Dominant CST3 variants cause adult onset leukodystrophy without amyloid angiopathy.

Leukodystrophies are rare genetic white matter disorders that have been regarded as mainly occurring in childhood. Recent years altered this perception, as a growing number of leukodystrophies was described to have an onset at adult ages. Still, many adult patients presenting with white matter changes remain without a specific molecular diagnosis. We describe a novel adult onset leukodystrophy in 16 patients from eight families carrying one of four different stop-gain or frameshift dominant variants in the CST3 gene. Clinical and radiological features differ markedly from the previously described Icelandic Cerebral Amyloid Angiopathy that was found in patients carrying p.Leu68Asn substitution in CST3. The clinical phenotype consists of recurrent episodes of hemiplegic migraine associated with transient unilateral focal deficits and slowly progressing motor symptoms and cognitive decline in mid-old adult ages. In addition, in some cases acute onset clinical deterioration led to a prolonged episode with reduced consciousness and even early death. Radiologically, pathognomonic changes are found at typical predilection sites involving the deep cerebral white matter sparing a periventricular and directly subcortical rim, the middle blade of corpus callosum, posterior limb of the internal capsule, middle cerebellar peduncles, cerebral peduncles, and specifically the globus pallidus. Histopathologic characterization in two autopsy cases did not reveal angiopathy, but instead micro- to macrocystic degeneration of the white matter. Astrocytes were activated at early stages and later on displayed severe degeneration and loss. In addition, despite loss of myelin, elevated numbers of partly apoptotic oligodendrocytes were observed. A structural comparison of the variants in CST3 suggests that specific truncations of Cystatin C result in an abnormal function, possibly by rendering the protein more prone to aggregation. Future studies are required to confirm the assumed effect on the protein and to determine pathophysiologic downstream events at the cellular level.

Proteomic dissection of vanishing white matter pathogenesis.

Vanishing white matter (VWM) is a leukodystrophy caused by biallelic pathogenic variants in eukaryotic translation initiation factor 2B. To date, it remains unclear which factors contribute to VWM pathogenesis. Here, we investigated the basis of VWM pathogenesis using the 2b5ho mouse model. We first mapped the temporal proteome in the cerebellum, corpus callosum, cortex, and brainstem of 2b5ho and wild-type (WT) mice. Protein changes observed in 2b5ho mice were then cross-referenced with published proteomic datasets from VWM patient brain tissue to define alterations relevant to the human disease. By comparing 2b5ho mice with their region- and age-matched WT counterparts, we showed that the proteome in the cerebellum and cortex of 2b5ho mice was already dysregulated prior to pathology development, whereas proteome changes in the corpus callosum only occurred after pathology onset. Remarkably, protein changes in the brainstem were transient, indicating that a compensatory mechanism might occur in this region. Importantly, 2b5ho mouse brain proteome changes reflect features well-known in VWM. Comparison of the 2b5ho mouse and VWM patient brain proteomes revealed shared changes. These could represent changes that contribute to the disease or even drive its progression in patients. Taken together, we show that the 2b5ho mouse brain proteome is affected in a region- and time-dependent manner. We found that the 2b5ho mouse model partly replicates the human disease at the protein level, providing a resource to study aspects of VWM pathogenesis by highlighting alterations from early to late disease stages, and those that possibly drive disease progression.

Is Osteogenesis Imperfecta Associated with Cardiovascular Abnormalities? A Systematic Review of the Literature.

Osteogenesis imperfecta (OI) is a rare genetic disorder caused by abnormal collagen type I production. While OI is primarily characterized by bone fragility and deformities, patients also have extraskeletal manifestations, including an increased risk of cardiovascular disease. This review provides a comprehensive overview of the literature on cardiovascular diseases in OI patients in order to raise awareness of this understudied clinical aspect of OI and support clinical guidelines. In accordance with the PRISMA guidelines, a systematic literature search in PubMed, Embase, Web of Science and Scopus was conducted that included articles from the inception of these databases to April 2023. Valvular disease, heart failure, atrial fibrillation, and hypertension appear to be more prevalent in OI than in control individuals. Moreover, a larger aortic root was observed in OI compared to controls. Various cardiovascular diseases appear to be more prevalent in OI than in controls. These cardiovascular abnormalities are observed in all types of OI and at all ages, including young children. As there are insufficient longitudinal studies, it is unknown whether these abnormalities are progressive in nature in OI patients. Based on these findings, we would recommend referring individuals with OI to a cardiologist with a low-threshold.

