CAG repeat mosaicism is gene specific in spinocerebellar ataxias.

Expanded CAG repeats in coding regions of different genes are the most common cause of dominantly inherited spinocerebellar ataxias (SCAs). These repeats are unstable through the germline, and larger repeats lead to earlier onset. We measured somatic expansion in blood samples collected from 30 SCA1, 50 SCA2, 74 SCA3, and 30 SCA7 individuals over a mean interval of 8.5 years, along with postmortem tissues and fetal tissues from SCA1, SCA3, and SCA7 individuals to examine somatic expansion at different stages of life. We showed that somatic mosaicism in the blood increases over time. Expansion levels are significantly different among SCAs and correlate with CAG repeat lengths. The level of expansion is greater in individuals with SCA7 who manifest disease compared to that of those who do not yet display symptoms. Brain tissues from SCA individuals have larger expansions compared to the blood. The cerebellum has the lowest mosaicism among the studied brain regions, along with a high expression of ATXNs and DNA repair genes. This was the opposite in cortices, with the highest mosaicism and lower expression of ATXNs and DNA repair genes. Fetal cortices did not show repeat instability. This study shows that CAG repeats are increasingly unstable during life in the blood and the brain of SCA individuals, with gene- and tissue-specific patterns.

Somatic instability of the FGF14-SCA27B GAA•TTC repeat reveals a marked expansion bias in the cerebellum.

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14. Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAA•TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAA•TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14. We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAA•TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure cerebellar involvement in SCA27B.

Amyloid-ß peptide signature associated with cerebral amyloid angiopathy in familial Alzheimer's disease with APPdup and Down syndrome.

Alzheimer's disease (AD) is characterized by extracellular amyloid plaques containing amyloid-ß (Aß) peptides, intraneuronal neurofibrillary tangles, extracellular neuropil threads, and dystrophic neurites surrounding plaques composed of hyperphosphorylated tau protein (pTau). Aß can also deposit in blood vessel walls leading to cerebral amyloid angiopathy (CAA). While amyloid plaques in AD brains are constant, CAA varies among cases. The study focuses on differences observed between rare and poorly studied patient groups with APP duplications (APPdup) and Down syndrome (DS) reported to have higher frequencies of elevated CAA levels in comparison to sporadic AD (sAD), most of APP mutations, and controls. We compared Aß and tau pathologies in postmortem brain tissues across cases and Aß peptides using mass spectrometry (MS). We further characterized the spatial distribution of Aß peptides with MS-brain imaging. While intraparenchymal Aß deposits were numerous in sAD, DS with AD (DS-AD) and AD with APP mutations, these were less abundant in APPdup. On the contrary, Aß deposits in the blood vessels were abundant in APPdup and DS-AD while only APPdup cases displayed high Aß deposits in capillaries. Investigation of Aß peptide profiles showed a specific increase in Aßx-37, Aßx-38 and Aßx-40 but not Aßx-42 in APPdup cases and to a lower extent in DS-AD cases. Interestingly, N-truncated Aß2-x peptides were particularly increased in APPdup compared to all other groups. This result was confirmed by MS-imaging of leptomeningeal and parenchymal vessels from an APPdup case, suggesting that CAA is associated with accumulation of shorter Aß peptides truncated both at N- and C-termini in blood vessels. Altogether, this study identified striking differences in the localization and composition of Aß deposits between AD cases, particularly APPdup and DS-AD, both carrying three genomic copies of the APP gene. Detection of specific Aß peptides in CSF or plasma of these patients could improve the diagnosis of CAA and their inclusion in anti-amyloid immunotherapy treatments.

Motor neuron pathology in CANVAS due to RFC1 expansions.

