Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Hippocampal aggregation signatures of pathogenic UBQLN2 in amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic variants in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia characterized by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial amyotrophic lateral sclerosis and/or frontotemporal dementia cases not caused by UBQLN2 pathogenic variants, particularly C9orf72-linked cases. This makes the mechanistic role of mutant ubiquilin 2 protein and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 44 genotypically diverse amyotrophic lateral sclerosis cases with or without frontotemporal dementia, including eight cases with UBQLN2 variants [resulting in p.S222G, p.P497H, p.P506S, p.T487I (two cases) and p.P497L (three cases)]. Using multiplexed (five-label) fluorescent immunohistochemistry, we mapped the co-localization of ubiquilin 2 with phosphorylated TDP-43, dipeptide repeat aggregates and p62 in the hippocampus of controls (n = 6), or amyotrophic lateral sclerosis with or without frontotemporal dementia in sporadic (n = 20), unknown familial (n = 3), SOD1-linked (n = 1), FUS-linked (n = 1), C9orf72-linked (n = 5) and UBQLN2-linked (n = 8) cases. We differentiate between (i) ubiquilin 2 aggregation together with phosphorylated TDP-43 or dipeptide repeat proteins; and (ii) ubiquilin 2 self-aggregation promoted by UBQLN2 pathogenic variants that cause amyotrophic lateral sclerosis and/or frontotemporal dementia. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wild-type ubiquilin 2 in amyotrophic lateral sclerosis with or without frontotemporal dementia, whereby mutant ubiquilin 2 is more prone than wild-type to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

Microglial proliferation and astrocytic protein alterations in the human Huntington's disease cortex.

Huntington's disease (HD) is a neurodegenerative disorder that severely affects the basal ganglia and regions of the cerebral cortex. While astrocytosis and microgliosis both contribute to basal ganglia pathology, the contribution of gliosis and potential factors driving glial activity in the human HD cerebral cortex is less understood. Our study aims to identify nuanced indicators of gliosis in HD which is challenging to identify in the severely degenerated basal ganglia, by investigating the middle temporal gyrus (MTG), a cortical region previously documented to demonstrate milder neuronal loss. Immunohistochemistry was conducted on MTG paraffin-embedded tissue microarrays (TMAs) comprising 29 HD and 35 neurologically normal cases to compare the immunoreactivity patterns of key astrocytic proteins (glial fibrillary acidic protein, GFAP; inwardly rectifying potassium channel 4.1, Kir4.1; glutamate transporter-1, GLT-1; aquaporin-4, AQP4), key microglial proteins (ionised calcium-binding adapter molecule-1, IBA-1; human leukocyte antigen (HLA)-DR; transmembrane protein 119, TMEM119; purinergic receptor P2RY12, P2RY12), and indicators of proliferation (Ki-67; proliferative cell nuclear antigen, PCNA). Our findings demonstrate an upregulation of GFAP+ protein expression attributed to the presence of more GFAP+ expressing cells in HD, which correlated with greater cortical mutant huntingtin (mHTT) deposition. In contrast, Kir4.1, GLT-1, and AQP4 immunoreactivity levels were unchanged in HD. We also demonstrate an increased number of IBA-1+ and TMEM119+ microglia with somal enlargement. IBA-1+, TMEM119+, and P2RY12+ reactive microglia immunophenotypes were also identified in HD, evidenced by the presence of rod-shaped, hypertrophic, and dystrophic microglia. In HD cases, IBA-1+ cells contained either Ki-67 or PCNA, whereas GFAP+ astrocytes were devoid of proliferative nuclei. These findings suggest cortical microgliosis may be driven by proliferation in HD, supporting the hypothesis of microglial proliferation as a feature of HD pathophysiology. In contrast, astrocytes in HD demonstrate an altered GFAP expression profile that is associated with the degree of mHTT deposition.

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.

Phosphorylation of the N-terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that efficiently phosphorylates full-length and N-terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation.

Characterization of neurofibrillary tangle immunophenotype signatures to classify tangle maturity in Alzheimer's disease.

