A flow cytometry-based in vitro assay reveals that formation of apolipoprotein E (ApoE)-amyloid beta complexes depends on ApoE isoform and cell type.

Apolipoprotein E (ApoE) is a secreted apolipoprotein with three isoforms, E2, E3, and E4, that binds to lipids and facilitates their transport in the extracellular environment of the brain and the periphery. The E4 allele is a major genetic risk factor for the sporadic form of Alzheimer's disease (AD), and studies of human brain and mouse models have revealed that E4 significantly exacerbates the deposition of amyloid beta (Aß). It has been suggested that this deposition could be attributed to the formation of soluble ApoE isoform-specific ApoE-Aß complexes. However, previous studies have reported conflicting results regarding the directionality and strength of those interactions. In this study, using a series of flow cytometry assays that maintain the physiological integrity of ApoE-Aß complexes, we systematically assessed the association of Aß with ApoE2, E3, or E4. We used ApoE secreted from HEK cells or astrocytes overexpressing ApoE fused with a GFP tag. As a source of soluble Aß peptide, we used synthetic Aß40 or Aß42 or physiological Aß secreted from CHO cell lines overexpressing WT or V717F variant amyloid precursor protein (APP). We observed significant interactions between the different ApoE isoforms and Aß, with E4 interacting with Aß more strongly than the E2 and E3 isoforms. We also found subtle differences depending on the Aß type and the ApoE-producing cell type. In conclusion, these results indicate that the strength of the ApoE-Aß association depends on the source of Aß or ApoE.

Isoform- and cell type-specific structure of apolipoprotein E lipoparticles as revealed by a novel Forster resonance energy transfer assay.

Apolipoprotein E (apoE) has an important role in the pathogenesis of Alzheimer's disease with its three isoforms having distinct effects on disease risk. Here, we assessed the conformational differences between those isoforms using a novel flow cytometry-Forster resonance energy transfer (FRET) assay. We showed that the conformation of intracellular apoE within HEK cells and astrocytes adopts a directional pattern; in other words, E4 adopts the most closed conformation, E2 adopts the most open conformation, and E3 adopts an intermediate conformation. However, this pattern was not maintained upon secretion of apoE from astrocytes. Intermolecular interactions between apoE molecules were isoform-specific, indicating a great diversity in the structure of apoE lipoparticles. Finally, we showed that secreted E4 is the most lipidated isoform in astrocytes, suggesting that increased lipidation acts as a folding chaperone enabling E4 to adopt a closed conformation. In conclusion, this study gives insights into apoE biology and establishes a robust screening system to monitor apoE conformation.

Genetic and phenotypic characterization of complex hereditary spastic paraplegia.

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease-causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15 Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson's disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes.

Exome sequencing reveals de novo WDR45 mutations causing a phenotypically distinct, X-linked dominant form of NBIA.

Neurodegeneration with brain iron accumulation (NBIA) is a group of genetic disorders characterized by abnormal iron deposition in the basal ganglia. We report that de novo mutations in WDR45, a gene located at Xp11.23 and encoding a beta-propeller scaffold protein with a putative role in autophagy, cause a distinctive NBIA phenotype. The clinical features include early-onset global developmental delay and further neurological deterioration (parkinsonism, dystonia, and dementia developing by early adulthood). Brain MRI revealed evidence of iron deposition in the substantia nigra and globus pallidus. Males and females are phenotypically similar, an observation that might be explained by somatic mosaicism in surviving males and germline or somatic mutations in females, as well as skewing of X chromosome inactivation. This clinically recognizable disorder is among the more common forms of NBIA, and we suggest that it be named accordingly as beta-propeller protein-associated neurodegeneration.

Mutations in the autoregulatory domain of ß-tubulin 4a cause hereditary dystonia.

Dystonia type 4 (DYT4) was first described in a large family from Heacham in Norfolk with an autosomal dominantly inherited whispering dysphonia, generalized dystonia, and a characteristic hobby horse ataxic gait. We carried out a genetic linkage analysis in the extended DYT4 family that spanned 7 generations from England and Australia, revealing a single LOD score peak of 6.33 on chromosome 19p13.12-13. Exome sequencing in 2 cousins identified a single cosegregating mutation (p.R2G) in the ß-tubulin 4a (TUBB4a) gene that was absent in a large number of controls. The mutation is highly conserved in the ß-tubulin autoregulatory MREI (methionine-arginine-glutamic acid-isoleucine) domain, highly expressed in the central nervous system, and extensive in vitro work has previously demonstrated that substitutions at residue 2, specifically R2G, disrupt the autoregulatory capability of the wild-type ß-tubulin peptide, affirming the role of the cytoskeleton in dystonia pathogenesis.

