Kinetic Properties of Glutamate Carboxypeptidase II Partially Purified from Leukodystrophy Patient's Serum.

Glutamate carboxypeptidase II (GCPII), a metallopeptidase, is a recently identified pharmacologically targeted protein that is predominantly expressed in the human central nervous system, where it degrades the most abundant neuropeptide in the brain, N-acetyl aspartate glutamate, releasing free glutamate. Dysregulated glutamate release is associated with numerous neurological disorders and brain inflammation. The present study was designed to evaluate the activity of GCPII in 60 serum samples from patients with leukodystrophy and 30 samples from a control group with an age of less than 10 years. Subsequently, the enzyme was purified from the serum of leukodystrophy patients for experimental studies using ion exchange and gel filtration techniques to enhance the enzyme purity and reduce impurities. Finally, the kinetic properties of the purified enzyme were measured. The results of the present study demonstrated a reduction in the efficacy of the enzyme in comparison to the control group at a significance level of P ≤ 0.00003. Additionally, the kinetic study of the purified enzyme revealed a Michaelis-Menten constant value of 0.012 µM and a maximum velocity of 1.1318 µmol min-1. As demonstrated by the Lineweaver-Burk plot, using folate as the substrate, the Km value indicates the high affinity of the enzyme for folate, which is a crucial consideration in the development of therapies for neurological diseases. Additionally, the enzyme exhibited optimal activity at 37 °C and pH 7.4, with an incubation time of 5 min. The significance of GCPII in patients with leukodystrophy is 2-fold: first, it may serve as an early diagnostic marker for leukodystrophy, and second, it could represent a potential therapeutic target for neurological disorders.

Atlas-based assessment of hypomyelination: Quantitative MRI in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a rare childhood hypomyelinating leukodystrophy. Quantification of the pronounced myelin deficit and delineation of subtle myelination processes are of high clinical interest. Quantitative magnetic resonance imaging (qMRI) techniques can provide in vivo insights into myelination status, its spatial distribution, and dynamics during brain maturation. They may serve as potential biomarkers to assess the efficacy of myelin-modulating therapies. However, registration techniques for image quantification and statistical comparison of affected pediatric brains, especially those of low or deviant image tissue contrast, with healthy controls are not yet established. This study aimed first to develop and compare postprocessing pipelines for atlas-based quantification of qMRI data in pediatric patients with PMD and evaluate their registration accuracy. Second, to apply an optimized pipeline to investigate spatial myelin deficiency using myelin water imaging (MWI) data from patients with PMD and healthy controls. This retrospective single-center study included five patients with PMD (mean age, 6 years ± 3.8) who underwent conventional brain MRI and diffusion tensor imaging (DTI), with MWI data available for a subset of patients. Three methods of registering PMD images to a pediatric template were investigated. These were based on (a) T1-weighted (T1w) images, (b) fractional anisotropy (FA) maps, and (c) a combination of T1w, T2-weighted, and FA images in a multimodal approach. Registration accuracy was determined by visual inspection and calculated using the structural similarity index method (SSIM). SSIM values for the registration approaches were compared using a t test. Myelin water fraction (MWF) was quantified from MWI data as an assessment of relative myelination. Mean MWF was obtained from two PMDs (mean age, 3.1 years ± 0.3) within four major white matter (WM) pathways of a pediatric atlas and compared to seven healthy controls (mean age, 3 years ± 0.2) using a Mann-Whitney U test. Our results show that visual registration accuracy estimation and computed SSIM were highest for FA-based registration, followed by multimodal, and T1w-based registration (SSIMFA = 0.67 ± 0.04 vs. SSIMmultimodal = 0.60 ± 0.03 vs. SSIMT1 = 0.40 ± 0.14). Mean MWF of patients with PMD within the WM pathways was significantly lower than in healthy controls MWFPMD = 0.0267 ± 0.021 vs. MWFcontrols = 0.1299 ± 0.039. Specifically, MWF was measurable in brain structures known to be myelinated at birth (brainstem) or postnatally (projection fibers) but was scarcely detectable in other brain regions (commissural and association fibers). Taken together, our results indicate that registration accuracy was highest with an FA-based registration pipeline, providing an alternative to conventional T1w-based registration approaches in the case of hypomyelinating leukodystrophies missing normative intrinsic tissue contrasts. The applied atlas-based analysis of MWF data revealed that the extent of spatial myelin deficiency in patients with PMD was most pronounced in commissural and association and to a lesser degree in brainstem and projection pathways.

