Disease-modifying trials in Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) increasingly incorporate fluid and imaging biomarkers to demonstrate target engagement, de-risk subsequent phases of drug development and provide mechanistic evidence of biological activity. Whether biomarker modulation predicts clinical benefit remains unclear. We conducted a PRISMA-compliant review of phase II and III disease-modifying trials (2020-2025) that included at least one biomarker intended to assess target engagement. Twenty-six trials met inclusion criteria (PD=19, MSA=4, DLB=3; n=2,195 participants). Target engagement was reported in 14 of 19 PD trials (12 phase II/2 phase III), 1 of 4 MSA trials, and 2 of 3 DLB trials (all phase II). Concordance between target engagement and clinical improvement was uncommon, as only 8/26 studies (7 phase II/1 phase III) demonstrated both target engagement and statistically significant clinical benefit, and just two of these (both phase II, one in PD and one in MSA) were rigorously controlled with low or moderate risk of bias. Nine trials showed target engagement without clinical benefit, whereas two reported clinical improvement without measurable target engagement. Baseline biomarker-guided patient selection was uncommon and rarely aligned with the compound's mechanism of action. Patient-reported outcomes were widely used but rarely improved. Across recent disease-modifying trials in PD, DLB, and MSA, target engagement was frequently demonstrated, yet clinical benefit rarely followed. These findings question the predictive value of currently used target-engagement biomarkers and highlight the need to biologically align trial populations with the mechanisms targeted by experimental molecular interventions.

Myotonic Dystrophy type 1 (DM1) is a multisystem disorder caused by CTG repeat expansions in the dystrophia myotonica protein kinase (DMPK) gene. Current prevalence estimates of 1 in 3,000-8,000 have traditionally relied on clinically ascertained and geographically restricted cohorts, underestimating population burden and failing to capture ancestry diversity. Using large-scale whole-genome sequencing (WGS) datasets, we aimed to identify DMPK repeat expansions across nearly one million participants, define their ancestry-specific distribution, and evaluate associated clinical phenotypes, with particular focus on cardiac manifestations and early-onset cataracts. We analysed WGS participants from three cohorts: Genomics England (n = 80,110), UK Biobank (n = 490,086), and the All of Us Research Program (n = 414,830). DMPK repeat lengths were estimated using ExpansionHunter. Alleles were classified as Normal (≤37 repeats), Intermediate (38-49 repeats), or Pathogenic (≥50 repeats). Genetic ancestry was assigned using cohort-specific methods. Linked health record data assessed cardiac phenotypes in UK Biobank and early-onset cataracts in Genomics England and UK Biobank. Across all cohorts (n = 985,026), pathogenic DMPK repeat expansions were identified in 645 individuals (0.07%; ∼1 in 1,527), a prevalence two-to-five times higher than previous estimates. Most expansions occurred in those of European ancestry, although expansions were detected across all groups, including up to 1 in 6,693 African and Asian ancestry. In UK Biobank, individuals with a pathogenic expansion had increased odds of any cardiac diagnosis (odds ratio [OR]: 2.06, 95% confidence interval [CI]: 1.58-2.67; p = 5.8×108) and greater cardiac diagnostic burden (risk ratio 2.46, CI: 1.92-3.15; p = 1.6×10-13). Conduction abnormalities-particularly atrioventricular and left bundle-branch block, atrial fibrillation, and flutter-showed the strongest association with expansion status (OR: 4.86, CI: 3.38-6.79; p = 2.7×10-18). No sex differences were observed among those with a DMPK expansion, although male sex was independently associated with conduction disease in the overall population. Approximately one-in-ten individuals with pathogenic expansions had a cataract diagnosis before age 55. Most individuals with an expansion had no prior DM1 diagnosis, highlighting the value of systematic genetic testing in these phenotypes to improve diagnosis, shorten the diagnostic interval, and mitigate harm. By integrating WGS data from nearly one million individuals, this study refines understanding of the population distribution and clinical consequences of DMPK repeat expansions, revealing a reservoir of undiagnosed and potentially untreated multisystemic disease. These findings support routine inclusion of DMPK analysis within specified cardiac and ophthalmic diagnostic pathways to enable earlier detection.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron degeneration. Microvascular dysfunction has been implicated by recent research in ALS pathogenesis and motor neuron vulnerability. The aim of this study is to investigate whether MRI markers of white matter hyperintensities (WMHs), reflecting small-vessel disease, are associated with motor and cognitive impairment, disease staging and prognosis in ALS. In this retrospective cohort study MRI scans from 399 ALS patients were evaluated for periventricular and deep white matter hyperintensities (pvWMH and dWMH) using visual grading scales together with semi-automated volumetric quantification of relative WMH volume (WMHrv). The primary outcome was survival. Secondary outcomes included disease staging, functional impairment, motor and cognitive outcome measures. WMH burden was associated with more advanced King's topographic staging [pvWMH (P < 0.001), dWMH (P = 0.002)] and MiToS functional staging [pvWMH (P < 0.001), dWMH (P = 0.017)]. High pvWMH burden was associated with shorter survival [exp(β) = 3.503; 95% CI = 1.131, 10.832; P = 0.030] after adjustment for known prognostic factors. WMH measures correlated with greater functional impairment on ALSFRS-R score [pvWMH (P < 0.001), dWMH (P = 0.003), WMHrv (P = 0.008)]. WMH burden correlated with more severe lower, but not upper, motor neuron impairment, as measured with the MRC sum score [pvWMH (P = 0.002), dWMH (P = 0.024), WMHrv (p=0.030)] and lower motor neuron score [pvWMH (P < 0.001), dWMH (P = 0.001)]. WMH burden was also associated with cognitive deficits on the Edinburgh Cognitive and Behavioural ALS Screen (ECAS), as indicated by total [pvWMH (P = 0.007), dWMH (P = 0.009), WMHrv (P = 0.037)] and ALS-specific ECAS scores [pvWMH (P = 0.001), dWMH (P = 0.020), WMHrv (P = 0.022)]. This large-scale MRI study links small-vessel disease to clinical staging, survival and ALS-specific motor (distinctively LMN) and cognitive impairments. These results suggest that WMHs may reflect a diffuse neurovascular pathology spanning cortical and spinal regions and may be relevant to ALS progression. Routine MRI assessment of WMHs could aid patient stratification and inform trial designs. Longitudinal studies are needed to clarify causal relationships and neurovascular-targeted therapeutic implications.

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.