ACS Chemical Neuroscience

The aggregation of amyloid-{beta} (A{beta}) peptides into insoluble deposits is a characteristic hallmark of Alzheimer's disease (AD) and related neurodegenerative disorders. While AD is the most common cause of dementia, there are currently no disease-modifying treatments which are both affordable and adverse-free. In this study, we report that melanin, a pigment which is commonly found in nature and is abundant in parts of the human brain, suppresses the aggregation of the 42-amino acid A{beta} variant (A{beta}42). Using biophysical and biochemical techniques, we show that melanin delays A{beta}42 aggregation while also reducing the amount of A{beta}42 that converts into aggregates. Using thioflavin T assays paired with chemical kinetics, we characterised the melanin-induced inhibition of A{beta}42 aggregation in vitro. Using MALDI-MS, we elucidate the molecular basis of this effect by showing that melanin prevents A{beta}42 dimerisation. We then demonstrate that melanin also reverts the aggregation process by dissolving pre-formed A{beta}42 fibrils. Finally, we show that melanin reduces A{beta}42 aggregation and rescues A{beta}42 toxicity in an SH-SY5Y neuroblastoma cell model. Our study shows that melanin disrupts the aggregation and cytotoxicity of A{beta}42, and suggests that compounds derived from human metabolites may offer promising avenues to combat amyloid formation.

Schizophrenia is a debilitating neuropsychiatric disease that lacks effective treatments for many symptom domains including negative, cognitive and sleep disturbances. Lack of clear disease etiology has hampered the development of new, effective treatments for the unmet needs of people with schizophrenia. Large scale human genetics have identified rare loss of function mutations that substantially increase risk of developing schizophrenia, including in GRIA3, the gene that encodes the GluA3 receptor subunit of the AMPA receptor (AMPAR). Several drug discovery programs have been aimed at developing AMPAR positive allosteric modulators (PAMs) as a novel treatment for schizophrenia. Despite intense drug discovery efforts, there are no FDA approved AMPAR PAMs. We therefore hypothesized that selectively targeting GluA3, the AMPAR subunit implicated by human genetics, could yield a safer and more effective AMPAR PAM for the potential treatment of schizophrenia. Using a combination of medicinal chemistry, in vitro, and in vivo studies, we discovered BRD3290, a GluA3-preferring AMPAR PAM with reasonable potency in heterologous cells, as well as favorable tolerability and brain exposure. Peripheral administration of BRD3290 engaged an established AMPAR PAM target engagement biomarker but did not improve performance of wildtype mice in the novel object recognition task (NOR), in contrast to the nonselective AMPAR PAM PF-4778574, which improved mouse NOR. These findings suggest that the GluA3 selectivity profile of BRD3290 was insufficient to enhance cognitive function in this mouse NOR paradigm. This work highlights the challenges of AMPAR subtype-selective modulation and provides molecular insights into the ability to develop subtype-selective AMPAR PAMs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/740780v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c14b74org.highwire.dtl.DTLVardef@140b2b3org.highwire.dtl.DTLVardef@942636org.highwire.dtl.DTLVardef@58bc24_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nerve Growth Factor (NGF) supports neuronal survival, plasticity, and growth by binding to p75NTR and TrkA receptors. There is a strong link between NGF and the cholinergic hippocampus, and changes in NGF pathways are observed in Alzheimer's disease (AD). Research shows NGF's neuroprotective and regenerative effects on cholinergic neurons, with positive results in AD models and clinical studies. However, NGF therapy faces challenges: limited blood-brain barrier crossing and dose-dependent pain. To address pain, a mutated NGF called CHF6467 was developed. Thanks to the R100E mutation, it prevents binding to p75NTR while maintaining TrkA affinity, which helps reduce pain side effects. Understanding CHF6467's mechanism remains a challenge. In this context, untargeted metabolomics is effective for uncovering unforeseen responses, making it suitable for assessing CHF6467's overall effects. Building on our previous research and recognizing the importance of amyloid-{beta} peptide as a critical pathological marker of AD, we used NMR-metabolomics to study the effect of CHF6467 on SH-SY5Y cells. The study examined metabolomic profiles of cells treated with CHF6467, with and without A{beta}(1-42), using NMR. Results show CHF6467 influences the metabolic changes caused by A{beta}(1-42), inducing biochemical effects consistent with the promotion of phospholipid precursors, reduced glutamate excitotoxicity, and restored energy metabolism.