Anything is better than nothing': exploring attitudes towards novel therapies in leukodystrophy clinical trials.

BACKGROUND/AIM: Leukodystrophies comprise a group of genetic white matter disorders that lead to progressive motor and cognitive impairment. Recent development of novel therapies has led to an increase in clinical trials for leukodystrophies. To enable recruitment of individuals with a leukodystrophy into clinical trials, clinical trial acceptability should be ascertained. We sought therefore, to identify the motivations for and barriers to clinical trial participation in addition to clinical trial features that may be of concern to individuals with a leukodystrophy and/or their carers. METHODS: Adults with a leukodystrophy and parents/carers of individuals with a leukodystrophy were recruited through the Australian Leukodystrophy Registry and through online advertisements. Qualitative semi-structured interviews were used to explore participants views on what clinical trials involve, the perceived risks and benefits of clinical trials, their desire to participate in clinical trials and their personal experience with leukodystrophy. Thematic analysis of data was performed with co-coding of interview transcripts. RESULTS: 5 interviews were held with parents of children with leukodystrophy, 4 with parents of adults with leukodystrophy and 3 with adults diagnosed with leukodystrophy. Motivations for clinical trial enrolment include access to potentially lifesaving novel treatments and improved prognostic outcomes. Participants were concerned about adverse clinical trial outcomes, including side effects and exacerbation of illness. Despite this, majority of participants were willing to try anything in clinical trials, demonstrating a high tolerance for first in human trials and trials utilising invasive treatment options. CONCLUSIONS: Interviewees communicated a strong desire to participate in interventional clinical trials involving novel therapies. To support enrolment into future leukodystrophy clinical trials we suggest the provision of transparent information regarding clinical trial treatments, consideration of alternative trial control measures, and inclusion of treating clinicians in the trial recruitment process. Clinicians play an integral role in initiating transparent conversations regarding trial risks and adverse outcomes.

Human iPSC-derived myelinating organoids and globoid cells to study Krabbe Disease.

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here, to understand the pathological mechanisms of Kd, we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis, astrogenesis, and oligodendrogenesis but manifest early myelination defects. Specifically, Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly, myelin is affected in the absence of autophagy and mTOR pathway dysregulation, suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions, Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding, Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether, this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd.

Terra incognita of glial cell dynamics in the etiology of leukodystrophies: Broadening disease and therapeutic perspectives.

Neuroglial cells, also known as glia, are primarily characterized as auxiliary cells within the central nervous system (CNS). The recent findings have shed light on their significance in numerous physiological processes and their involvement in various neurological disorders. Leukodystrophies encompass an array of rare and hereditary neurodegenerative conditions that were initially characterized by the deficiency, aberration, or degradation of myelin sheath within CNS. The primary cellular populations that experience significant alterations are astrocytes, oligodendrocytes and microglia. These glial cells are either structurally or metabolically impaired due to inherent cellular dysfunction. Alternatively, they may fall victim to the accumulation of harmful by-products resulting from metabolic disturbances. In either situation, the possible replacement of glial cells through the utilization of implanted tissue or stem cell-derived human neural or glial progenitor cells hold great promise as a therapeutic strategy for both the restoration of structural integrity through remyelination and the amelioration of metabolic deficiencies. Various emerging treatment strategies like stem cell therapy, ex-vivo gene therapy, infusion of adeno-associated virus vectors, emerging RNA-based therapies as well as long-term therapies have demonstrated success in pre-clinical studies and show promise for rapid clinical translation. Here, we addressed various leukodystrophies in a comprehensive and detailed manner as well as provide prospective therapeutic interventions that are being considered for clinical trials. Further, we aim to emphasize the crucial role of different glial cells in the pathogenesis of leukodystrophies. By doing so, we hope to advance our understanding of the disease, elucidate underlying mechanisms, and facilitate the development of potential treatment interventions.