Analysis of Parkinson's disease brain-derived DNA for alpha-synuclein coding somatic mutations.

BACKGROUND: Although alpha-synuclein (SNCA) is crucial to the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), mutations in the gene appear to be rare. We have recently hypothesized that somatic mutations in early development could contribute to PD. METHODS: Expanding on our recent negative small study, we used high-resolution melting (HRM) analysis to screen SNCA coding exons for somatic point mutations in DNA from 539 PD and DLB cerebellar samples, with two additional regions (frontal cortex, substantia nigra) for 20 PD cases. We used artificial mosaics to determine sensitivity where possible. RESULTS: We did not detect any evidence of somatic coding mutations. Three cases were heterozygous for known silent polymorphisms. The protocol we used was sensitive enough to detect 5% to 10% mutant DNA. CONCLUSION: Using DNA predominantly from cerebellum, but also from frontal cortex and substantia nigra (n = 20 each), we have not detected any somatic coding SNCA point mutations.

Kohlschütter-Tönz syndrome: mutations in ROGDI and evidence of genetic heterogeneity.

Kohlschütter-Tönz syndrome (KTS) is a rare autosomal recessive disorder characterized by amelogenesis imperfecta, psychomotor delay or regression and seizures starting early in childhood. KTS was established as a distinct clinical entity after the first report by Kohlschütter in 1974, and to date, only a total of 20 pedigrees have been reported. The genetic etiology of KTS remained elusive until recently when mutations in ROGDI were independently identified in three unrelated families and in five likely related Druze families. Herein, we report a clinical and genetic study of 10 KTS families. By using a combination of whole exome sequencing, linkage analysis, and Sanger sequencing, we identify novel homozygous or compound heterozygous ROGDI mutations in five families, all presenting with a typical KTS phenotype. The other families, mostly presenting with additional atypical features, were negative for ROGDI mutations, suggesting genetic heterogeneity of atypical forms of the disease.

The pallidopyramidal syndromes: nosology, aetiology and pathogenesis.

PURPOSE OF REVIEW: The aims of this review is to suggest a new nomenclature and classification system for the diseases currently categorized as neurodegeneration with brain iron accumulation (NBIA) or dystonia-parkinsonism, and to discuss the mechanisms implicated in the pathogenesis of these diseases. RECENT FINDINGS: NBIA is a disease category encompassing syndromes with iron accumulation and prominent dystonia-parkinsonism. However, as there are many diseases with similar clinical presentations but without iron accumulation and/or known genetic cause, the current classification system and nomenclature remain confusing. The pathogenetic mechanisms of these diseases and the causes of gross iron accumulation and significant burden of neuroaxonal spheroids are also elusive. Recent genetic and functional studies have identified surprising links between NBIA, Parkinson's disease and lysosomal storage disorders (LSD) with the common theme being a combined lysosomal-mitochondrial dysfunction. We hypothesize that mitochondria and lysosomes form a functional continuum with a predominance of mitochondrial and lysosomal pathways in NBIA and LSD, respectively, and with Parkinson's disease representing an intermediate form of disease. SUMMARY: During the past 18 months, important advances have been made towards understanding the genetic and pathological underpinnings of the pallidopyramidal syndromes with important implications for clinical practice and future treatment developments.

Pathogenic Parkinson's disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation.

LRRK2 is one of the most important genetic contributors to Parkinson's disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.

α-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson's disease and multiple system atrophy?

