CLN3 disease, or Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), is a rare, genetic neurodegenerative condition, typically manifesting in the first decade of life and progressing in severity, with death typically occurring in early adulthood. Despite two decades of natural history research, a clear timeline of CLN3 disease symptom onset and progression remains poorly defined, limiting optimal patient management and therapeutic development. We conducted a literature review and analysed the natural history data to better understand the age of core symptom onset and chronological disease progression. A literature review was undertaken using a pre-defined search strategy focused on CLN3 disease natural history studies, where age at onset for one or more core symptoms was reported in cohorts of ≥ 15 subjects. For each symptom, weighted mean age at onset and weighted standard deviation were calculated, with 95

Neuronal ceroid lipofuscinosis type 2 (CLN2) disease, a lysosomal storage disorder, causes early childhood psychomotor regression, vision loss, seizures, and rapid progressive gray matter loss. However, the link between neurodegenerative processes induced by lysosomal pathophysiology and the clinical phenotype remains unclear. This study investigated the longitudinal association of gray matter atrophy on MRI with in-depth clinical phenotyping in 27 patients receiving intraventricular enzyme therapy (ntimepoints = 170; biannual clinical assessments and MRIs). Longitudinal changes in cortical thickness and subcortical volumes were modeled via linear mixed effects regression. We used linear regression to correlate 24-week (Δ24) changes in clinical assessments with global cortical thickness and applied multivariate data-driven statistics to model how specific brain regions are associated with clinical domains. Our analysis revealed a significant reduction in the mean cortical thickness over time (β = -0.002, p = 0.021), corresponding to an annual loss of 4.2%, compared to natural history controls with 12.5%, respectively. Regional analysis revealed a widespread pattern of cortical and subcortical gray matter atrophy. Global cortical thickness reductions over 24 weeks (Δ24) were significantly associated with changes in the Hamburg motor and language scale Δ24, Weill Cornell scale Δ24, and Movement Disorder Inventory Δ24. Multivariate statistics identified a significant latent dimension relating regional morphometric abnormalities to worse clinical outcomes, accounting for 82% of the shared variance. Leveraging connectome data, we demonstrated that atrophy was linked to brain network architecture. Given their strong associations with clinical outcomes, MRI-based brain morphometric measures are promising CLN2 disease biomarkers to aid diagnosis, monitor disease progression, and guide therapy.

Microglia are the resident immune cells of the human central nervous system and play key roles in development, homeostasis, and disease. These functions are mediated by a broad repertoire of cell-surface receptors, including G protein-coupled receptors such as the ADP receptor P2Y12 and GPR34, a receptor for lysophosphatidylserine. While GPR34 deficiency has been linked to impaired microglial phagocytosis, its regulation in relation to amyloid-β (Aβ) and tau pathology in Alzheimer’s disease (AD) remains unclear. We performed a quantitative analysis of microglial density, morphology, and GPR34 expression in the medial temporal lobe cortex (MTLC) of elderly human body and tissue donors across the AD spectrum. Using fluorescence in situ hybridization and immunolabeling, we analyzed 187,670 microglial cells and correlated microglial parameters with the severity and spatial proximity of Aβ plaques and tau inclusions. In parallel, we analyzed human single-nucleus RNA sequencing data from 236,002 cells to assess GPR34 expression across microglial subtypes, brain regions, and neuropathological stages. Microglial density and overall morphology in the MTLC were largely preserved, independent of local Aβ or hyperphosphorylated tau burdens. Apart from a moderate shortening of microglial processes in the immediate vicinity of Aβ plaques, no consistent pathology-associated morphological changes were detected. GPR34 expression showed pronounced cell-to-cell variability and differed across microglial subtypes and brain regions, but neither expression intensity nor the proportion of GPR34-positive microglia correlated consistently with Braak stage or Thal phase. These findings suggest that GPR34 regulation in human microglia is highly context-dependent and shaped by regional and cellular heterogeneity rather than AD-associated pathology alone.

