Autopsy Brain Tissue Research Articles

Brain and serum lipidomic profiles implicate Lands cycle acyl chain remodeling association with APOEε4 and mild cognitive impairment.

At least one-third of the identified risk alleles from Genome-Wide Association Studies (GWAS) of Alzheimer's disease (AD) are involved in lipid metabolism, lipid transport, or direct lipid binding. In fact, a common genetic variant (ε4) in a cholesterol and phospholipid transporter, Apolipoprotein E (APOEε4), is the primary genetic risk factor for late-onset AD. In addition to genetic variants, lipidomic studies have reported severe metabolic dysregulation in human autopsy brain tissue, cerebrospinal fluid, blood, and multiple mouse models of AD. We aimed to identify an overarching metabolic pathway in lipid metabolism by integrating analyses of lipidomics and transcriptomics from the Religious Order Study and Rush Memory Aging Project (ROSMAP) using differential analysis and network correlation analysis. Coordinated differences in lipids were found to be dysregulated in association with both mild cognitive impairment (MCI) and APOEε4 carriers. Interestingly, these correlations were weakened when adjusting for education. Indeed, the cognitively non-impaired APOEε4 carriers have higher education levels in the ROSMAP cohort, suggesting that this lipid signature may be associated with a resilience phenotype. Network correlation analysis identified multiple differential lipids within a single module that are substrates and products in the Lands Cycle for acyl chain remodeling. In addition, our analyses identified multiple genes in the Lands Cycle acyl chain remodeling pathway, which were associated with cognitive decline independent of amyloid-β (Aβ) load and tau tangle pathologies. Our studies highlight the critical differences in acyl chain remodeling in brain tissue from APOEε4 carriers and individual non-carriers with MCI. A coordinated lipid profile shift in dorsolateral prefrontal cortex from both APOEε4 carriers and MCI suggests differences in lipid metabolism occur early in disease stage and highlights lipid homeostasis as a tractable target for early disease modifying intervention.

In pursuit of degenerative brain disease diagnosis: Dementia biomarkers detected by DNA aptamer-attached portable graphene biosensor.

Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aβ), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aβ, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aβ, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.

Enhanced Creutzfeldt-Jakob disease surveillance in the older population: Assessment of a protocol for screening brain tissue donations for prion disease.

Human prion diseases, including Creutzfeldt-Jakob disease (CJD), occur in sporadic, genetic, and acquired forms. Variant Creutzfeldt-Jakob disease (vCJD) first reported in 1996 in the United Kingdom (UK), resulted from contamination of food with bovine spongiform encephalopathy. There is a concern that UK national surveillance mechanisms might miss some CJD cases (including vCJD), particularly in the older population where other neurodegenerative disorders are more prevalent. We developed a highly sensitive protocol for analysing autopsy brain tissue for the misfolded prion protein (PrPSc ) associated with prion disease, which could be used to screen for prion disease in the elderly. Brain tissue samples from 331 donors to the Edinburgh Brain and Tissue Bank (EBTB), from 2005 to 2022, were analysed, using immunohistochemical analysis on fixed tissue, and five biochemical tests on frozen specimens from six brain regions, based on different principles for detecting PrPSc . An algorithm was established for classifying the biochemical results. To test the effectiveness of the protocol, several neuropathologically confirmed prion disease controls, including vCJD, were included and blinded in the study cohort. On unblinding, all the positive control cases had been correctly identified. No other cases tested positive; our analysis uncovered no overlooked prion disease cases. Our algorithm for classifying cases was effective for handling anomalous biochemical results. An overall analysis suggested that a reduced biochemical protocol employing only three of the five tests on only two brain tissue regions gave sufficient sensitivity and specificity. We conclude that this protocol may be useful as a UK-wide screening programme for human prion disease in selected brains from autopsies in the elderly. Further improvements to the protocol were suggested by enhancements of the in vitro conversion assays made during the course of this study.

Integrated Analysis of Immune Infiltration and Hub Pyroptosis-Related Genes for Multiple Sclerosis.