Exploring the globoid cell leukodystrophy protein network and therapeutic interventions.

Globoid cell leukodystrophy is a severe rare disorder characterized by white matter degradation, resulting in a progressive loss of physical and mental abilities and has extremely limited therapeutic interventions. Therefore, this study aimed to delve into the Globoid cell leukodystrophy associated intricate network of differentially expressed genes (p < 0.05, |Fc|> 1) to identify potential druggable targets and possible therapeutic interventions using small molecules. The disease-associated neuronal protein circuit was constructed and analyzed, identifying 53 nodes (minimum edge cutoff 1), among which five (FOS, FOSB, GDNF, GFRA1, and JUN) were discerned as potential core protein nodes. Although our research enumerates the potential small molecules to target various protein nodes in the proposed disease network, we particularly underscore T-5224 to inhibit c-Jun activity as JUN was identified as one of the pivotal elements within the disease-associated neuronal protein circuit. The evaluation of T-5224 binding energy (- 11.0 kcal/mol) from docking study revealed that the compound to exhibit a notable affinity towards Jun/CRE complex. Moreover, the structural integrity of complex was affirmed through comprehensive molecular dynamics simulations, indicating a stable hydrophilic interaction between T-5224 and the Jun/CRE complex, thereby enhancing protein compactness and reducing solvent accessibility. This binding energy was further substantiated by free binding analysis, revealing a substantial thermodynamics complex state (- 448.00 ± 41.73 kJ/mol). Given that this investigation is confined to a computational framework, we additionally propose a hypothetical framework to ascertain the feasibility of inhibiting the Jun/CRE complex with T-5224 against Globoid cell leukodystrophy, employing a combination of in vitro and in vivo methodologies as a prospective avenue of this study.

Deciphering glial contributions to CSF1R-related disorder via single-nuclear transcriptomic profiling: a case study.

CSF1R-related disorder (CSF1R-RD) is a neurodegenerative condition that predominantly affects white matter due to genetic alterations in the CSF1R gene, which is expressed by microglia. We studied an elderly man with a hereditary, progressive dementing disorder of unclear etiology. Standard genetic testing for leukodystrophy and other neurodegenerative conditions was negative. Brain autopsy revealed classic features of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), including confluent white matter degeneration with axonal spheroids and pigmented glial cells in the affected white matter, consistent with CSF1R-RD. Subsequent long-read sequencing identified a novel deletion in CSF1R that was not detectable with short-read exome sequencing. To gain insight into potential mechanisms underlying white matter degeneration in CSF1R-RD, we studied multiple brain regions exhibiting varying degrees of white matter pathology. We found decreased CSF1R transcript and protein across brain regions, including intact white matter. Single nuclear RNA sequencing (snRNAseq) identified two disease-associated microglial cell states: lipid-laden microglia (expressing GPNMB, ATG7, LGALS1, LGALS3) and inflammatory microglia (expressing IL2RA, ATP2C1, FCGBP, VSIR, SESN3), along with a small population of CD44+ peripheral monocyte-derived macrophages exhibiting migratory and phagocytic signatures. GPNMB+ lipid-laden microglia with ameboid morphology represented the end-stage disease microglia state. Disease-associated oligodendrocytes exhibited cell stress signatures and dysregulated apoptosis-related genes. Disease-associated oligodendrocyte precursor cells (OPCs) displayed a failure in their differentiation into mature myelin-forming oligodendrocytes, as evidenced by upregulated LRP1, PDGFRA, SOX5, NFIA, and downregulated NKX2-2, NKX6.2, SOX4, SOX8, TCF7L2, YY1, ZNF488. Overall, our findings highlight microglia-oligodendroglia crosstalk in demyelination, with CSF1R dysfunction promoting phagocytic and inflammatory microglia states, an arrest in OPC differentiation, and oligodendrocyte depletion.