CANVAS caused by RFC1 biallelic expansions is a major cause of inherited sensory neuronopathy. Detection of RFC1 expansion is challenging and CANVAS can be associated with atypical features. We clinically and genetically characterized 50 patients, selected based on the presence of sensory neuronopathy confirmed by EMG. We screened RFC1 expansion by PCR, repeat-primed PCR, and Southern blotting of long-range PCR products, a newly developed method. Neuropathological characterization was performed on the brain and spinal cord of one patient. Most patients (88%) carried a biallelic (AAGGG)n expansion in RFC1. In addition to the core CANVAS phenotype (sensory neuronopathy, cerebellar syndrome and vestibular impairment), we observed chronic cough (97%), oculomotor signs (85%), motor neuron involvement (55%), dysautonomia (50%), and parkinsonism (10%). Motor neuron involvement was found for 24 of 38 patients (63.1%). First motor neuron signs, such as brisk reflexes, extensor plantar responses, and/or spasticity, were present in 29% of patients, second motor neuron signs, such as fasciculations, wasting, weakness, or a neurogenic pattern on EMG in 18%, and both in 16%. Mixed motor and sensory neuronopathy was observed in 19% of patients. Among six non-RFC1 patients, one carried a heterozygous AAGGG expansion and a pathogenic variant in GRM1. Neuropathological examination of one RFC1 patient with an enriched phenotype, including parkinsonism, dysautonomia, and cognitive decline, showed posterior column and lumbar posterior root atrophy. Degeneration of the vestibulospinal and spinocerebellar tracts was mild. We observed marked astrocytic gliosis and axonal swelling of the synapse between first and second motor neurons in the anterior horn at the lumbar level. The cerebellum showed mild depletion of Purkinje cells, with empty baskets, torpedoes, and astrogliosis characterized by a disorganization of the Bergmann's radial glia. We found neuronal loss in the vagal nucleus. The pars compacta of the substantia nigra was depleted, with widespread Lewy bodies in the locus coeruleus, substantia nigra, hippocampus, entorhinal cortex, and amygdala. We propose new guidelines for the screening of RFC1 expansion, considering different expansion motifs. Here, we developed a new method to more easily detect pathogenic RFC1 expansions. We report frequent motor neuron involvement and different neuronopathy subtypes. Parkinsonism was more prevalent in this cohort than in the general population, 10% versus the expected 1% (P < 0.001). We describe, for the first time, the spinal cord pathology in CANVAS, showing the alteration of posterior columns and roots, astrocytic gliosis and axonal swelling, suggesting motor neuron synaptic dysfunction.

Identification of Umbre Orthobunyavirus as a Novel Zoonotic Virus Responsible for Lethal Encephalitis in 2 French Patients with Hypogammaglobulinemia.

BACKGROUND: Human encephalitis represents a medical challenge from a diagnostic and therapeutic point of view. We investigated the cause of 2 fatal cases of encephalitis of unknown origin in immunocompromised patients. METHODS: Untargeted metatranscriptomics was applied on the brain tissue of 2 patients to search for pathogens (viruses, bacteria, fungi, or protozoans) without a prior hypothesis. RESULTS: Umbre arbovirus, an orthobunyavirus never previously identified in humans, was found in 2 patients. In situ hybridization and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) showed that Umbre virus infected neurons and replicated at high titers. The virus was not detected in cerebrospinal fluid by RT-qPCR. Viral sequences related to Koongol virus, another orthobunyavirus close to Umbre virus, were found in Culex pipiens mosquitoes captured in the south of France where the patients had spent some time before the onset of symptoms, demonstrating the presence of the same clade of arboviruses in Europe and their potential public health impact. A serological survey conducted in the same area did not identify individuals positive for Umbre virus. The absence of seropositivity in the population may not reflect the actual risk of disease transmission in immunocompromised individuals. CONCLUSIONS: Umbre arbovirus can cause encephalitis in immunocompromised humans and is present in Europe.

Clinical and neuropathological diversity of tauopathy in MAPT duplication carriers.