Studies on overall immune infiltration and pyroptosis in patients with multiple sclerosis (MS) are limited. This study explored immune cell infiltration and pyroptosis in MS using bioinformatics and experimental validation. The GSE131282 and GSE135511 microarray datasets including brain autopsy tissues from controls and MS patients were downloaded for bioinformatic analysis. The gene expression-based deconvolution method, CIBERSORT, was used to determine immune infiltration. Differentially expressed genes (DEGs) and functional enrichments were analyzed. We then extracted pyroptosis-related genes (PRGs) from the DEGs by using machine learning strategies. Their diagnostic ability for MS was evaluated in both the training set (GSE131282 dataset) and validation set (GSE135511 dataset). In addition, messenger RNA (mRNA) expression of PRGs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in cortical tissue from an experimental autoimmune encephalomyelitis (EAE) model of MS. Moreover, the functional enrichment pathways of each hub PRG were estimated. Finally, co-expressed competitive endogenous RNA (ceRNA) networks of PRGs in MS were constructed. Among the infiltrating cells, naive CD4+ T cells (P=0.006), resting NK cells (P=0.002), activated mast cells (P=0.022), and neutrophils (P=0.002) were significantly higher in patients with MS than in controls. The DEGs of MS were screened. Analysis of enrichment pathways showed that the pathways of transcriptional regulatory mechanisms and ion channels associating with pyroptosis. Four PRGs genes CASP4, PLCG1, CASP9 and NLRC4 were identified. They were validated in both the GSE135511 dataset and the EAE model by using qRT-PCR. CASP4 and NLRC4 were ultimately identified as stable hub PRGs for MS. Single-gene Gene Set Enrichment Analysis showed that they mainly participated in biosynthesis, metabolism, and organism resistance. ceRNA networks containing CASP4 and NLRC4 were constructed. MS was associated with immune infiltration. CASP4 and NLRC4 were key biomarkers of pyroptosis in MS.

Landscape of Double-Stranded DNA Breaks in Postmortem Brains from Alzheimer's Disease and Non-Demented Individuals.

Alzheimer's disease (AD) brains accumulate DNA double-strand breaks (DSBs), which could contribute to neurodegeneration and dysfunction. The genomic distribution of AD brain DSBs is unclear. To map genome-wide DSB distributions in AD and age-matched control brains. We obtained autopsy brain tissue from 3 AD and 3 age-matched control individuals. The donors were men between the ages of 78 to 91. Nuclei extracted from frontal cortex tissue were subjected to Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay with an antibody against γH2AX, a marker of DSB formation. γH2AX-enriched chromatins were purified and analyzed via high-throughput genomic sequencing. The AD brains contained 18 times more DSBs than the control brains and the pattern of AD DSBs differed from the control brain pattern. In conjunction with published genome, epigenome, and transcriptome analyses, our data revealed aberrant DSB formation correlates with AD-associated single-nucleotide polymorphisms, increased chromatin accessibility, and upregulated gene expression. To our knowledge, this study is the first to characterize the AD brain DSB landscape. Our data suggest in AD, an accumulation of DSBs at ectopic genomic loci could contribute to an aberrant upregulation of gene expression.

Circulating SARS-CoV-2+ megakaryocytes are associated with severe viral infection in COVID-19

AbstractSeveral independent lines of evidence suggest that megakaryocytes are dysfunctional in severe COVID-19. Herein, we characterized peripheral circulating megakaryocytes in a large cohort of inpatients with COVID-19 and correlated the subpopulation frequencies with clinical outcomes. Using peripheral blood, we show that megakaryocytes are increased in the systemic circulation in COVID-19, and we identify and validate S100A8/A9 as a defining marker of megakaryocyte dysfunction. We further reveal a subpopulation of S100A8/A9+ megakaryocytes that contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein and RNA. Using flow cytometry of peripheral blood and in vitro studies on SARS-CoV-2–infected primary human megakaryocytes, we demonstrate that megakaryocytes can transfer viral antigens to emerging platelets. Mechanistically, we show that SARS-CoV-2–containing megakaryocytes are nuclear factor κB (NF-κB)-activated, via p65 and p52; express the NF-κB–mediated cytokines interleukin-6 (IL-6) and IL-1β; and display high surface expression of Toll-like receptor 2 (TLR2) and TLR4, canonical drivers of NF-κB. In a cohort of 218 inpatients with COVID-19, we correlate frequencies of megakaryocyte subpopulations with clinical outcomes and show that SARS-CoV-2–containing megakaryocytes are a strong risk factor for mortality and multiorgan injury, including respiratory failure, mechanical ventilation, acute kidney injury, thrombotic events, and intensive care unit admission. Furthermore, we show that SARS-CoV-2+ megakaryocytes are present in lung and brain autopsy tissues from deceased donors who had COVID-19. To our knowledge, this study offers the first evidence implicating SARS-CoV-2+ peripheral megakaryocytes in severe disease and suggests that circulating megakaryocytes warrant investigation in inflammatory disorders beyond COVID-19.