Imipramine is known as an older generation tricyclic antidepressant drug. It has been identified in prior studies that imipramine blocks Apolipoprotein E4 (ApoE4)-induced amyloid-{beta}(A{beta}) aggregation and is associated with an improved AD diagnosis [Johnson et al. Alzheimers Research Therapy, 2022, 14, 88]. Using NMR methods such as 1H-1H NOESY and Saturation Transfer Difference Spectroscopy, we demonstrate the binding of A{beta} monomers to imipramine when the full-length A{beta} (1-42) sequence is considered. The more abundant but less toxic form, A{beta} (1-40) does not show interaction with imipramine.

Sleep disorders and wakefulness constitute a pronounced and pervasive health risk that contributes to numerous cerebral pathologies such as stroke, depression, and neurodegenerative disorders. The hippocampus (HC) plays vital roles in learning and memory and is especially susceptible to the pathological insults imposed by sleep deprivation (SD). While SD disrupts neuronal network signaling and triggers inflammatory processes, the impact of SD on metabolism within the HC presently remains poorly understood. In this study, we assessed the progressive and cumulative effects of partial SD in the live mouse HC CA1 region using two-photon microscopy. Using a custom-designed implantable cannula enabling direct HC visualization and administration of dyes and pharmacological agents directly to HC tissue, we longitudinally monitored spontaneous neuronal activity and partial pressure of oxygen (pO2) within HC CA1 tissue before and during a 7-day period of partial SD and after a 7- day period of recovery. Our results revealed a progressive decline in spontaneous neural activity, alongside sustained elevation in tissue pO2; during the 7-day partial SD period. Notably, after 7 days of recovery, neuronal activity did not return to baseline, and tissue oxygenation continued to increase. Our findings emphasize chronic SD's cumulative and enduring impairments to neuronal function and metabolism in the HC and reveal uncoupling between metabolic and functional recovery that outlasts the sleep restriction period.

Wallerian degeneration of anucleated axonal segments is driven by SARM1, which depletes axonal NAD+, disrupting energy metabolism and triggering self-destruction. SARM1 inhibition is an emerging therapeutic target for traumatic nerve injuries. Boldine, a natural aporphine alkaloid from Peumus boldus, modulates connexin hemichannels, oxidative stress, and inflammation. Building on our published work showing boldines neuroprotective effects in nerve injury models, we hypothesized that boldine also inhibits SARM1 directly. A fluorescence polarization assay revealed that boldine inhibits SARM1 NADase activity with an IC50 of approximately 7.5 uM. AI-assisted structural modeling (AlphaFold3-based Boltz-1 with GNINA docking) predicted two boldine binding sites on SARM1: the TIR catalytic site (Kd [~] 13.5 uM) and the ARM-TIR regulatory interface (Kd [~] 12 uM). In a sciatic nerve explant model, boldine preserved the integrity of anucleated axonal segments at 3 and 7 days post-transection relative to vehicle controls. These findings suggest boldine may act as a dual-site SARM1 inhibitor and support its development as a neuroprotective therapy after traumatic axonal injury.