Abstract Background Multiple documented underlying etiologies may lead to bronchiectasis, but the European Bronchiectasis Registry found that in 38% of patients the cause is unknown, referred to as idiopathic. We wanted to resolve the role of CFTR dysfunction in people with idiopathic bronchiectasis by nasal potential difference (NPD) measurements. Methods NPD was examined in people with cystic fibrosis (CF), healthy controls, 40 people with idiopathic bronchiectasis recruited from the local outpatient clinic and in 60 people with idiopathic bronchiectasis with the suspected etiology of CF who had been referred from 2010 – 2022 to our electrophysiological laboratory to make a diagnosis by NPD. Results The unselected MHH cohort and the preselected diagnostic cohort of people with idiopathic bronchiectasis matched in their basal NPD potential with healthy controls. Conversely, after inhibition of the sodium conductance with amiloride, the distribution of the CFTR-mediated depolarization potential upon exposure to chloride-free solution and isoproterenol was in between those of healthy controls and CF patients with exocrine pancreatic insufficiency and overlapped with that of patients with exocrine pancreatic sufficiency. This intermediate phenotype was characteristic for the whole study population of 100 people with idiopathic bronchiectasis irrespectively of whether clinical features of CFTR dysfunction had been recognized before in an individual. Taking the Sermet Score that was developed to discriminate patients with CF from non-CF patients by NPD, the bronchiectasis population was significantly distinct from both healthy people and people with CF. Conclusions A CFTR activity of the nasal surface epithelium in the lower quartile is typical for people with idiopathic bronchiectasis, but further CFTR-independent inherited susceptibilities and external insults are necessary to materialize the emergence of bronchiectasis. Trial registration Clinical trial number: not applicable.

Synonymous single nucleotide variants (sSNVs), traditionally seen as neutral, are now recognized for their biological impact. To assess their relevance, we developed SyMetrics, a framework that integrates predictors of splicing, RNA stability, evolutionary conservation, codon usage, synonymous variation effects, sequence properties, and allele frequency. We analyzed all possible sSNVs across the human genome, and our machine-learning model achieved 97% accuracy in distinguishing deleterious from benign variants, with a ROC-AUC of 0.89, outperforming individual predictors. Our estimates indicate that about 1.98 ± 0.17% of sSNVs absent from population databases are damaging (roughly 900 000 sSNVs), with an odds ratio of 3.87 for deleteriousness compared to common sSNVs (P < 0.05). To validate predictions, we performed functional assays on selected sSNVs in the AVPR2 gene and additionally used available large scale mutagenesis screens of RAD51C and BAP1 variants. In a clinical cohort, we identified 15 predicted deleterious sSNVs in genes linked to patient phenotypes; 9 were classified as (likely) pathogenic while 6 were variants of uncertain significance (VUS) per American College of Medical Genetics guidelines. For three VUS, segregation data supported their suspected inheritance patterns (de novo, X-linked). Our findings underscore the functional importance of sSNVs. To support further research and clinical applications, we provide a Python package and web application (https://symetrics.org/) for evaluating these variants comprehensively.

Abstract Background CLN3 disease, also called Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), or Batten disease, is an ultra‑rare, neurodegenerative lysosomal storage disorder generally affecting individuals during the first decade of life. There can be a delay in diagnosis or misdiagnosis due to a lack of awareness, and when the most common presenting symptom of visual loss is attributed to more common conditions affecting vision. Methods We used a previously published Expert Mapping Tool (EMT) to identify multidisciplinary professionals with diagnostic or clinical management expertise, as well as patient advocates with experience of CLN3 disease. A systematic literature review of published evidence using the Preferred Reporting Items for Systematic Reviews and Meta‑Analyses (PRISMA) guidance was conducted independently and simultaneously to develop key clinical care statements. Each statement was based on the strength of the evidence. The statements formed the basis of an international modified-Delphi consensus process using a virtual meeting platform (Within3). Experts were asked to agree or disagree with each statement and suggest any changes. Statements that reached a consensus of 75% or over are the guiding statements within this manuscript. The processes and manuscript have been independently assessed using the Appraisal of Guidelines for Research and Evaluation (AGREE II) criteria. Results Thirty‑nine international experts from eight specialities were identified, including a patient advocate. Fifty‑three recommendation statements were developed covering eleven domains: General statements, Diagnostics, Clinical Recommendations and Management, Assessments, Social Considerations, Ocular Management, Epilepsy/Seizures, Nutrition, Respiratory Health, Sleep and Rest, and End-of-Life Care. Consensus was reached after one round of voting for all except three statements. The overall AGREE II score for developing these recommendations was 6.4, where 1 represents the lowest and 7 is the highest quality. Conclusion Currently, there are no comprehensive clinical recommendations for CLN3 disease. These recommendations provide a comprehensive, evidence- and consensus‑based tool that can be used by all healthcare professionals involved in the management of CLN3 disease and other similar neurodegenerative conditions. The goal is to address the unmet clinical need for CLN3 disease management and complement other information available.

