Postmortem Human Brain Tissue Research Articles

Unfolded protein response markers Grp78 and eIF2alpha are upregulated with increasing alpha-synuclein levels in Lewy body disease.

Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.

Human post-mortem organotypic brain slice cultures: a tool to study pathomechanisms and test therapies

Human brain experimental models recapitulating age- and disease-related characteristics are lacking. There is urgent need for human-specific tools that model the complex molecular and cellular interplay between different cell types to assess underlying disease mechanisms and test therapies. Here we present an adapted ex vivo organotypic slice culture method using human post-mortem brain tissue cultured at an air-liquid interface to also study brain white matter. We assessed whether these human post-mortem brain slices recapitulate the in vivo neuropathology and if they are suitable for pathophysiological, experimental and pre-clinical treatment development purposes, specifically regarding leukodystrophies. Human post-mortem brain tissue and cerebrospinal fluid were obtained from control, psychiatric and leukodystrophy donors. Slices were cultured up to six weeks, in culture medium with or without human cerebrospinal fluid. Human post-mortem organotypic brain slice cultures remained viable for at least six weeks ex vivo and maintained tissue structure and diversity of (neural) cell types. Supplementation with cerebrospinal fluid could improve slice recovery. Patient-derived organotypic slice cultures recapitulated and maintained known in vivo neuropathology. The cultures also showed physiologic multicellular responses to lysolecithin-induced demyelination ex vivo, indicating their suitability to study intrinsic repair mechanisms upon injury. The slice cultures were applicable for various experimental studies, as multi-electrode neuronal recordings. Finally, the cultures showed successful cell-type dependent transduction with gene therapy vectors. These human post-mortem organotypic brain slice cultures represent an adapted ex vivo model suitable for multifaceted studies of brain disease mechanisms, boosting translation from human ex vivo to in vivo. This model also allows for assessing potential treatment options, including gene therapy applications. Human post-mortem brain slice cultures are thus a valuable tool in preclinical research to study the pathomechanisms of a wide variety of brain diseases in living human tissue.

A Scalable Histological Method to Embed and Section Multiple Brains Simultaneously.

The preparation and processing of rodent brains for evaluation by immunohistochemistry is time-consuming. A large number of mouse brains are routinely used in experiments in neuroscience laboratories to evaluate several models of human diseases. Thus, methods are needed to reduce the time associated with processing brains for histology. A scalable method was developed to embed, section, and stain multiple mouse brains using supplies found in any common histology laboratory. Section collection schemes can be scaled to provide identical bregma locations between adjacent sections for immunohistochemistry, facilitating comprehensive, high-quality immunohistochemistry. As a result, sectioning and staining times are considerably reduced as sections from multiple blocks are stained simultaneously. This method improves on previous procedures and allows multiple embedding and subsequent immunostaining of brains easily with a dramatically reduced time requirement. Furthermore, we expand this method for use in numerous mouse tissues, rat brain tissue, and post-mortem human brain and arterial tissues. In summary, this procedure allows the processing of many rodent or human tissues from perfusion through microscopy in 10 days or less.

Automated deep learning segmentation of high-resolution 7 Tesla postmortem MRI for quantitative analysis of structure-pathology correlations in neurodegenerative diseases

Abstract Postmortem MRI allows brain anatomy to be examined at high resolution and to link pathology measures with morphometric measurements. However, automated segmentation methods for brain mapping in postmortem MRI are not well developed, primarily due to limited availability of labeled datasets, and heterogeneity in scanner hardware and acquisition protocols. In this work, we present a high-resolution dataset of 135 postmortem human brain tissue specimens imaged at 0.3 mm3 isotropic using a T2w sequence on a 7T whole-body MRI scanner. We developed a deep learning pipeline to segment the cortical mantle by benchmarking the performance of nine deep neural architectures, followed by post-hoc topological correction. We evaluate the reliability of this pipeline via overlap metrics with manual segmentation in 6 specimens, and intra-class correlation between cortical thickness measures extracted from the automatic segmentation and expert-generated reference measures in 36 specimens. We also segment four subcortical structures (caudate, putamen, globus pallidus, and thalamus), white matter hyperintensities, and the normal appearing white matter, providing a limited evaluation of accuracy. We show generalizing capabilities across whole-brain hemispheres in different specimens, and also on unseen images acquired at 0.28 mm3 and 0.16 mm3 isotropic T2*w fast low angle shot (FLASH) sequence at 7T. We report associations between localized cortical thickness and volumetric measurements across key regions, and semi-quantitative neuropathological ratings in a subset of 82 individuals with Alzheimer’s disease (AD) continuum diagnoses. Our code, Jupyter notebooks, and the containerized executables are publicly available at the project webpage (https://pulkit-khandelwal.github.io/exvivo-brain-upenn/).

Primary mitochondrial diseases: The intertwined pathophysiology of bioenergetic dysregulation, oxidative stress and neuroinflammation.

Primary mitochondrial diseases (PMDs) are rare genetic disorders resulting from mutations in genes crucial for effective oxidative phosphorylation (OXPHOS) that can affect mitochondrial function. In this review, we examine the bioenergetic alterations and oxidative stress observed in cellular models of primary mitochondrial diseases (PMDs), shedding light on the intricate complexity between mitochondrial dysfunction and cellular pathology. We explore the diverse cellular models utilized to study PMDs, including patient-derived fibroblasts, induced pluripotent stem cells (iPSCs) and cybrids. Moreover, we also emphasize the connection between oxidative stress and neuroinflammation. The central nervous system (CNS) is particularly vulnerable to mitochondrial dysfunction due to its dependence on aerobic metabolism and the correct functioning of OXPHOS. Similar to other neurodegenerative diseases affecting the CNS, individuals with PMDs exhibit several neuroinflammatory hallmarks alongside neurodegeneration, a pattern also extensively observed in mouse models of mitochondrial diseases. Based on histopathological analysis of postmortem human brain tissue and findings in mouse models of PMDs, we posit that neuroinflammation is not merely a consequence of neurodegeneration but a potential pathogenic mechanism for disease progression that deserves further investigation. This recognition may pave the way for novel therapeutic strategies for this group of devastating diseases that currently lack effective treatments. In summary, this review provides a comprehensive overview of bioenergetic alterations and redox imbalance in cellular models of PMDs while underscoring the significance of neuroinflammation as a potential driver in disease progression.

Interaction of exogenous acetylcholinesterase and butyrylcholinesterase with amyloid-β plaques in human brain tissue

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are associated with amyloid-β (Aβ) plaques and exhibit altered biochemical properties in human Alzheimer's disease (AD), as well as in the transgenic 5XFAD mouse model of AD amyloidosis. In the brains of the 5XFAD mouse model devoid of BChE enzyme (5XFAD/BChE-KO), incubation of tissue sections with exogenous BChE purified from human plasma (pl-BChE) leads to its association with Aβ plaques and its biochemical properties are comparable to those reported for endogenous BChE associated with plaques in both human AD and in 5XFAD mouse brain tissue. We sought to determine whether these observations in 5XFAD/BChE-KO mice also apply to human brain tissues. To do so, endogenous ChE activity in human AD brain tissue sections was quenched with 50 % aqueous acetonitrile (MeCNaq) leaving the tissue suitable for further studies. Quenched sections were then incubated with recombinant AChE (r-AChE) or pl-BChE and stained for each enzymes' activity. Exogenous r-AChE or pl-BChE became associated with Aβ plaques, and when bound, had properties that were comparable to the endogenous ChE enzymes associated with plaques in AD brain tissues without acetonitrile treatment. These findings in human AD brain tissue extend previous observations in the 5XFAD/BChE-KO mouse model and demonstrate that exogenously applied r-AChE and pl-BChE have high affinity for Aβ plaques in human brain tissues. This association alters the biochemical properties of these enzymes, most likely due a conformational change. If incorporation of AChE and BChE in Aβ plaques facilitates AD pathogenesis, blocking this association could lead to disease-modifying approaches to AD. This work provides a method to study the mechanism of AChE and BChE interaction with Aβ plaque pathology in post-mortem human brain tissue.

Discovery of CVN293, a Brain Permeable KCNK13 (THIK-1) Inhibitor Suitable for Clinical Assessment.

The potassium (K+) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1β from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.