Major depressive disorder and bipolar depression are disabling illnesses associated with impaired neuroplasticity, mitochondrial dysfunction, and altered cellular bioenergetics. Available pharmacotherapies often have delayed onset, incomplete efficacy, and limited effects on underlying plasticity and energetic systems. Non-selective thyroid hormone therapies provide evidence that enhancing central thyroid hormone signaling may improve depressive symptoms, but systemic cardiovascular, skeletal, and metabolic effects limit their broader use. Rapid-acting treatments such as esketamine and psychedelics further support neuroplasticity as a therapeutic strategy, although psychotomimetic, hallucinogenic, and implementation burdens may constrain widespread use. Elunetirom is a brain-targeted, fatty acid amide hydrolase-sensitive prodrug of LL-340001, a potent thyroid hormone receptor activator as demonstrated in transfected cell-line and brain-slice target-engagement assays. We evaluated elunetirom and LL-340001 in primary cortical and mature hippocampal neuronal cultures. LL-340001 increased MAP-2-positive neuron number, neurite length, branching, and neurite extremities in immature cortical neurons. In mature hippocampal neurons, elunetirom rapidly increased neurite network and synapse number within 24 hours, with effects persisting through 72 hours, while LL-340001 preserved synapse number following amyloid-{beta}1-42 challenge. LL-340001 also increased nuclear PGC-1 and NRF2, the number of functional mitochondria measured by MitoTracker, and ATP content. Pharmacologic inhibition indicated that the LL-340001-induced increase in cortical MAP-2-positive neuron number was sensitive to TrkB inhibition but not 5-HT2A antagonism. Together, these findings indicate that elunetirom and LL- 340001 engage complementary neuroplasticity and mitochondrial biogenesis-related programs in neuronal systems and support further investigation of brain-targeted thyromimetic signaling as a mechanistically differentiated approach for major depressive disorder and bipolar depression.

Psilocybin is a psychedelic which has been shown to induce neural plasticity through activation of intracellular serotonergic 5-HT2A receptors. It also produces brain-wide changes in structural and functional connectivity and holds promise as a therapeutic compound for treating anxiety and depression. Despite links between psilocybin-induced plasticity, the psychedelic experience, and reduction in depressive symptoms, little is known about the effects of psilocybin on the function of the highly plastic hippocampus, a region crucial for memory whose dysfunction is linked to neural disorders such as depression and anxiety. In this study, we investigated the acute and lasting effects of psilocybin on rodent sharp-wave ripples (SWRs), transient high frequency oscillations observable in the hippocampal local field potential which are linked to memory consolidation. We found that a 10 mg/kg dose of psilocybin robustly decreased the peak SWR frequency and increased the duration of SWRs immediately following administration compared to control sessions the day before and after. Psilocybin also perturbed sleep architecture, resulting in a pronounced reduction in non-rapid eye movement (NREM) sleep which lasted for hours. Therefore, psilocybin could impact memory processing by modulating hippocampal SWRs.

Membrane surfaces can accelerate amyloid $\beta$ (A$\beta$) aggregation, yet the role of membrane curvature in this process remains poorly understood. Here, we used multi-million atom all-atom molecular dynamics simulations to compare the adsorption, conformational dynamics, and oligomerization of four A$\beta$42 peptides at a planar neuronal membrane and a highly curved lipid vesicle. For both systems, all peptides adsorbed within the first 2 $\mu$s, but their subsequent behavior differed substantially. The curved membrane exhibited a larger area per lipid and more extensive hydrophobic packing defects, allowing A$\beta$42 to penetrate more deeply and form strong contacts with lipid tails through its central hydrophobic core and C-terminal region. These interactions disrupted a solution-formed dimer and limited peptide-peptide association during the simulated interval. Additionally, vesicle-bound peptides adopted more extended conformations with increased $\beta$-structure and $\beta$-hairpin formation compared with peptides at the planar membrane. A$\beta$42 adsorption was also corelated to lipid reorganization in the vesicle. In contrast, the planar membrane supported weaker adsorption and stable dimer-to-trimer growth but showed little large-scale lipid segregation. These findings reveal that curvature reshapes the early A$\beta$42 aggregation landscape by strengthening peptide-lipid interactions, altering aggregation-prone conformations, and reorganizing membrane domains. Membrane geometry should therefore be considered alongside lipid composition in mechanistic models of A$\beta$42 oligomerization and membrane-associated toxicity.