INTRODUCTION: Tau aggregation into neurofibrillary tangles in Alzheimer's disease (AD) is a dynamic process involving changes in tau phosphorylation, isoform composition, and morphology. To facilitate studies of tangle maturity, we developed an image analysis pipeline to study antibody labeling signatures that can distinguish tangle maturity levels in AD brain tissue. METHODS: Using fluorescent immunohistochemistry, we co-labeled AD brain tissue with four antibodies that bind different tau epitopes. Mean fluorescence intensity of each antibody was measured, and spectral clustering was used to identify tangle immunophenotypes. RESULTS: Five distinct tangle populations were identified, and different tangle maturity immunophenotypes were identified with increasing Braak stage. Early tangle immunophenotypes were more prevalent in later affected regions and advanced immunophenotypes were associated with ghost morphology. DISCUSSION: Our findings indicate that tangle populations characterized by advanced tau immunophenotypes are associated with higher Braak stage and more mature morphology, providing a new framework for defining tangle maturity levels using tau antibody signatures. HIGHLIGHTS: Populations of neurofibrillary tangles exist in Alzheimer's disease. The immunophenotype of neurofibrillary tangle populations relates to their maturity. The most advanced immunophenotypes are associated with higher Braak stage. The most advanced immunophenotypes are associated with ghost morphology. The most immature immunophenotypes are associated with later affected regions.

A panel of TDP-43-regulated splicing events verifies loss of TDP-43 function in amyotrophic lateral sclerosis brain tissue.

TDP-43 dysfunction is a molecular hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major hypothesis of TDP-43 dysfunction in disease is the loss of normal nuclear function, resulting in impaired RNA regulation and the emergence of cryptic exons. Cryptic exons and differential exon usage are emerging as promising markers of lost TDP-43 function in addition to revealing biological pathways involved in neurodegeneration in ALS/FTD. In this brief report, we identified markers of TDP-43 loss of function by depleting TARDBP from post-mortem human brain pericytes, a manipulable in vitro primary human brain cell model, and identifying differential exon usage events with bulk RNA-sequencing analysis. We present these data in an interactive database (https://www.scotterlab.auckland.ac.nz/research-themes/tdp43-lof-db-v2/) together with seven other TDP-43-depletion datasets we meta-analysed previously, for user analysis of differential expression and splicing signatures. Differential exon usage events that were validated by qPCR were then compiled into a 'differential exon usage panel' with other well-established TDP-43 loss-of-function exon markers. This differential exon usage panel was investigated in ALS and control motor cortex tissue to verify whether, and to what extent, TDP-43 loss of function occurs in ALS. We find that profiles of TDP-43-regulated cryptic exons, changed exon usage and changed 3' UTR usage discriminate ALS brain tissue from controls, verifying that TDP-43 loss of function occurs in ALS. We propose that TDP-43-regulated splicing events that occur in brain tissue will have promise as predictors of disease.

ALS/FTD mutations in UBQLN2 impede autophagy by reducing autophagosome acidification through loss of function.

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.

N-terminal mutant huntingtin deposition correlates with CAG repeat length and symptom onset, but not neuronal loss in Huntington's disease.

Huntington's disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake an immunohistochemical screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs). Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabeled human brain tissue. Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with a customized automated image analysis pipeline on the TMAs. A 2.5-12 fold increase in the number of Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. MAB5374, MW1, and EPR5526 Htt aggregate numbers were positively correlated with CAG repeat length, and negatively correlated with the age of symptom onset in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and greater Htt cortical aggregate deposition is associated with an earlier age of symptom onset in HD. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD do not specifically immunolabel Htt aggregates in HD human brain tissue exclusively, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

Persistent cortical and white matter inflammation after therapeutic hypothermia for ischemia in near-term fetal sheep.