Microduplications of the 17q21.31 chromosomal region encompassing the MAPT gene, which encodes the Tau protein, were identified in patients with a progressive disorder initially characterized by severe memory impairment with or without behavioral changes that can clinically mimic Alzheimer disease. The unique neuropathological report showed a primary tauopathy, which could not be unanimously classified in a given known subtype, showing both 4R- and 3R-tau inclusions, mainly within temporal cortical subregions and basal ganglia, without amyloid deposits. Recently, two subjects harboring the same duplication were reported with an atypical extrapyramidal syndrome and gait disorder. To decipher the phenotypic spectrum associated with MAPT duplications, we studied ten carriers from nine families, including two novel unrelated probands, gathering clinical (n = 10), cerebrospinal fluid (n = 6), MRI (n = 8), dopamine transporter scan (n = 4), functional (n = 5), amyloid (n = 3) and Tau-tracer (n = 2) PET imaging data as well as neuropathological examination (n = 4). Ages at onset ranged from 37 to 57 years, with prominent episodic memory impairment in 8/10 patients, associated with behavioral changes in four, while two patients showed atypical extrapyramidal syndrome with gait disorder at presentation, including one with associated cognitive deficits. Amyloid imaging was negative but Tau imaging showed significant deposits mainly in both mesiotemporal cortex. Dopaminergic denervation was found in 4/4 patients, including three without extrapyramidal symptoms. Neuropathological examination exclusively showed Tau-immunoreactive lesions. Distribution, aspect and 4R/3R tau aggregates composition suggested a spectrum from predominantly 3R, mainly cortical deposits well correlating with cognitive and behavioral changes, to predominantly 4R deposits, mainly in the basal ganglia and midbrain, in patients with prominent extrapyramidal syndrome. Finally, we performed in vitro seeding experiments in HEK-biosensor cells. Morphological features of aggregates induced by homogenates of three MAPT duplication carriers showed dense/granular ratios graduating between those induced by homogenates of a Pick disease and a progressive supranuclear palsy cases. These results suggest that MAPT duplication causes a primary tauopathy associated with diverse clinical and neuropathological features.

Pathway from TDP-43-Related Pathology to Neuronal Dysfunction in Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration.

Transactivation response DNA binding protein 43 kDa (TDP-43) is known to be a pathologic protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 is normally a nuclear protein, but affected neurons of ALS or FTLD patients exhibit mislocalization of nuclear TDP-43 and cytoplasmic inclusions. Basic studies have suggested gain-of-neurotoxicity of aggregated TDP-43 or loss-of-function of intrinsic, nuclear TDP-43. It has also been hypothesized that the aggregated TDP-43 functions as a propagation seed of TDP-43 pathology. However, a mechanistic discrepancy between the TDP-43 pathology and neuronal dysfunctions remains. This article aims to review the observations of TDP-43 pathology in autopsied ALS and FTLD patients and address pathways of neuronal dysfunction related to the neuropathological findings, focusing on impaired clearance of TDP-43 and synaptic alterations in TDP-43-related ALS and FTLD. The former may be relevant to intraneuronal aggregation of TDP-43 and exocytosis of propagation seeds, whereas the latter may be related to neuronal dysfunction induced by TDP-43 pathology. Successful strategies of disease-modifying therapy might arise from further investigation of these subcellular alterations.

Human subiculo-fornico-mamillary system in Alzheimer's disease: Tau seeding by the pillar of the fornix.

