Acta Neuropathologica Communications

BackgroundMisfolded -synuclein (-syn) is believed to contribute to neurodegeneration in Lewy body disease (LBD) based on considerable evidence including a gene-dosage effect observed in relation to point mutations and multiplication of SNCA in familial Parkinsons disease. A contradictory concept proposes early loss of the physiological -syn as the major driver of neurodegeneration. There is a paucity of data on SNCA transcripts in various -syn immunoreactive cytopathologies. MethodsSNCA transcripts in neurons without and with various -syn immunoreactive cytopathologies in the substantia nigra and amygdala in LBD (n = 5) were evaluated using RNAscope combined with immunofluorescence for disease-associated -syn. Single-nucleus RNA sequencing was performed to elucidate cell-type specific SNCA expression in non-diseased frontal cortex (n = 3). ResultsSNCA transcripts in neurons with punctate -syn immunoreactivity were preserved both in the substantia nigra and amygdala but were reduced in neurons with compact -syn inclusions. Only single SNCA transcripts were detected in astrocytes with or without -syn immunoreactivity in the amygdala. Single-nucleus RNA sequencing revealed that excitatory and inhibitory neurons, oligodendrocyte progenitor cells, oligodendrocytes, and homeostatic microglia expressed SNCA transcripts, while expression was largely absent in astrocytes and microglia. ConclusionsThe preserved cellular SNCA expression in the more abundant non-Lewy body type -syn cytopathologies provides a pool for local protein production that can aggregate and serve as a seed for misfolded -syn. Successful segregation of disease-associated -syn is associated with the exhaustion of SNCA production in the terminal cytopathology, the Lewy body. Our observations support a therapeutic strategy incorporating a finely tuned dual approach targeting the elimination of misfolded -syn along with the reduction of the SNCA transcription to avoid feeding of pathological -syn seeding.

BackgroundChitotriosidase (Chit-1) and chitinase-3-like protein 1 (CHI3L1) protein levels are increased in the cerebrospinal fluid (CSF) of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD). Few studies have examined the spatial expression of chitinase expressing cells with respect to neuropathologic hallmarks of disease. MethodsRNA-sequencing was used to examine Chit-1 and CHI3L1 gene expression in the spinal cord and motor cortex. Immunohistochemistry was used to characterize the distribution of Chit-1 and CHI3L1 expressing cells in ALS, C9-ALS, FTLD, AD, and non-neurologic disease controls. Immunofluorescence confocal microscopy was used to correlate distribution of Chit-1 and CHI3L1 expressing cells to TDP pathology. ResultsChit-1 gene expression was increased in the spinal cord, and CHI3L1 expression was increased in both the spinal cord and motor cortex of sALS and C9-ALS patients when compared to controls. Highest levels of Chit-1+ glia were in cortical regions that contain hallmark neuropathology for each neurodegenerative disease. CHI3L1+ glia were only significantly increased in sALS. Neither Chit-1+ nor CHI3L1+ glia were in close proximity to pTDP containing neurons in the motor cortex gray matter; however, there was a significant co-localization of glial pTDP with Chit-1 and CHI3L1 in the motor cortex white matter. ConclusionsChit-1 and CHI3L1 expressing cells were most abundant in the white matter of cortical regions affected by each neurodegenerative disease and the spinal cord. Chit-1 or CHI3L1 expressing cells in the white matter also contained phosphorylated TDP-43. We also observed correlations between levels of Chit-1 or CHI3L1 expressing cells in the white matter to disease duration. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSPrior studies identified elevated levels of Chit-1 and CHI3L1 proteins in the CSF of various neurodegenerative conditions, though few studies examined levels of Chit-1 and CHI3L1 expressing cells both spatially and in relation to disease pathology. What this study addsWe performed an extensive spatial characterization of Chit-1 and CHI3L1 protein levels across multiple regions and neurodegenerative conditions. This study also correlates Chit-1 and CHI3L1 expression to TDP pathology and other clinical parameters of disease duration. How this study might affect research, practice or policyOur findings indicate that the majority of Chit-1 and CHI3L1 expressing glia are located in the cortical subpial layer and the white matter, suggesting a role for chitinases in modulating neuroinflammatory mechanisms or reparative/regenerative responses in the white matter of ALS and other neurodegenerative diseases. This study suggests new therapeutic opportunities for targeting chitinase expressing cells in neurodegenerative diseases.

