Human Postmortem Brain Samples Research Articles

Peptidoglycan accumulates in distinct brain regions and cell types over lifetime but is absent in newborns

Peptidoglycan (PGN) is a large complex polymer critical to structure and function of all bacterial species. Intact PGN and its fragments are inflammatory, contributing to infectious and autoimmune disease. Recent studies show that PGN physiologically contributes to immune setpoints, and importantly also to mouse brain development and behavior. However, for the human brain, it remains unknown whether PGN and its fragments differentially gain access to distinct brain regions, which cell types accumulate it, and whether PGN brain load varies with age. Therefore, we investigated human postmortem brain samples of donors with an extensive age range, from newborns to nonagenarians. We examined two monoclonal antibodies against PGN which were validated using dot blot analysis, competition assays and immunofluorescence experiments on bacteria sacculi, which jointly showed specific detection of Gram-positive PGN. As positive reference tissue, brain tissue from sepsis patients, and human liver were used, both showing the expected high PGN levels. In adult brain tissue of different age (34- to 94-year-old) and sex, we detected PGN signals in seven different brain regions, with highest loads in the occipital cortex, hippocampal formation, frontal cortex, the periventricular region and the olfactory bulb. Age-dependent increase of signals was not evident by microscopic observations and only weak correlation was found by statistical analysis in this cohort. PGN was found intracellularly in the cytoplasm surrounding the cell nucleus in astrocytes, oligodendrocytes, neurons, and endothelial cells, but not in macrophages like microglia. PGN was absent in brain tissues of three human newborns (stillbirth to four weeks old). For comparison, three brain regions from non-human primates of varying age (newborn to 21 years) were immunohistochemically stained. The highest PGN-load was observed in brain tissue from 18- to 21-year-old macaques.This first systematic evaluation of PGN in human postmortem brain suggests that PGN accumulates during lifetime until it reaches a plateau by homeostatic turnover and highlights the ubiquitous presence of PGN in human brain tissues, and their ability to participate in physiological as well as pathological processes throughout life.

Emerging signs of Alzheimer-like tau hyperphosphorylation and neuroinflammation in the brain post recovery from COVID-19.

Coronavirus disease 2019 (COVID-19) has been suggested to increase the risk of memory decline and Alzheimer's disease (AD), the main cause of dementia in the elderly. However, direct evidence about whether COVID-19 induces AD-like neuropathological changes in the brain, especially post recovery from acute infection, is still lacking. Here, using postmortem human brain samples, we found abnormal accumulation of hyperphosphorylated tau protein in the hippocampus and medial entorhinal cortex within 4-13 months post clinically recovery from acute COVID-19, together with prolonged activation of glia cells and increases in inflammatory factors, even though no SARS-COV-2 invasion was detected in these regions. By contrast, COVID-19 did not change beta-amyloid deposition and hippocampal neuron number, and had limited effects on AD-related pathological phenotypes in olfactory circuits including olfactory bulb, anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. These results provide neuropathological evidences linking COVID-19 with prognostic increase of risk for AD.

Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W.

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.

Low-cost and scalable projected light-sheet microscopy for the high-resolution imaging of cleared tissue and living samples.

Light-sheet fluorescence microscopy (LSFM) is a widely used technique for imaging cleared tissue and living samples. However, high-performance LSFM systems are typically expensive and not easily scalable. Here we introduce a low-cost, scalable and versatile LSFM framework, which we named 'projected light-sheet microscopy' (pLSM), with high imaging performance and small device and computational footprints. We characterized the capabilities of pLSM, which repurposes readily available consumer-grade components, optimized optics, over-network control architecture and software-driven light-sheet modulation, by performing high-resolution mapping of cleared mouse brains and of post-mortem pathological human brain samples, and via the molecular phenotyping of brain and blood-vessel organoids derived from human induced pluripotent stem cells. We also report a method that leverages pLSM for the live imaging of the dynamics of sparsely labelled multi-layered bacterial pellicle biofilms at an air-liquid interface. pLSM can make high-resolution LSFM for biomedical applications more accessible, affordable and scalable.