N, N-dimethyltryptamine (DMT) and 5-methoxy-N, N-dimethyltryptamine (5-MeO-DMT) are structurally related tryptamine psychedelics with emerging therapeutic potential, yet their comparative acute pharmacology and longer-term neuroplastic effects remain incompletely defined. Here we show that DMT produces a bell-shaped dose-response curve in the mouse head-twitch response (HTR) assay, whereas 5-MeO-DMT elicits a monotonic increase. Selective antagonism at 5-HT2A or 5-HT1D receptors, or agonism at 5-HT1A, robustly attenuates HTR for both compounds without abolishing their ability to reduce marble burying, a screening assay for OCD-like behavior. Acutely, both agents elevate TrkB phosphorylation in a region-specific manner, with broader engagement by DMT across default-mode-network and hippocampal territories. Twelve days after a single dose, both compounds increase synaptic proteins (PSD-95, synaptophysin; SV2A for DMT), while DMT uniquely lowers hippocampal BDNF and reprograms frontal-cortex glutathione and energy metabolism. These findings demonstrate that acute hallucinogenic-like activity and selected therapeutic-like behavioral and plasticity outcomes can be pharmacologically dissociated, informing the rational design of more tolerable, scalable psychedelic-based treatments.

Allosteric modulation of G protein-coupled receptors (GPCR) is a rapidly advancing area of drug discovery with considerable therapeutic potential. In this study, we used various bioluminescence resonance energy transfer (BRET) assay configurations to investigate the differential activity of LY3154207, a dopamine D1 receptor (D1R)- positive allosteric modulator (PAM), in potentiating D1R-mediated signaling pathways. Our data showed that LY3154207 demonstrated clear bias toward {beta}-arrestin recruitment over G-protein engagement. Among the G-protein subtypes, LY3154207 preferentially potentiated Gs signaling induced by DA. Interestingly, when tested for its effect on the Gs-selective D1R agonist DHX and the Golf-selective agonist TAV, LY3154207 further enhanced the selectivity of these compounds, suggesting a probe-dependent effect. We also studied the structural mechanism underlying this subtype selectivity and found that W123 and R130 in the intracellular loop 2 (ICL2) of D1R were crucial for the activity of LY3154207. Furthermore, the N domain of the G-protein appeared to make minor contributions to this G-protein subtype selectivity of LY3154207. Lastly, our data demonstrated that LY3154207 induced cAMP production in the absence of the orthosteric ligand, indicating that this compound is an ago-PAM.