Charcot-Marie-Tooth Disease With Leukodystrophy: An Atypical Presentation.

This case report presents a 23-year-old male diagnosed with Charcot-Marie-Tooth (CMT) disease, who exhibited additional neurological symptoms suggestive of leukodystrophy. The patient experienced recurrent episodes of slurred speech, imbalance, and a recent tonic-clonic seizure, prompting admission. Neurological examination and imaging revealed bilateral white matter changes, raising suspicion of leukoencephalopathy. Further investigations confirmed a nonsense mutation c.64C>T (p.Arg22*) in the gap junction beta 1 (GJB1) gene. This case underscores the complexity of Charcot-Marie-Tooth disease type 1 (CMTX1) with atypical central nervous system (CNS) manifestations, highlighting the importance of comprehensive diagnostic evaluations and a multidisciplinary approach to management.

Exploring the Cost-Effectiveness of Newborn Screening for Metachromatic Leukodystrophy (MLD) in the UK.

Metachromatic leukodystrophy (MLD) is a fatal inherited lysosomal storage disease that can be detected through newborn bloodspot screening. The feasibility of the screening assay and the clinical rationale for screening for MLD have been previously demonstrated, so the aim of this study is to determine whether the addition of screening for MLD to the routine newborn screening program in the UK is a cost-effective use of National Health Service (NHS) resources. A health economic analysis from the perspective of the NHS and Personal Social Services was developed based on a decision-tree framework for each MLD subtype using long-term outcomes derived from a previously presented partitioned survival and Markov economic model. Modelling inputs for parameters related to epidemiology, test characteristics, screening and treatment costs were based on data from three major UK specialist MLD hospitals, structured expert opinion and published literature. Lifetime costs and quality-adjusted life years (QALYs) were discounted at 1.5% to account for time preference. Uncertainty associated with the parameter inputs was explored using sensitivity analyses. This health economic analysis demonstrates that newborn screening for MLD is a cost-effective use of NHS resources using a willingness-to-pay threshold appropriate to the severity of the disease; and supports the inclusion of MLD into the routine newborn screening programme in the UK.

Developmental delay can precede neurologic regression in early onset metachromatic leukodystrophy.

OBJECTIVE: Metachromatic leukodystrophy (MLD) is a rare neurodegenerative disorder. Emerging therapies are most effective in the presymptomatic phase, and thus defining this window is critical. We hypothesize that early development delay may precede developmental plateau. With the advent of presymptomatic screening platforms and transformative therapies, it is essential to define the onset of neurologic disease. METHODS: The specific ages of gain and loss of developmental milestones were captured from the medical records of individuals affected by MLD. Milestone acquisition was characterized as: on target (obtained before the age limit of 90th percentile plus 2 standard deviations compared to a normative dataset), delayed (obtained after 90th percentile plus 2 standard deviations), or plateau (skills never gained). Regression was defined as the age at which skills were lost. LI-MLD was defined by age at onset before 2.5 years. RESULTS: Across an international cohort, 351 subjects were included (n = 194 LI-MLD subcohort). The median age at presentation of the LI-MLD cohort was 1.4 years (25th-75th %ile: 1.0-1.5). Within the LI-MLD cohort, 75/194 (39%) had developmental delay (or plateau) prior to MLD clinical presentation. Among the LI-MLD cohort with a minimum of 1.5 years of follow-up (n = 187), 73 (39.0%) subjects never attained independent ambulation. Within LI-MLD + delay subcohort, the median time between first missed milestone target to MLD decline was 0.60 years (maximum distance from delay to onset: 1.9 years). INTERPRETATION: Early developmental delay precedes regression in a subset of children affected by LI-MLD, defining the onset of neurologic dysfunction earlier than previously appreciated. The use of realworld data prior to diagnosis revealed an early deviation from typical development. Close monitoring for early developmental delay in presymptomatic individuals may help in earlier diagnosis with important consequences for treatment decisions.