The autophagy proteome in the brain.

As one of the most important cellular housekeepers, autophagy directly affects cellular health, homeostasis, and function. Even though the mechanisms behind autophagy are well described, how molecular alterations and dysfunctions can lead to pathology in disease contexts still demands deeper investigation. Proteomics is a widely employed tool used to investigate molecular alterations associated with pathological states and has proven useful in identifying alterations in protein expression levels and post-translational modifications in autophagy. In this narrative review, we expand on the molecular mechanisms behind autophagy and its regulation, and further compile recent literature associating autophagy disturbances in context of brain disorders, utilizing discoveries from varying models and species from rodents and cellular models to human post-mortem brain samples. To outline, the canonical pathways of autophagy, the effects of post-translational modifications on regulating each step of autophagy, and the future directions of proteomics in autophagy will be discussed. We further aim to suggest how advancing proteomics can help further unveil molecular mechanisms with regard to neurological disorders.

A small TAT-TrkB peptide prevents BDNF receptor cleavage and restores synaptic physiology in Alzheimer's disease

In Alzheimer’s disease (AD), amyloid β (Aβ)-triggered cleavage of TrkB-FL impairs Brain-derived neurotrophic factor (BDNF) signaling, thereby compromising neuronal survival, differentiation, as well as synaptic transmission and plasticity. Using cerebrospinal fluid and post-mortem human brain samples, we show that TrkB-FL cleavage occurs from the early stages of the disease and increases as function of pathology severity. To explore the therapeutic potential of this disease mechanism, we designed small TAT-fused peptides and screened their ability to prevent TrkB-FL receptor cleavage. Among these, a TAT-TrkB peptide with a lysine-lysine linker prevented TrkB-FL cleavage both in vitro and in vivo and rescued synaptic deficits induced by oligomeric Aβ in hippocampal slices. Furthermore, this TAT-TrkB peptide improved the cognitive performance, ameliorated synaptic plasticity deficits and prevented Tau pathology progression in vivo in the 5XFAD mouse model of AD. No evidence of liver or kidney toxicity was found. We provide proof-of-concept evidence for the efficacy and safety of this therapeutic strategy and anticipate that this TAT-TrkB peptide has the potential of a disease-modifying drug that can prevent and/or reverse cognitive deficits in patients with AD.

Single-cell multiregion dissection of Alzheimer’s disease

Alzheimer’s disease is the leading cause of dementia worldwide, but the cellular pathways that underlie its pathological progression across brain regions remain poorly understood1–3. Here we report a single-cell transcriptomic atlas of six different brain regions in the aged human brain, covering 1.3 million cells from 283 post-mortem human brain samples across 48 individuals with and without Alzheimer’s disease. We identify 76 cell types, including region-specific subtypes of astrocytes and excitatory neurons and an inhibitory interneuron population unique to the thalamus and distinct from canonical inhibitory subclasses. We identify vulnerable populations of excitatory and inhibitory neurons that are depleted in specific brain regions in Alzheimer’s disease, and provide evidence that the Reelin signalling pathway is involved in modulating the vulnerability of these neurons. We develop a scalable method for discovering gene modules, which we use to identify cell-type-specific and region-specific modules that are altered in Alzheimer’s disease and to annotate transcriptomic differences associated with diverse pathological variables. We identify an astrocyte program that is associated with cognitive resilience to Alzheimer’s disease pathology, tying choline metabolism and polyamine biosynthesis in astrocytes to preserved cognitive function late in life. Together, our study develops a regional atlas of the ageing human brain and provides insights into cellular vulnerability, response and resilience to Alzheimer’s disease pathology.

APOER2 splicing repertoire in Alzheimer's disease: Insights from long-read RNA sequencing.