We report a British family with young-onset Parkinson's disease (PD) and a G51D SNCA mutation that segregates with the disease. Family history was consistent with autosomal dominant inheritance as both the father and sister of the proband developed levodopa-responsive parkinsonism with onset in their late thirties. Clinical features show similarity to those seen in families with SNCA triplication and to cases of A53T SNCA mutation. Post-mortem brain examination of the proband revealed atrophy affecting frontal and temporal lobes in addition to the caudate, putamen, globus pallidus and amygdala. There was severe loss of pigmentation in the substantia nigra and pallor of the locus coeruleus. Neuronal loss was most marked in frontal and temporal cortices, hippocampal CA2/3 subregions, substantia nigra, locus coeruleus and dorsal motor nucleus of the vagus. The cellular pathology included widespread and frequent neuronal α-synuclein immunoreactive inclusions of variable morphology and oligodendroglial inclusions similar to the glial cytoplasmic inclusions of multiple system atrophy (MSA). Both inclusion types were ubiquitin and p62 positive and were labelled with phosphorylation-dependent anti-α-synuclein antibodies In addition, TDP-43 immunoreactive inclusions were observed in limbic regions and in the striatum. Together the data show clinical and neuropathological similarities to both the A53T SNCA mutation and multiplication cases. The cellular neuropathological features of this case share some characteristics of both PD and MSA with additional unique striatal and neocortical pathology. Greater understanding of the disease mechanism underlying the G51D mutation could aid in understanding of α-synuclein biology and its impact on disease phenotype.

THAP1 mutations and dystonia phenotypes: genotype phenotype correlations.

THAP1 mutations have been shown to be the cause of DYT6. A number of different mutation types and locations in the THAP1 gene have been associated with a range of severity and dystonia phenotypes, but, as yet, it has been difficult to identify clear genotype phenotype patterns. Here, we screened the THAP1 gene in a further series of dystonia cases and evaluated the mutation pathogenicity in this series as well as previously reported mutations to investigate possible phenotype-genotype correlations. THAP1 mutations have been identified throughout the coding region of the gene, with the greatest concentration of variants localized to the THAP1 domain. In the additional cases analyzed here, a further two mutations were found. No obvious, indisputable genotype-phenotype correlation emerged from these data. However, we managed to find a correlation between the pathogenicity of mutations, distribution, and age of onset of dystonia. THAP1 mutations are an important cause of dystonia, but, as yet, no clear genotype-phenotype correlations have been identified. Greater mutation numbers in different populations will be important and mutation-specific functional studies will be essential to identify the pathogenicity of the various THAP1 mutations. © 2012 Movement Disorder Society.

Muscle biopsy in myositis: What the rheumatologist needs to know.

The appropriate analysis of skeletal muscle tissues is a key element in many diagnostic procedures and can deliver valuable information about the organ that is affected. Although arguably the frequency of muscle biopsy may be declining in certain domains where genetic analysis is now the first line of diagnostic evaluation, it still has an important role in assessment of patients with neuromuscular disorders such as congenital myopathies, muscular dystrophies, metabolic and inflammatory diseases. Here, we have comprehensively discussed the aspects of a modern and fruitful approach to muscle biopsy histopathological studies in rheumatological disorders. We have focussed on the neuromuscular involvement in myositis and its differential diagnoses in both adult and paediatric settings. We have also covered the clinical indications for the biopsy, technical aspects and practical points relevant for the rheumatologists. Finally, we have critically discussed the current and future opportunities that a muscle biopsy may offer and its limitations.

Integrating protein networks and machine learning for disease stratification in the Hereditary Spastic Paraplegias.

The Hereditary Spastic Paraplegias are a group of neurodegenerative diseases characterized by spasticity and weakness in the lower body. Owing to the combination of genetic diversity and variable clinical presentation, the Hereditary Spastic Paraplegias are a strong candidate for protein-protein interaction network analysis as a tool to understand disease mechanism(s) and to aid functional stratification of phenotypes. In this study, experimentally validated human data were used to create a protein-protein interaction network based on the causative genes. Network evaluation as a combination of topological analysis and functional annotation led to the identification of core proteins in putative shared biological processes, such as intracellular transport and vesicle trafficking. The application of machine learning techniques suggested a functional dichotomy linked with distinct sets of clinical presentations, indicating that there is scope to further classify conditions currently described under the same umbrella-term of Hereditary Spastic Paraplegias based on specific molecular mechanisms of disease.

Pembrolizumab for the treatment of progressive multifocal leukoencephalopathy following anti-CD19 CAR-T therapy: a case report.

Progressive multifocal leukoencephalopathy (PML) is an opportunistic brain infection with few treatment options and poor survival when reversal of the underlying immune dysfunction is not achievable. JC polyomavirus reactivation resulting in PML can rarely complicate chimeric antigen receptor T-cell (CAR-T) therapy. We describe successful treatment of PML with Programmed death-1 (PD-1) blockade using pembrolizumab, 4 months following axicabtagene ciloleucel. Radiological features of immune reconstitution inflammatory syndrome without clinical deterioration were seen. Evidence of anti-viral immune reconstitution by in vitro detection of JC-specific T-cells and sustained neurological recovery in this patient suggest PD-1 blockade may be an effective treatment approach for PML post-CAR-T.