BACKGROUND: Therapeutic hypothermia significantly improves outcomes after moderate-severe hypoxic-ischemic encephalopathy (HIE), but it is partially effective. Although hypothermia is consistently associated with reduced microgliosis, it is still unclear whether it normalizes microglial morphology and phenotype. METHODS: Near-term fetal sheep (n = 24) were randomized to sham control, ischemia-normothermia, or ischemia-hypothermia. Brain sections were immunohistochemically labeled to assess neurons, microglia and their interactions with neurons, astrocytes, myelination, and gitter cells (microglia with cytoplasmic lipid granules) 7 days after cerebral ischemia. Lesions were defined as areas with complete loss of cells. RNAscope® was used to assess microglial phenotype markers CD86 and CD206. RESULTS: Ischemia-normothermia was associated with severe loss of neurons and myelin (p < 0.05), with extensive lesions, astrogliosis and microgliosis with a high proportion of gitter cells (p < 0.05). Microglial wrapping of neurons was present in both the ischemia groups. Hypothermia improved neuronal survival, suppressed lesions, gitter cells and gliosis (p < 0.05), and attenuated the reduction of myelin area fraction. The "M1" marker CD86 and "M2" marker CD206 were upregulated after ischemia. Hypothermia partially suppressed CD86 in the cortex only (p < 0.05), but did not affect CD206. CONCLUSIONS: Hypothermia prevented lesions after cerebral ischemia, but only partially suppressed microglial wrapping and M1 marker expression. These data support the hypothesis that persistent upregulation of injurious microglial activity may contribute to partial neuroprotection after hypothermia, and that immunomodulation after rewarming may be an important therapeutic target.

Normal aging, motor neurone disease, and Alzheimer's disease are characterized by cortical changes in inflammatory cytokines.

The role of increased brain inflammation in the development of neurodegenerative diseases is unclear. Here, we have compared cytokine changes in normal aging, motor neurone disease (MND), and Alzheimer's disease (AD). After an initial analysis, six candidate cytokines, interleukin (IL)- 4, 5, 6, 10, macrophage inhibitory protein (MIP)-1α, and fibroblast growth factor (FGF)-2, showing greatest changes were assayed in postmortem frozen human superior frontal gyri (n = 12) of AD patients, aging and young adult controls along with the precentral gyrus (n = 12) of MND patients. Healthy aging was associated with decreased anti-inflammatory IL-10 and FGF-2 levels. AD prefrontal cortex was associated with increased levels of IL-4, IL-5, and FGF-2, with the largest increase seen for FGF-2. Notwithstanding differences in the specific frontal lobe gyrus sampled, MND patients' primary motor cortex (precentral gyrus) was associated with increased levels of IL-5, IL-6, IL-10, and FGF-2 compared to the aging prefrontal cortex (superior frontal gyrus). Immunocytochemistry showed that FGF-2 is expressed in neurons, astrocytes, and microglia in normal aging prefrontal cortex, AD prefrontal cortex, and MND motor cortex. We report that healthy aging and age-related neurodegenerative diseases have different cortical inflammatory signatures that are characterized by increased levels of anti-inflammatory cytokines and call into question the view that increased inflammation underlies the development of age-related neurodegenerative diseases.

Prevalence of young-onset dementia: nationwide analysis of routinely collected data.

INTRODUCTION: Young-onset dementia prevalence is understudied internationally. Previous studies have been limited by low case numbers, reliance on single sources of routinely collected health data for case identification and inclusion of a limited age range. Our objective was to determine the 1-year period prevalence of diagnosed dementia in people aged 0-64 in the entire New Zealand population using routinely collected health data. METHODS: A population-based descriptive study was carried out in New Zealand (population 4.8 million) using routinely collected deidentified health data from 2016 to 2020. Dementia cases in seven linked health datasets in the New Zealand Integrated Data Infrastructure were identified using diagnostic codes and/or use of antidementia medication. Prevalence for each of the four study years was calculated by age, sex and ethnicity. RESULTS: From a total population of 4 027 332-4 169 754 individuals aged 0-64, we identified 3396-3474 cases of 'all-cause' dementia in each of the study years (prevalence crude range: 83-84/100 000 people aged 0-64; 139-141/100 000 people aged 30-64 years; 204-207/100 000 people aged 45-64 years). Age-standardised prevalence was higher in males than females. Age-standardised and sex-standardised prevalence was higher in Maori and Pacific People than European and Asian. DISCUSSION: By using a large study population and multiple national health datasets, we have minimised selection bias and estimated the national prevalence of diagnosed young-onset dementia with precision. Young-onset dementia prevalence for the total New Zealand population was similar to reported global prevalence, validating previous estimates. Prevalence differed by ethnicity, which has important implications for service planning.

An imaging mass spectrometry atlas of lipids in the human neurologically normal and Huntington's disease caudate nucleus.