Circadian disruption, notably sleep disturbances, serves as an early indicator of Alzheimers disease (AD), preceding cognitive symptoms like memory loss. The suprachiasmatic nucleus (SCN) governs biological rhythms and receives direct retinal input via melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize with environmental light cycles. The anatomical and functional basis for circadian disruption in AD remains unclear. Here, we explored the multi-level relationships between gene expression, the SCN connectome, and regulations of sleep and circadian rhythms in the APP/PS1 mouse model. The sleep architecture of APP/PS1 mice displayed significantly reduced rapid eye movement sleep (REM), associated with a reduced daily core body temperature amplitude and locomotor hyperactivity. Lastly, APP/PS1 mice showed an impaired response to acute light pulse stimulation and present hyperactivity of mRGCs at a young age and hypoactivity of these cells at older ages. These physiological functions are known to be, at least in part, regulated by the SCN, the main target of mRGCs. We noted several modifications in SCN connectomics using serial blockface electron microscopy (SBEM), including a reduction of the dendro-dendritic chemical synapse (DDCS) network that receives a large part of the retinal input and is thought to be crucial for synchronicity between SCN neurons. In addition, we observed multiple signs of dystrophy, including modifications of the shape of dendrites and cell soma, accumulation of aggregated lysosomes, and swelling of axons. At the same time, we investigated the changes in gene expression using spatial transcriptomics. The SCN presents changes in the expression of genes associated with synapse formation, cell adhesion, and neurite growth. These results suggest that, despite the absence of amyloid plaques in the ventral hypothalamus, the SCN of APP/PS1 mice still undergo profound gene expression changes, impacting connectomics and physiological functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/744599v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ceedb0org.highwire.dtl.DTLVardef@156cfaaorg.highwire.dtl.DTLVardef@5bc262org.highwire.dtl.DTLVardef@36df4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dopamine has long been regarded as the key neurotransmitter governing motor function through the direct and indirect striatal outflow pathways. However, the tripeptide ophthalmate (ophthalmic acid, OA), a glutathione analog, has recently emerged as another regulator of motor function. While the neural mechanisms by which OA regulates motor function remain unknown, we explored how OA behaves as a striatal neuromodulator. Using radiotracer-based uptake and release assays in mouse striatal tissue, combined with whole-cell electrophysiological recordings, we found that OA is taken up through a saturable, glutathione-competitive transport mechanism and is released in a Ca{superscript 2}-dependent manner upon depolarization, consistent with regulated exocytotic release. OA enhanced depolarization-evoked release of {gamma}-aminobutyric acid (GABA) but not glutamate, indicating selective modulation of distinct neurotransmitter systems. OA and dopamine reciprocally regulated one another: OA enhanced dopamine release, while D2 dopamine receptor activation suppressed OA release. Whole-cell recordings from medium spiny neurons (MSNs) showed that OA increased the amplitude of evoked AMPA receptor-mediated currents and shifted short-term plasticity from facilitation toward depression at excitatory synapses onto direct-pathway MSNs (dMSNs). These findings are consistent with an increase in presynaptic glutamate release probability, while sparing synapses onto indirect-pathway MSNs (iMSNs). Together, these findings establish OA as a striatal neuromodulator that interacts with other neurotransmitter systems and preferentially potentiates direct-pathway transmission. This novel discovery identifies a candidate mechanism within the basal ganglia circuitry that governs motor control, with major relevance to Parkinsons disease and other movement disorders.

