Basal forebrain cholinergic neurons play a key role in cognition. This neuronal system is highly dependent on nerve growth factor (NGF) for its synaptic integrity and the phenotypic maintenance of its cell bodies. Basal forebrain cholinergic neurons progressively degenerate in Alzheimer's disease and Down's syndrome, and their atrophy contributes to the manifestation of dementia. Paradoxically, in Alzheimer's disease brains, the synthesis of NGF is not affected and there is abundance of the NGF precursor (proNGF). We have shown that this phenomenon is due to a deficit in NGF's extracellular metabolism which compromises proNGF maturation and exacerbates its subsequent degradation. We hypothesized that a similar imbalance should be present in Down's syndrome. Using a combination of quantitative reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting and zymography, we have investigated signs of NGF metabolic dysfunction in post-mortem brains from the temporal (n = 14), frontal (n = 34) and parietal (n = 20) cortex obtained from Down's syndrome subjects and age-matched controls (age range 31-68 years). We further examined primary cultures of human fetal Down's syndrome cortex (17-21 gestational age weeks). We report a significant increase in proNGF levels in human Down's syndrome brains, with a concomitant reduction in the levels of plasminogen and tissue plasminogen activator mRNA as well as an increment in neuroserpin expression; enzymes that partake in proNGF maturation. Down's syndrome brains also exhibited elevated zymogenic activity of MMP9, the major NGF-degrading protease. Our results indicate a failure in proNGF maturation in Down's syndrome brains and a likely enhanced proteolytic degradation of NGF, changes which can compromise the trophic support of basal forebrain cholinergic neurons. The alterations in proNGF and MMP9 were also present in cultures of Down's syndrome fetal cortex; suggesting that this trophic compromise may be amenable to rescue, before frank dementia onset. Our study thus provides a novel paradigm for cholinergic neuroprotection in Alzheimer's disease and Down's syndrome.

In Parkinson's disease, cholinergic neurons of the pedunculopontine nucleus, located in the upper pons, undergo extensive degeneration, contributing to severe hypokinetic-motor and non-motor symptoms. We previously demonstrated that loss of these neurons is associated with mitochondrial dysfunction, driven by high mitochondrial DNA deletion burden. Increased mitochondrial DNA replication was also observed in these neurons, contrasting with reduced mitochondrial DNA copy number previously reported in Parkinson's-affected surviving nigral-dopaminergic neurons. Here, ultra-deep whole-genome sequencing combined with stringent quality control was used to characterise mitochondrial DNA alterations within single-cell pedunculopontine-cholinergic neurons isolated from Parkinson's-affected and neurologically-normal post-mortem specimens. Analyses included mitochondrial DNA deletion spectrum, -size distribution, genomic location, heteroplasmy levels, breakpoint classification, and point-mutation frequency. Thermodynamic modelling was used to investigate whether the stability of mitochondrial DNA secondary structures surrounding deletion breakpoints could provide insight into the mechanisms underlying mitochondrial DNA deletion formation in Parkinson's disease. In parallel, nuclear-encoded mitochondrial pathway responses were assessed using a customised single-cell multiplexed multi-target gene expression assay, comparing Parkinson's and control post-mortem neurons. Parkinson's-affected neurons exhibited substantial mitochondrial DNA damage, characterised by large-scale mitochondrial DNA deletions predominantly locating within mitochondrial DNA's major arc. Mitochondrial DNA deletions ranged ∼50-5,500 base-pairs, with most exceeding 1,000 base-pairs. Clonal expansion of large-scale mitochondrial DNA deletions, likely arising from replication-associated errors, represented the dominant mutant species. Small mitochondrial DNA deletions (<50bp) were rare, suggesting that free radical-induced strand breakage was not the primary driver of somatic mitochondrial DNA damage. Point-mutation frequency was similar between Parkinson's and control neurons but showed greater functional impact on electron transport chain complex-I subunits in Parkinsonian neurons. Thermodynamic modelling revealed reduced stability of mitochondrial DNA secondary structures flanking mitochondrial DNA deletion breakpoints in Parkinsonian post-mortem neurons, with an average decrease of ∼2 kJ/mol across two major mitochondrial DNA deletions formation models. The increased mitochondrial DNA damage was accompanied by marked upregulation of the nuclear-encoded mitophagy regulator PINK1, specifically in PD patients with extended survival, suggesting activation of a compensatory cytoprotective response. Together, these findings provide comprehensive single-cell resolution profiling of mitochondrial genomic alterations in pedunculopontine-cholinergic neurons in Parkinson's, displaying extensive mitochondrial DNA structural disruption dominated by large-scale mitochondrial DNA deletions. Our results further suggest that upregulation of mitophagy-related pathways may represent an adaptive mechanism to mitigate progressive mitochondrial genomic instability and preserve neuronal energy homeostasis.