Huntington's disease (HD) is a fatal disorder associated with germline trinucleotide repeat expansions in the HTT gene and characterised by striatal neurodegeneration. No efficacious interventions are available for HD, highlighting a major unmet medical need. The molecular mechanisms underlying HD are incompletely understood despite its monogenic aetiology. However, direct interactions between HTT and membrane lipids suggest that lipidomic perturbations may be implicated in the neuropathology of HD. In this study, we employed matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) to generate a comprehensive, unbiased and spatially resolved lipidomic atlas of the caudate nucleus (CN) in human post-mortem tissue from neurologically normal (n = 10) and HD (n = 13) subjects. Fourier transform-ion cyclotron resonance mass spectrometry and liquid chromatography-tandem mass spectrometry were used for lipid assignment. Lipidomic specialisation was observed in the grey and white matter constituents of the CN and these features were highly conserved between subjects. While the majority of lipid species were highly conserved in HD, compared to age-matched controls, CN specimens from HD cases in our cohort spanning a range of neuropathological grades showed a lower focal abundance of the neuroprotective docosahexaenoic and adrenic acids, several cardiolipins, the ganglioside GM1 and glycerophospholipids with long polyunsaturated fatty acyls. HD cases showed a higher focal abundance of several sphingomyelins and glycerophospholipids with shorter monosaturated fatty acyls. Moreover, we demonstrate that MALDI-IMS is tractable as a primary discovery modality comparing heterogeneous human brain tissue, provided that appropriate statistical approaches are adopted. Our findings support further investigation into the potential role of lipidomic aberrations in HD.

fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

Sociodemographic and Clinical Characteristics of 1350 Patients With Young Onset Dementia: A Comparison With Older Patients.

OBJECTIVE: To determine the sociodemographic and clinical characteristics of a large cohort of patients with young onset dementia (YOD) (aged below 65), and whether they differ from older (age 65+) adults with dementia. METHODS: Retrospective cross-sectional study. Participants were New Zealanders who were assessed with International Residential Assessment Instrument (interRAI) assessments (including community-dwelling adults and those in long-term care) from 2016 to 2019 and had a diagnosis of dementia. Outcomes were sociodemographic and clinical characteristics captured in the interRAI assessment. RESULTS: People with YOD were more likely to be male, of non-European ethnicity, and live in a dwelling other than a private home or be homeless. They were more likely to exhibit problematic behaviors and neuropsychiatric symptoms but were less frail and less dependent for activities of daily living. Financial strain and loneliness were more common in people with YOD. Carers of people with YOD were more likely to feel distress, anger, or depression, and families of people with YOD were more likely to feel overwhelmed. CONCLUSIONS: YOD patients have different needs than older adults with dementia. These differences must be considered by clinicians and organizations that provide care and support to people living with dementia.

Pyridine alkaloids with activity in the central nervous system.

This review discusses all pyridine alkaloids with CNS activity, their therapeutic potential, and the interesting array of sources whence they originate.

Why people donate their brain to science: a systematic review.

The acquisition of brain tissue for research purposes is an important endeavour in research on ageing, pathological diagnosis, and the advancement of treatment of neurological or neurodegenerative diseases. While some tissue samples can be obtained from a living patient, the procurement of a whole brain requires the donation from people after their death. In order to promote positive attitudes towards brain donation, it is essential to understand why people do or do not donate their brain to medical research. In 2018 we undertook a systematic review of the international literature concerning people's attitudes, motivations, and feelings about brain donation. Five electronic databases were searched: Scopus, PsycINFO, Embase, Medline, and Google Scholar. Search terms included: ("brain donor*" OR "brain donation" OR "brain banking" OR "banking on brain") AND (attitude* OR motivation* OR decision*") AND (LIMIT-TO "human") AND (LIMIT-TO (LANGUAGE, "English")). Articles were analysed using the Framework for Assessing Qualitative Evaluations and a meta-ethnographic approach. Fourteen articles were included for review. The findings suggest four universal factors informing a person's decision to donate their brain: (1) contextual knowledge, (2) conceptual understandings, (3) family/friends matter, and (4) personal experience, time and process. The findings also indicate that the way healthcare professionals present themselves can influence people's feelings and attitudes towards brain donation. Healthcare and research professionals who are involved in brain donation processes must be mindful of the complex and multiple factors that influence donation outcomes. Effective and sensitive communication with potential donors and their family/friends is paramount.