Cortical interneuron development is affected in 4H leukodystrophy.

4H leukodystrophy is a rare genetic disorder classically characterized by hypomyelination, hypodontia and hypogonadotropic hypogonadism. With the discovery that 4H is caused by mutations that affect RNA polymerase III, mainly involved in the transcription of small non-coding RNAs, patients with atypical presentations with mainly a neuronal phenotype were also identified. Pathomechanisms of 4H brain abnormalities are still unknown and research is hampered by a lack of preclinical models. We aimed to identify cells and pathways that are affected by 4H mutations using induced pluripotent stem cell models. RNA sequencing analysis on induced pluripotent stem cell-derived cerebellar cells revealed several differentially expressed genes between 4H patients and control samples, including reduced ARX expression. As ARX is involved in early brain and interneuron development, we studied and confirmed interneuron changes in primary tissue of 4H patients. Subsequently, we studied interneuron changes in more depth and analysed induced pluripotent stem cell-derived cortical neuron cultures for changes in neuronal morphology, synaptic balance, network activity and myelination. We showed a decreased percentage of GABAergic synapses in 4H, which correlated to increased neuronal network activity. Treatment of cultures with GABA antagonists led to a significant increase in neuronal network activity in control cells but not in 4H cells, also pointing to lack of inhibitory activity in 4H. Myelination and oligodendrocyte maturation in cultures with 4H neurons was normal, and treatment with sonic hedgehog agonist SAG did not improve 4H related neuronal phenotypes. Quantitative PCR analysis revealed increased expression of parvalbumin interneuron marker ERBB4, suggesting that the development rather than generation of interneurons may be affected in 4H. Together, these results indicate that interneurons are involved, possibly parvalbumin interneurons, in disease mechanisms of 4H leukodystrophy.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Marchiafava-Bignami disease: why not Marchiafava-Bignami-Carducci disease?

The Marchiafava-Bignami disease has a curious backstory, namely, the publication in 1898 of the Contribution to the Study of Nonsuppurative Encephalitis (Carducci A in Riv Psicol Psichiat Neuropat 8-9:125-135, 1898), in which the neo-graduate Agostino Carducci described the disease that the pathologists Ettore Marchiafava and Amico Bignami would report 5 years later.

Age-related changes in plasma biomarkers and their association with mortality in COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19)-induced mortality occurs predominantly in older patients. Several immunomodulating therapies seem less beneficial in these patients. The biological substrate behind these observations is unknown. The aim of this study was to obtain insight into the association between ageing, the host response and mortality in patients with COVID-19. METHODS: We determined 43 biomarkers reflective of alterations in four pathophysiological domains: endothelial cell and coagulation activation, inflammation and organ damage, and cytokine and chemokine release. We used mediation analysis to associate ageing-driven alterations in the host response with 30-day mortality. Biomarkers associated with both ageing and mortality were validated in an intensive care unit and external cohort. RESULTS: 464 general ward patients with COVID-19 were stratified according to age decades. Increasing age was an independent risk factor for 30-day mortality. Ageing was associated with alterations in each of the host response domains, characterised by greater activation of the endothelium and coagulation system and stronger elevation of inflammation and organ damage markers, which was independent of an increase in age-related comorbidities. Soluble tumour necrosis factor receptor 1, soluble triggering receptor expressed on myeloid cells 1 and soluble thrombomodulin showed the strongest correlation with ageing and explained part of the ageing-driven increase in 30-day mortality (proportion mediated: 13.0%, 12.9% and 12.6%, respectively). CONCLUSIONS: Ageing is associated with a strong and broad modification of the host response to COVID-19, and specific immune changes likely contribute to increased mortality in older patients. These results may provide insight into potential age-specific immunomodulatory targets in COVID-19.

Heterozygous NOTCH1 Variants Cause CNS Immune Activation and Microangiopathy.