Mutations in the UBQLN2 gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The neuropathology of such UBQLN2-linked cases of ALS/FTD is characterised by aggregates of the ubiquilin 2 protein in addition to aggregates of the transactive response DNA-binding protein of 43 kDa (TDP-43). ALS and FTD without UBQLN2 mutations are also characterised by TDP-43 aggregates, that may or may not colocalise with wildtype ubiquilin 2. Despite this, the relative contributions of TDP-43 and ubiquilin 2 to disease pathogenesis remain largely under-characterised, as does their relative deposition as aggregates across the central nervous system (CNS). Here we conducted multiplex immunohistochemistry of three UBQLN2 p.T487I-linked ALS/FTD cases, three non-UBQLN2-linked (sporadic) ALS cases, and eight non-neurodegenerative disease controls, covering 40 CNS regions. We then quantified ubiquilin 2 aggregates, TDP-43 aggregates, and aggregates containing both proteins in regions of interest to determine how UBQLN2-linked and non-UBQLN2-linked proteinopathy differ. We find that ubiquilin 2 aggregates that are negative for TDP-43 are predominantly small and punctate, and are abundant in the hippocampal formation, spinal cord, all tested regions of neocortex, medulla, and substantia nigra in UBQLN2-linked ALS/FTD but not sporadic ALS. Curiously, the striatum harboured small punctate ubiquilin 2 aggregates in all cases examined, while large diffuse striatal ubiquilin 2 aggregates were specific to UBQLN2-linked ALS/FTD. Overall, ubiquilin 2 is mainly deposited in clinically unaffected regions throughout the CNS such that symptomology in UBQLN2-linked cases maps best to the aggregation of TDP-43.

Dominantly inherited mutation D395G in the gene encoding valosin-containing protein causes vacuolar tauopathy, a type of behavioural-variant frontotemporal dementia, with marked vacuolation and abundant filamentous tau inclusions made of all six brain isoforms. Here we report that tau inclusions were concentrated in layers II/III of the frontotemporal cortex in a case of vacuolar tauopathy. By electron cryo-microscopy, tau filaments had the chronic traumatic encephalopathy (CTE) fold. Tau inclusions of vacuolar tauopathy share this cortical location and the tau fold with CTE, subacute sclerosing panencephalitis and amyotrophic lateral sclerosis/parkinsonism-dementia complex, which are believed to be environmentally induced. Vacuolar tauopathy is the first inherited disease with the CTE tau fold.

Lewy bodies (LBs) and Lewy neurites are pathological hallmarks of Parkinsons disease and other progressive neurodegenerative disorders known as Lewy body diseases (LBD). These proteinaceous deposits are immunopositive for alpha-synuclein (aSyn) and several other proteins, as neurofilament components. The structural organization and composition of aSyn inclusions is still unclear and needs to be addressed in greater detail, as this may open novel avenues for our understanding of the disease-relevant pathological events. In this study, we investigated the molecular architecture of aSyn inclusions, both in cell models and in human brain tissue, using state-of-art super resolution X10 Expansion microscopy (ExM). This approach physically expands specimens embedded into a swellable gel, preserving their biological information. Then, the specimen can be analyzed using standard epifluorescence microscopes, thereby obtaining nanoscale information. The combination of different cell models, mouse and human brain tissue enabled us to distinguish different types aSyn assemblies (e.g. ring shape or tubular structures), and a conserved pattern of aSyn inclusions surrounded/encaged by intermediate filament proteins. Overall, X10 ExM enabled us to gain insight into the architecture and biology of aSyn inclusions and constitutes a powerful tool in the quest to understanding underlying disease mechanisms in synucleinopathies.

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised 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 ALS or FTD cases not caused by UBQLN2 mutations, particularly C9orf72-linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 41 genotypically diverse ALS cases with or without FTD, including five cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, and two cases with p.T487I). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=20), unknown familial (n=3), SOD1-linked (n=1), FUS-linked (n=1), C9orf72-linked (n=5), and UBQLN2-linked (n=5) cases. We differentiate between i) ubiquilin 2 aggregation together with pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 gene mutations that cause ALS/FTD. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype 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.