CLN5 Batten disease, caused by biallelic mutations in CLN5, is a rare, early-onset neurodegenerative lysosomal storage disorder that has no cure and lacks validated biomarkers, hindering accurate diagnosis and assessment of therapeutic response. We recently identified CLN5 as the synthase of bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid crucial for lysosome function and lipid catabolism. This suggested BMP and its precursor lysophosphatidylglycerol (LPG) as clinically relevant biomarkers. It also prompted in vivo confirmation of CLN5 as the biologically relevant lysosomal BMP synthase. Here we show that murine and ovine disease models lacking CLN5 show significant and universal depletion of BMP and elevation of LPG across tissues and brain regions, consistent with the biochemical function of CLN5. Additionally, lysosomal lysates from murine models of CLN5 Batten disease lack the ability to synthesize BMP from its precursor LPG, establishing CLN5 as the main BMP synthase in vivo. Of importance, CLN5 patient-derived fibroblasts show BMP depletion and LPG elevation. Translating these results towards clinical utility, we demonstrate BMP and LPG to be accessible biomarkers for CLN5 Batten disease in both plasma and dried blood spots, enabling early diagnosis and patient screening.

ABSTRACT Background CLN2 disease, Neuronal Ceroid Lipofuscinosis (NCL) type 2, is a rare, genetic neurodegenerative condition predominantly affecting children. CLN2 disease is characterized by seizures, language and motor decline, vision loss, and premature death. Currently, the only regulatory-approved therapy is the enzyme replacement therapy (ERT) Cerliponase alfa, administered fortnightly via intracerebroventricular infusion as a lifelong treatment. While ERT has been shown to slow motor and language decline, it is not curative and does not fully address disease progression, including retinal degeneration. To better understand the lived experience of affected families, and perspectives on current and emerging treatments, we conducted a community survey of parents and caregivers of individuals with CLN2 disease. Methods A 25-question anonymous, voluntary survey was distributed through the BDSRA Foundation and international partner patient advocacy organisations via email and social media. Eligible participants included current and bereaved parents or primary caregivers of individuals with CLN2 disease, regardless of treatment history. The survey explored treatment experiences, unmet needs, and knowledge of and attitudes toward emerging therapeutic approaches, particularly gene-based therapies. Results Ninety-eight respondents from 19 countries completed the survey. Fifty-seven respondents reported current or prior use of ERT, with 94.7% (n=54/57) actively receiving treatment at the time of survey. ERT was perceived to provide greatest benefit for motor function and seizure control; however, respondents reported substantial treatment burden (mean burden score 4.8/7, n=66). Despite treatment availability, 94.9% of respondents (n=75/79) indicated a need for alternative therapeutic options and 94.8% (73/77) expressed interest in learning more about gene therapy. Overall, 72.4% (n=55/76) reported they were likely or very likely to consider participation in an investigational gene therapy trial. Key factors influencing decision-making included potential safety risks (57.9%, n=44/76), preclinical safety and efficacy evidence (54.0%, n=41/76), and whether ERT discontinuation would be required to participate (54.0%, n=44/76). Conclusion While ERT has altered the treatment landscape for CLN2 disease, this survey highlights the ongoing disease burden and treatment challenges experienced by families. Findings demonstrate strong community interest in next-generation therapies that may reduce treatment burden and provide more comprehensive disease modification, including effects on both central nervous system (CNS) and ocular manifestations.

