Abstract Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and increasingly recognized as a key contributor to neurodegeneration (neuroferroptosis). Current pharmacological approaches primarily target downstream ferroptotic events and do not address upstream epigenetic regulation. MicroRNA (miRNA)-mediated modulation represents a potential strategy to overcome this limitation, while its application is hindered by the lack of delivery vectors that combine efficient nucleic acid delivery with physiological compatibility. In this study, guanidinium-functionalized aminoglycosides (GAs) are investigated as a natural product-derived small-molecule carrier for miRNA delivery. GAs form stable nanocomplexes with miR-7a, an epigenetic regulator of neuroferroptosis, through multivalent guanidinium-mediated interactions, enabling efficient cellular uptake. Among the molecules evaluated, neomycin G (NEO G) exhibited enhanced miRNA binding, improved intracellular delivery, and functional activity under viscosity conditions closely recapitulating the extracellular milieu, wherein conventional delivery vectors typically display reduced efficacy. Delivery of miR-7a mimic using NEO G results in effective downregulation of Krüppel-like factor 4 (Klf4), a transcription factor implicated in neuroferroptosis, and confers significant neuroprotection via modulation of the miR-7a-Klf4 axis. These findings identify GAs as a biocompatible and effective platform for miRNA delivery and highlight their potential utility in miRNA therapeutic strategies targeting neuroferroptosis.
Abstract Integrated neural signaling and gut microbiome analysis are crucial for the mechanistic understanding of environmental exposure and health. Photochemical smog is a societal challenge; formaldehyde is a component of photochemical smog and a common indoor air pollutant. Although depression-like behavior has been linked to formaldehyde exposure, the effect of exposure on real-time dopamine release remains unknown despite its significance in depression. Here, we reveal the effects of formaldehyde exposure on real-time dopamine concentration dynamics in the nucleus accumbens, elicited by electrically stimulating the medial forebrain bundle. We used fast-scan cyclic voltammetry and carbon fiber microelectrodes to quantify real-time dopamine concentration dynamics. In addition, we extracted DNA from the mice’s fecal samples and conducted 16S rRNA gene sequencing to measure the gut microbiome. We observed a decrease in real-time dopamine concentration dynamics in the nucleus accumbens associated with formaldehyde exposure, with peak dopamine concentration being 2700 ± 500 nM (no exposure), 2100 ± 400 nM (3-h exposure), and 1600 ± 500 nM (7-day exposure). By quantifying dopamine release in the nucleus accumbens evoked by electrically stimulating the medial forebrain bundle, we elucidated how formaldehyde exposure affects the associated neuronal circuitry. These experiments are the first, to the best of our knowledge, to demonstrate a significant decrease (p < 0.05) in real-time dopamine release in the nucleus accumbens following formaldehyde exposure in a duration-dependent manner. Additionally, gut microbiome analysis suggested a decrease in Lactobacillus, which has immunomodulatory functions, after 7 days of formaldehyde exposure.
Phosphodiesterase 7B (PDE7B) is involved in the cAMP-PKA signaling pathway and has been implicated in neurodegenerative disorders. Accordingly, PDE7B represents a promising target for the development of therapeutics to treat such diseases. Recent efforts have led to the discovery of pyrimidinone-based compounds with high affinity and selectivity for PDE7B. In this study, we developed a novel pyrimidinone-based positron emission tomography (PET) probe, [11C]5. In vitro autoradiography using rat brain sections revealed specific binding of [11C]5 in the striatum and thalamus, consistent with the known distribution of PDE7B. Baseline PET scans showed relatively low brain uptake of [11C]5, except in the olfactory bulb. Pretreatment with elacridar, a potent dual inhibitor of drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), enhanced the brain uptake of [11C]5, resulting in further specific in vivo binding in the olfactory bulb and striatum. In conclusion, although the brain permeability of [11C]5 was limited by P-gp and BCRP, [11C]5 demonstrated specific binding to PDE7B, both in vitro and in vivo, supporting its potential utility for imaging PDE7B in the central nervous system.