Neuropathology of microbleeds in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

Cerebral microbleeds (CMBs) detected on magnetic resonance imaging are common in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The neuropathologic correlates of CMBs are unclear. In this study, we characterized findings relevant to CMBs in autopsy brain tissue of 8 patients with genetically confirmed CADASIL and 10 controls within the age range of the CADASIL patients by assessing the distribution and extent of hemosiderin/iron deposits including perivascular hemosiderin leakage (PVH), capillary hemosiderin deposits, and parenchymal iron deposits (PID) in the frontal cortex and white matter, basal ganglia and cerebellum. We also characterized infarcts, vessel wall thickening, and severity of vascular smooth muscle cell degeneration. CADASIL subjects had a significant increase in hemosiderin/iron deposits compared with controls. This increase was principally seen with PID. Hemosiderin/iron deposits were seen in the majority of CADASIL subjects in all brain areas. PVH was most pronounced in the frontal white matter and basal ganglia around small to medium sized arterioles, with no predilection for the vicinity of vessels with severe vascular changes or infarcts. CADASIL subjects have increased brain hemosiderin/iron deposits but these do not occur in a periarteriolar distribution. Pathogenesis of these lesions remains uncertain.

Human microRNA‐298 plays a vital role in maintaining APP and tau homeostasis and IL‐induced inflammation: Implication in Alzheimer’s disease.

AbstractBackgroundAlzheimer’s disease (AD) is marked by neurofibrillary tangles mainly composed of hyperphosphorylated microtubule‐associated protein tau; senile plaques comprising amyloid β (Aβ) peptides; and neuroinflammation. How these pathological changes are regulated remains unclear. Genetic, epigenetic, and environmental factors play important roles in disease progression. MicroRNA (miRNA), a group of small non‐coding RNAs, regulates multiple biological and pathological processes by binding to target mRNAs and altering translation levels. Disrupting this process could contribute to AD pathogenesis. We hypothesize that miR‐298 plays a vital role in maintaining APP and tau homeostasis and IL‐induced inflammation, particularly gliosis.MethodWe performed miR‐298 treatments and blockade by antisense‐miR298 (antagomiRs) in 3’UTR‐reporter clone‐transfected cultures and protein levels from target native mRNAs (Chopra et al‐Mol. Psychiatry‐2021). We transfected human astrocyte cells with miR‐298 mimics or antagomiRs. We analyzed culture media and lysates for protein and mRNA. We performed ELISA and real‐time‐qPCR assays to determine cytokines, APP and tau levels. We analyzed autopsy brain tissues from non‐cognitively impaired (NCI) and AD subjects by measuring levels of miR‐298, target mRNAs and proteins in cortical samples, as we described (Wang et al‐Mol. Psychiatry‐2022).ResultWe identified putative sites for miR‐298 in mRNAs encoding APP, BACE1, MAPT, IL1α, and IL6. Treatment by miR‐298 significantly increased production and secretion of multiple pro‐inflammatory cytokines, specifically IL‐1, IL‐6 and TNF‐α in human astrocyte cultures. Furthermore, treatment by miR‐298 reduced tau, APP and BACE1 protein and mRNA levels in astrocyte cultures. Levels of miR‐298 varied in postmortem human temporal lobe between AD patients and age‐matched NCI controls.ConclusionMiR‐298 induced a variety of pro‐inflammatory cytokines without administration of other challenges. Induction of multiple cytokines may be due to combinations of direct miRNA activity and regulation of upstream signaling pathways. MiR‐298 may play an important biological role in AD by reducing APP, BACE1 and tau expression while increasing the physiologic production of IL cytokines. This apparent paradox is resolved by noting that early‐stage (prodromal) gliosis may contribute to the clearance of toxic aggregates. We plan to test miR‐298 in AD animal models, including human tau‐overexpressing transgenic mice.We appreciate NIH‐NIA grants: (R01AG051086, R21AG056007, R56AG072810, P01AG014449, P30AG072976).