Towards a Treatment for Leukodystrophy Using Cell-Based Interception and Precision Medicine.

Cell-based interception and precision medicine is a novel approach aimed at improving healthcare through the early detection and treatment of diseased cells. Here, we describe our recent progress towards developing cell-based interception and precision medicine to detect, understand, and advance the development of novel therapeutic approaches through a single-cell omics and drug screening platform, as part of a multi-laboratory collaborative effort, for a group of neurodegenerative disorders named leukodystrophies. Our strategy aims at the identification of diseased cells as early as possible to intercept progression of the disease prior to severe clinical impairment and irreversible tissue damage.

International Society for Cell & Gene Therapy Stem Cell Engineering Committee report on the current state of hematopoietic stem and progenitor cell-based genomic therapies and the challenges faced.

Genetic manipulation of hematopoietic stem cells (HSCs) is being developed as a therapeutic strategy for several inherited disorders. This field is rapidly evolving with several novel tools and techniques being employed to achieve desired genetic changes. While commercial products are now available for sickle cell disease, transfusion-dependent ß-thalassemia, metachromatic leukodystrophy and adrenoleukodystrophy, several challenges remain in patient selection, HSC mobilization and collection, genetic manipulation of stem cells, conditioning, hematologic recovery and post-transplant complications, financial issues, equity of access and institutional and global preparedness. In this report, we explore the current state of development of these therapies and provide a comprehensive assessment of the challenges these therapies face as well as potential solutions.

Metachromatic leukodystrophy in infant presenting as acute febrile illness: a case report.

Introduction and Importance: Metachromatic leukodystrophy (MLD) is a rare genetic disorder affecting the central and peripheral nervous systems. It results from ARSA enzyme deficiency, causing sulfatide accumulation and myelin damage. Early diagnosis is crucial, and this case highlights the diagnostic challenges and rapid health deterioration associated with MLD. Case Presentation: A 14-month-old male, initially presenting with fever and crying during micturition, experienced a devastating health decline. Previously, he had achieved developmental milestones but rapidly lost motor and cognitive skills. Extensive investigations led to an MLD diagnosis, complicated by severe malnutrition. Despite medical interventions, his condition worsened, leading to cardiopulmonary arrest and a tragic end. Clinical Discussion: MLD is an exceedingly rare genetic disease with systemic effects, as illustrated by severe metabolic acidosis in this case. Early diagnosis, through comprehensive investigations like MRI, is critical, but MLD's rapid progression poses challenges in management. Therapeutic options remain limited, emphasizing the importance of a multidisciplinary approach. Conclusion: This case emphasizes the insidious nature of MLD, highlighting the need for considering rare genetic conditions in unexplained neurological regression. It underscores the urgency of improved awareness, early diagnosis, and comprehensive care for individuals affected by such devastating disorders. Despite the challenges, the medical community's dedication to providing care and support remains unwavering.

Exploring the effect of disease causing mutations in metal binding sites of human ARSA in metachromatic leukodystrophy.