Menthol, a prevalent additive in tobacco and electronic cigarette products, has been reported to interact with nicotine at both neuropharmacological and behavioral levels. Although menthol alone is not reinforcing, converging evidence suggests that it may modulate the pharmacological and behavioral effects of nicotine through nicotinic acetylcholine receptor (nAChR) mechanisms. Using an inhalation-based rodent model that mimics human exposure to electronic nicotine delivery systems, we examined how menthol aerosol influences nicotine-induced hypothermia, discriminative stimulus effects, and the potential involvement of α4β2* nAChRs in male rats. Plasma nicotine, cotinine, and trans-3'-hydroxycotinine (3'-OH-cotinine) concentrations were also measured to assess pharmacokinetic interactions. Menthol aerosol prolonged and potentiated nicotine-induced hypothermia, and this effect was attenuated by the α4β2* receptor antagonist dihydro-β-erythroidine (DHβE). In drug discrimination (DD) assays, coinhalation of menthol enhanced nicotine-appropriate responding, suggesting facilitation of nicotine's interoceptive stimulus properties. These effects were also attenuated by DHβE, consistent with involvement of α4β2* nAChR-mediated mechanisms. In addition, the behavioral effects were accompanied by alterations in nicotine pharmacokinetics, including increased levels of nicotine metabolites. Collectively, these findings suggest that menthol modulates nicotine's pharmacological and behavioral effects, potentially through α4β2* nAChR-dependent mechanisms. By prolonging nicotine's physiological and interoceptive effects, menthol may increase the salience of nicotine-related cues during inhalation exposure. These interactions could contribute to enhanced abuse liability associated with menthol-containing nicotine products, although further studies are required to establish causal mechanisms. This study provides mechanistic insight into menthol-nicotine interactions and highlights considerations relevant to mentholated e-cigarette products.
Extracellular protein aggregates have long been viewed primarily as toxic deposits, yet this framing fails to explain a critical clinical observation: removal of amyloid-β oligomers does not halt or reverse Alzheimer's disease progression. Here, we propose that this paradox reflects the emergence of a self-sustaining intracellular signaling state, initiated by aggregate-driven receptor clustering but capable of persisting independently of the original extracellular trigger. We argue that oligomeric assemblies act as multivalent scaffolds that cross-link cell-surface receptors, inducing nanoscale clustering and the formation of signaling-competent membrane platforms. In Alzheimer's disease, this mechanism links amyloid-β to a receptor complex involving PrPc and mGluR5, leading to Fyn activation and downstream tau hyperphosphorylation. Critically, redistributed tau enhances Fyn recruitment to postsynaptic compartments, establishing a positive feedback loop in which kinase activity and tau pathology become mutually reinforcing, and progressively decoupled from the initiating amyloid signal. This self-amplifying circuit provides a mechanistic basis for the limited efficacy of amyloid-targeted therapies and reframes the therapeutic window problem in neurodegeneration. Beyond Alzheimer's disease, we propose that clustering-driven feedback loops may represent a generalizable principle in proteinopathies involving extracellular or membrane-associated aggregates─such as α-synuclein─though the applicability of this principle appears to depend on whether the aggregating protein directly engages cell-surface receptor systems. This framework shifts the focus from aggregate burden to membrane organization and feedback topology as determinants of disease progression, and identifies the tau-Fyn feedback loop as a candidate therapeutic target in proteinopathies.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons, with familial ALS (fALS) frequently caused by mutations in Cu/Zn superoxide dismutase (SOD1). The G93A mutation, one of the most aggressive forms, promotes the formation of cytotoxic protein aggregates through cross-β-sheet structures, leading to neuronal dysfunction and death. In this study, we investigated the therapeutic potential of NABi (natural Aβ binder and Aβ-aggregation inhibitor), a stable small engineered protein composed of the N-terminal 90 amino acids of SOD1, originally developed to target amyloid-β aggregation in Alzheimer's disease. Given the shared β-sheet-rich aggregation mechanisms between amyloid-β and mutant SOD1 proteins, we hypothesized that NABi could serve as a dual-action therapeutic for both diseases. Through an integrated approach involving structural, biochemical, and cellular analyses, we demonstrate that NABi exhibits a 4-fold greater binding affinity for SOD1G93A compared to SOD1WT, selectively targeting the mutant protein via specific hydrophobic interactions. Structural modeling using AlphaFold2 reveals that the G93A mutation exposes hydrophobic residues that create an optimal binding interface for NABi. Functionally, NABi effectively inhibits SOD1G93A aggregation, as demonstrated by filter trap assays and immunofluorescence microscopy, while maintaining the protein in a soluble, nontoxic state. Importantly, coexpression of NABi reduces SOD1G93A-induced cytotoxicity by approximately 4-fold, significantly enhancing neuronal survival. These findings establish NABi as a promising therapeutic candidate for SOD1G93A-associated familial ALS, demonstrating its capacity to selectively target pathological protein conformations while preserving normal cellular function. Our results support the development of NABi as an innovative pan-therapeutic approach targeting shared aggregation pathways across multiple neurodegenerative diseases.