Evaluation of pathologies associated with hyperhomocysteinemia in human autopsy brain tissue

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) are one of the leading causes of dementia; VCID affects roughly 10‐40% of all dementia patients. A major, yet underrecognized, modifiable risk factor for VCID is hyperhomocysteinemia (HHcy). Defined as elevated levels of plasma homocysteine (a non‐protein‐forming amino acid), most late‐life HHcy is caused by impaired B vitamin absorption. Although HHcy has been recognized as a risk factor for VCID, studies aimed at identifying pathologies associated with HHcy have been lacking.MethodTo determine pathologies associated with HHcy, we identified 31 autopsied research volunteers with antemortem homocysteine levels; 13 cases had normal plasma homocysteine levels (>14μmol/L) and 18 had high plasma homocysteine levels (<14μmol/L). We then measured levels of homocysteine and related metabolites in both plasma samples taken closest to autopsy and frontal cortex. Next, we determined whether the level of plasma homocysteine was associated with several markers identified via immunohistochemistry, including Aβ, PHF‐1, IBA‐1 and GFAP. Plasma and brain protein markers for inflammation and angiogenesis were also measured to determine associations with plasma homocysteine. Finally, we used the Human Neuroinflammation NanoString panel to determine gene expression changes of inflammatory markers associated with high homocysteine levels in the frontal cortex and occipital lobe.ResultPlasma metabolite analysis showed patients who had elevated levels of homocysteine also had increased levels of several homocysteine cycle metabolites such as cysteine, S‐adenosyl‐homocysteine, cystathionine and choline. Flt1, an angiogenic marker, and IL5, an inflammatory marker, had a positive correlation with increased plasma homocysteine. No correlation between IBA‐1 or GFAP immunohistochemistry with plasma homocysteine was found. Gene expression showed that most genes were downregulated in the presence of high plasma homocysteine, including many significant genes involved in apoptosis, growth factor and cytokine signaling, and the innate and adaptive immune response.ConclusionThese preliminary data show that increased plasma homocysteine correlates with protein inflammatory and angiogenic markers and with a significant downregulation of inflammation‐related gene expression markers in the brain. Overall, this could reflect impaired normal immune function, providing possible mechanisms by which hyperhomocysteinemia induces cognitive deficits and cerebrovascular damage.

Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features.

The amplification of CNS damage in Alzheimer's disease due to SARS-CoV2 infection

Pre-existing Alzheimer's disease is a risk factor for severe/fatal COVID-19 and infection by SARS-CoV2 virus has been associated with an increased incidence of un-masked Alzheimer's disease. The molecular basis whereby SARS-CoV2 may amplify Alzheimer's disease is not well understood. This study analyzed the molecular changes in autopsy brain tissues from people with pre-existing dementia who died of COVID-19 (n = 5) which was compared to equivalent tissues of people who died of COVID-19 with no history of dementia (n = 8), Alzheimer's disease pre-COVID-19 (n = 10) and aged matched controls (n = 10) in a blinded fashion. Immunohistochemistry analyses for hyperphosphorylated tau protein, α-synuclein, and β-amyloid-42 confirmed the diagnoses of Alzheimer's disease (n = 4), and Lewy body dementia (n = 1) in the COVID-19 group. The brain tissues from patients who died of COVID-19 with no history of dementia showed a diffuse microangiopathy marked by endocytosis of spike subunit S1 and S2 in primarily CD31+ endothelia with strong co-localization with ACE2, Caspase-3, IL6, TNFα, and Complement component 6 that was not associated with SARS-CoV2 RNA. Microglial activation marked by increased TMEM119 and MCP1 protein expression closely paralleled the endocytosed spike protein. The COVID-19 tissues from people with no pre-existing dementia showed, compared to controls, 5-10× fold increases in expression of neuronal NOS and NMDAR2 as well as a marked decrease in the expression of proteins whose loss is associated with worsening Alzheimer's disease: MFSD2a, SHIP1, BCL6, BCL10, and BACH1. In COVID-19 tissues from people with dementia the widespread spike-induced microencephalitis with the concomitant microglial activation co-existed in the same areas where neurons had hyperphosphorylated tau protein suggesting that the already dysfunctional neurons were additionally stressed by the SARS-CoV2 induced microangiopathy. ACE2+ human brain endothelial cells treated with high dose (but not vaccine equivalent low dose) spike S1 protein demonstrated each of the molecular changes noted in the in vivo COVID-19 and COVID-19/Alzheimer's disease brain tissues. It is concluded that fatal COVID-19 induces a diffuse microencephalitis and microglial activation in the brain due to endocytosis of circulating viral spike protein that amplifies pre-existing dementia in at least two ways: 1) modulates the expression of proteins that may worsen Alzheimer's disease and 2) stresses the already dysfunctional neurons by causing an acute proinflammatory/hypercoagulable/hypoxic microenvironment in areas with abundant hyperphosphorylated tau protein and/or βA-42.