Subventricular zone lipidomic architecture loss in Huntington's disease.

The human subventricular zone (SVZ) has a defined cytological and neurochemical architecture, with four constituent laminae that act in concert to support its neurogenic activity. Lipidomic specialisation has previously been demonstrated in the neurologically normal human SVZ, with enrichment of functionally important lipid classes in each lamina. The SVZ is also responsive to neurodegenerative disorders, where thickening of the niche and enhanced proliferation of resident cells were observed in Huntington's disease (HD) brains. In this study, we hypothesised lipidomic changes in the HD SVZ. Using matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry, this analysis shows differences in the lipidomic architecture in the post-mortem Vonsattel grade III cases. Relative to matched, neurologically normal specimens (N = 4), the lipidomic signature of the HD SVZ (N = 4) was characterized by loss of sulfatides and triglycerides in the myelin layer, with an ectopic and focal accumulation of sphingomyelins and ceramide-1-phosphate observed in this lamina. A striking loss of lipidomic patterning was also observed in the ependymal layer, where the local abundance of phosphatidylinositols was significantly reduced in HD. This comprehensive spatially resolved lipidomic analysis of the human HD SVZ identifies alterations in lipid architecture that may shed light on the mechanisms of SVZ responses to neurodegeneration in HD. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Transcriptome sequencing reveals aberrant alternative splicing in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the gene-encoding Huntingtin (HTT). Transcriptome dysregulation is a major feature of HD pathogenesis, as revealed by a large body of work on gene expression profiling of tissues from human HD patients and mouse models. These studies were primarily focused on transcriptional changes affecting steady-state overall gene expression levels using microarray based approaches. A major missing component, however, has been the study of transcriptome changes at the post-transcriptional level, such as alternative splicing. Alternative splicing is a critical mechanism for expanding regulatory and functional diversity from a limited number of genes, and is particularly complex in the mammalian brain. Here we carried out a deep RNA-seq analysis of the BA4 (Brodmann area 4) motor cortex from seven human HD brains and seven controls to systematically discover aberrant alternative splicing events and characterize potential associated splicing factors in HD. We identified 593 differential alternative splicing events between HD and control brains. Using two expanded panels with a total of 108 BA4 tissues from patients and controls, we identified four splicing factors exhibiting significantly altered expression levels in HD patient brains. Moreover, follow-up molecular analyses of one splicing factor PTBP1 revealed its impact on disease-associated splicing patterns in HD. Collectively, our data provide genomic evidence for widespread splicing dysregulation in HD brains, and suggest the role of aberrant alternative splicing in the pathogenesis of HD.

Differential Fatty Acid-Binding Protein Expression in Persistent Radial Glia in the Human and Sheep Subventricular Zone.

Fatty acid-binding proteins (FABPs) are a family of transport proteins that facilitate intracellular transport of fatty acids. Despite abundant expression in the brain, the role that FABPs play in the process of cell proliferation and migration in the subventricular zone (SVZ) remains unclear. Our results provide a detailed characterisation of FABP3, 5, and 7 expression in adult and fetal human and sheep SVZ. High FABP5 expression was specifically observed in the adult human SVZ and co-labelled with polysialylated neural cell adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP), GFAPδ, and proliferating cell nuclear antigen (PCNA), indicating a role for FABP5 throughout the full maturation process of astrocytes and neuroblasts. Some FABP5+ cells had a radial glial-like appearance and co-labelled with the radial glia markers vimentin (40E-C) and GFAP. In the fetal human brain, FABP5 was expressed by radial glia cells throughout the ventricular zone. In contrast, radial glia-like cells in sheep highly expressed FABP3. Taken together, these differences highlight the species-specific expression profile of FABPs in the SVZ. In this study, we demonstrate the distribution of FABP in the adult human SVZ and fetal ventricular zone and reveal its expression on persistent radial glia that may be involved in adult neurogenesis.

Unique and shared inflammatory profiles of human brain endothelia and pericytes.

BACKGROUND: Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed. METHODS: To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1ß, TNFα, LPS, IFN-γ, TGF-ß1, IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1ß. RESULTS: Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1ß. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1ß, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro. CONCLUSIONS: Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.