An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein.

Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson's disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread.

Sex-dependent calcium hyperactivity due to lysosomal-related dysfunction in astrocytes from APOE4 versus APOE3 gene targeted replacement mice.

BACKGROUND: The apolipoprotein E (APOE) gene exists in three isoforms in humans: APOE2, APOE3 and APOE4. APOE4 causes structural and functional alterations in normal brains, and is the strongest genetic risk factor of the sporadic form of Alzheimer's disease (LOAD). Research on APOE4 has mainly focused on the neuronal damage caused by defective cholesterol transport and exacerbated amyloid-ß and Tau pathology. The impact of APOE4 on non-neuronal cell functions has been overlooked. Astrocytes, the main producers of ApoE in the healthy brain, are building blocks of neural circuits, and Ca2+ signaling is the basis of their excitability. Because APOE4 modifies membrane-lipid composition, and lipids regulate Ca2+ channels, we determined whether APOE4 dysregulates Ca2+signaling in astrocytes. METHODS: Ca2+ signals were recorded in astrocytes in hippocampal slices from APOE3 and APOE4 gene targeted replacement male and female mice using Ca2+ imaging. Mechanistic analyses were performed in immortalized astrocytes. Ca2+ fluxes were examined with pharmacological tools and Ca2+ probes. APOE3 and APOE4 expression was manipulated with GFP-APOE vectors and APOE siRNA. Lipidomics of lysosomal and whole-membranes were also performed. RESULTS: We found potentiation of ATP-elicited Ca2+responses in APOE4 versus APOE3 astrocytes in male, but not female, mice. The immortalized astrocytes modeled the male response, and showed that Ca2+ hyperactivity associated with APOE4 is caused by dysregulation of Ca2+ handling in lysosomal-enriched acidic stores, and is reversed by the expression of APOE3, but not of APOE4, pointing to loss of function due to APOE4 malfunction. Moreover, immortalized APOE4 astrocytes are refractory to control of Ca2+ fluxes by extracellular lipids, and present distinct lipid composition in lysosomal and plasma membranes. CONCLUSIONS: Immortalized APOE4 versus APOE3 astrocytes present: increased Ca2+ excitability due to lysosome dysregulation, altered membrane lipidomes and intracellular cholesterol distribution, and impaired modulation of Ca2+ responses upon changes in extracellular lipids. Ca2+ hyperactivity associated with APOE4 is found in astrocytes from male, but not female, targeted replacement mice. The study suggests that, independently of Aß and Tau pathologies, altered astrocyte excitability might contribute to neural-circuit hyperactivity depending on APOE allele, sex and lipids, and supports lysosome-targeted therapies to rescue APOE4 phenotypes in LOAD.

Toxic Protein Spread in Neurodegeneration: Reality versus Fantasy.

Over the past decade, the importance of the propagation of amyloidogenic proteins such as α-synuclein and tau in the pathogenesis of neurodegenerative diseases has been supported by numerous neuropathological and experimental studies. While these proteins behave similarly to prions, recent evidence suggests the existence of fundamental differences, as they can propagate in the absence of endogenous template, they do not exhibit a strict 'strain' behavior, and they may not be transmissible between individuals. We therefore propose to name these proteins 'prionoids'. In this review we critically assess the extent of the overlap between these two entities and evaluate how the propagation of prionoids can fit into the wider system dysfunction seen in the brains of patients with Alzheimer's and Parkinson's diseases.

Pathogenic PS1 phosphorylation at Ser367.

The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic function of PS1/γ-secretase can be modulated by its 'phosphorylated' and 'dephosphorylated' states. However, the functional outcome of PS1 phosphorylation and its significance for Alzheimer's disease (AD) pathogenesis is poorly understood. Here, comprehensive analysis using FRET-based imaging reveals that activity-driven and Protein Kinase A-mediated PS1 phosphorylation at three domains (domain 1: T74, domain 2: S310 and S313, domain 3: S365, S366, and S367), with S367 being critical, is responsible for the PS1 pathogenic 'closed' conformation, and resulting increase in the Aß42/40 ratio. Moreover, we have established novel imaging assays for monitoring PS1 conformation in vivo, and report that PS1 phosphorylation induces the pathogenic conformational shift in the living mouse brain. These phosphorylation sites represent potential new targets for AD treatment.

Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism-dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.