The metal ion chelator TPEN is widely used to study the role of mobile zinc in biology; however, its high affinity for other metal ions raises questions about the specificity when interpreting biological effects of TPEN. Using the Cu(I)-selective chelator PSP-2, we found that the chelation of copper, and not zinc, likely stimulates axon regeneration after optic nerve injury, thus challenging the previous viewpoint that dysregulation of mobile zinc contributes to regenerative failure upon optic nerve damage. Spectroscopic and electrochemical measurements revealed that TPEN sequesters subattomolar buffered Cu(I) through a novel redox-trapping mechanism, which was corroborated by fluorescence imaging studies with a Cu(I)-selective probe in live cells. These findings highlight the ambiguity of TPEN-induced biological effects and identify PSP-2 as a versatile tool for dissecting the role of copper in biological processes.
Huntington's disease (HD) is known for its abnormal polyQ stretch in the first exon of the HTT gene. HD is listed among the fatal autosomal dominant neurodegenerative disorders targeting the central nervous system and interfering with motor, cognitive, and psychiatric functions. The HTT gene codes for Huntingtin (Htt), a large three-domain protein with a polyQ stretch present in the N-terminal domain. In diseased cells, inclusion bodies are enriched in Htt fragments containing an extended polyQ region. The expansion of the polyQ correlates with aggregate size and onset of HD. However, the exact role of the full-length Htt in aggregate formation has not been fully known. Using coarse-grained molecular dynamics simulations at the near-atom level, our study indicates that the conformational dynamics of Htt─twisting and open-close motions of the domains─are dependent on the length of polyQ. In the presence of HAP40, the global conformational landscape is restricted, and the degree of dependence on polyQ length is low. The intra- and intercontact analyses suggest that the polyQ region can loop like a hairpin with a higher probability for a larger polyQ length. However, the looping probability is reduced in the presence of HAP40 due to increased contacts between polyQ and other regions of Htt. Thus, our findings indicate that polyQ has a significant role in the disease's pathogenesis, which includes altered functional activity and structural modifications leading to inclusion body formation. These effects can be controlled with the help of HAP40.
We report on the development of a highly water-soluble pegylated imidazodiazepine ETK-II-83. The compound binds selectively to GABAA receptors among forty-five other receptors expressed in the brain with a higher affinity for the α5-containing GABAA receptors. The compound is converted in vitro by liver microsomes into ETK-III-9. The metabolic reaction selectively oxidizes the polyethylene glycol groups of ETK-II-83 forming eventually the stable 2-hydroxyethyl amide-bearing ETK-III-9. The conversion is fast with a half-life of 5.33 min. ETK-III-9 strongly binds to α3/α5 containing GABAA receptors. A pharmacokinetic study with ETK-II-83 confirmed metabolic instability resulting in low AUCs for blood and brain. Interestingly, higher brain concentrations than blood concentrations (KP = 5.17) were observed. The in vivo formation of ETK-III-9 inside ETK-II-83-treated animals was rapid and peaked at 40 min. Almost equal amounts of ETK-III-9 were found in blood and brain with half-lives of 219 and 202 min, respectively. We also conducted a pharmacokinetic study with ETK-III-9 at the same dose and observed three times higher AUCs for blood and brain. Importantly, the free brain concentration of ETK-III-9 in ETK-II-83-administrated animals was higher than its affinity for the GABAA receptors. This was confirmed by investigating ETK-II-83 with two anxiety mouse models. For the elevated plus maze, ETK-III-9- and ETK-II-83-treated animals spent significantly more time in the open arm than vehicle-treated animals, without any differences in the overall distance traveled. ETK-III-9- and ETK-II-83-treated animals also buried fewer marbles than vehicle control animals. To demonstrate that these effects were not caused by sedation or inhibition of sensorimotor inhibition, we conducted open field and rotarod tests. For both tests, ETK-III-9- and ETK-II-83-treated animals did not behave differently from vehicle-treated mice in contrast to diazepam-treated animals. The innovation is a highly water-soluble prodrug ETK-II-83 that metabolizes to ETK-III-9, an α3/α5 GABAA receptor-selective compound that reduces anxiety in rodent models.