The arylsulfatase A (ARSA) gene is observed to be deficient in patients with metachromatic leukodystrophy (MLD), a type of lysosomal storage disease. MLD is a severe neurodegenerative disorder characterized by an autosomal recessive inheritance pattern. This study aimed to map the most deleterious mutations at the metal binding sites of ARSA and the amino acids in proximity to the mutated positions. We utilized an array of computational tools, including PredictSNP, MAPP, PhD-SNP, PolyPhen-1, PolyPhen-2, SIFT, SNAP, and ConSurf, to identify the most detrimental mutations potentially implicated in MLD collected from UniProt, ClinVar, and HGMD. Two mutations, D29N and D30H, as being extremely deleterious based on assessments of pathogenicity, conservation, biophysical characteristics, and stability analysis. The D29 and D30 are located at the metal-interacting regions of ARSA and found to undergo post-translational modification, specifically phosphorylation. Henceforth, the in-depth effect of metal binding upon mutation was examined using molecular dynamics simulations (MDS) before and after phosphorylation. The MDS results exhibited high deviation for the D29N and D30H mutations in comparison to the native, and the same was confirmed by significant residue fluctuation and reduced compactness. These structural alterations suggest that such mutations may influence protein functionality, offering potential avenues for personalized therapeutic and providing a basis for potential mutation-specific treatments for severe MLD patients.

Psychotic attack during the clinical course of megalencephalic leukoencephalopathy with subcortical cysts: a case report.

OBJECTIVES: Van der Knaap disease or megalencephalic leukoencephalopathy with subcortical cysts (MLC), is a slowly progressive neurodegenerative disease with macrocephaly. We present a case of MLC with a confirmed homozygous MLC1 mutation who presented with a psychotic attack at a very young age. CASE PRESENTATION: An 11-year-old girl was admitted to the emergency room with delusions, hallucinations, and irritability. She was diagnosed with MLC at 18 months old. Her psychotic symptoms were improved with the administration of risperidone treatment added to her valproic acid treatment for EEG abnormality. CONCLUSION: In this case study we reported the first episode of a psychotic attack during the clinical course of MLC. This case indicates the possibility that MLC influences the onset of the psychotic attack. Further investigation with more patients is needed to identify the relationship.

Lentivirus-modified hematopoietic stem cell gene therapy for advanced symptomatic juvenile metachromatic leukodystrophy: A long-term follow-up pilot study.

Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA). Lentivirus-modified autologous hematopoietic stem cell gene therapy (HSCGT) has recently been approved for clinical use in pre- and early-symptomatic children with MLD to increase ARSA activity. Unfortunately, this advanced therapy is not available for most patients with MLD who have progressed to more advanced symptomatic stages at diagnosis. Patients with late-onset juvenile MLD typically present with a slower neurological progression of symptoms and represent a significant burden to the economy and healthcare system, whereas those with early-onset infantile MLD die within a few years of symptom onset. We conducted a pilot study to determine the safety and benefit of HSCGT in patients with post-symptomatic juvenile MLD and report preliminary results. The safety profile of HSCGT was favorable in this long-term follow-up over nine years. The most common adverse events (AEs) within two months of HSCGT were related to busulfan conditioning, and all AEs resolved. No HSCGT-related AEs and no evidence of distorted hematopoietic differentiation during long-term follow-up for up to 9.6 years. Importantly, to date, patients have maintained remarkably improved ARSA activity with a stable disease state, including increased Functional Independence Measure (FIM) score and decreased magnetic resonance imaging (MRI) lesion score. This long-term follow-up pilot study suggests that HSCGT is safe and provides clinical benefit to patients with post-symptomatic juvenile MLD.

A novel missense variant in HIKESHI: Clinical phenotype, in vitro functional testing, and potential for gene therapy.