NOTCH1 belongs to the NOTCH family of proteins that regulate cell fate and inflammatory responses. Somatic and germline NOTCH1 variants have been implicated in cancer, Adams-Oliver syndrome, and cardiovascular defects. We describe 7 unrelated patients grouped by the presence of leukoencephalopathy with calcifications and heterozygous de novo gain-of-function variants in NOTCH1. Immunologic profiling showed upregulated CSF IP-10, a cytokine secreted downstream of NOTCH1 signaling. Autopsy revealed extensive leukoencephalopathy and microangiopathy with vascular calcifications. This evidence implicates that heterozygous gain-of-function variants in NOTCH1 lead to a chronic central nervous system (CNS) inflammatory response resulting in a calcifying microangiopathy with leukoencephalopathy. ANN NEUROL 2022;92:895-901.

Indoleamine 2,3-dioxygenase (IDO)-1 and IDO-2 activity and severe course of COVID-19.

COVID-19 is a pandemic with high morbidity and mortality. In an autopsy cohort of COVID-19 patients, we found extensive accumulation of the tryptophan degradation products 3-hydroxy-anthranilic acid and quinolinic acid in the lungs, heart, and brain. This was not related to the expression of the tryptophan-catabolizing indoleamine 2,3-dioxygenase (IDO)-1, but rather to that of its isoform IDO-2, which otherwise is expressed rarely. Bioavailability of tryptophan is an absolute requirement for proper cell functioning and synthesis of hormones, whereas its degradation products can cause cell death. Markers of apoptosis and severe cellular stress were associated with IDO-2 expression in large areas of lung and heart tissue, whereas affected areas in brain were more restricted. Analyses of tissue, cerebrospinal fluid, and sequential plasma samples indicate early initiation of the kynurenine/aryl-hydrocarbon receptor/IDO-2 axis as a positive feedback loop, potentially leading to severe COVID-19 pathology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Heterogeneity of white matter astrocytes in the human brain.

Astrocytes regulate central nervous system development, maintain its homeostasis and orchestrate repair upon injury. Emerging evidence support functional specialization of astroglia, both between and within brain regions. Different subtypes of gray matter astrocytes have been identified, yet molecular and functional diversity of white matter astrocytes remains largely unexplored. Nonetheless, their important and diverse roles in maintaining white matter integrity and function are well recognized. Compelling evidence indicate that impairment of normal astrocytic function and their response to injury contribute to a wide variety of diseases, including white matter disorders. In this review, we highlight our current understanding of astrocyte heterogeneity in the white matter of the mammalian brain and how an interplay between developmental origins and local environmental cues contribute to astroglial diversification. In addition, we discuss whether, and if so, how, heterogeneous astrocytes could contribute to white matter function in health and disease and focus on the sparse human research data available. We highlight four leukodystrophies primarily due to astrocytic dysfunction, the so-called astrocytopathies. Insight into the role of astroglial heterogeneity in both healthy and diseased white matter may provide new avenues for therapies aimed at promoting repair and restoring normal white matter function.

Dominant-acting CSF1R variants cause microglial depletion and altered astrocytic phenotype in zebrafish and adult-onset leukodystrophy.

Tissue-resident macrophages of the brain, including microglia, are implicated in the pathogenesis of various CNS disorders and are possible therapeutic targets by their chemical depletion or replenishment by hematopoietic stem cell therapy. Nevertheless, a comprehensive understanding of microglial function and the consequences of microglial depletion in the human brain is lacking. In human disease, heterozygous variants in CSF1R, encoding the Colony-stimulating factor 1 receptor, can lead to adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) possibly caused by microglial depletion. Here, we investigate the effects of ALSP-causing CSF1R variants on microglia and explore the consequences of microglial depletion in the brain. In intermediate- and late-stage ALSP post-mortem brain, we establish that there is an overall loss of homeostatic microglia and that this is predominantly seen in the white matter. By introducing ALSP-causing missense variants into the zebrafish genomic csf1ra locus, we show that these variants act dominant negatively on the number of microglia in vertebrate brain development. Transcriptomics and proteomics on relatively spared ALSP brain tissue validated a downregulation of microglia-associated genes and revealed elevated astrocytic proteins, possibly suggesting involvement of astrocytes in early pathogenesis. Indeed, neuropathological analysis and in vivo imaging of csf1r zebrafish models showed an astrocytic phenotype associated with enhanced, possibly compensatory, endocytosis. Together, our findings indicate that microglial depletion in zebrafish and human disease, likely as a consequence of dominant-acting pathogenic CSF1R variants, correlates with altered astrocytes. These findings underscore the unique opportunity CSF1R variants provide to gain insight into the roles of microglia in the human brain, and the need to further investigate how microglia, astrocytes, and their interactions contribute to white matter homeostasis.

DTYMK is essential for genome integrity and neuronal survival.

Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.

Early-Onset Vascular Leukoencephalopathy Caused by Bi-Allelic NOTCH3 Variants.

OBJECTIVE: Heterozygous NOTCH3 variants are known to cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), with patients typically presenting in adulthood. We describe three patients presenting at an early age with a vascular leukoencephalopathy. Genome sequencing revealed bi-allelic variants in the NOTCH3 gene. METHODS: Clinical records and available MRI and CT scans of three patients from two unrelated families were retrospectively reviewed. RESULTS: The patients presented at 9 to 14 months of age with developmental delay, seizures, or both. The disease course was characterized by cognitive impairment and variably recurrent strokes, migraine attacks, and seizures. MRI findings pointed at a small vessel disease, with extensive cerebral white matter abnormalities, atrophy, lacunes in the basal ganglia, microbleeds, and microcalcifications. The anterior temporal lobes were spared. Bi-allelic cysteine-sparing NOTCH3 variants in exons 1, 32, and 33 were found. INTERPRETATION: This study indicates that bi-allelic loss-of-function NOTCH3 variants may cause a vascular leukoencephalopathy, distinct from CADASIL.

ACE2 Protein Expression During Childhood, Adolescence, and Early Adulthood.

Purpose and context. Angiotensin-converting enzyme 2 is the entry receptor for SARS-CoV and SARS-CoV-2. Variations in ACE2 expression might explain age-related symptomatology of COVID-19, that is, more gastro-intestinal symptoms and less pulmonary complaints. This study qualitatively investigated ACE2 protein expression in various organs from the fetal to the young adolescent stage. Method. Autopsy samples from lung, heart, liver, stomach, small intestine, pancreas, kidney, adrenals, and brain (when available) were obtained from twenty subjects aged 24 weeks gestational age through 28 years. Formalin-fixed paraffin-embedded 4-um-thick tissue sections were stained against ACE2. Key results. We showed that the extent of ACE2 expression is age-related. With age, expression increases in lungs and decreases in intestines. In the other examined organs, ACE2 protein expression did not change with age. In brain tissue, ACE2 was expressed in astrocytes and endothelial cells. Conclusions. Age-related ACE2 expression differences could be one substrate of the selective clinical vulnerability of the respiratory and gastro-intestinal system to SARS-CoV-2 infection during infancy.

Childhood onset nexilin dilated cardiomyopathy: A heterozygous and a homozygous case.

Pathogenic heterozygous NEXN variants are associated with progressive dilated cardiomyopathy (DCM) usually presenting around 50 years of age. We describe an asymptomatic boy who had transient DCM at 3 months of age, that resolved by 4 months. Presently, at 11 years of age, he has normal cardiac function with signs of mild DCM on cardiac MRI. Genetic diagnostics revealed a paternally derived, heterozygous 1949_1951del class 4 variant in NEXN. His father had mild DCM with mildly reduced systolic function. The second patient presented with fetal hydrops at 33 weeks gestation requiring emergency caesarian delivery. Postnatally she required ventilation and continuous inotropic support for left ventricle systolic dysfunction. She died after 2 weeks when therapy was withdrawn. Homozygous c.1174C > T,p.(R392*) class 4 variants in the NEXN gene were found via WES. Microscopic investigation showed endomyocardial fibroelastosis. Her parents, both heterozygous carriers, had normal cardiac function and the family history was normal. These patients show a new clinical spectrum of pediatric cardiac disease seen in heterozygous and homozygous NEXN variants, ranging from mild, transient DCM to a severe, fatal neonatal DCM. These patients support the inclusion of the NEXN gene in the investigation of pediatric patients with DCM, even in cases with transient DCM.

Homozygous UBA5 Variant Leads to Hypomyelination with Thalamic Involvement and Axonal Neuropathy.