BackgroundUnderstanding the molecular basis of Parkinsons disease (PD) phenotypic heterogeneity maybe improved by in vivo access to deep brain tissue. Deep brain stimulation (DBS) surgery offers a unique opportunity: microelectrodes traversing the subthalamic nucleus (STN) carry adherent brain tissue upon withdrawal, providing a source of RNA from subcortical regions in living patients without additional invasive procedures. AimTo develop and validate a low-input RNA extraction and transcriptomic profiling protocol using DBS microelectrodes in post-mortem human brain, to confirm that recovered RNA is of brain rather than blood origin, and to characterise its sub-regional and cellular identity. MethodologyDBS microelectrodes were inserted without image guidance or guide tube into three unfixed post-mortem human brains targeting the STN trajectory. In clinical practice, a guide tube shields the electrode from cortical tissue; its absence here means tissue from the full insertion trajectory may contribute to recovered RNA. Thirty-eight microelectrodes were evaluated across single and pooled strategies. RNA was extracted using a modified RNeasy Micro low-input protocol; libraries prepared using NEBNext Single Cell/Low Input RNA Library Kit and sequenced on Illumina NovaSeq 6000 (paired-end, 2x150 bp). Tissue identity was validated against GTEx v10 (54 tissues) and Allen Human Brain Atlas (ABA, 19 subcortical regions including STN) using Pearson correlation with permutation testing (1,000 permutations) and BH-FDR correction. Transcriptional overlap between subcortical and cortical reference regions was quantified and subcortical-enriched gene filtering performed. ResultsTwenty-five RNA isolates were obtained from 38 microelectrodes; 54.5% of Bioanalyzer-assessed samples achieved RIN [&ge;]5 (median 7.1; range: 5.9-7.8). Fifteen libraries passed sequencing QC (mean depth 43.0 {+/-} 14.1 million read pairs; mean Q30 83.2 {+/-} 5.6 %). PCA of rlog transformed expression data resolved samples by donor identity. All samples confirmed brain tissue origin by GTEx {tau}-index tissue specificity correlation (n=996 brain and blood specific markers). Brain correlations were highest for frontal cortex (mean r= 0.683 {+/-} 0.072), anterior cingulate cortex (mean r= 0.658 {+/-} 0.069), amygdala (mean r= 0.615 {+/-} 0.068), and basal ganglia including caudate (mean r= 0.553 {+/-} 0.059), putamen (mean r= 0.546 {+/-} 0.059), and nucleus accumbens (mean r= 0.544 {+/-} 0.057); all BH-FDR < 0.05. Cerebellum showed the lowest brain correlations (cerebellar hemisphere: mean r = 0.327 {+/-} 0.039; cerebellum: mean r = 0.314 {+/-} 0.037). Whole blood correlation was strongly negative (mean r= -0.250 {+/-} 0.079), confirming non-haematological origin. ABA genome-wide analysis confirmed positive STN correlation (r=0.54-0.62; permutation p<0.001). MuSiC cell-type deconvolution against the Allen Brain Atlas HMBA-BG snRNA-seq reference identified oligodendrocytes (33.5 {+/-} 17.2%), frontal cortical neurons (9.1 {+/-} 7.0%), STR D1 MSNs (6.0 {+/-} 4.9%), and dopaminergic neurons (1.1{+/-} 2.7%) as the principal cell types, independently corroborating bulk transcriptomic findings at single-cell resolution. ConclusionThis study validates a low-input RNA extraction protocol for DBS microelectrodes, confirming brain-specific transcriptomic profiles consistent with STN-adjacent subcortical sampling. The 96% transcriptional overlap between cortical and subcortical regions, combined with absence of a guide tube in this post-mortem model, limits sub-regional specificity; clinical application with a guide tube would enrich the subcortical signal. These findings provide the methodological framework for in vivo molecular profiling of the human basal ganglia during DBS surgery in PD patients.

BackgroundWe previously demonstrated that 1,10-phenanthroline-5-amine (PAA) significantly reduced the number and size of amyloid plaques in one-year-old APP/TAU mice. The primary objective of the present study was to validate these findings in the APP/PS1 mouse model using a larger cohort of animals. A second objective was to determine whether PAA binds directly to amyloid plaques in brain tissue sections. MethodsFor the in vivo studies, APP/PS1 mice received daily oral PAA or vehicle treatment and were euthanized at one year of age. Brains were collected, fixed, cryosectioned, and stained with hydroxyquinoline oxalate (HQ-O) to visualize amyloid plaques. For the in vitro studies, brain tissue sections were incubated in a PAA solution. Double labeling with PAA and HQ-O was performed on the same tissue sections to compare plaque labeling patterns. ResultsDaily oral administration of PAA produced a significant reduction in both the number and size of amyloid plaques compared with untreated control mice. In vitro incubation of tissue sections with PAA resulted in red fluorescent labeling of all amyloid plaques. Double-labeling studies showed that PAA labeled plaques are more extensive than HQ-O in frozen tissue sections, whereas no such difference was observed in paraffin-embedded sections. ConclusionsThese findings extend our previous observation that chronic oral administration of PAA significantly reduces amyloid plaque burden in vivo. In addition, the in vitro studies demonstrate that PAA binds directly to amyloid plaques. The mechanism of PAA binding may involve interactions with transition metals incorporated within amyloid plaques and/or the sialic acid moieties of plaque-associated gangliosides.