Converging evidence implicates dysregulation of serotonin in emotion-related psychiatric disorders. Leading pharmacotherapies for such disorders, including general anxiety and posttraumatic stress disorders, target the serotonin transporter (SERT); yet, selective serotonin reuptake inhibitors (SSRIs) exhibit limited efficacy, suggesting that additional mechanisms contribute to these clinically prevalent psychopathologies. Recent evidence suggests that organic cation transporter 3 (OCT3) contributes to serotonin homeostasis; however, OCT3 contributions to serotonin clearance in basolateral amygdala (BLA), a central hub for emotional processing, are unknown. We utilized transgenic mice, in vivo neurochemical, and behavioral approaches to test OCT3 contributions to serotonin clearance in BLA and fear behaviors. Depletion of OCT3 from serotonin neurons prolonged serotonin clearance in BLA across a range of concentrations, in comparison to depletion of SERT from serotonin neurons, which prolonged serotonin clearance in BLA only at relatively low concentrations. The SSRI fluvoxamine prolonged serotonin clearance in BLA of wildtype mice and OCT3 knockdown mice to a similar degree, and this effect was lost in mice with SERT depletion. Behaviorally, depletion of SERT or OCT3 from serotonin neurons did not impact fear learning; however, depletion of SERT trended to attenuate cued fear memory, and depletion of OCT3 modestly attenuated cued and contextual fear memory. These findings indicate that OCT3 could serve as a novel therapeutic target for psychiatric disorders related to emotional dysregulation and encourage research into nonconventional treatments for these prevalent disorders.
The progressive loss of midbrain dopamine neurons leading to motor deficits is the primary cause of Parkinson's disease (PD). Emerging reports on the role of NURR1 in the differentiation and maintenance of dopamine neurons; the association of reduced levels/familial mutants of NURR1 in PD subjects makes it a promising candidate for developing novel therapeutics. In the present study, while examining the effects of known deubiquitinase inhibitors, we have identified that the USP10/13 inhibitor, spautin-1, upregulated NURR1 levels in a transcription-dependent manner. Further, USP13, but not USP10, was found to be essential in spautin-1-mediated NURR1 regulation. Reports indicate that USP13 deubiquitinates SKP2 and thereby prevents it from degradation. Also, SKP2 is an E3 ligase for P57/kip2 and reduced SKP2 expression leads to enhanced P57/kip2 levels. In line with the above findings, cells treated with spautin-1 exhibited reduced SKP2 with a concomitant increase in P57/kip2 levels. Our results indicate that spautin-1-mediated regulation of the USP13-SKP2-P57/kip2 axis could play a crucial role in NURR1 upregulation since overexpression of SKP2, or knockdown of P57/kip2, abrogated spautin-1-mediated effects. Further, spautin-1 mediated neurite outgrowth of dopaminergic cells was mitigated under NURR1 knockdown conditions, indicating that small molecule activators of NURR1 could promote neurite outgrowth. Lastly, the ability of spautin-1 in enhancing tyrosine hydroxylase staining in vivo in PD mouse model suggests that pharmacological activation of NURR1 could mitigate biochemical manifestations of PD.