Methamphetamine and HIV-1 Tat proteins synergistically induce microglial autophagy via activation of the Nrf2/NQO1/HO-1 signal pathway

Methamphetamine (METH) is a psychostimulant that is abused throughout the world. METH is a highly addictive drug commonly used by persons living with HIV, and its use can result in cognitive impairment and memory deficits. METH and human immunodeficiency virus-1 transactivator of transcription (HIV-1Tat) have toxic and synergistic effects on the nervous system; however, the mechanism of their synergistic effects has not been clarified. We used BV2 cells, primary microglia, Nrf2-KO C57BL/6J mice, and autopsied brain tissues of METH-abusing, HIV infection, and METH-abusing individuals comorbid with HIV to explore the regulatory role of Nrf2/NQO1/HO-1 signal pathway on microglia autophagy. Our results showed that microglia were significantly activated by METH and HIV-1Tat protein. METH and HIV-1Tat protein combination significantly increase the autophagy-related proteins (LC3-II, Beclin-1, ATG5, and ATG7) expression in microglia and striatum of C57BL/6J mice. After silencing or knocking out the Nrf2 gene, the expression levels of autophagy-related proteins were significantly increased. In human brain tissue, microglia were activated, Nrf2, LC3-II, and Beclin-1 expression levels were raised, and the p62 expression level was decreased. Our results suggested that METH and HIV or HIV-1Tat synergistically affect autophagy. And the Nrf2 pathway plays a vital role in regulating the synergistic induction of microglial autophagy by METH and HIV-1Tat protein. This study may provide a theoretical basis and new ideas for effective targets for pharmacological intervention in HIV-infected patients with drug abuse.

Interpretable deep learning of myelin histopathology in age-related cognitive impairment

Age-related cognitive impairment is multifactorial, with numerous underlying and frequently co-morbid pathological correlates. Amyloid beta (Aβ) plays a major role in Alzheimer’s type age-related cognitive impairment, in addition to other etiopathologies such as Aβ-independent hyperphosphorylated tau, cerebrovascular disease, and myelin damage, which also warrant further investigation. Classical methods, even in the setting of the gold standard of postmortem brain assessment, involve semi-quantitative ordinal staging systems that often correlate poorly with clinical outcomes, due to imperfect cognitive measurements and preconceived notions regarding the neuropathologic features that should be chosen for study. Improved approaches are needed to identify histopathological changes correlated with cognition in an unbiased way. We used a weakly supervised multiple instance learning algorithm on whole slide images of human brain autopsy tissue sections from a group of elderly donors to predict the presence or absence of cognitive impairment (n = 367 with cognitive impairment, n = 349 without). Attention analysis allowed us to pinpoint the underlying subregional architecture and cellular features that the models used for the prediction in both brain regions studied, the medial temporal lobe and frontal cortex. Despite noisy labels of cognition, our trained models were able to predict the presence of cognitive impairment with a modest accuracy that was significantly greater than chance. Attention-based interpretation studies of the features most associated with cognitive impairment in the top performing models suggest that they identified myelin pallor in the white matter. Our results demonstrate a scalable platform with interpretable deep learning to identify unexpected aspects of pathology in cognitive impairment that can be translated to the study of other neurobiological disorders.