Age-related accumulation of neuromelanin (NM) has been implicated in the intrinsic vulnerability of catecholaminergic neurons in Parkinson's disease. Postmortem studies in Parkinson's disease patients have revealed elevated intracellular and extracellular NM in the substantia nigra pars compacta, whereas experimental enhancement of NM synthesis in animal models disrupts neuronal homeostasis and accelerates neurodegeneration. Low-intensity focused ultrasound combined with microbubbles (LIFU-MB) induces focal opening of the blood-brain barrier. It has been shown to influence protein aggregation and modulate pigment-related processes, thereby raising the possibility that it may also impact NM dynamics in the brain. Here, we investigated the possibility that repeated LIFU-MB-mediated blood-brain barrier opening could modify NM accumulation in a non-human primate model that overexpresses human tyrosinase, leading to elevated NM accumulation in the substantia nigra pars compacta. Comprehensive characterization revealed that these animals exhibit several features compatible with early pathological changes in Parkinson's disease including extracellular NM release, increased iron accumulation, nigral inflammatory changes, and intracellular inclusion formation. Importantly, these alterations occur in the absence of substantial dopaminergic cell loss and behavioral deficits. LIFU-MB-treated animals showed a marked reduction in both intracellular and extracellular NM, accompanied by a trend toward decreased free ferric iron. These results indicate that early, non-invasive LIFU-MB intervention can counteract mechanisms linked to excessive NM accumulation. Moreover, this approach provides a translational framework to explore the broader feasibility of reducing intracellular pigments and pathological aggregates in the brain. Given its clinical safety, capacity to reach deep brain structures, and non-invasive nature, LIFU-MB warrants further investigation as a possible therapeutic approach in Parkinson´s disease and other neurodegenerative disorders.

RNA modifications, especially N6-methyladenosine (m6A), have emerged as pivotal regulators of gene expression in neurological disorders, yet their roles in ischaemic stroke remain unclear. Here, we performed a CRISPR-Cas9 loss-of-function screen targeting 118 RNA modification-related genes in neuronal cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) and validated the findings in a mouse model of middle cerebral artery occlusion/reperfusion (MCAO/R). We identified Fto as a top negatively selected gene, with its expression progressively reduced and global m6A levels increased during ischaemic injury. Neuron-specific Fto knockout aggravated infarct size, neurological deficits, cognitive impairment, and neuronal apoptosis, accompanied by activation of multiple signalling pathways revealed by RNA-seq analysis. Integrated m6A-seq and RNA-seq profiling uncovered Atf3 as a hypermethylated and upregulated hub gene downstream of Fto deficiency, linking m6A dysregulation to enhanced neuronal apoptosis. In vitro assays confirmed that FTO knockdown increased ATF3 mRNA methylation and expression under OGD/R conditions, whereas Fto overexpression in vivo alleviated ischaemic brain injury, improved neurological outcomes, reduced Atf3 expression, and lowered global m6A levels. Together, our findings reveal the Fto/m6A/ATF3 axis as a critical mediator of ischaemic injury and highlight Fto restoration as a potential therapeutic strategy for stroke.

Chloride homeostasis, and thereby the efficacy of chloride-permeable GABAA receptor signaling, depends on the dynamic balance between NKCC1 (Na+-K+-2Cl- cotransporter 1) and KCC2 (K+-Cl- cotransporter 2), which respectively mediate chloride influx and extrusion to tightly control intracellular chloride concentration ([Cl-]i). Here, we uncover an unexpected mechanism by which NKCC1 regulates KCC2 membrane stability and function. Co-immunoprecipitation experiments from mouse brain lysates revealed that KCC2 associates with NKCC1 and SPAK (STE20/SPS1-related proline-alanine-rich kinase), indicating that these proteins are within a common molecular interaction network. Furthermore, we show that NKCC1 and KCC2 form co-clusters in the plasma membrane of hippocampal neurons and that freely diffusing KCC2 molecules become trapped within NKCC1 membrane clusters. These observations suggest that NKCC1 coordinates SPAK- and PP1-dependent regulation of KCC2. Guided by molecular modeling, we designed peptides targeting specific interactions within this complex. Peptides that activate SPAK by engaging NKCC1's PP1-binding motif decrease KCC2 surface expression and impair chloride extrusion, whereas a SPAK-inhibiting peptide that prevents SPAK recruitment to NKCC1, stabilizes KCC2 in membrane clusters, and enhances chloride extrusion. An optimized peptide analog suitable for in vivo use preserves KCC2 clustering under hyperexcitable conditions, reduces seizure frequency and severity in an acute pentylenetetrazol-induced (PTZ) seizure model, and suppresses ictal activity in chronically epileptic human tissue. Together, these findings identify NKCC1 as a key regulator of KCC2 membrane stability, reveal NKCC1-KCC2 coupling as a critical determinant of inhibitory signaling, and establish this complex as a potential therapeutic target for restoring chloride homeostasis in epilepsy and related neurological disorders.