Disrupted alternative splicing plays a determinative role in neurological diseases, either as a direct cause or as a driver in disease susceptibility. Transcriptomic profiling of aged human postmortem brain samples has uncovered hundreds of aberrant mRNA splicing events in Alzheimer's disease (AD) brains, associating dysregulated RNA splicing with disease. We previously identified a complex array of alternative splicing combinations across apolipoprotein E receptor 2 (APOER2), a transmembrane receptor that interacts with both the neuroprotective ligand Reelin and the AD-associated risk factor, APOE. Many of the human APOER2 isoforms, predominantly featuring cassette splicing events within functionally important domains, are critical for the receptor's function and ligand interaction. However, a comprehensive repertoire and the functional implications of APOER2 isoforms under both physiological and AD conditions are not fully understood. Here, we present an in-depth analysis of the splicing landscape of human APOER2 isoforms in normal and AD states. Using single-molecule, long-read sequencing, we profiled the entire APOER2 transcript from the parietal cortex and hippocampus of Braak stage IV AD brain tissues along with age-matched controls and investigated several functional properties of APOER2 isoforms. Our findings reveal diverse patterns of cassette exon skipping for APOER2 isoforms, with some showing region-specific expression and others unique to AD-affected brains. Notably, exon 15 of APOER2, which encodes the glycosylation domain, showed less inclusion in AD compared to control in the parietal cortex of females with an APOE ɛ3/ɛ3 genotype. Also, some of these APOER2 isoforms demonstrated changes in cell surface expression, APOE-mediated receptor processing, and synaptic number. These variations are likely critical in inducing synaptic alterations and may contribute to the neuronal dysfunction underlying AD pathogenesis.

DNA Damage and Senescence in the Aging and Alzheimer's Disease Cortex Are Not Uniformly Distributed.

Alzheimer's disease (AD) is a neurodegenerative illness with a typical age of onset exceeding 65 years of age. The age dependency of the condition led us to track the appearance of DNA damage in the frontal cortex of individuals who died with a diagnosis of AD. The focus on DNA damage was motivated by evidence that increasing levels of irreparable DNA damage are a major driver of the aging process. The connection between aging and the loss of genomic integrity is compelling because DNA damage has also been identified as a possible cause of cellular senescence. The number of senescent cells has been reported to increase with age, and their senescence-associated secreted products are likely contributing factors to age-related illnesses. We tracked DNA damage with 53BP1 and cellular senescence with p16 immunostaining of human post-mortem brain samples. We found that DNA damage was significantly increased in the BA9 region of the AD cortex compared with the same region in unaffected controls (UCs). In the AD but not UC cases, the density of cells with DNA damage increased with distance from the pia mater up to approximately layer V and then decreased in deeper areas. This pattern of DNA damage was overlaid with the pattern of cellular senescence, which also increased with cortical depth. On a cell-by-cell basis, we found that the intensities of the two markers were tightly linked in the AD but not the UC brain. To test whether DNA damage was a causal factor in the emergence of the senescence program, we used etoposide treatment to damage the DNA of cultured mouse primary neurons. While DNA damage increased after treatment, after 24 h, no change in the expression of senescence-associated markers was observed. Our work suggests that DNA damage and cellular senescence are both increased in the AD brain and increasingly coupled. We propose that in vivo, the relationship between the two age-related processes is more complex than previously thought.

Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD.