Metabolites and Biomarker Compounds of Neurodegenerative Diseases in Cerebrospinal Fluid.

Despite recent advances in diagnostic procedures for neurological disorders, it is still difficult to definitively diagnose some neurodegenerative diseases without neuropathological examination of autopsied brain tissue. As pathological processes in the brain are frequently reflected in the components of cerebrospinal fluid (CSF), CSF samples are sometimes useful for diagnosis. After CSF is secreted from the choroid plexus epithelial cells in the ventricles, some flows in the brain, some is mixed with intracerebral interstitial fluid, and some is excreted through two major drainage pathways, i.e., the intravascular periarterial drainage pathway and the glymphatic system. Accordingly, substances produced by metabolic and pathological processes in the brain may be detectable in CSF. Many papers have reported changes in the concentration of substances in the CSF of patients with metabolic and neurological disorders, some of which can be useful biomarkers of the disorders. In this paper, we show the significance of glucose- and neurotransmitter-related CSF metabolites, considering their transporters in the choroid plexus; summarize the reported candidates of CSF biomarkers for neurodegenerative diseases, including amyloid-β, tau, α-synuclein, microRNAs, and mitochondrial DNA; and evaluate their potential as efficient diagnostic tools.

Neuropathology and virus in brain of SARS-CoV-2 infected non-human primates

Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren’t well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with “long COVID”. Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.

Brain virtual histology with X-ray phase-contrast tomography Part II:3D morphologies of amyloid-β plaques in Alzheimer's disease models.

While numerous transgenic mouse strains have been produced to model the formation of amyloid-β (Aβ) plaques in the brain, efficient methods for whole-brain 3D analysis of Aβ deposits have to be validated and standardized. Moreover, routine immunohistochemistry performed on brain slices precludes any shape analysis of Aβ plaques, or require complex procedures for serial acquisition and reconstruction. The present study shows how in-line (propagation-based) X-ray phase-contrast tomography (XPCT) combined with ethanol-induced brain sample dehydration enables hippocampus-wide detection and morphometric analysis of Aβ plaques. Performed in three distinct Alzheimer mouse strains, the proposed workflow identified differences in signal intensity and 3D shape parameters: 3xTg displayed a different type of Aβ plaques, with a larger volume and area, greater elongation, flatness and mean breadth, and more intense average signal than J20 and APP/PS1. As a label-free non-destructive technique, XPCT can be combined with standard immunohistochemistry. XPCT virtual histology could thus become instrumental in quantifying the 3D spreading and the morphological impact of seeding when studying prion-like properties of Aβ aggregates in animal models of Alzheimer's disease. This is Part II of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part I shows how in-line XPCT enables 3D myelin mapping in the whole rodent brain and in human autopsy brain tissue.

Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation

The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases.

Coronin 1A facilitates calcium mobilization and promotes astrocyte reactivity in HIV-1 neuropathogenesis.

Astrocyte reactivity, a phenomenon observed in a variety of neurodegenerative disorders, can have both beneficial and detrimental manifestations which significantly affect neuronal physiology. In neuroAIDS, reactive astrocytes have been observed to severely affect the neuronal population present in their vicinity. Calcium signaling plays a central role in mediating astrocyte reactivity. Coronin 1A, an actin‐binding protein, majorly reported in hematopoietic cells, regulates cell activity in a calcium‐dependent manner, but its role in astrocyte physiology and reactivity is largely unknown. Using a well‐characterized primary culture of human astroglia and neurons, we explored the roles of coronin 1A in astrocyte physiology and its involvement in facilitating astrocyte reactivity. In this study, we report coronin 1A expression in human primary astrocytes and autopsy brain sections, and that it plays activity‐dependent roles by facilitating calcium mobilization from the intracellular stores. HIV‐1 Tat, a potent neurotoxicant that turns astrocytes reactive, augments coronin 1A expression, apart from affecting GFAP and pro‐inflammatory molecules. Also, the autopsy brain tissue of HIV‐1 infected individuals has a higher expression of coronin 1A. Downregulation of coronin 1A attenuated the HIV‐1 Tat‐induced deleterious effects of reactive astrocytes, measured as the upregulated expression of GFAP, pro‐inflammatory molecules, and enhanced release of IL‐6, and hence reduced astrocyte‐mediated neurodegeneration. Our findings also suggest that out of a pool of dysregulated miRNAs studied by us, hsa‐miR‐92b‐5p regulates coronin 1A expression under the effect of HIV‐1 Tat. These findings highlight the novel roles of coronin 1A in regulating astrocyte activity in stimulated conditions and astrocyte reactivity observed in HIV‐1 neuropathogenesis.