Millions of chronic pain patients are managed with opioids, yet the neuropsychological implications of stable long-term daily opioid consumption remain unknown. Here, we contrasted the psychosocial profiles and brain structure and function of 70 chronic back pain patients prescribed opioids (CBP+O, average current daily opioid exposure of 7.8 years) with tightly matched 70 opioid-naïve patients (CBP-O) and 30 healthy subjects. Despite CBP+O exhibiting only modestly worse psychosocial profiles and small differences in brain morphology relative to CBP-O, CBP+O showed stark differences in brain activity (ALFF). A large portion (38% of the variance) of this pattern was captured by a model using 19 receptor/transporter distributions. The patients' (across all 140 CBP) psychosocial profiles were summarized by three principal components (PCs, explaining 70% of variance): functional disability, pain quality, and negative affect. These PCs were respectively associated with serotonergic (5HT1a and 5HT6) and opioidergic (mu-opioid receptor, MOR) receptor-related activities. In additional analyses and new data, 5HT1a- and MOR-related activities tracked opioid dose, abstinence status, and tapering success, while 5HT6-related activity tracked pain intensity. Overall, our findings uncover molecularly defined brain circuits in opioid use and chronic pain.

Ischemic stroke remains a leading cause of mortality and disability worldwide, yet current therapeutic strategies, such as thrombolysis and thrombectomy, are constrained by narrow treatment windows and incomplete microvascular repair. While angiogenesis is pivotal for long-term functional recovery, the epigenetic mechanisms governing post-stroke vascular remodeling remain poorly understood. Here, we demonstrate that the long non-coding RNA (lncRNA) Malat1 acts as a critical endothelial regulator of angiogenesis and neurological restoration. Using an endothelium-specific Malat1 overexpression mouse model (EC-Malat1 Tg), we demonstrate endothelial Malat1 upregulation enhances angiogenesis, restores cerebral blood flow, and improves sensorimotor and cognitive function following ischemic stroke. Mechanistically, fluorescence-activated cell sorting (FACS)-based RNA sequencing of brain endothelial cells identified novel downstream targets of Malat1, including Hapln2, Bcl6, Esr1, Sox17, Tfrc, Hsp90aa1, and Tek. Among these, Tek (Tie2) showed robust endothelial co-localization and direct interaction with Malat1. Notably, while Malat1 overexpression markedly increased Tek protein expression, adeno-associated virus (AAV)-mediated endothelial knockdown of Tek abrogated the pro-angiogenic and neurovascular reparative effects of Malat1. Collectively, our data establishes the Malat1-Tek axis as a novel signaling pathway essential for driving post-ischemic cerebrovascular remodeling and long-term functional recovery. Targeting Malat1 represents a novel restorative therapeutic approach for ischemic stroke.

Aberrant interval timing in the millisecond-to-second range is a consistent feature of neurological and neuropsychiatric disorders involving striatal dysfunction, yet its neurochemical substrates in the human brain remain poorly characterised. Striatal dopamine has long been implicated in temporal processing, but evidence from animal models and human pharmacological studies has proved difficult to reconcile, in part because endogenous dopamine dynamics in humans have been inaccessible at relevant timescales. Patients with Parkinson's disease (N=5, 62-73 years; two women) completed a temporal bisection task (300 trials), judging whether visual stimulus intervals (500-1100 ms) were closer to a learned short (500 ms) or long (1100 ms) reference. Dopamine and serotonin concentrations were measured in the caudate nucleus during awake deep brain stimulation surgery using fast-scan cyclic voltammetry. Elastic net machine-learning models trained on in vitro voltammetry data were applied to human recordings to derive dopamine and serotonin time series at 10 Hz. We used cluster-based permutation analysis to identify stimulus-aligned time windows in which dopamine or serotonin differed between short and long judgements, and entered mean concentrations from these windows as predictors of temporal performance in trial-level hierarchical mixed-effects models. Slower, steady-state dopaminergic and serotonergic tone, quantified as the mean concentration within successive multi-minute periods, was also assessed as a predictor of temporal behaviour. Finally, behavioural performance was compared with a healthy control cohort (N=17, 50-66 years; seven women). Elevated phasic dopamine (625-670 ms after stimulus onset) increased the likelihood of short judgements and was associated with reduced temporal overestimation bias. This association was independent of objective clock time, with the identical analysis yielding non-significant results for the short versus the long stimulus intervals. By contrast, tonic dopamine levels covaried with sensitivity in discriminating stimulus intervals (temporal precision). Lower tonic dopamine was associated with poorer temporal precision both within patients over the experimental session and in patients relative to healthy controls. No corresponding effects were observed for serotonin, indicating dopamine specificity across phasic and tonic timescales. By resolving endogenous striatal dopamine with sub-second precision during conscious human behaviour, this study links temporally distinct dopaminergic dynamics to dissociable aspects of timing behaviour in Parkinson's disease. This challenges accounts of dopamine as a single modulatory influence and situates these findings within the literature on dissociable effects of phasic and tonic dopaminergic signalling, reconciling discrepancies between human and animal evidence and advancing understanding of the neurochemical basis of human time perception.