Cellular senescence is a hallmark of aging and has been implicated in Alzheimer's disease (AD) pathogenesis. Cholesterol accumulation drives cellular senescence; however, the underlying mechanisms are unclear. ATP-binding cassette transporter A1 (ABCA1) plays an important role in cholesterol homeostasis. ABCA1 expression and its trafficking is afiltered in APOE4 and AD cellular and mouse models. However, whether ABCA1 trafficking is involved in cellular senescence in APOE4 and AD remains unknown. We examined the association between cellular senescence and ABCA1 expression in human postmortem brain samples using transcriptomic, histological, and biochemical analyses. An unbiased proteomic screening was performed to identify targets that mediate cellular ABCA1 trafficking. APOE4-TR mice, immortalized, primary and induced pluripotent stem cell (iPSC) models were used to examine the cholesterol-ABCA1-senescence pathways. Bulk and single nuclei transcriptomic profiling of the human dorsolateral prefrontal cortex from the Religious Order Study/Memory Aging Project (ROSMAP) revealed upregulation of cellular senescence transcriptome signatures in AD, which was strongly correlated with ABCA1 expression. Immunofluorescence and immunoblotting analyses confirmed increased ABCA1 expression in AD brain tissues, which was associated with lipofuscin-stained lipids and mTOR phosphorylation. Using discovery proteomics, caveolin-1, a sensor of cellular cholesterol accumulation, was identified to promote ABCA1 endolysosomal trafficking. Greater caveolin-1 expression was found in both APOE4-TR mouse models and AD human brains. Cholesterol induced mTORC1 activation was regulated by ABCA1 expression or its lysosomal trapping. Reducing cholesterol by cyclodextrin in APOE4-TR mice reduced ABCA1 lysosome trapping and increased ABCA1 recycling to efflux cholesterol to HDL particles, reducing mTORC1 activation and senescence-associated neuroinflammation. In human iPSC-derived astrocytes, the reduction of cholesterol by cyclodextrin attenuated inflammatory responses. Cholesterol accumulation in APOE4 and AD induced caveolin-1 expression, which traps ABCA1 in lysosomes to activate mTORC1 pathways and induce cellular senescence. This study provided novel insights into how cholesterol accumulation in APOE4 and AD accelerates senescence.

Antipsychotic-induced epigenomic reorganization in frontal cortex of individuals with schizophrenia

Genome-wide association studies have revealed >270 loci associated with schizophrenia risk, yet these genetic factors do not seem to be sufficient to fully explain the molecular determinants behind this psychiatric condition. Epigenetic marks such as post-translational histone modifications remain largely plastic during development and adulthood, allowing a dynamic impact of environmental factors, including antipsychotic medications, on access to genes and regulatory elements. However, few studies so far have profiled cell-specific genome-wide histone modifications in postmortem brain samples from schizophrenia subjects, or the effect of antipsychotic treatment on such epigenetic marks. Here, we conducted ChIP-seq analyses focusing on histone marks indicative of active enhancers (H3K27ac) and active promoters (H3K4me3), alongside RNA-seq, using frontal cortex samples from antipsychotic-free (AF) and antipsychotic-treated (AT) individuals with schizophrenia, as well as individually matched controls (n=58). Schizophrenia subjects exhibited thousands of neuronal and non-neuronal epigenetic differences at regions that included several susceptibility genetic loci, such as NRG1, DISC1, and DRD3. By analyzing the AF and AT cohorts separately, we identified schizophrenia-associated alterations in specific transcription factors, their regulatees, and epigenomic and transcriptomic features that were reversed by antipsychotic treatment; as well as those that represented a consequence of antipsychotic medication rather than a hallmark of schizophrenia in postmortem human brain samples. Notably, we also found that the effect of age on epigenomic landscapes was more pronounced in frontal cortex of AT-schizophrenics, as compared to AF-schizophrenics and controls. Together, these data provide important evidence of epigenetic alterations in the frontal cortex of individuals with schizophrenia, and remark for the first time on the impact of age and antipsychotic treatment on chromatin organization.

Neuronal-specific methylome and hydroxymethylome analysis reveal significant loci associated with alcohol use disorder.