Alzheimer's disease (AD) is characterized by progressive neurodegeneration and cognitive decline, largely driven by amyloid-β (Aβ) aggregation and dysregulated acetylcholinesterase (AChE) activity. While current pharmacological interventions utilize AChE inhibitors and Aβ antagonists, their efficacy is frequently hampered by monotherapeutic limitations, poor blood-brain barrier (BBB) permeability, and a lack of controlled release mechanisms. Herein, we report the engineering of the liposomal formulation coloaded with Donepezil (DNP) and the photothermal agent indocyanine green (ICG), followed by conjugation of ApoE3 protein (LIDA) designed for multimodal AD therapy. Surface-conjugated ApoE3 serves a bifunctional role by facilitating BBB penetration and actively inhibiting Aβ oligomerization. Upon 808 nm laser irradiation, ICG-mediated photothermal induction triggers the spatiotemporal release of DNP, significantly enhancing AChE inhibition. Furthermore, LIDA treatment effectively mitigates Aβ-induced cytotoxicity and mitochondrial dysfunction in SH-SY5Y cells. By integrating targeted delivery with NIR responsiveness, this study presents a novel, multivalent strategy to combat the complex pathological landscape of AD.
Neural function emerges from the interplay between electrical activity and neurochemical signaling, yet most implantable neural interfaces primarily record electrophysiological signals and lack molecular specificity for neurotransmitter monitoring. Here, we developed a flexible multimodal neural probe integrating an on-chip three-electrode electrochemical aptamer sensor with electrophysiological recording sites for combined monitoring of extracellular dopamine (DA) dynamics and neuronal activity. Fabrication of the electrochemical interface was systematically optimized by controlling gold nanostructure (AuNS) electrodeposition from 0.50 to 0.70 V. Electrodeposition at 0.65 V provided the best balance between increased electrochemically active surface area (ECSA), surface morphology, fabrication reproducibility, and electrical isolation, yielding an approximately 4.7-fold ECSA enhancement with a probe-to-probe coefficient of variation (CV%) of 2.02%. A methylene blue-labeled DA aptamer was assembled onto the AuNS working electrodes for sequence-specific molecular recognition, while integrated Ag/AgCl reference and nanostructured platinum counter electrodes completed the on-chip sensing system. Electrochemical impedance analysis further demonstrated reproducible interfacial characteristics following stepwise functionalization. The electrophysiological electrodes exhibited a mean impedance of 366.02 ± 18.63 kΩ at 1 kHz with an interprobe CV% of 5.09%. Square-wave voltammetry frequency was experimentally optimized from 10 to 200 Hz, with 100 Hz providing the best balance between analytical response, background current, and reproducibility. Under optimized conditions, the sensor demonstrated sequence-specific DA recognition, selectivity against electroactive interferents, and an experimentally determined detection limit of 10 fM. Two absolute linear response regions were identified at 0.5-10 pM (R2 = 0.9945) and 0.5-10 nM (R2 = 0.9949), while the broader detectable concentration range extended from 10 fM to 1 μM. Flow-injection and brain-phantom experiments demonstrated reversible DA sensing. In acute rat caudate-putamen experiments, intravenous nomifensine induced DA-associated electrochemical responses accompanied by increased neuronal firing and γ/high-γ local field potential activity, supporting this platform for acute multimodal neurochemical and electrophysiological interrogation in vivo.
Single-cell proteomics enables direct measurement of cellular heterogeneity during dynamic biological processes, but its application to fragile and highly adherent neuronal models remains challenging. Here, we developed and applied an optimized single-cell proteomics workflow to characterize proteome remodeling during nerve growth factor (NGF)-induced differentiation of PC12 cells. To enable reliable single-cell analysis, we implemented gentle dissociation, antiaggregation strategies, and thermal inkjet-based cell dispensing, achieving high accuracy in single-cell isolation. Inclusion of n-dodecyl-β-d-maltoside (DDM) improved recovery of membrane-associated and low-solubility proteins. Coupled with LC-ion mobility-mass spectrometry, this workflow enabled quantification of 2,000-3,000 proteins per cell across the differentiation time course. Single-cell proteomic analysis revealed progressive and heterogeneous proteome remodeling during differentiation. While undifferentiated cells formed a relatively homogeneous population, later stages (Days 4-6) exhibited increased variability, including multimodal protein abundance distributions and separation into distinct subpopulations. Dimensionality reduction, clustering, and non-negative matrix factorization identified multiple coexisting proteomic states within the same time points, reflecting asynchronous differentiation trajectories. These subpopulations were characterized by coordinated differences in pathways related to intracellular trafficking, protein translation, cytoskeletal organization, and neuronal maturation. Comparison with bulk proteomics demonstrated that proteins associated with differentiated neuronal states, including those involved in neurite formation and structural remodeling, are underrepresented in population-averaged measurements but are enriched within specific single-cell subpopulations. Temporal and cluster-resolved analyses further revealed distinct protein expression trajectories, including early decreases in cell cycle and metabolic pathways and later increases in neuronal structural and regulatory proteins. Together, this study establishes an optimized workflow for single-cell proteomics of neuronal systems and demonstrates that NGF-induced PC12 differentiation proceeds through heterogeneous and divergent proteomic states that are not resolved by bulk analysis.