In Alzheimer's disease (AD), Tau and Aß aggregates involve sequentially connected regions, sometimes distantly separated. These alterations were studied in the pillar of the fornix (PoF), an axonal tract, to analyse the role of axons in their propagation. The PoF axons mainly originate from the subicular neurons and project to the mamillary body. Forty-seven post-mortem cases at various Braak stages (Tau) and Thal phases (Aß) were analysed by immunohistochemistry. The distribution of the lesions showed that the subiculum was affected before the mamillary body, but neither Tau aggregation nor Aß deposition was consistently first. The subiculum and the mamillary body contained Gallyas positive neurofibrillary tangles, immunolabelled by AT8, TG3, PHF1, Alz50 and C3 Tau antibodies. In the PoF, only thin and fragmented threads were observed, exclusively in the cases with neurofibrillary tangles in the subiculum. The threads were made of Gallyas negative, AT8 and TG3 positive Tau. They were intra-axonal and devoid of paired helical filaments at electron microscopy. We tested PoF homogenates containing Tau AT8 positive axons in a Tau P301S biosensor HEK cell line and found a seeding activity. There was no Aß immunoreactivity detected in the PoF. We could follow microcryodissected AT8 positive axons entering the mamillary body; contacts between Tau positive endings and Aß positive diffuse or focal deposits were observed in CLARITY-cleared mamillary body. In conclusion, we show that non-fibrillary, hyperphosphorylated Tau is transported by the axons of the PoF from the subiculum to the mamillary body and has a seeding activity. Either Tau aggregation or Aß accumulation may occur first in this system: this inconstant order is incompatible with a cause-and-effects relationship. However, both pathologies were correlated and intimately associated, indicating an interaction of the two processes, once initiated.

Increased prevalence of granulovacuolar degeneration in C9orf72 mutation.

Granulovacuolar degeneration (GVD) is usually found in Alzheimer's disease (AD) cases or in elderly individuals. Its severity correlates positively with the density of neurofibrillary tangles (NFTs). Mechanisms underlying GVD formation are unknown. We assessed the prevalence and distribution of GVD in cases with TDP-43-related frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS-TDP). Consecutively autopsied cases with FTLD/ALS-TDP and C9orf72 mutations (FTLD/ALS-C9; N = 29), cases with FTLD/ALS-TDP without C9orf72 mutations (FTLD/ALS-nonC9; N = 46), and age-matched healthy controls (N = 40) were studied. The prevalence of GVD was significantly higher in the FTLD/ALS-C9 cases (26/29 cases) than in the FTLD/ALS-nonC9 cases (15/46 cases; Fisher exact test; p < 2×10-6) or in the control group (12/40 individuals; p < 1×10-6). Average Braak stages and ages of death were not significantly different among the groups. The CA2 sector was most frequently affected in the FTLD/ALS-C9 group, whereas the CA1/subiculum was the most vulnerable area in the other groups. Extension of GVD correlated with the clinical duration of the disease in the FTLD/ALS-C9 cases but not in the FTLD/ALS-nonC9 cases. The GVD-containing neurons frequently had dipeptide repeat (DPR) protein inclusions. GVD granules labeled with antibodies directed against charged multivesicular body protein 2B or casein kinase 1δ were attached to DPR inclusions within GVD. Our results suggest that development of GVD and DPR inclusions is related to common pathogenic mechanisms and that GVD is not only associated with NFTs seen in AD cases or aging individuals.

Alzheimer's senile plaque as shown by microcryodissection, a new technique for dissociating tissue structures.

Extracellular accumulation of Aß peptides and intracellular aggregation of hyperphosphorylated tau proteins are the two hallmark lesions of Alzheimer disease (AD). The senile plaque is made of a core of extracellular Aß surrounded by phospho-tau positive neurites. It includes multiple components such as axons, synapses, glial fibers and microglia. To visualize the relationships of those elements, an original technique was developed, based on the dilation of interstitial water during freezing. Samples of neocortex, hippocampus and striatum were taken from formalin-fixed brains (one control case; three cases with severe Alzheimer disease). The samples were subjected to various numbers of freezing/thawing cycles (from 0 to 320) with an automated system we devised. The samples were embedded in paraffin, cut and stained with haematoxylin-eosin or immunostained against Aß, phospho-tau, and antigens enriched in axons, synapses, macrophages or astrocytes. Microcryodissection induced the dissociation of tissue components, especially in the grey matter where the neuropil formed an oriented "mesh". The size of the empty spaces separating the fiber bundles and cells increased with the number of cycles. The amyloid core of the senile plaque separated from its neuritic crown at around 300 freezing/thawing cycles. The dissected core remained associated with macrophages containing Aß in their cytoplasm. Phospho-tau positive axons were distinctly seen projecting from the neuritic crown to the isolated amyloid core, where they ended in large synapses. The microcryodissection showed astrocytic processes stuck directly to the core. The original method we developed-microcryodissection-helped understanding how histological components were assembled in the tissue.

Pathological α-synuclein distribution in subjects with coincident Alzheimer's and Lewy body pathology.

We investigated the distribution patterns of Lewy body-related pathology (LRP) and the effect of coincident Alzheimer disease (AD) pathology using a data-driven clustering approach that identified groups with different LRP pathology distributions without any diagnostic or researcher's input in two cohorts including: Parkinson disease patients without (PD, n = 141) and with AD (PD-AD, n = 80), dementia with Lewy bodies subjects without AD (DLB, n = 13) and demented subjects with AD and LRP pathology (Dem-AD-LB, n = 308). The Dem-AD-LB group presented two LRP patterns, olfactory-amygdala and limbic LRP with negligible brainstem pathology, that were absent in the PD groups, which are not currently included in the DLB staging system and lacked extracranial LRP as opposed to the PD group. The Dem-AD-LB individuals showed relative preservation of substantia nigra cells and dopamine active transporter in putamen. PD cases with AD pathology showed increased LRP. The cluster with occipital LRP was associated with non-AD type dementia clinical diagnosis in the Dem-AD-LB group and a faster progression to dementia in the PD groups. We found that (1) LRP pathology in Dem-AD-LB shows a distribution that differs from PD, without significant brainstem or extracranial LRP in initial phases; (2) coincident AD pathology is associated with increased LRP in PD indicating an interaction; (3) LRP and coincident AD pathology independently predict progression to dementia in PD, and (4) evaluation of LRP needs to acknowledge different LRP spreading patterns and evaluate substantia nigra integrity in the neuropathological assessment and consider the implications of neuropathological heterogeneity for clinical and biomarker characterization.

Differential induction and spread of tau pathology in young PS19 tau transgenic mice following intracerebral injections of pathological tau from Alzheimer's disease or corticobasal degeneration brains.

Filamentous tau pathologies are hallmark lesions of several neurodegenerative tauopathies including Alzheimer's disease (AD) and corticobasal degeneration (CBD) which show cell type-specific and topographically distinct tau inclusions. Growing evidence supports templated transmission of tauopathies through functionally interconnected neuroanatomical pathways suggesting that different self-propagating strains of pathological tau could account for the diverse manifestations of neurodegenerative tauopathies. Here, we describe the rapid and distinct cell type-specific spread of pathological tau following intracerebral injections of CBD or AD brain extracts enriched in pathological tau (designated CBD-Tau and AD-Tau, respectively) in young human mutant P301S tau transgenic (Tg) mice (line PS19) ~6-9 months before they show onset of mutant tau transgene-induced tau pathology. At 1 month post-injection of CBD-Tau, tau inclusions developed predominantly in oligodendrocytes of the fimbria and white matter near the injection sites with infrequent intraneuronal tau aggregates. In contrast, injections of AD-Tau in young PS19 mice induced tau pathology predominantly in neuronal perikarya with little or no oligodendrocyte involvement 1 month post-injection. With longer post-injection survival intervals of up to 6 months, CBD-Tau- and AD-Tau-induced tau pathology spread to different brain regions distant from the injection sites while maintaining the cell type-specific pattern noted above. Finally, CA3 neuron loss was detected 3 months post-injection of AD-Tau but not CBD-Tau. Thus, AD-Tau and CBD-Tau represent specific pathological tau strains that spread differentially and may underlie distinct clinical and pathological features of these two tauopathies. Hence, these strains could become targets to develop disease-modifying therapies for CBD and AD.