Background: Alcohol use disorder (AUD) is a complex condition associated with adverse health consequences that affect millions of individuals worldwide. Epigenetic modifications, including DNA methylation (5mC), have been associated with AUD and other alcohol-related traits. Epigenome-wide association studies (EWAS) have identified differentially methylated genes associated with AUD in human peripheral and brain tissue. More recently, epigenetic studies of AUD have also evaluated DNA hydroxymethylation (5hmC) in the human brain. However, most of the epigenetic work in postmortem brain tissue has examined bulk tissue. In this study, we investigated neuronal-specific 5mC and 5hmC alterations at CpG sites associated with AUD in the human orbitofrontal cortex (OFC). Methods: Neuronal nuclei from the OFC were evaluated in 34 human postmortem brain samples (10 AUD, 24 non-AUD). Reduced representation oxidative bisulfite sequencing was used to assess 5mC and 5hmC at the genome-wide level. Differential 5mC and 5hmC were evaluated using the methylKit R package and significance was set at false discovery rate < 0.05 and differential methylation > 2. Functional enrichment analyses were performed, and gene-level convergence was evaluated in an independent dataset that assessed 5mC and 5hmC of AUD in bulk cortical tissue. Results: We identified 417 5mC and 3635hmC significant differential CpG sites associated with AUD, with 59% in gene promoters. Some of the identified genes have been previously implicated in alcohol consumption, including SYK, DNMT3A for 5mC, GAD1, DLX1, DLX2, for 5hmC and GATA4 in both. Convergence with a previous AUD 5mC and 5hmC study was observed for 28 genes. We also identified 5 and 35 differential regions for 5mC and 5hmC, respectively. Lastly, GWAS enrichment analysis showed an association with AUD for differential 5mC genes. Discussion: This study reveals neuronal-specific methylome and hydroxymethylome dysregulation associated with AUD, identifying both previously reported and potentially novel gene associations with AUD. Our findings provide new insights into the epigenomic dysregulation of AUD in the human brain.

α-Synuclein oligomers potentiate neuroinflammatory NF-κB activity and induce Cav3.2 calcium signaling in astrocytes

BackgroundIt is now realized that Parkinson’s disease (PD) pathology extends beyond the substantia nigra, affecting both central and peripheral nervous systems, and exhibits a variety of non-motor symptoms often preceding motor features. Neuroinflammation induced by activated microglia and astrocytes is thought to underlie these manifestations. α-Synuclein aggregation has been linked with sustained neuroinflammation in PD, aggravating neuronal degeneration; however, there is still a lack of critical information about the structural identity of the α-synuclein conformers that activate microglia and/or astrocytes and the molecular pathways involved.MethodsTo investigate the role of α-synuclein conformers in the development and maintenance of neuroinflammation, we used primary quiescent microglia and astrocytes, post-mortem brain tissues from PD patients and A53T α-synuclein transgenic mice that recapitulate key features of PD-related inflammatory responses in the absence of cell death, i.e., increased levels of pro-inflammatory cytokines and complement proteins. Biochemical and -omics techniques including RNAseq and secretomic analyses, combined with 3D reconstruction of individual astrocytes and live calcium imaging, were used to uncover the molecular mechanisms underlying glial responses in the presence of α-synuclein oligomers in vivo and in vitro.ResultsWe found that the presence of SDS-resistant hyper-phosphorylated α-synuclein oligomers, but not monomers, was correlated with sustained inflammatory responses, such as elevated levels of endogenous antibodies and cytokines and microglial activation. Similar oligomeric α-synuclein species were found in post-mortem human brain samples of PD patients but not control individuals. Detailed analysis revealed a decrease in Iba1Low/CD68Low microglia and robust alterations in astrocyte number and morphology including process retraction. Our data indicated an activation of the p38/ATF2 signaling pathway mostly in microglia and a sustained induction of the NF-κB pathway in astrocytes of A53T mice. The sustained NF-κB activity triggered the upregulation of astrocytic T-type Cav3.2 Ca2+ channels, altering the astrocytic secretome and promoting the secretion of IGFBPL1, an IGF-1 binding protein with anti-inflammatory and neuroprotective potential.ConclusionsOur work supports a causative link between the neuron-produced α-synuclein oligomers and sustained neuroinflammation in vivo and maps the signaling pathways that are stimulated in microglia and astrocytes. It also highlights the recruitment of astrocytic Cav3.2 channels as a potential neuroprotective mediator against the α-synuclein-induced neuroinflammation.Graphical

