polymorphic position, directly binds P4 and P5 through polar contacts, providing a rationale for HLA-DRB allelic bias underpinning accommodation of A{beta}1-15. We also describe the A{beta}1-15:HLA-DRB1 surface topology, informing a candidate binding surface for potential T-cell recognition. Collectively, these findings contribute a structural framework for further research in the context of A{beta}-specific CD4+ T-cell autoreactivity in AD.

IntroductionThe proteasome is a critical cellular degradative machinery impaired in late-stage Alzheimers disease (AD). However, the status and activity of the proteasome in early-stage sporadic AD (sAD) is unknown. MethodsA cellular model of human early-stage sAD was generated from sAD patient iPSC-derived cortical neurons by dual-SMAD inhibition. The iPSCs, neuroprogenitors, and cortical neurons were validated by the expressions of key markers. The level of total intraneuronal A{beta} was measured by ELISA. Composition and native proteolytic activities of the proteasome in control and sAD cortical neurons were measured using complementary fluorogenic probes. ResultsControl and sAD patients iPSCs expressed pluripotent markers OCT4, NANOG, and SSEA4 which induced into neuroprogenitors expressing NESTIN and PAX6. The neuroprogenitors terminally differentiated into cortical neurons expressing neuronal markers MAP2 and TUJ1, and cortical layer marker TBR1. The level of intraneuronal A{beta} in the sAD cortical neurons was significantly higher compared to control. Control and sAD cortical neurons expressed native 30S, 26S, and 20S proteasome assemblies with the sAD cortical neurons displaying higher 20S assemblies. Increased active 20S assemblies was associated with higher {beta}1, {beta}2, and {beta}5 proteolytic sites activities. DiscussionThe significant elevation in the proteolytic activities of the {beta}1, {beta}2, and {beta}5 subunits of 20S proteasome in sAD cortical neurons suggests that this may be a possible compensatory response to elevated intraneuronal A{beta}. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/734021v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d8c382org.highwire.dtl.DTLVardef@b92e8org.highwire.dtl.DTLVardef@1d9c699org.highwire.dtl.DTLVardef@7d826d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Aging is a major risk factor for exacerbated neuroinflammation and neurodegenerative diseases, yet the underlying lipid metabolic mechanisms remain incompletely understood. Here, we employed high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) combined with quantitative peak-area analysis and conceptual kinetic modeling to investigate age-dependent sphingolipid remodeling in the rat brain following intracerebro-ventricular (ICV) LPS challenge. In old rats, MALDI-MSI revealed pronounced and progressive sphingolipid dysregulation compared with young animals. Quantitative analysis showed a dramatic [~]10-fold reduction in SM(d36:1) and [~]4-fold reduction in SM(d42:2), accompanied by significant accumulation of long-chain sulfatides (2.12-fold increase in C24:1-sulfatide and 1.45-fold increase in C24(OH)-sulfatide) at both 24 h and 72 h post-LPS. Spatial imaging demonstrated that these changes were markedly amplified in white matter regions and became more widespread and intense at 72 h. A simplified Michaelis-Menten kinetic model successfully recapitulated the experimental data, identifying increased nSMase2 activity (higher Vmax) as the primary driver of accelerated sphingomyelin hydrolysis and subsequent ceramide rerouting into sulfatide synthesis. This metabolic shift generates excess ceramide that promotes Drp1-mediated mitochondrial fission, elevates mitochondrial ROS production, and disrupts bioenergetics, establishing a feed-forward loop linking sphingolipid remodeling to mitochondrial oxidative stress and white matter vulnerability in the aged brain. These findings provide the first spatially and temporally resolved demonstration of age-dependent sphingolipid metabolic reprogramming during neuroinflammation. By integrating multimodal MALDI-MSI, quantitative lipidomics, and kinetic modeling, this study reveals a previously underappreciated nSMase2-ceramide-mitochondrial axis in neuroinflammaging. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/734865v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@17ec6fforg.highwire.dtl.DTLVardef@3aabcforg.highwire.dtl.DTLVardef@1e5d671org.highwire.dtl.DTLVardef@61b997_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cryo-electron microscopy (cryo-EM) has revealed disease-specific tau filament folds, yet the local sequence elements that determine them remain poorly understood. Here we focused on the 329HHK331 motif near an inter-protofilament interface in Alzheimer's disease (AD)-type tau filaments, and analyzed recombinant dGAE filaments of wild-type (WT) and mutants in this motif. All mutants formed amyloid-like filaments in vitro, but their morphologies differed. In cultured cells, WT filaments efficiently seeded WT tau aggregation. Filaments with three-residue changes (deletion or Ala substitution) showed almost no seeding activity, whereas two-residue deletions retained partial activity. Cryo-EM showed that WT dGAE filaments form a quadruple helical filament of two protofilament dimers. Each dimer comprises two C-shaped protofilaments, centered on a 333GGG335-mediated inter-protofilament interaction and supported by flanking 329HHK331-336QVE338 contacts. Three-residue alterations abolished interactions with the QVE motif at the protofilament interface, thereby displacing 333GGG335 and forming non-C-shaped protofilament structures that are intrinsically poor templates for tau seeding. By contrast, two-residue deletions maintained the C-shaped protofilament structure because the remaining residue formed alternative inter-protofilament interactions. These findings suggest that the 329HHK331 region is a key determinant of the AD-like C-shaped protofilament fold and link this motif to tau seeding, providing insight into disease-specific tau filament formation.

Proteolytic processing of the amyloid precursor protein (APP) generates a 99-amino acid precursor, {beta}-carboxyl-terminal fragments (APP-CTF{beta} or C99). Upon {gamma}-secretase inhibition, APP-CTF{beta} accumulates and induces synaptic defects, resulting in neuronal hyperactivity. However, mechanistic insights in the critical role of APP-CTF{beta} has not been completely elucidated. Here, we show that in primary neurons expressing human APP-CTF{beta} (C99) variants, acute {gamma}-secretase inhibition selectively increases evoked synaptic vesicle release in cells, whereas deletion of the C-terminus abolishes this effect. Using single-molecule approaches and reconstituted membrane systems, we demonstrate that accumulation of APP-CTF{beta} promotes its oligomerisation. In particular, APP-CTF{beta} oligomers augment synaptic vesicle tethering via their C-terminal domain. This effect is driven by the interaction with synaptic vesicle proteins, independent of the YENPTY binding motif. Additionally, APP-CTF{beta} oligomers were associated with alterations in membrane lipid organization. Together, our findings identify APP-CTF{beta} oligomerization as a constitutional gain-of-function mechanism that enhances presynaptic vesicle tethering and release, providing mechanistic insight into how altered APP processing regulates synaptic activity.