Introduction This study assessed the real-world effectiveness and safety of the enzyme replacement therapy (ERT), cerliponase alfa, to treat neuronal ceroid lipofuscinosis type 2 (CLN2) disease. Methods Data from the DEM-CHILD database were analyzed, comparing patients who initiated ERT outside clinical trials with natural history (NH) controls. Treated patients were matched 1:1 with NH controls on baseline age and combined motor-language (ML) score on the CLN2 clinical rating scale. Rate of ML score decline, time to unreversed 2-point decline or score of 0, and time to unreversed score of 0 were assessed. Safety was assessed in treated patients. Results Twenty-four ERT-treated patients were eligible (mean [SD] follow-up: 106.7 [64.1] weeks); 21 matched to a NH control, with baseline mean (SD) age of 4.7 (1.9) years and mean (SD) ML score of 3.9 (1.6) points. ERT-treated patients had reduced likelihood of an unreversed ML 2-point decline or score of 0 (HR 0.08; 95% CI 0.02, 0.28; p < 0.0001), and unreversed ML score of 0 (HR 0.07; 95% CI 0.01, 0.40; p = 0.003) versus NH controls. Mean (SD) rate of ML score decline was 0.46 (0.43) versus 1.88 (1.45) points/48 weeks for ERT-treated and NH groups, respectively (mean difference: 1.42; 95% CI 0.74, 2.10; p = 0.0003). Sixteen patients (67%) had treatment-related adverse events; the most common were pyrexia (50%), vomiting (33%), and nausea (21%). No ERT-treated patients died. Discussion Cerliponase alfa for real-world CLN2 disease treatment slowed decline in motor and language function and demonstrated an acceptable safety profile.

Succinate is a pivotal tricarboxylic acid cycle metabolite but also specifically activates the Gi- and Gq-coupled succinate receptor 1 (SUCNR1). Contradictory roles of succinate and succinate-SUCNR1 signaling include reports about its anti- or pro-inflammatory effects. The link between cellular metabolism and localization-dependent SUCNR1 signaling qualifies as a potential cause for the reported conflicts. To systematically address this connection, we used a diverse set of methods, including several bioluminescence resonance energy transfer-based biosensors, dynamic mass redistribution measurements, second messenger and kinase phosphorylation assays, calcium imaging, and metabolic analyses. Different cellular metabolic states were mimicked using glucose (Glc) or glutamine (Gln) as available energy substrates to provoke differential endogenous succinate (SUC) production. We show that SUCNR1 signaling, localization, and metabolism are mutually dependent, with SUCNR1 showing distinct spatial and energy substrate-dependent Gi and Gq protein activation. We found that Gln-consumption associated with a higher rate of oxidative phosphorylation causes increased extracellular SUC concentrations, accompanied by a higher rate of SUCNR1 internalization, reduced miniGq protein recruitment to the plasma membrane, and lower Ca2+ signals. In Glc, under basal conditions, SUCNR1 causes stronger Gq than Gi protein activation, while the opposite is true upon stimulation with an agonist. In addition, SUCNR1 specifically interacts with miniG proteins in endosomal compartments. In THP-1 cells, polarized to M2-like macrophages, endogenous SUCNR1-mediated Gi signaling stimulates glycolysis, while Gq signaling inhibits the glycolytic rate. Our results suggest that the metabolic context determines spatially dependent SUCNR1 signaling, which in turn modulates cellular energy homeostasis and mediates adaptations to changes in SUC concentrations.

The neuronal ceroid-lipofuscinoses (NCLs), collectively also called Batten disease, constitute one of the most common groups of inherited neurodegenerative disorders in children but may also occur in adults. The childhood forms present clinically as progressive mental and motor deterioration and loss of vision, while the rare adult-onset forms are dominated by dementia. To date, 13 genetically distinct forms of NCL have been identified, all characterized by the accumulation of abnormal lipofuscin-like material in the lysosomes of nerve cells, associated with progressive and selective destruction of neurons particularly in the cerebral and cerebellar cortex and, less constantly, in the retina. The distribution of the stored material resembles that of the "wear-and-tear" pigment lipofuscin that accumulates during normal ageing.