A comparison of Aß amyloid pathology staging systems and correlation with clinical diagnosis.

Current neuropathological Alzheimer's disease (AD) criteria from the National Institute on Aging-Alzheimer's Association (NIA-AA) incorporate two staging systems for Aß pathology, namely the Thal Aß phase (TAP) and the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) methods. The goal of this study was to compare and contrast results obtained with these two different staging systems for Aß pathology since this is critical for future correlations of Aß amyloid imaging data with Aß neuropathology data based on immunohistochemical detection of Aß deposits. A total of 123 cases, divided into 82 training and 41 validation cases, with a diagnosis of either unremarkable adult brain (normal) or AD and CERAD scores ranging from none to frequent were included. There was no clear and consistent relationship between CERAD and the TAP Aß scores with the exception of scores for the highest plaque burdens (i.e., CERAD C3 and TAP A3) in the cases studied here. However, we developed an algorithm that relates CERAD scores to TAP scores with high agreement (94 % in training and 98 % in the validation set). In addition, TAP scores were a better predictor of dementia (sensitivity of 94 % specificity 87.7 %) than CERAD scores (sensitivity of 57 % specificity 100 %). Yet, further research is needed to define strategies to relate CERAD and TAP Aß plaque scores to compare their utility and for determining the clinical associations of these different amyloid staging systems with aging and AD.

Evaluation of SP3 for antibody-free quantification of tau in CSF mimic and brain by mass spectrometry.

Tubulin-associated unit (tau) has an important role in the pathogenesis and the diagnosis of Alzheimer's disease (AD) and other tauopathies. In view of the diversity of tau proteoforms, antibody-free methods represent a good approach for unbiased quantification. We adapted and evaluated the single-pot, solid-phase-enhanced sample-preparation (SP3) protocol for antibody-free extraction of the tau protein in cerebro-spinal fluid (CSF) mimic and in human brain. A total of 13 non-modified peptides were quantified by high-resolution mass spectrometry (HRMS) after digestion of tau by trypsin. We significantly improved the basic SP3 protocol by carefully optimizing the organic solvents and incubation time for tau binding, as well as the digestion step for the release directly from the SP3 beads of the 13 tau peptides. These optimizations proved to be primarily beneficial for the most hydrophilic tau peptides, increasing the sequence coverage of recombinant tau. Mean recovery in CSF mimic of the 13 non-modified peptides was of 53%, with LODs ranging from 0.75 to 10 ng/mL. Next, we tested the optimized SP3 protocol on pathological tau extracted from the soluble fraction from an AD brain sample (middle frontal gyrus). We could successfully identify and quantify biologically relevant tau peptides including representative peptides of two isoforms and two phospho-peptides (pTau217 and pTau181).

Automated deep learning segmentation of neuritic plaques and neurofibrillary tangles in Alzheimer disease brain sections using a proprietary software.

Neuropathological diagnosis of Alzheimer disease (AD) relies on semiquantitative analysis of phosphorylated tau-positive neurofibrillary tangles (NFTs) and neuritic plaques (NPs), without consideration of lesion heterogeneity in individual cases. We developed a deep learning workflow for automated annotation and segmentation of NPs and NFTs from AT8-immunostained whole slide images (WSIs) of AD brain sections. Fifteen WSIs of frontal cortex from 4 biobanks with varying tissue quality, staining intensity, and scanning formats were analyzed. We established an artificial intelligence (AI)-driven iterative procedure to improve the generation of expert-validated annotation datasets for NPs and NFTs thereby increasing annotation quality by >50%. This strategy yielded an expert-validated annotation database with 5013 NPs and 5143 NFTs. We next trained two U-Net convolutional neural networks for detection and segmentation of NPs or NFTs, achieving high accuracy and consistency (mean Dice similarity coefficient: NPs, 0.77; NFTs, 0.81). The workflow showed high generalization performance across different cases. This study serves as a proof-of-concept for the utilization of proprietary image analysis software (Visiopharm) in the automated deep learning segmentation of NPs and NFTs, demonstrating that AI can significantly improve the annotation quality of complex neuropathological features and enable the creation of highly precise models for identifying these markers in AD brain sections.