Molecular profiling of the hippocampus of children with autism spectrum disorder

Several lines of evidence point to a key role of the hippocampus in Autism Spectrum Disorders (ASD). Altered hippocampal volume and deficits in memory for person and emotion related stimuli have been reported, along with enhanced ability for declarative memories. Mouse models have demonstrated a critical role of the hippocampus in social memory dysfunction, associated with ASD, together with decreased synaptic plasticity. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix molecules, represent a potential key link between neurodevelopment, synaptic plasticity, and immune system signaling. There is a lack of information regarding the molecular pathology of the hippocampus in ASD. We conducted RNAseq profiling on postmortem human brain samples containing the hippocampus from male children with ASD (n = 7) and normal male children (3–14 yrs old), (n = 6) from the NIH NeuroBioBank. Gene expression profiling analysis implicated molecular pathways involved in extracellular matrix organization, neurodevelopment, synaptic regulation, and immune system signaling. qRT-PCR and Western blotting were used to confirm several of the top markers identified. The CSPG protein BCAN was examined with multiplex immunofluorescence to analyze cell-type specific expression of BCAN and astrocyte morphology. We observed decreased expression of synaptic proteins PSD95 (p < 0.02) and SYN1 (p < 0.02), increased expression of the extracellular matrix (ECM) protease MMP9 (p < 0.03), and decreased expression of MEF2C (p < 0.03). We also observed increased BCAN expression with astrocytes in children with ASD, together with altered astrocyte morphology. Our results point to alterations in immune system signaling, glia cell differentiation, and synaptic signaling in the hippocampus of children with ASD, together with alterations in extracellular matrix molecules. Furthermore, our results demonstrate altered expression of genes implicated in genetic studies of ASD including SYN1 and MEF2C.

The association between neuropsychiatric effects of substance use and occurrence of endoplasmic reticulum and unfolded protein response: A systematic review

IntroductionThis systematic review aimed to assess the association between neuropsychiatric effects of substance use and occurrence of ER stress and unfolded protein response (UPR) through comprehensive electronic search of existing literature and review of their findings. MethodsA comprehensive electronic literature search was carried out on research articles published between 1950 to July 2023 through major databases, such as Scopus, Web of Science, Google Scholar, PubMed, PsycINFO, EMBASE, Medline and Cochrane Library. ResultsA total of 21 research articles were selected for review, which were comprised of sixteen animal studies, four human studies and one study on postmortem human brain samples. The selected studies revealed that alcohol, methamphetamine, cocaine, opioid and kratom exposures contributed to neuropsychiatric effects: such as decline in learning and memory function, executive dysfunction, alcohol, methamphetamine, opioid, and kratom dependence. These effects were associated with activation and persistent of ER stress and UPR with elevation of BiP and CHOP expression and the direction of ER stress is progressing towards the PERK-eIF2α-ATF4-CHOP pathway and neuronal apoptosis and neurodegeneration at various regions of the brain. In addition, regular kratom use in humans also contributed to elevation of p-JNK expression, denoting progress of ER stress towards the IRE1-ASK1-JNK-p-JNK pathway which was linked to kratom use disorder. However, treatment with certain compounds or biological agents could reverse the activation of ER stress. ConclusionsThe neuropsychiatric effects of alcohol, methamphetamine, cocaine, opioid and kratom use may be associated with persistent ER stress and UPR.

Cholesterol accumulation promotes ABCA1 degradation in astrocytes via Caveolin‐1 mediated endocytosis in ApoE4

AbstractBackgroundApolipoprotein E4 (ApoE4) dysregulates cholesterol metabolism in the brain promoting the accumulation of lipid droplets, synaptic dysfunction and associates with a greater Alzheimer’s disease risk. We previously demonstrated that ApoE4 reduces the function of ATP‐binding cassette transporters A1 (ABCA1) which has important roles in cholesterol transport. The mechanisms of how ApoE4 induces less ABCA1 activity are poorly understood.MethodABCA1 binding proteins were analyzed with mass spectrum after immunoprecipitation. Immortalized astrocytes, primary astrocytes, mouse brain and human postmortem brain samples were used to test the association of ABCA1 and caveolin‐1 levels. Small interfere RNA was used to knockdown caveolin‐1 expression and 3H labelled cholesterol was used to test the cholesterol efflux of ABCA1.ResultHere, we enriched ABCA1 binding protein complexes from ABCA1‐overexpressing cells treated with recombinant ApoE3 or ApoE4 proteins for a proteomic screen implicating endocytosis as a major pathway differentiating ApoE3 from ApoE4. Among the protein targets discovered, caveolin‐1 expression increased after ApoE4 treatment. Caveolin‐1 and ABCA1 interactions were confirmed using immunoprecipitation experiments. Cholesterol accumulation in astrocytes induced caveolin‐1 expression and promoted ABCA1 degradation. Greater caveolin‐1 and lower ABCA1 expression was observed in brains of 8, 18 and 22 months‐old ApoE4‐TR mice compared with the corresponding ApoE3‐TR mice. Isolation of single cell types from adult mouse ApoE‐TR brains confirmed lower ABCA1 and greater caveolin‐1 expression was largely driven by astrocytes and not microglia or neurons enriched fractions.ConclusionIn conclusion, cholesterol accumulation induced by ApoE4 promotes ABCA1 degradation in astrocytes partly through a caveolin‐1 dependent endocytosis pathway.

Tpr Misregulation in Hippocampal Neural Stem Cells in Mouse Models of Alzheimer's Disease.

Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer's disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-β plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localization of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis.

Imaging and quantifying microglial activation in vivo and ex vivo using diffusion MRI – with validation by immunohistochemistry

AbstractBackgroundAlzheimer disease (AD) is the most common type of dementia.There is growing evidence that the amyloid plaques in postmortem AD human brains are surrounded by activated microglia. Using diffusion basis spectrum imaging (DBSI), we have previously observed increased cellular diffusivity in AD patients, suggesting increased microglia activation as a key pathology in AD. To better understand the underlying mechanism and further establish the noninvasive and nonradioactive DBSI biomarkers of microglia activation, we conducted a realistic Monte Carlo (MC) simulation to model diffusion MRI signals associated with microglia in various activation states.MethodFirst, realistic microglia diffusion signals were generated using MC simulation with real 3D microglia geometry obtained from neuromorpho.org (Fig. 1A). We simulated different pathophysiological scenarios to validate DBSI’s capability in quantitatively detecting microglia activation (Fig. 1B). Second, we conducted and ex vivo MRI scans on human postmortem AD brain samples, followed by immunohistochemical (IHC) staining of microglia (Iba‐1) in aligned sections from the same tissue. DBSI cell apparent diffusion coefficients (ADC), reflecting microglia activation, were estimated and compared to quantitative histological assessments of microglia activation. Finally, in vivo DBSI was conducted on 200 participants who were classified into healthy control, preclinical AD, and early symptomatic AD groups based on cognitive assessments and measures of CSF amyloid (Aβ42) and pTau181.ResultTwo exemplary regions in grey matter (GM) and white matter (WM) of a postmortem AD brain were selected (Fig. 1C). We found that GM had significantly higher DBSI‐derived cell ADC than WM (Fig. 1D). Iba‐1 IHC revealed that WM had significantly larger average microglial area (p&lt;0.005) and bigger max microglia larea (Fig. 1E‐F). MC Simulation was conducted on 700 microglia models. Microglial activation would result in an increased DBSI cell ADC (Fig. 1G). According to MC simulation, increased DBSI‐derived cell ADC in preclinical and early symptomatic AD indicated microglial activation in preclinical and early symptomatic AD (Fig. 1H).ConclusionDBSI‐derived cell ADC increases with microglial activation as assessed by quantitative Iba‐1 IHC. This finding is supported by MC simulation using real 3D microglia geometry. The study indicates that DBSI has the potential to provide non‐invasive MRI biomarkers of microglia activation.