The enzyme ubiquitin-like modifier activating enzyme 5 (UBA5) plays an important role in activating ubiquitin-fold modifier 1 (UFM1) and its associated cascade. UFM1 is widely expressed and known to facilitate the post-translational modification of proteins. Variants in UBA5 and UFM1 are involved in neurodevelopmental disorders with early-onset epileptic encephalopathy as a frequently seen disease manifestation. Using whole exome sequencing, we detected a homozygous UBA5 variant (c.895C > T p. [Pro299Ser]) in a patient with severe global developmental delay and epilepsy, the latter from the age of 4 years. Magnetic resonance imaging showed hypomyelination with atrophy and T2 hyperintensity of the thalamus. Histology of the sural nerve showed axonal neuropathy with decreased myelin. Functional analyses confirmed the effect of the Pro299Ser variant on UBA5 protein function, showing 58% residual protein activity. Our findings indicate that the epilepsy currently associated with UBA5 variants may present later in life than previously thought, and that radiological signs include hypomyelination and thalamic involvement. The data also reinforce recently reported associations between UBA5 variants and peripheral neuropathy.

COVID-19: Histopathological correlates of imaging patterns on chest computed tomography.

BACKGROUND AND OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) pneumonia present with typical findings on chest computed tomography (CT), but the underlying histopathological patterns are unknown. Through direct regional correlation of imaging findings to histopathological patterns, this study aimed to explain typical COVID-19 CT patterns at tissue level. METHODS: Eight autopsy cases were prospectively selected of patients with PCR-proven COVID-19 pneumonia with varying clinical manifestations and causes of death. All had been subjected to chest CT imaging 24-72 h prior to death. Twenty-seven lung areas with typical COVID-19 patterns and two radiologically unaffected pulmonary areas were correlated to histopathological findings in the same lung regions. RESULTS: Two dominant radiological patterns were observed: ground-glass opacity (GGO) (n = 11) and consolidation (n = 16). In seven of 11 sampled areas of GGO, diffuse alveolar damage (DAD) was observed. In four areas of GGO, the histological pattern was vascular damage and thrombosis, with (n = 2) or without DAD (n = 2). DAD was also observed in five of 16 samples derived from areas of radiological consolidation. Seven areas of consolidation were based on a combination of DAD, vascular damage and thrombosis. In four areas of consolidation, bronchopneumonia was found. Unexpectedly, in samples from radiologically unaffected lung parenchyma, evidence was found of vascular damage and thrombosis. CONCLUSION: In COVID-19, radiological findings of GGO and consolidation are mostly explained by DAD or a combination of DAD and vascular damage plus thrombosis. However, the different typical CT patterns in COVID-19 are not related to specific histopathological patterns. Microvascular damage and thrombosis are even encountered in the radiologically normal lung.

Care Pathway for Foetal Joint Contractures, Foetal Akinesia Deformation Sequence, and Arthrogryposis Multiplex Congenita.

INTRODUCTION: The majority of arthrogryposis multiplex congenita (AMC) and lethal forms of AMC such as foetal akinesia deformation sequence (FADS) cases are missed prenatally. We have demonstrated the additional value of foetal motor assessment and evaluation in a multidisciplinary team for the period 2007-2016. An applied care pathway was developed for foetuses presenting with joint contracture(s) in one anatomic region (e.g., talipes equinovarus [TEV]), more than one body part with non-progressive contractures and motility (AMC) and with deterioration over time (FADS). METHODS: The multidisciplinary team of Amsterdam University Medical Centre Expertise Centre FADS and AMC developed the care pathway. Additional tools are provided including a motor assessment by ultrasound examination and a post-mortem assessment form. RESULTS: An eight-step care pathway is presented with a proposed timing for prenatal sonographic examination, genetic examinations, multidisciplinary meetings, prenatal and postnatal counselling of the parents by a specialist also treating after birth, and the follow-up of prenatal and postnatal findings with counselling for future pregnancies. DISCUSSION/CONCLUSION: The scheduled serial structural and motor sonograpahic assessment together with follow-up examinations and genetic analysis should be tailored per prenatal centre per available resources. The multidisciplinary care pathway may pave the way to increase the detection rate and diagnosis of isolated contracture(s), TEV with underlying genetic causes, and the rare phenotypes AMC/FADS and prompt treatment after birth within expertise teams.