In human dorsal root ganglia (DRG), and trigeminal (TG) neurons, the various voltage-gated sodium channel (Nav) isoforms play critical roles in the firing of action potentials, which drive electrical impulses that encode somatosensations including, itch, and pain. The SCN11A gene encodes the tetrodotoxin (TTX)-resistant voltage-gated sodium channel Nav1.9, characterized by unique gating properties. Unlike other isoforms, the Nav1.9 channel activates and inactivates slowly and has a hyperpolarized voltage-dependence of activation and depolarized voltage-dependence of inactivation. This leads to a large window current that has been suggested to function as a regulator of the resting membrane potential of neurons. Mutations in Nav1.9 channels lead to congenital insensitivity to pain (gain-of-function) or familial episodic pain syndrome (loss-of-function) suggesting the channel is a critical mediator of pain. Despite its relevance in pain pathophysiology, most existing data relies on rodent models or heterologous expression systems, leaving the specific pharmacology and biophysical behavior of these channels in human primary neurons largely unknown. In this study, we pharmacologically isolated and characterized native Nav1.9 channel currents in human DRG and TG neurons to compare their biophysical profiles. Our findings reveal significant kinetic and voltage-dependent differences between the two populations. Specifically, Nav1.9 channels in TG neurons exhibit a right-shifted steady-state inactivation curve, a larger window current, and faster activation kinetics compared to those in DRG neurons. In addition, conditions that simulate inflammatory states in-vivo greatly potentiates the Nav1.9 currents consistent with similar observations in rodent models. By detailing these distinct biophysical properties, this research offers crucial insights into Nav1.9 channel function relevant for drug discovery efforts aimed at developing analgesics for both acute and chronic pain.

The frequent coexistence of -synuclein (-syn) and amyloid-{beta} (A{beta}) aggregates in neurodegenerative diseases suggests that heterotypic interactions between these amyloidogenic proteins may influence disease progression, yet their molecular consequences remain poorly understood. Here, we investigated how distinct aggregation states of A{beta}42, monomers and preformed fibrils (PFFs), modulate -syn fibril formation, structure, and downstream neuronal pathology. Thioflavin T kinetics showed that A{beta}42 monomers delayed -syn fibril formation, whereas A{beta}42 PFFs exhibited a trend toward accelerated aggregation, indicating aggregation state-dependent effects on -syn aggregation. Negative-stain TEM, proteinase K digestion, and solid-state NMR spectroscopy further demonstrated that both A{beta}42 monomers and PFFs altered -syn fibril structure, generating distinct fibril conformations depending on the A{beta}42 concentration and aggregation state. To determine whether these conformational differences influence pathological activity, -syn PFF variants generated in the presence of different concentrations of A{beta}42 monomers or PFFs were applied to dopaminergic neuronal cells. -Syn fibrils formed in the presence of A{beta}42 PFFs showed greater capacity to induce intraneuronal -syn aggregation than -syn PFFs, whereas fibrils formed in the presence of A{beta}42 monomers exhibited similar or reduced seeding capacity relative to -syn PFFs. Together, our findings demonstrate that heterotypic interactions with A{beta}42 reshape -syn aggregation pathways and fibril conformations, generating structurally distinct -syn fibril populations with different neuronal seeding activities. These results provide a molecular framework for understanding how cross-talk between amyloidogenic proteins may contribute to structural and pathological heterogeneity in mixed neurodegenerative diseases.

The dopamine D2 receptor is a principal target of antipsychotic and antiparkinsonian drugs, yet well-characterized fluorescent ligands for the D2 receptor remain scarce compared with its relative, the D3 receptor. Here we show that fluorescent ligands developed for the D3 subtype can be repurposed as tools for studying the D2 receptor pharmacology. Using a machine-learning-assisted, live-cell fluorescence microscopy assay, we screened twelve fluorescent probes and identified eight ligands that bound specifically to the D2 receptor at 10 nM. Four ligands exhibited classical saturable binding, yielding Kd in the nanomolar range, whereas the remaining probes displayed unsaturated, time-dependent binding profiles. Kinetic analyses revealed substantial differences in association and dissociation behavior, highlighting the importance of time-resolved measurements. Competition binding with dopamine and haloperidol yielded affinity estimates consistent with literature values. These results expand the toolkit of validated D2 receptor fluorescent probes and support cross-receptor repurposing as a strategy for fluorescent ligand development.