Borrelia burgdorferi Co-Localizing with Amyloid Markers in Alzheimer's Disease Brain Tissues.

Background: Infections by bacterial or viral agents have been hypothesized to influence the etiology of neurodegenerative diseases.Objective: This study examined the potential presence of Borrelia burgdorferi spirochete, the causative agent of Lyme disease, in brain autopsy tissue of patients diagnosed with either Alzheimer’s (AD) or Parkinson’s diseases.Methods: Brain tissue sections from patients with age-matched controls were evaluated for antigen and DNA presence of B. burgdorferi using various methods. Positive Borrelia structures were evaluated for co-localization with biofilm and AD markers such as amyloid and phospho-tau (p-Tau) using immunohistochemical methods.Results: The results showed the presence of B. burgdorferi antigen and DNA in patients with AD pathology and among those, one of them was previously diagnosed with Lyme disease. Interestingly, a significant number of Borrelia-positive aggregates with a known biofilm marker, alginate, were found along with the spirochetal structures. Our immunohistochemical data also showed that Borrelia-positive aggregates co-localized with amyloid and phospho-tau markers. To further prove the potential relationship of B. burgdorferi and amyloids, we infected two mammalian cell lines with B. burgdorferi which resulted in a significant increase in the expression of amyloid-β and p-Tau proteins in both cells lines post-infection.Conclusion: These results indicate that B. burgdorferi can be found in AD brain tissues, not just in spirochete but a known antibiotics resistant biofilm form, and its co-localized amyloid markers. In summary, this study provides evidence for a likely association between B. burgdorferi infections and biofilm formation, AD pathology, and chronic neurodegenerative diseases.

Genetic expression changes and pathologic findings associated with hyperhomocysteinemia in human autopsy brain tissue.

Vascular contributions to cognitive impairment and dementia (VCID) are a leading cause of dementia. An underappreciated, modifiable risk factor for VCID is hyperhomocysteinemia (HHcy), defined by elevated levels of plasma homocysteine, most often due to impaired B vitamin absorption in aged persons. Studies aimed at identifying neuropathologic features and gene expression profiles associated with HHcy have been lacking. A subset of research volunteers from the University of Kentucky Alzheimer's Disease Research Center longitudinal cohort came to autopsy and had ante mortem plasma homocysteine levels available. Brain tissue and blood plasma drawn closest to death were used to measure homocysteine and related metabolites in the current pilot study. Genetic expression profiles of inflammatory markers were evaluated using the Human Neuroinflammation NanoString panel. Further analyses included an evaluation of plasma homocysteine effects on amyloid beta, tau, ionized calcium-binding adaptor molecule 1, and glial fibrillary acidic protein immunohistochemistry in the frontal and occipital cortices. Analytes and other study outcomes were evaluated in relation to ante mortem HHcy status: We identified 13 persons with normal ante mortem plasma homocysteine levels (<14µmol/L) and 18 who had high plasma homocysteine levels (≥14µmol/L). Participants with HHcy demonstrated increased levels of several plasma homocysteine cycle metabolites such as total cysteine, S-adenosyl-homocysteine, cystathionine, and choline. Inflammatory gene expression profiles showed a general downregulation in the setting of elevated plasma homocysteine. HHcy was associated with more and longer microglial processes, but smaller and fewer astrocytes, especially in participants of older age at death. HHcy in older participants was also associated with occipital cortex microhemorrhages and increased severity of atherosclerosis throughout the cerebral vasculature. Increased plasma homocysteine and older age were associated with the downregulation of inflammatory gene expression markers in association with significant glial and vascular pathology changes. Impaired immune function is a plausible mechanism by which HHcy increases cerebrovascular damage leading to impaired cognitive function.