Pseudo‐temporal models of Alzheimer’s Disease progression across brain‐derived transcriptomics, proteomics, and metabolomics

AbstractBackgroundWe applied a pseudo‐temporal model to multi‐omic data from individuals with heterogeneous neuropathology to examine molecular changes across the spectrum of Alzheimer’s Disease (AD) progression.MethodsManifold learning algorithms ‘order’ samples based on similarity of expression or abundance patterns to calculate a ‘trajectory’ of disease progression. We applied this approach to RNAseq (N = 1078), TMT‐proteomics (N = 602), and metabolomics (N = 500) profiles of human postmortem brain samples from the ROS/MAP study within the Accelerating Medicine Partnership‐AD consortia (AMP‐AD); 318 individuals were included in all three ‐omics datasets. Analyses were stratified by sex. We examined associations between pseudotime, a quantitative measure of progress along each trajectory, and neuropathological and clinical endpoints. We calculated differential expression or abundance along each trajectory. For transcriptomic and proteomic data, we used gene set enrichment analyses to identify significant GO terms changing along the trajectory and grouped them into 19 distinct AD‐relevant biological domains. We similarly annotated changing metabolic pathways along metabolomic trajectories.ResultsPseudotime estimates were significantly associated with AD status such that “early” (low pseudotime) samples were enriched for controls, and “late” (high pseudotime) samples were enriched for cases, in all ‐omics. Omic‐specific pseudotimes were weakly correlated with each other (Pearson’s r for: RNAseq vs proteomics: 0.39; RNAseq vs metabolomics: 0.28; proteomics vs metabolomics: 0.41). Proteins involved in mitochondrial metabolism and synaptic function were downregulated in early stages in both sexes, while transcriptomic trajectories indicated early sex‐specific differences in synaptic function and immune response. In both sexes, metabolic amino acid and lipid pathways were perturbed in early stages, though specific pathways were different; early Phosphatidylcholine and Leucine, Isoleucine, and Valine Metabolism perturbations were especially prominent in women, while early Glutathione Metabolism perturbations were more prominent in men.ConclusionsDespite differences in the biological nature of the data used to order trajectories, pseudotime was associated with multiple measures of AD progression across all three ‐omics. Each analysis provided a unique snapshot of molecular change across AD progression. Sex‐specific differences, especially those noted early in disease trajectories, may provide crucial insights into key processes driving disease in women and men.

Exploration of SUMO2/3 Expression Levels and Autophagy Process in Fragile X-Associated Tremor/Ataxia Syndrome: Addressing Study Limitations and Insights for Future Research.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in adult FMR1 premutation carriers. The neuropathological hallmark of FXTAS is an intranuclear inclusion in neurons and astrocytes. Nearly 200 different proteins have been identified in FXTAS inclusions, being the small ubiquitin-related modifier 2 (SUMO2), ubiquitin and p62 the most highly abundant. These proteins are components of the protein degradation machinery. This study aimed to characterize SUMO2/3 expression levels and autophagy process in human postmortem brain samples and skin fibroblast cultures from FXTAS patients. Results revealed that FXTAS postmortem brain samples are positive for SUMO2/3 conjugates and supported the idea that SUMO2/3 accumulation is involved in inclusion formation. Insights from RNA-sequencing data indicated that SUMOylation processes are significantly upregulated in FXTAS samples. In addition, the analysis of the autophagy flux showed the accumulation of p62 protein levels and autophagosomes in skin fibroblasts from FXTAS patients. Similarly, gene set analysis evidenced a significant downregulation in gene ontology terms related to autophagy in FXTAS samples. Overall, this study provides new evidence supporting the role of SUMOylation and autophagic processes in the pathogenic mechanisms underlying FXTAS.