Focal segmental glomerulosclerosis (FSGS) is a common glomerular pathology characterized by podocyte injury, which can lead to kidney failure. Among the factors contributing to podocyte damage are mutations in nuclear pore complexes (NPCs), which regulate nuclear-cytoplasmic transport of proteins and RNAs. Defective NPCs can accumulate in highly differentiated, nondividing cells such as podocytes. However, their role in podocyte dysfunction is largely unexplored, particularly as a potential therapeutic target. To address this, we investigated the effects of selinexor (KPT-330), a drug that inhibits XPO1-mediated nuclear-cytoplasmic protein export. In HeLa cells, KPT-330 restored compromised NPC function. Munich Wistar Fröemter (MWF) rats, a model for spontaneous FSGS development, aged 10 wk, were treated with KPT-330 for 10 wk and then observed for another 20 wk. Improvements in kidney function were observed at the end of the 10-wk treatment period, with serum creatinine significantly lower in the KPT-330 group (34.11 ± 1.77 μmol/L) versus the vehicle group (39.25 ± 3.54 μmol/L, P < 0.01). Serum cystatin C levels remained lower in the KPT-330 group (3.62 ± 0.39 μg/mL) versus vehicle (4.19 ± 0.44 μg/mL, P < 0.05) after an additional 20 wk without treatment. Hyperlipidemia was significantly reduced immediately after the end of the 10-wk KPT-330 treatment compared with vehicle (triglyceride: 1.23 ± 0.34 mmol/L vs. 1.92 ± 0.4 mmol/L, P < 0.01; total cholesterol: 1.47 ± 0.08 mmol/L vs. 2.96 ± 0.44 mmol/L, P < 0.0001). However, histopathological parameters, including glomerulosclerosis, podocyte numbers, and activation of parietal epithelial cells, showed that kidney damage continued to progress. Thus, KPT-330 has beneficial effects on kidney function, but was not sufficient to halt the histological progression of glomerular damage.NEW & NOTEWORTHY Focal segmental glomerulosclerosis (FSGS) involves podocyte injury, potentially linked to dysfunctional nuclear pore complexes (NPCs). We show that selinexor (KPT-330), a nuclear export inhibitor, restores NPC function in vitro. In an FSGS rat model, selinexor improves kidney function, lowers serum creatinine and cystatin C levels, and reduces serum lipid levels. However, histological damage persists, indicating partial but not complete protection. These findings highlight NPC-targeted therapies as a potential strategy for treating FSGS.

Most genes are not yet fully annotated, and the extent of their transcript diversity and the roles and significance of specific isoforms is not understood. This information is therefore lacking for disease genes. The CLN3 gene underlies classic juvenile CLN3 disease, also known as juvenile neuronal ceroid lipofuscinosis, a rare paediatric neurodegenerative disorder. The most common cause of this biallelic disorder is a 1-kb intragenic deletion that removes two internal coding exons (exons 7 and 8). Here, we report findings from the first long-read RNA sequencing targeting CLN3 in blood samples derived from control individuals and from patients clinically and genetically diagnosed with juvenile CLN3 disease. We find that CLN3 transcription is complex, with >80 different transcripts encoding >35 different open reading frames (ORF) of different lengths, and no dominantly expressed transcript. The 1-kb deletion has direct consequences on this. This is consistent across patients, with total loss of some transcripts including those encoding the canonical 438 amino acid protein and other significant smaller isoforms. The highest expressed disease transcripts include those lacking exons 7 and 8 and encoding a 181 amino acid protein isoform, and other novel isoforms that lack additional exons and encode longer ORFs. The different effects on transcription of other CLN3 disease-causing variants are revealed in single patients. Together, these findings confirm the complexity of transcription at the CLN3 locus, reveal the impact of the 1-kb deletion and other variants on isoform abundance, and highlight the importance of understanding the contribution of these isoforms to CLN3 function in health and disease. Moreover, they impact the future design and development of personalised therapeutics and the design and generation of disease models. Finally, they underline the importance of full annotation for disease genes. ### Competing Interest Statement The authors have declared no competing interest. * NCLs : Neuronal ceroid lipofuscinoses MFS : major facilitator superfamily ORFs : open-reading frames UTRs : untranslated regions TPM : transcripts per million ID : identifier CDS : coding sequence NMD : nonsense-mediated decay Medical Research Council, https://ror.org/03x94j517, MR/V033956 USA Children’s Brain Disease Foundation, , BrightFocus Foundation, , A2021009F Tenure Track Clinician Scientist Fellowship, , MR/N008324/1 Fight for Sight, https://ror.org/03133qe46, 5045-5046 Sight Research UK, , SAC051 Moorfields Eye Charity, https://ror.org/017hm5p20, GR001203 Federal Ministry of Education and Research, https://ror.org/04pz7b180, 01GL2404A

Lysosomes are implicated in a wide spectrum of human diseases, including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration, and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in patients remains challenging. Here, we report the development of the 'tagless LysoIP' method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell-derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients' lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms.