Herein, we report the structure-activity relationship (SAR) to develop novel mGlu5 negative allosteric modulator (NAM) scaffolds devoid of the aryl/heterobiaryl acetylene moiety found in many historic mGlu5 NAMs, which has been linked to metabolic liabilities and hepatotoxicity. This endeavor utilized a scaffold-hopping strategy from the predecessor compound VU6031545, in which we replace an ether-linked tetrahydrofuran with various carbon-linked heteroaryl motifs to generate highly potent and selective mGlu5 NAMs. One such compound, VU6035386, displayed low nanomolar potency against human mGlu5 and was highly brain penetrant. Moreover, VU6035386 showed a vast improvement in predicted human hepatic clearance versus predecessor compound VU6031545.

DNA polymerase-β (DNA pol-β) plays a critical role in β-amyloid-induced neurodegeneration by mediating aberrant DNA replication in postmitotic neurons. In previous work, we demonstrated that 5-methoxyflavone inhibits DNA pol-β, though computational analyses suggested potential binding to the primase p58 subunit. Through molecular modeling, here, we designed (S)-2-cyclohexyl-5-methoxy-2H-chromene (S-chromene), a novel flavone-derived inhibitor exhibiting strong electrostatic complementarity with DNA pol-β but weak interaction with primase p58, suggesting enhanced selectivity. (R)-2-cyclohexyl-5-methoxy-2H-chromene (R-chromene) exhibited indistinguishable binding properties from S-chromene. The compound was obtained as a racemic mixture (chromene). Since the separated enantiomers were unstable, all biological assays used the racemate. DNA polymerase activity assay confirmed that chromene inhibited selectively DNA pol-β without affecting the primase/DNA pol-α complex activity. Also, the compound amplified methylmethanesulfonate toxicity in wild-type but not DNA pol-β-null fibroblasts, validating target-engagement. In cultured neurons, chromene effectively prevented β-amyloid-induced DNA replication and apoptosis. Ours is the first demonstration of a chromene acting as a selective DNA pol-β inhibitor endowed with a unique mechanism of neuroprotection.

Parkinson's disease (PD) is a debilitating neurological disorder characterized by the buildup of abnormal protein clumps, primarily composed of a protein called Alpha-Synuclein (α-Syn). Under physiological conditions, α-Syn exists as a dynamic, intrinsically disordered protein that resists aggregation through transient intramolecular interactions. However, under pathological conditions, this protein can misfold and stack into rigid, fibrous structures known as amyloid fibrils. These fibrillar deposits accumulate within neurons and are central to PD pathogenesis. This review focuses on understanding what keeps monomeric α-Syn in its innocuous form and what causes it to shift into a disease-associated state. In particular, we explore the role of subtle, often-overlooked intramolecular forces, like cation-π, π-π, and CH-π interactions, that may help stabilize the protein and prevent aggregation. We also examine how genetic mutations linked to familial forms of PD influence these internal interactions and drive the formation of partially folded intermediate forms that can trigger fibril growth. Some familial mutations accelerate α-Syn aggregation, while others slow it down, but the reasons behind these different outcomes are not fully understood. By analyzing how these mutations alter the protein's early structure and behavior, this review aims to shed light on the first steps of α-Syn misfolding. A deeper understanding of these mechanisms could support the development of new therapies designed to stabilize the soluble form of monomeric α-Syn and slow or prevent disease progression.

The escalating global burden of Alzheimer's disease (AD), projected to reach $16.9 trillion by 2050 with disproportionate impacts on low- and middle-income countries and racial minorities, underscores an urgent need for accessible early detection tools. Current therapies offer limited symptomatic relief but fail to halt neurodegeneration. Serum exosomal lipids, which reflect brain pathophysiology through blood-brain barrier crossing vesicles, present promising minimally invasive biomarkers. However, a standardized framework for their systematic development is lacking. We propose a structured three-phase approach comprising discovery, analytical validation, and clinical utility assessment. The discovery phase employs nontargeted lipidomics of serum exosomes from AD patients and controls integrated with machine learning to identify dysregulated pathways and prioritize candidate biomarkers. Analytical validation involves targeted quantification using UPLC-MS/MS to optimize sensitivity and specificity within complex matrices, with rigorous performance evaluation via receiver operating characteristic (ROC) curve analysis and area under the curve (AUC) analysis in independent case-control cohorts establishing preliminary diagnostic cut-offs. Clinical utility assessment requires longitudinal evaluation in treated AD cohorts to correlate biomarker dynamics with disease progression or therapeutic response, refine diagnostic thresholds, and explore presymptomatic risk prediction. Implementing this framework demands multidisciplinary collaboration and strict ethical adherence. This strategy paves the way for clinically validated serum exosomal lipid biomarkers to enable presymptomatic detection and personalized risk stratification, ultimately mitigating AD's devastating socioeconomic impact.

Neuroinflammation is evident in Alzheimer's disease (AD) brains, exacerbating the pathology and ensuing cognitive deficits in patients. The prostaglandin-E2 receptor EP2 emerged as a neuroinflammatory target in several neurodegenerative diseases, including AD. Antagonism of EP2 mitigates neuroinflammation and cognitive deficits in status epilepticus and stroke models. Here, we investigated the efficacy of a potent and selective EP2 antagonist TG11-77.HCl on the cognitive behavior and neuroinflammation in a two-hit 5xFAD mouse model of AD. We exposed adult 5xFAD mice on B6SJL genetic background and their nontransgenic littermates to a low dose of lipopolysaccharide and administered TG11-77.HCl or the vehicle in the drinking water for 12 weeks. Mice were subjected to Morris water maze and Y-maze testing during their last week of drug treatment. Blood samples were subjected to complete blood count (CBC) analysis and brain tissues were processed to examine the levels of inflammatory transcripts and glial marker expression (mRNA), followed by the quantification of congophilic amyloid deposition and microglial activation (IBA+) in the brain by immunohistochemistry. TG11-77.HCl treatment enhanced the spatial memory performance and ameliorated mRNA expression of proinflammatory mediators, chemokines, and cytokines in the neocortex of 5xFAD males only and attenuated astroglia and microglia activation in both male and female 5xFAD mice and the congophilic amyloid load in 5xFAD males only. CBC analysis revealed no changes in peripheral inflammation, irrespective of sex, on treatment with TG11-77.HCl. This study reveals sex-specific protection of selective EP2 antagonism in a two-hit mouse model of AD and supports a prudent therapeutic strategy against neuroinflammation and associated cognitive impairment in AD.

Understanding how drug exposure alters synaptic proteins is essential for uncovering the molecular mechanisms underlying addiction and identifying new therapeutic targets. Synaptoneurosomes (SNs), subcellular fractions enriched in pre- and postsynaptic components, provide a powerful resource for studying synaptic adaptations in the brain. Here, we applied a deep, discovery-based proteomic strategy to profile SNs isolated from the dorsal striatum (DS) of rats 1 h after acute methamphetamine (Meth) administration. To maximize proteome coverage, we combined tandem mass tag (TMT) labeling, offline high-pH fractionation, and low-pH nanoflow liquid chromatography coupled to high-resolution (orbitrap) mass spectrometry (HRMS). This approach enabled identification of ∼6100 cytosolic and membrane proteins from ∼500 ng of SN proteome digest─representing the most comprehensive DS SN proteome reported to date. A comparative analysis between Meth-treated and saline control animals revealed 147 differentially abundant proteins (81 with increased abundance; 66 with decreased abundance) enriched in pathways related to dopamine biosynthesis, synaptic vesicle cycling, mitochondrial energy metabolism, and proteasomal degradation. These findings highlight coordinated molecular remodeling of striatal synapses in response to Meth and demonstrate the utility of deep SN proteomics in addiction research. Deep, low-input SN proteomics nominates vesicle acidification, mitochondrial ATP supply, and proteasome function as testable pathway-level targets in addiction.

Tau undergoes fibrillogenesis in a group of neurodegenerative diseases, termed tauopathies. Each tauopathy is characterized by tau fibrils with disease-specific conformations, highlighting the complexity of tau self-assembly. This has led to debate surrounding the precise mechanisms that govern the self-assembly of tau in disease, especially the involvement of disulfide bonding (DSB) between cysteine residues. In this study, we use a truncated form of tau, dGAE, capable of forming filaments identical to those in disease. We reveal the impact of DSB on dGAE assembly and propagation by resolving the global mechanisms that dominate its assembly. We found evidence of surface-mediated secondary nucleation and fragmentation being active in dGAE assembly. The inhibition of DSB during dGAE assembly leads to an enhanced aggregation rate through a reduced lag phase but with no effect on the global assembly mechanisms. We suggest this is due to the formation of a dominant, seed-competent species in the absence of DSB that facilitates elongation and secondary nucleation, resulting in enhanced assembly. In vitro seeding assays reveal the recruitment of endogenous tau in a cell model only when using dGAE species formed under conditions that inhibit DSB. Our results further support the use of the in vitro dGAE tau aggregation model for investigating the mechanism of tau assembly, show the effect of varying conditions on tau assembly, and how these conditions affect the resultant species. Further studies may utilize dGAE and its aggregates to investigate tau seeding, propagation, and to highlight or test potential targets for therapies that reduce the spread of pathological tau throughout the brain.

The gut microbiota influences neurodegenerative disease progression, including Alzheimer's disease (AD), through microbial metabolites like amyloids in bacterial biofilms, such as the curli protein in Eshcherichia coli biofilm. In this context, the study focuses on two key aspects, namely, (i) how cross-kingdom bacterial biofilm proteins accelerate Aβ42 aggregation and induce neurotoxicity and (ii) whether a nanochaperone with hydrophobic sheets and hydrophilic polyphenolic moieties could inhibit cross-seeded aggregation. Considering this, we chemically synthesized and further characterized gallic acid-conjugated molybdenum disulfide quantum dots (GA@MoS2 QDs, ∼9.6 ± 4.2 nm) using spectroscopy and microscopy techniques, which showed ∼1.84-fold reduction in E. coli biofilm thickness, indicating interaction with biofilm components. The presence of the curli protein in E. coli was confirmed by dot blot and MALDI-TOF studies. Subsequent biophysical studies showed that isolated E. coli biofilm protein accelerated Aβ42 aggregation (heterotypic) by ∼6.76-fold, while GA@MoS2 QDs reduced this heterotypic aggregation by ∼9.49-fold reduction in Aβ42+ECBFP fluorescence relative to Aβ42 aggregates. In vitro studies with SH-SY5Y cells showed that heterotypic protein aggregation led to increased ROS production, intracellular calcium influx, and apoptosis induction, which were mitigated by GA@MoS2 QDs. The neuroprotective effect of GA@MoS2 QDs was also studied on Caenorhabditis elegans. Overall, the present studies suggested that the bacterial amyloid proteins may play a crucial role in Aβ42 aggregation, suggesting that targeting coaggregation could provide a novel therapeutic approach for the treatment of early onset AD.