Current research into the actions of psychedelic drugs is focused on 5-HT2A receptor-coupled signaling pathways, particularly β-arrestin2 signaling. Here, we investigated the involvement of β-arrestin2 in psychedelic drug-induced mouse markers of hallucinogenic effects, molecular plasticity, and structural plasticity; specifically, the head-twitch response, cortical expression of plasticity-related genes (cFos, Egr1, Egr2, and Arc), and dendritogenesis (primary cortical neuron cultures), respectively. Psilocin-induced head-twitches, plasticity gene expression, and dendritogenesis were all blocked by the 5-HT2A receptor antagonist MDL-100,907. The head-twitch response to psilocin was not different between wildtype and β-arrestin2 knockout (KO) mice, and the head-twitch responses to 2,5-dimethoxy-4-iodoamphetamine (DOI) and lysergic acid diethylamide (LSD) were similarly unaffected by β-arrestin2 KO. Psilocin-evoked gene expression showed a trend to be less in β-arrestin2 KO compared to wildtype mice, but the gene expression response to DOI was clearly not altered by β-arrestin2 KO. Finally, psilocin-induced dendritogenesis was attenuated in β-arrestin2 KO versus wild-type cultured neurons, and similar findings were obtained with DOI. In summary, the current study found no convincing evidence to support a role for β-arrestin2 signaling in either the head-twitch or plasticity-related gene responses to the psychedelic drugs tested. However, our data suggest a role for the β-arrestin2 pathway in psychedelic drug-evoked dendritogenesis of cultured neurons. Thus, the β-arrestin2 pathway unlikely mediates the hallucinogenic effects of psychedelic drugs but may contribute to neural plasticity changes in certain models.
The spinal cord exhibits a complex cytoarchitecture and neuronal circuitry organization that challenges in vitro replication of its functional integrity. Here we employed a previously published protocol to generate spinal cord organoids (SCOs) from human-induced pluripotent stem cells (hiPSCs). We characterized phenotypic changes in functional neuronal properties in SCO neurons at early developmental stages (15-32 days in culture) using a quantitative electrophysiological approach. Using the whole-cell patch-clamp technique to evaluate electrophysiological properties, we found that SCO neurons exhibited progressive maturation, as evidenced by hyperpolarized resting membrane potentials, increased inward current amplitude, refined action potential kinetics, and the early emergence of mature-type firing patterns. In particular, we show that the spike frequency adaptation phenomenon, which prevails in motor neurons, appears at early stages of SCO neuron development. Immunohistochemical assessment confirmed the expression of key transcription factors in motor neurons (ISLET1 and HB9) and immature spinal interneurons (LHX1/5 and PAX2). Collectively, our findings demonstrate that neurons in hiPSC-derived SCOs exhibit physiological differentiation, which is important for using SCOs to investigate human spinal cord development and advance translational research in CNS disorders and cell replacement therapies.
Repeated psychological stress is a major risk factor for psychiatric disorders. Sphingosine 1-phosphate (S1P), a bioactive sphingolipid, is known to contribute to regulating central nervous system functions. However, the relationship between the onset of psychological stress-induced behavioral disorders and S1P metabolism in the brain remains poorly understood. Because prefrontal cortex (PFC) and hippocampus are key brain regions involved in psychological stress responses, we investigated whether repeated social defeat stress (SDS) alters S1P metabolism in these regions. The S1P levels in the PFC, but not in the hippocampus, of mice susceptible to 4-day SDS were markedly lower than those in the control mice and were positively correlated with sociability. Additionally, mRNA expressions of an S1P-degrading enzyme Plpp3 and an inflammatory mediator Hmgb1 were increased in the PFC of 4-day SDS-susceptible mice. These results suggest that impaired S1P signaling in the PFC is associated with the onset of psychosocial stress-induced social avoidance.