A 7-month-old boy presented to our clinic with developmental delay, Magnetic Resonance Imaging (MRI) features of delayed myelination and diffusion restriction, and a homozygous variant of uncertain significance (c.4T>G, p.Phe2Val) in HIKESHI, a gene associated with autosomal-recessive hypomyelinating leukodystrophy 13. We hypothesized that the variant is disease-causing and aimed to rescue the cellular phenotype with vector-mediated gene replacement. HIKESHI mediates heat-induced nuclear accumulation of heat-shock proteins, including HSP70, to protect cells from stress. We generated skin fibroblasts from the proband and proband's mother (heterozygous) to compare protein expression and subcellular localization of HSP70 under heat stress conditions, and the effect of vector-mediated overexpression of HIKESHI in the proband's cells under the same heat stress conditions. Western blot analysis revealed absent HIKESHI protein from proband fibroblasts, contrasted with ample expression in parental cells. Under heat stress conditions, while the mother's cells displayed appropriate nuclear localization of HSP70, the proband's cells displayed impaired nuclear translocalization. When patient fibroblasts were provided exogenous HIKESHI, the transfected proband's cells showed restored heat-induced nuclear translocalization of HSP70 under conditions of heat stress. These functional data establish that the patient's variant is a pathogenic loss-of-function mutation, thus confirming a diagnosis of hypomyelinating leukodystrophy 13 and that vector-mediated gene replacement may be an effective treatment approach for patients with this disorder.

Human post-mortem organotypic brain slice cultures: a tool to study pathomechanisms and test therapies.

Human brain experimental models recapitulating age- and disease-related characteristics are lacking. There is urgent need for human-specific tools that model the complex molecular and cellular interplay between different cell types to assess underlying disease mechanisms and test therapies. Here we present an adapted ex vivo organotypic slice culture method using human post-mortem brain tissue cultured at an air-liquid interface to also study brain white matter. We assessed whether these human post-mortem brain slices recapitulate the in vivo neuropathology and if they are suitable for pathophysiological, experimental and pre-clinical treatment development purposes, specifically regarding leukodystrophies. Human post-mortem brain tissue and cerebrospinal fluid were obtained from control, psychiatric and leukodystrophy donors. Slices were cultured up to six weeks, in culture medium with or without human cerebrospinal fluid. Human post-mortem organotypic brain slice cultures remained viable for at least six weeks ex vivo and maintained tissue structure and diversity of (neural) cell types. Supplementation with cerebrospinal fluid could improve slice recovery. Patient-derived organotypic slice cultures recapitulated and maintained known in vivo neuropathology. The cultures also showed physiologic multicellular responses to lysolecithin-induced demyelination ex vivo, indicating their suitability to study intrinsic repair mechanisms upon injury. The slice cultures were applicable for various experimental studies, as multi-electrode neuronal recordings. Finally, the cultures showed successful cell-type dependent transduction with gene therapy vectors. These human post-mortem organotypic brain slice cultures represent an adapted ex vivo model suitable for multifaceted studies of brain disease mechanisms, boosting translation from human ex vivo to in vivo. This model also allows for assessing potential treatment options, including gene therapy applications. Human post-mortem brain slice cultures are thus a valuable tool in preclinical research to study the pathomechanisms of a wide variety of brain diseases in living human tissue.

Distinct neurological phenotypes associated with biallelic loss of NOTCH3 function: evidence for recessive inheritance.

BACKGROUND: NOTCH3 variants are known to be linked to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, some null NOTCH3 variants with homozygous inheritance cause neurological symptoms distinct from CADASIL. The aim of this study was to expand the clinical spectrum of this distinct condition and provide further evidence of its autosomal recessive inheritance. METHODS AND RESULTS: Whole exome sequencing (WES) was performed on a proband who exhibited livedo racemosa, ataxia, cognitive decline, seizures, and MRI white matter abnormalities without anterior temporal pole lesions. Segregation analysis was conducted with Sanger sequencing. WES of the proband identified a novel homozygous NOTCH3 null variant (c.2984delC). The consanguineous parents were confirmed as heterozygous variant carriers. In addition, three heterozygous NOTCH3 null variants were reported as incidental findings in three unrelated cases analyzed in our center. CONCLUSION: The findings of this study suggest an autosomal recessive inheritance pattern in this early-onset leukoencephalopathy, in contrast to CADASIL's dominant gain-of-function mechanism; which is a clear example of genotype-phenotype correlation. Comprehensive genetic analysis provides valuable insights into disease mechanisms and facilitates diagnosis and family planning for NOTCH3-associated neurological disorders.