Disturbed brain ether lipid metabolism and histology in Sjögren-Larsson syndrome.

Sjögren-Larsson syndrome (SLS) is a rare neurometabolic syndrome caused by deficient fatty aldehyde dehydrogenase. Patients exhibit intellectual disability, spastic paraplegia, and ichthyosis. The accumulation of fatty alcohols and fatty aldehydes has been demonstrated in plasma and skin but never in brain. Brain magnetic resonance imaging and spectroscopy studies, however, have shown an abundant lipid peak in the white matter of patients with SLS, suggesting lipid accumulation in the brain as well. Using histopathology, mass spectrometry imaging, and lipidomics, we studied the morphology and the lipidome of a postmortem brain of a 65-year-old female patient with genetically confirmed SLS and compared the results with a matched control brain. Histopathological analyses revealed structural white matter abnormalities with the presence of small lipid droplets, deficient myelin, and astrogliosis. Biochemically, severely disturbed lipid profiles were found in both white and gray matter of the SLS brain, with accumulation of fatty alcohols and ether lipids. Particularly, long-chain unsaturated ether lipid species accumulated, most prominently in white matter. Also, there was a striking accumulation of odd-chain fatty alcohols and odd-chain ether(phospho)lipids. Our results suggest that the central nervous system involvement in SLS is caused by the accumulation of fatty alcohols leading to a disbalance between ether lipid and glycero(phospho)lipid metabolism resulting in a profoundly disrupted brain lipidome. Our data show that SLS is not a pure leukoencephalopathy, but also a gray matter disease. Additionally, the histopathological abnormalities suggest that astrocytes and microglia might play a pivotal role in the underlying disease mechanism, possibly contributing to the impairment of myelin maintenance.

TUBB4A mutations result in specific neuronal and oligodendrocytic defects that closely match clinically distinct phenotypes.

Hypomyelinating leukodystrophies are heritable disorders defined by lack of development of brain myelin, but the cellular mechanisms of hypomyelination are often poorly understood. Mutations in TUBB4A, encoding the tubulin isoform tubulin beta class IVA (Tubb4a), result in the symptom complex of hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC). Additionally, TUBB4A mutations are known to result in a broad phenotypic spectrum, ranging from primary dystonia (DYT4), isolated hypomyelination with spastic quadriplegia, and an infantile onset encephalopathy, suggesting multiple cell types may be involved. We present a study of the cellular effects of TUBB4A mutations responsible for H-ABC (p.Asp249Asn), DYT4 (p.Arg2Gly), a severe combined phenotype with hypomyelination and encephalopathy (p.Asn414Lys), as well as milder phenotypes causing isolated hypomyelination (p.Val255Ile and p.Arg282Pro). We used a combination of histopathological, biochemical and cellular approaches to determine how these different mutations may have variable cellular effects in neurons and/or oligodendrocytes. Our results demonstrate that specific mutations lead to either purely neuronal, combined neuronal and oligodendrocytic or purely oligodendrocytic defects that closely match their respective clinical phenotypes. Thus, the DYT4 mutation that leads to phenotypes attributable to neuronal dysfunction results in altered neuronal morphology, but with unchanged tubulin quantity and polymerization, with normal oligodendrocyte morphology and myelin gene expression. Conversely, mutations associated with isolated hypomyelination (p.Val255Ile and p.Arg282Pro) and the severe combined phenotype (p.Asn414Lys) resulted in normal neuronal morphology but were associated with altered oligodendrocyte morphology, myelin gene expression, and microtubule dysfunction. The H-ABC mutation (p.Asp249Asn) that exhibits a combined neuronal and myelin phenotype had overlapping cellular defects involving both neuronal and oligodendrocyte cell types in vitro. Only mutations causing hypomyelination phenotypes showed altered microtubule dynamics and acted through a dominant toxic gain of function mechanism. The DYT4 mutation had no impact on microtubule dynamics suggesting a distinct mechanism of action. In summary, the different clinical phenotypes associated with TUBB4A reflect the selective and specific cellular effects of the causative mutations. Cellular specificity of disease pathogenesis is relevant to developing targeted treatments for this disabling condition.