Emerging evidence suggests that dysregulated cellular stress responses, particularly the formation and persistence of stress granules (SGs), may significantly contribute to Alzheimer's disease (AD) pathogenesis. SGs are dynamic, membrane-less ribonucleoprotein assemblies that sequester stalled translation preinitiation complexes during cellular stress. Under physiological conditions, SGs are transient and disassemble upon stress resolution, often facilitated by heat shock proteins (HSPs). However, under chronic stress, persistent or aberrant SGs can form, which may seed pathological protein aggregation and exacerbate proteostatic imbalance, neuroinflammation, and neurodegeneration. Despite the growing body of evidence linking HDAC6 to SG dynamics and AD pathology, the precise mechanistic relationship remains unresolved. This review highlights the current understanding of HDAC6-mediated SG regulation in AD, identifies critical knowledge gaps, and discusses the therapeutic potential of selective HDAC6 inhibition in modulating SG pathology and neuroinflammation.
Gain-of-function mutations in the LRRK2 protein elevate kinase activity, and heterozygous or homozygous carriers of mutant LRRK2 have an increased risk of Parkinson's disease (PD), together suggesting that inhibition of mutant LRRK2 kinase activity may be a promising therapeutic target. Conversely, LRRK2 knockout mice and people with homozygous loss-of-function LRRK2 mutations exhibit prominent pathologic changes in lung and kidney, and nonselective inhibition of wild-type and mutant LRRK2 kinase at therapeutic doses causes on-target toxicity in rodent and nonhuman primate models, most prominently in lung. Numerous LRRK2 variants have been described that cause increased phosphorylation of endogenous substrates. The G2019S mutation is the most prevalent, occurring in up to 40% of PD patients in certain populations, with the vast majority being heterozygous. We previously disclosed a series of G2019S LRRK2-selective kinase inhibitors designed to avoid on-target side effects. In this study, we examined the effect of a LRRK2 inhibitor specific for the G2019S variant, STF-3600, in WT, and heterozygous or homozygous G2019S LRRK2 knock-in (KI) mice. This inhibitor did not cause vacuolization in lungs of WT or heterozygous G2019S LRRK2 KI mice but did cause lung toxicity in homozygous G2019S LRRK2 KI mice that was similar to what was observed with the nonselective inhibitor, MLi-2. This study underscores the potential of G2019S LRRK2-selective kinase inhibitors to be safe and effective for heterozygous G2019S LRRK2 PD carriers.
With the recent FDA approval of antibody-based therapeutics for Alzheimer's disease (AD), the need for diagnostic tools capable of detecting the disease at the earliest stages has become increasingly urgent. Although small molecules offer advantages in terms of production, stability, and accessibility, no imaging agent currently enables reliable detection of early-stage aggregates of the amyloid-β (Aβ) peptide, among the earliest biomarkers in AD progression. Herein, we report a series of bis(styryl)benzene (BSB) probes derived from the methoxy-X04 scaffold and a systematic structure-activity investigation examining how targeted molecular modifications, including hydroxyl positioning, (Me)HN or (Me)2N substitution, and incorporation of a 1,4-dimethyl-1,4,7-triazacyclononane (tacn) moiety, modulate Aβ binding, cytotoxicity, and brain uptake. These methoxy-X04 analogues retain Aβ affinity while exhibiting markedly improved cytotoxicity profiles and logD values consistent with in vivo applicability. Amyloid-β binding was evaluated using age-dependent in situ staining of 5xFAD mouse brain sections, supported by in vitro fluorescence-based oligomer and fibril assays and in silico analyses that reveal tunable selectivity toward early versus late Aβ aggregates. In vivo brain uptake studies of BSB5 and Me2tacnBSB5 further support the utility of these probes as modular Aβ-targeting fragments for the development of early-stage AD imaging agents.