Krabbe disease (KD) is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene which causes accumulation of the toxic sphingolipid psychosine. GALC variants are associated with increased risk of Lewy body diseases (LBD), an umbrella term for age-associated neurodegenerative diseases in which the protein -synuclein aggregates into Lewy bodies. To explore whether -synuclein in KD has pathological similarities to that in LBD, we compared post-mortem KD tissue to that of infant control cases and identified alterations to -synuclein localisation and expression of modifications associated with LBD. To determine whether -synuclein in KD displayed pathogenic properties associated with LBD we evaluated its seeding capacity using the real-time quaking-induced conversion assay. Strikingly, seeded aggregation of -synuclein resulted in the formation of fibrillar aggregates similar to those observed in LBD, confirming the prion-like capacity of KD-derived -synuclein. These observations constitute the first report of prion-like -synuclein in the brain tissue of infants and challenge the putative view that -synuclein pathology is merely an age-associated phenomenon, instead suggesting it can result from alterations to biological processes such as sphingolipid homeostasis. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in LBD.

Early diagnosis of Parkinsons disease (PD) remains challenging because motor symptoms appear only after extensive neurodegeneration, and a definitive diagnosis still relies on post-mortem neuropathology. Increasing evidence implicates the enteric nervous system (ENS) in prodromal disease stages, but routine ENS-based diagnosis is limited by the complexity of intestinal tissue organization and the need for specific labeling strategies. Here, we developed a label-free autofluorescence (AF) imaging workflow combined with unbiased morphometric analysis to identify neurodegenerative alterations in fixed human colonic tissue. Using a correlative multiscale imaging approach, we generated a database of almost 800 high-resolution confocal images from myenteric and submucosal plexuses of controls, PD, and Alzheimers disease (AD) patients. Blind evaluation by four expert histologists showed reliable identification of control tissue but lower sensitivity for pathological cases, reflecting the heterogeneous distribution of ENS lesions. Semi-quantitative and morphometric image analyses identified a distinct population of enlarged enteric neurons, termed large neural cells (LNCs), strongly enriched in PD and AD compared with controls. LNCs contained autofluorescent cytoplasmic inclusions and frequently prominent nucleoli, both features largely absent from control tissue independent of aging. Co-localization with the amyloid-binding probe Amytracker (AmyT) demonstrated that AF granules correspond to {beta}-sheet-rich protein aggregates rather than merely age-related lipofuscin granules. Similar alterations were detected in intact three-dimensional (3-D) colonic biopsies, demonstrating the feasibility of volumetric ENS imaging without tissue clearing. Together, our results establish label-free AF imaging as a rapid and clinically compatible strategy for detecting enteric neurodegenerative pathology. This approach provides a framework for the future development of ENS-based biomarkers and supports the use of volumetric intestinal imaging for early diagnosis of neurodegenerative diseases.

The choroid plexus epithelial cells (CPECs) at the blood-cerebrospinal fluid (CSF) interface possess an exceptionally high mitochondrial content to support CNS homeostasis. Oncocytic CPECs (O-CPECs), characterized by enlarged and granular eosinophilic cytoplasm composed of excessive abnormal mitochondria, likely contribute to an energetic failure of this energy-demanding tissue. The relationship between O-CPECs and other CPEC pathologies in humans, such as Biondi body (BB) amyloid inclusions, remains poorly defined. In the present study, using H&E-stained sections from 68 postmortem cases, we classified O-CPECs by quantitative size criteria and cytological features, and found an increase in the prevalence of O-CPECs with age after adjusting for sex and tissue source. After excluding two influential control cases, there was evidence for a further increase associated with Alzheimers disease. Using antibodies to ATP synthase beta chain to classify O-CPECs, and thioflavin-S to identify BBs, we revealed an increased prevalence of BBs in O-CPECs compared to neighboring non-oncocytic cells. Small multiple BB inclusions were responsible for the increase in O-CPECs, while the prevalence of larger inclusions was decreased in O-CPECs. Together, our data support a clear age-associated oncocytic transformation of CPECs and implicate mitochondrial dysfunction-amyloid interactions.

Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntingtons disease (HD). Although lowering mHTT by stimulating autophagy has been considered a possible therapeutic strategy, the role and competence of autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown. Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex of HD brains staged from HD2 to HD4 by Vonsattel neuropathological criteria compared to controls. Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins p62/SQSTM1 and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. Importantly, no blockage in autophagosome formation and autophagosome-lysosome fusion was detected, thus pinpointing autophagy substrate clearance deficits as a basis for autophagic flux declines. The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but failure at advanced stages contributes to progressive HTT build-up and potential neurotoxicity. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, may have therapeutic benefits in HD patients.

Progressive supranuclear palsy (PSP) is a sporadic neurodegenerative tauopathy variably affecting brainstem and cortical structures and characterized by tau inclusions in neurons and glia. The precise mechanism whereby these protein aggregates lead to cell death remains unclear. To investigate the contribution of these different cellular abnormalities to PSP pathogenesis, we performed single-nucleus RNA sequencing and analyzed 45,559 high quality nuclei targeting the subthalamic nucleus and adjacent structures from human post-mortem PSP brains with varying degrees of pathology compared to controls. Cell-type specific differential expression and pathway analysis identified both common and discrete changes in numerous pathways previously implicated in PSP and other neurodegenerative disorders. This included EIF2 signaling, an adaptive pathway activated in response to diverse stressors, which was the top activated pathway in vulnerable cell types. Using immunohistochemistry, we found that activated eIF2 was positively correlated with tau pathology burden in vulnerable brain regions. Multiplex immunofluorescence localized activated eIF2 positivity to hyperphosphorylated tau (p-tau) positive neurons and ALDH1L1-positive astrocytes, supporting the increased transcriptomic EIF2 activation observed in these vulnerable cell types. In conclusion, these data provide insights into cell-type-specific pathological changes in PSP and support the hypothesis that failure of adaptive stress pathways play a mechanistic role in the pathogenesis and progression of PSP.

Background: Although PSEN1 is best known for its role in Alzheimer's disease, it also regulates neural development and cerebellar morphogenesis. Medulloblastoma (MB) is the most common malignant pediatric brain tumor and arises from disrupted cerebellar developmental programs. The clinical significance of PSEN1 in MB remains unknown. We investigated the prognostic value and transcriptional correlates of PSEN1 expression across molecular subgroups and subtypes of MB. Methods: Public bulk and single-cell transcriptomic datasets were used to examine PSEN1 expression, associations with overall survival (OS), and transcriptional correlates in MB. The SHH -associated transcriptional pattern was evaluated in an independent cohort, and PSEN1 expression was further examined in the developing human cerebellum and across pediatric brain tumor types. Genes strongly correlated with PSEN1 in SHH MB were subjected to Gene Ontology (GO) enrichment analysis. Results: High PSEN1 expression was consistently associated with significantly longer OS exclusively in SHH MB. The PSEN1-associated transcriptional pattern was reproduced in an independent SHH cohort. PSEN1 was expressed across developing cerebellar cell populations and pediatric brain tumor types, with MB showing intermediate expression among the tumor entities examined. In SHH MB, PSEN1 was associated with a coordinated transcriptional program enriched for RNA homeostasis, intracellular membrane trafficking, protein quality control, lipid and calcium signaling, and developmental pathways. Conclusions: High PSEN1 expression identifies a favorable-prognosis subset of SHH MB and is associated with a distinct transcriptional program related to endomembrane organization and cellular homeostasis rather than canonical SHH signaling. These findings suggest that PSEN1 may mark a developmentally distinct tumor state and generate new hypotheses regarding subtype-specific developmental programs in MB.

Traumatic brain injury (TBI) is a major cause of mortality and long-term disability worldwide, giving rise to complex neurological complications that impact millions of individuals each year. Cellular stress and neuronal injury vary dramatically across cortical layers, vascular niches, and between the ipsilateral (injured) or contralateral (uninjured) hemispheres. There is a critical need for quantitative measures that capture the spatial distribution of injury-induced cellular changes, as well as the gene regulatory elements that drive them. Here, we developed OmicGlaze, an experimental and computational workflow for systematically profiling the spatial transcriptome and epigenome of mouse brains following mild traumatic brain injury. We established a spatial scoring system, and identified region-specific biological processes post injury, including changes in neuronal activities, cellular stress, immune response, and gliosis. Spatial assay for transposase-accessible chromatin with sequencing (Spatial ATAC-seq) generated the first epigenetic map of traumatic brain injury near single-cell resolution. Notably, we identified the Activator Protein-1 family transcription factor Atf3 as a key gene regulator of injury-induced cellular stress. Together, these spatial multi-omics analyses revealed gene regulatory network in TBI and provided a broadly applicable framework for dissecting cellular and molecular mechanisms underlying complex neurological disorders.

Kufs disease/CLN4 is an autosomal dominant neurodegenerative disorder that affects young adults, caused by mutations in the DNAJC5 gene that encodes the synaptic vesicle co-chaperone Cysteine String Protein (CSP/DNAJC5). The Leu115Arg and Leu116{Delta} mutations in humans are known to independently cause the disease, although the underlying mechanisms are unknown. To investigate the disease mechanisms in vivo, we generated three independent mouse lines overexpressing different versions of CSP/DNAJC5 under the neuron-specific Thy1 promoter: wild-type (WT), Leu115Arg, and Leu116{Delta}. Mice expressing mutant CSP/DNAJC5 are viable and do not show any significant increase in morbidity or mortality. However, we observed the presence of pathological lipofuscinosis in the mutants, indicated by autofluorescent punctate structures labeled with antibodies against ATP synthase subunit C, which were absent in the WT transgenic line. Additionally, transmission electron microscopy revealed intracellular structures resembling granular osmiophilic deposits (GRODs), observed in Kufs disease patients, in the mutants but not in non-transgenic controls or the WT transgenic mice. Notably, conventional, or conditional knockout mice lacking CSP/DNAJC5 did not exhibit any signs of increased lipofuscinosis or GRODs. Our novel mouse models thus provide a valuable tool to investigate the molecular mechanisms underlying Kufs disease/CLN4. We conclude that DNAJC5 mutations cause neuronal lipofuscinosis through a cell-autonomous gain of a novel but pathological function of CSP/DNAJC5.

Focal cortical dysplasias (FCDs) are local malformations of the human neocortex and a leading cause of intractable epilepsy. FCDs are classified into different subtypes including FCD IIa and IIb, characterized by a blurred gray-white matter boundary or a transmantle sign indicating abnormal white matter myelination. Recently, we have shown that myelination is also compromised in the gray matter of FCD IIa of the temporal lobe. Since myelination is key for brain function which is imbalanced in epilepsy, in the current study we investigated myelination in the gray matter of FCD IIa and IIb from the frontal lobe. We found that in particular FCD IIb showed myelination disturbances such as increased numbers of myelinating oligodendrocytes (OLs) and an irregular and disorganized myelination pattern covering an enlarged area in comparison to FCD IIa and controls. Interestingly, both FCD types presented with larger axon diameters when compared to controls. A significant correlation of axon diameter and myelin sheath thickness was found for FCD IIb and controls, whereas in FCD IIa large caliber axons were less myelinated. On the level of gene expression, FCD IIb presented with a significant up-regulation of myelin-associated mRNA synthesis in comparison to FCD IIa and by enhanced binding-capacities of the transcription factor MYRF to promoters of myelin-associated genes reflecting the need for more myelin due to increased axon diameters. These data show that FCD IIa and IIb are characterized by divergent signs of maladaptive myelination which may contribute to the epileptic phenotype. Main pointsO_LIIn the gray matter of the frontal lobe, FCD IIa and FCD IIb are characterized by divergent signs of maladaptive myelination. C_LIO_LIFCD IIa presents with an ordinary radial fiber pattern, but with a reduced thickness of the myelin sheath around large diameter axons and with an attenuation of the myelin synthesis machinery. C_LIO_LIFCD IIb is characterized by an irregular and disorganized myelin fiber pattern, a higher density of myelinating oligodendrocytes and an elevated transcriptional turnover of myelin-associated genes. C_LI

Microglia, the innate immune cells of the brain, are activated by damage or disease. In mouse models of amyotrophic lateral sclerosis (ALS), microglia shift from neurotrophic to neurotoxic states with disease progression. It remains unclear how human microglia change relative to the TAR DNA-binding protein 43 (TDP-43) aggregation that occurs in 97% of ALS cases. Here we examine spatial relationships between microglial activation and TDP-43 pathology in brain tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from the Neurological Foundation Human Brain Bank was obtained from 10 control and 10 ALS cases in parallel with brain tissue from a bigenic NFFH-tTA/tetO-hTDP-43{Delta}NLS (rNLS) mouse model of ALS at disease onset, early disease, and late disease stages. The spatiotemporal relationship between microglial activation and ALS pathology was determined by investigating microglial functional marker expression in brain regions with low and high TDP-43 burden at end-stage human disease: hippocampus and motor cortex, respectively. Sections were immunohistochemically labelled with a two-round multiplexed antibody panel against; microglial functional markers (L-ferritin, HLA-DR, CD74, CD68, and Iba1), a neuronal marker (NeuN), an astrocyte marker (GFAP), and pathological phosphorylated TDP-43 (pTDP-43). Single-cell levels of microglial functional markers were quantified using custom analysis pipelines and mapped to anatomical regions and ALS pathology. We identified a significant increase in microglial Iba1 and CD68 expression in the human ALS motor cortex, with microglial CD68 being significantly correlated with pTDP-43 pathology load. We also identified two subpopulations of microglia enriched in the ALS motor cortex that were defined by high L-ferritin expression. A similar pattern of microglial changes was observed in the rNLS mouse, with an increase first in CD68 and then in L-ferritin expression, with both occurring only after pTDP-43 inclusions were detectable. Our data strongly suggest that microglia are phagocytic at early-stage ALS but transition to a dysfunctional state at end-stage disease, and that these functional states are driven by pTDP-43 aggregation. Overall, these findings enhance our understanding of microglial phenotypes and function in ALS.

Astrocytes are central to central nervous system (CNS) homeostasis and memory consolidation. They also display remarkably heterogeneous morphological phenotypes, functions and molecular profiles between and within distinct regions of the human brain. Yet, their role in regional vulnerability in neurodegenerative diseases (NDDs) remains poorly understood. To elucidate this subregional heterogeneity of astrocytes in the hippocampus and parahippocampal cortex and its implication for neurodegeneration, we employed high-content neuropathology, integrating chromogenic immunohistochemistry (cIHC) and digital pathology, to map and quantify the expression of functional astrocytic markers (GFAP, ALDH1L1, AQP4, GPC5 and ALDH7A1) of healthy individuals, of patients with Alzheimers disease (AD) or Parkinsons disease with dementia (PDD). We found that astrocytic markers followed distinct expression patterns in AD and PDD. In AD, GFAP was strongly reduced in specific hippocampal regions, whereas AQP4 was increased and GPC5 expression, although regionally stable, was locally associated with amyloid and tau pathology. In PDD, astrocytic responses were characterized by selective decreases in ALDH1L1 and ALDH7A1, with GFAP and GPC5 remaining largely unaffected. Notably, GFAP and GPC5 expression delineated distinct astrocytic subtypes that were differentially distributed across hippocampal subfields and showed specific responses to AD and PD pathologies. These findings provide new insights into the landscape of astrocytic heterogeneity in the human hippocampus and beyond, revealing disease- and region-specific astrocytic signatures. Importantly, they underscore the value of incorporating novel markers such as GPC5 to fully capture astrocytic diversity and to better understand astrocyte contributions to neurodegeneration.

BackgroundLate-onset Alzheimers disease (LOAD) accounts for more than 95% of AD cases. Previously, we have described that 6% of AD patients present CAG intermediate alleles in the huntingtin gene (HTT IAs). The caudate nucleus, the most affected region in Huntingtons disease, is highly sensitive to these HTT CAG expansions, as they can induce epigenetic changes, including altered microRNA profiles. All this implies a potential source of gene expression deregulation, affecting disease onset and/or progression in LOAD patients with HTT IAs. MethodsWe genotyped HTT CAG repeats and Apolipoprotein E (APOE) in postmortem brain frozen samples from 323 LOAD patients and 335 healthy controls. From them, we selected caudate samples of HTT IA carrier and non-carrier LOAD patients and controls, with neuropathological study, and performed next-generation microRNA sequencing, in silico target prediction and pathway analysis, followed by molecular and histopathological studies. ResultsOur study revealed that the presence of HTT IAs decreases survival in LOAD patients after disease onset. MicroRNA profiles in the caudate nucleus are altered in all LOAD compared to the control cases but are more pronounced in HTT IAs carriers. In silico analysis suggests that the microRNAs expressed differentially in HTT IAs carriers regulate key components of the spliceosome, affecting splicing factors of the SRSF family or the nuclear FUS-SFPQ complex, which was confirmed by different techniques. This leads to an increase in Tau 3R protein, conducting to a higher presence of ghost tangles, the last state of neurofibrillary tangles, in LOAD patients with HTT IAs. In addition, they also present a higher number of HTT-positive neurons in a CAG repeat expansion-dependent manner. ConclusionsOur findings demonstrate a synergistic effect of HTT IAs and miRNAs deregulation in the evolution of tau pathology, which could be related to an accelerated misprocessing and subsequent aggregation of the Tau 3R isoform, favoring a subsequent faster disease progression. The incorporation of genetic screening for HTT alleles into clinical practice would allow a more accurate classification of LOAD patients, facilitating the design of personalized therapeutic interventions and improving the prospects for the management of this debilitating disease.

In Alzheimers disease (AD), a central role is given to the extracellular deposition of A{beta} peptides, remotely produced by the proteolysis of the amyloid precursor protein (APP). This contrasts with other neurodegenerative diseases which are characterized by the intraneuronal aggregation of full-length proteins such as huntingtin, -synuclein or TDP-43. Importantly, the distribution of APP around amyloid plaques is poorly characterized. Here, we combined an extensive set of methodological and analytical tools to investigate neuropathological features of APP in the human AD hippocampus and in two mouse models of AD. We report that APP remarkably accumulates in the surrounding of dense-core amyloid plaques together with the secretases necessary to produce A{beta} peptides. In addition, the Nter domain, but not the Cter domain of APP is enriched in the core of amyloid plaques uncovering a potential pathological role of the secreted APP-Nter in dense-core plaques. To investigate the subcellular compartment in which APP accumulates, we labelled neuritic and synaptic markers and report an enrichment in presynaptic proteins (Syt1, VAMP2) and phosphorylated-Tau. Ultrastructural analysis of APP accumulations reveals abundant multivesicular bodies containing presynaptic vesicles proteins and autophagosomal built-up of APP. Altogether, our data supports a role of presynaptic APP in AD pathology and highlights APP accumulations as a potential source of A{beta} and Nter peptides to fuel amyloid plaques.

DNA damage and DNA damage repair (DDR) dysfunction are insults with broad implications on cellular physiology, including in proteostasis, and have been recently implicated in many neurodegenerative diseases. Alpha-synuclein (aSyn), a pre-synaptic and nuclear protein associated with neurodegenerative disorders known as synucleinopathies, has been implicated in DNA double strand break (DSB) repair function. Consistently, DSB induction has been demonstrated in cell and animal models of synucleinopathy. Nevertheless, the types of DNA damage and the contribution of DNA damage towards Lewy body (LB) formation in synucleinopathies are unknown. Here, we demonstrate the increase of DSB in neuronal and non-neuronal cellular populations of post-mortem temporal cortex tissue from dementia with Lewy body (DLB) patients and demonstrate increases in DSBs early at a presymptomatic age of aSyn transgenic mice. Strikingly, in postmortem DLB tissue, DNA damage-derived ectopic cytoplasmic genomic material (eCGM) was evident within the majority of LBs examined. The observed cellular pathology was consistent with nucleoproteasomal upregulation of associated DNA damage repair proteins, particularly in base excision repair and DSB repair pathways. Collectively our study demonstrates the early occurrence of DNA damage and associated nucleoproteasomal changes in response to nuclear aSyn pathology. Furthermore, the data suggests a potential involvement for DNA damage derived eCGM for the facilitation of cytoplasmic aSyn aggregates. Ultimately, uncovering pathological mechanisms underlying DNA damage in DLB sheds light into novel disease mechanisms and opens novel possibilities for diagnosing and treating synucleinopathies.