Amyloid-{beta}42 (A{beta}42) aggregation is highly sensitive to experimental conditions, making reproducibility a persistent challenge in Alzheimers disease research. Among the many variables that influence aggregation, the impact of peptide production remains poorly understood. Direct comparison of recombinant and chemically synthesised A{beta}42 prepared under carefully controlled conditions reveals that, despite following similar aggregation mechanism and forming the same predominant fibril structures, synthetic A{beta}42 aggregates more slowly and exhibits reduced seeding efficiency. Consequently, synthetic A{beta}42 produces fewer oligomeric species and displays lower cellular toxicity. Mass spectrometric analyses identify low-abundance sequence imperfections introduced during peptide synthesis as the origin of these differences. By linking synthesis-derived imperfections to variations in A{beta}42 behaviour, this work reveals a previously underappreciated source of discrepancies in amyloid studies. In addition, we provide a framework for evaluating the impact of sequence impurities on biophysical studies that are sensitive to peptide composition.

The psychiatric utility of ketamine is limited by its dissociative and systemic side effects. Recently, to enable precision ketamine pharmacotherapy, we introduced SonoKet, ketamine-loaded acoustically activatable liposomes that enable focused ultrasound (FUS)-targeted ketamine delivery to millimeter-sized brain regions. In initial studies, we observed that SonoKet uncaging targeted ketamine to the ultrasound-treated brain region, while inducing greater electrophysiologic and behavioral functional effects than dose-matched free ketamine. To further define these uncaging-potentiated neuromodulatory effects, we used solid-phase microextraction (SPME) coupled to LC-MS/MS to investigate the effect of ultrasound and SonoKet uncaging on key neurotransmitters in real-time. SPME probes were used to sample ketamine, its metabolites, and glutamate, GABA, serotonin (5-HT), and dopamine in the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and retrosplenial cortex (RsC) of awake rats. Sampling occurred before and after intravenous administration of either SonoKet, free ketamine, or saline, with FUS targeted to either a frontolimbic or caudal brain region. FUS alone did not yield significant changes in neurotransmitter concentration, nor did it affect the pharmacodistribution of free ketamine. In contrast, FUS generally increased the neurotransmitter response to free ketamine, suggesting an ultrasonic potentiation of ketamine neuromodulation. SonoKet (0.75 mg/kg) uncaging with FUS elicited further elevations in glutamate, GABA, and 5-HT within the FUS-targeted region, along with an increase in dopamine in the NAc when the frontolimbic region was sonicated. These increases were similar to or higher than those induced by 10 mg/kg free ketamine alone or 0.75 mg/kg free ketamine combined with FUS, especially with frontolimbic SonoKet uncaging. Altogether, FUS potentiates ketamine-induced neuromodulation, with spatially specific and synergistically greater effects when ketamine is spatially localized via ultrasonic uncaging. This strategy could augment ketamine pharmacotherapy for psychiatric diseases, while limiting its dissociative and abuse liabilities. HighlightsO_LIFocused ultrasound potentiated ketamine-driven glutamate, serotonin, and dopamine release C_LIO_LILocalized ketamine delivery with SonoKet uncaging drove synergistically greater region-specific neurochemical responses C_LIO_LIUncaging boosts ketamine effects at a fraction of the ketamine dose C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/741494v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1745214org.highwire.dtl.DTLVardef@1b8e06borg.highwire.dtl.DTLVardef@95a840org.highwire.dtl.DTLVardef@15924ed_HPS_FORMAT_FIGEXP M_FIG C_FIG