Somatic instability of the FGF14 -SCA27B GAA•TTC repeat reveals a marked expansion bias in the cerebellum.

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14 . Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAA•TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAA•TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14 . We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAA•TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure and late-onset cerebellar involvement in SCA27B.

MAPT H2 haplotype and risk of Pick's disease in the Pick's disease International Consortium: a genetic association study.

BACKGROUND: Pick's disease is a rare and predominantly sporadic form of frontotemporal dementia that is classified as a primary tauopathy. Pick's disease is pathologically defined by the presence in the frontal and temporal lobes of Pick bodies, composed of hyperphosphorylated, three-repeat tau protein, encoded by the MAPT gene. MAPT has two distinct haplotypes, H1 and H2; the MAPT H1 haplotype is the major genetic risk factor for four-repeat tauopathies (eg, progressive supranuclear palsy and corticobasal degeneration), and the MAPT H2 haplotype is protective for these disorders. The primary aim of this study was to evaluate the association of MAPT H2 with Pick's disease risk, age at onset, and disease duration. METHODS: In this genetic association study, we used data from the Pick's disease International Consortium, which we established to enable collection of data from individuals with pathologically confirmed Pick's disease worldwide. For this analysis, we collected brain samples from individuals with pathologically confirmed Pick's disease from 35 sites (brainbanks and hospitals) in North America, Europe, and Australia between Jan 1, 2020, and Jan 31, 2023. Neurologically healthy controls were recruited from the Mayo Clinic (FL, USA, or MN, USA between March 1, 1998, and Sept 1, 2019). For the primary analysis, individuals were directly genotyped for the MAPT H1-H2 haplotype-defining variant rs8070723. In a secondary analysis, we genotyped and constructed the six-variant-defined (rs1467967-rs242557-rs3785883-rs2471738-rs8070723-rs7521) MAPT H1 subhaplotypes. Associations of MAPT variants and MAPT haplotypes with Pick's disease risk, age at onset, and disease duration were examined using logistic and linear regression models; odds ratios (ORs) and ß coefficients were estimated and correspond to each additional minor allele or each additional copy of the given haplotype. FINDINGS: We obtained brain samples from 338 people with pathologically confirmed Pick's disease (205 [61%] male and 133 [39%] female; 338 [100%] White) and 1312 neurologically healthy controls (611 [47%] male and 701 [53%] female; 1312 [100%] White). The MAPT H2 haplotype was associated with increased risk of Pick's disease compared with the H1 haplotype (OR 1·35 [95% CI 1·12 to 1·64], p=0·0021). MAPT H2 was not associated with age at onset (ß -0·54 [95% CI -1·94 to 0·87], p=0·45) or disease duration (ß 0·05 [-0·06 to 0·16], p=0·35). Although not significant after correcting for multiple testing, associations were observed at p less than 0·05: with risk of Pick's disease for the H1f subhaplotype (OR 0·11 [0·01 to 0·99], p=0·049); with age at onset for H1b (ß 2·66 [0·63 to 4·70], p=0·011), H1i (ß -3·66 [-6·83 to -0·48], p=0·025), and H1u (ß -5·25 [-10·42 to -0·07], p=0·048); and with disease duration for H1x (ß -0·57 [-1·07 to -0·07], p=0·026). INTERPRETATION: The Pick's disease International Consortium provides an opportunity to do large studies to enhance our understanding of the pathobiology of Pick's disease. This study shows that, in contrast to the decreased risk of four-repeat tauopathies, the MAPT H2 haplotype is associated with an increased risk of Pick's disease in people of European ancestry. This finding could inform development of isoform-related therapeutics for tauopathies. FUNDING: Wellcome Trust, Rotha Abraham Trust, Brain Research UK, the Dolby Fund, Dementia Research Institute (Medical Research Council), US National Institutes of Health, and the Mayo Clinic Foundation.

Changes in dental plaque following hospitalisation in a critical care unit: an observational study.

INTRODUCTION: Previous research has suggested that deterioration in oral health can occur following hospitalisation. The impact of such deterioration could increase the risk of oral disease, reduce quality of life and increase the potential for healthcare-associated infections (HCAI) such as healthcare-associated pneumonia (HAP). However, the strength of the evidence is limited by, amongst other factors, the few observational studies published that assess oral health longitudinally. In view of the microbiological component of oral diseases and HCAIs, the objective of this study was to investigate the microbiological changes in dental plaque following hospitalisation in a Critical Care Unit (CCU): (1) total number of cultivable bacteria and (2) presence and changes in specific HAP pathogens. METHODS: We conducted a prospective, longitudinal observational study in the CCU of University College Hospital, London. Study participants were recruited within 24 hours of admission. Dental plaque samples were collected from up to six sites per patient. The primary outcome was microbiological change from baseline to seven days with additional analysis for participants still present at day 14. RESULTS: 50 patients were recruited with 36 available for review at one week, with early discharge accounting for much of the loss to follow-up. The median total viable count of the plaque microbiota at baseline was 4.40 × 105 cfu/ml and increased at week one to 3.44 × 106 cfu/ml. The total viable microbe counts increased by a median of 2.26 × 106 cfu/ml from baseline to week one (95% CI: 3.19 × 106, 1.24 × 107) and this was statistically significant (P < 0.01). Specific HAP bacteria were detected in 26% of participants sampled, although accounted for a relatively low proportion of the total viable bacteria. CONCLUSION: Total bacterial count of dental plaque increases during hospitalisation in CCU. This finding, together with the colonisation of dental plaque by HAP bacteria strengthens the evidence for a deterioration in oral health in CCU and a risk factor for negative health and quality of life outcomes.

'Keeping the plates spinning': a qualitative study of the complexity, barriers, and facilitators to caregiving in heart failure with preserved ejection fraction.

AIMS: Heart failure with preserved ejection fraction (HFpEF) accounts for 50% of all heart failure cases; yet remains poorly understood, diagnosed, and managed, which adds complexity to the carer role. No study to date has investigated the experiences of informal carers of people with HFpEF. The aim of this study was to explore the role and experiences of informal carers of people with HFpEF. METHODS AND RESULTS: A qualitative study using semi-structured interviews involving carers alone, patients alone, or carer/patient dyads. The interviews were part of a larger programme of research in HFpEF. Participants were recruited from three regions of England. Interviews were recorded, transcribed verbatim, and analysed thematically. Twenty-two interviews were conducted with 38 participants, 17 were informal carers. Three inter-related themes were identified: Theme 1, the complex nature of informal caregiving ('spinning plates'); Theme 2, the barriers to caregiving ('the spinning falters'); and Theme 3, the facilitators of caregiving ('keeping the plates spinning'). CONCLUSIONS: Informal carers play an important role in supporting people with HFpEF. The experience of caregiving in HFpEF is similar to that described for Heart Failure with reduced Ejection Fraction, but complicated by challenges of limited information and support specific to HFpEF, and high burden of multi-morbidity. Healthcare providers should assess the needs of informal carers as part of patient care in HFpEF. Carers and patients would benefit from improved information and co-ordinated management of HFpEF and multi-morbidities. Helping carers 'keep the plates spinning' will require innovative approaches and co-ordination across the care continuum.