Inflammation-induced TRPV4 channels exacerbate blood–brain barrier dysfunction in multiple sclerosis

BackgroundBlood–brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS.Main textIn human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium.ConclusionCollectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Altered hippocampal doublecortin expression in Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative disorder characterized by motor and non-motor symptoms. Motor symptoms include bradykinesia, resting tremors, muscular rigidity, and postural instability, while non-motor symptoms include cognitive impairments, mood disturbances, sleep disturbances, autonomic dysfunction, and sensory abnormalities. Some of these symptoms may be influenced by the proper hippocampus functioning, including adult neurogenesis. Doublecortin (DCX) is a microtubule-associated protein that plays a pivotal role in the development and differentiation of migrating neurons. This study utilized postmortem human brain tissue of PD and age-matched control individuals to investigate DCX expression in the context of adult hippocampal neurogenesis. Our findings demonstrate a significant reduction in the number of DCX-expressing cells within the subgranular zone (SGZ), as well as a decrease in the nuclear area of these DCX-positive cells in postmortem brain tissue obtained from PD cases, suggesting an impairment in the adult hippocampal neurogenesis. Additionally, we found that the nuclear area of DCX-positive cells correlates with pH levels. In summary, we provide evidence supporting that the process of hippocampal adult neurogenesis is likely to be compromised in PD patients before cognitive dysfunction, shedding light on potential mechanisms contributing to the neuropsychiatric symptoms observed in affected individuals. Understanding these mechanisms may offer novel insights into the pathophysiology of PD and possible therapeutic avenues.

A Survey of Community Perceptions on Brain Donation for Research.

Postmortem brain donation for medical research is a little-known form of organ donation. While most brain research is carried out using animal models, many neurological diseases are uniquely human. Greater availability of human postmortem brain tissue from diseased individuals and controls would therefore improve the development of treatments for neurological and neuropsychiatric diseases. Globally, organ donation for medical research is dwarfed by organ donation for transplantation. In 2021, 36% of Australians were registered organ donors for transplantation, with public "in-principle" support even higher, at 76%. In contrast, there are little data on Australian or international brain donation rates for research. A 30-item online survey was conducted to ascertain knowledge of, and attitudes toward, brain donation in Australia. Of the respondents, 12/237 (5%) were current brain donors and excluded from further analysis. Of the remaining 225, 75% were registered organ donors for transplant. The vast majority (n = 189/225, 84%) of respondents supported or strongly supported the principle of brain donation. However, of those registered for transplantation or whole-body donors, 93/170 (55%) were not aware that brain donation was possible, while 50%, alternatively or also, thought that registering as an organ donor for transplantation rendered them a brain donor by default. Only 9/225 (4%) respondents indicated that they would definitely not donate their brain in the future, while 27 remained unsure. There is prominent public support for brain donation in Australia, with 84% of respondents willing to donate their brain. Yet, the extent of public misconceptions on brain donation for research suggests the need for further education on all types of organ donation, so individuals may make informed decisions.

Profiling Small RNA From Brain Extracellular Vesicles in Individuals With Depression.

Major depressive disorder (MDD) is a leading cause of disability with significant mortality risk. Despite progress in our understanding of the etiology of MDD, the underlying molecular changes in the brain remain poorly understood. Extracellular vesicles (EVs) are lipid-bound particles that can reflect the molecular signatures of the tissue of origin. We aimed to optimize a streamlined EV isolation protocol from postmortem brain tissue and determine whether EV RNA cargo, particularly microRNAs (miRNAs), have an MDD-specific profile. EVs were isolated from postmortem human brain tissue. Quality was assessed using western blots, transmission electron microscopy, and microfluidic resistive pulse sensing. EV RNA was extracted and sequenced on Illumina platforms. Functional follow-up was performed in silico. Quality assessment showed an enrichment of EV markers, as well as a size distribution of 30 to 200nm in diameter, and no contamination with cellular debris. Small RNA profiling indicated the presence of several RNA biotypes, with miRNAs and transfer RNAs being the most prominent. Exploring miRNA levels between groups revealed decreased expression of miR-92a-3p and miR-129-5p, which was validated by qPCR and was specific to EVs and not seen in bulk tissue. Finally, in silico functional analyses indicate potential roles for these 2 miRNAs in neurotransmission and synaptic plasticity. We provide a streamlined isolation protocol that yields EVs of high quality that are suitable for molecular follow-up. Our findings warrant future investigations into brain EV miRNA dysregulation in MDD.

The lncRNA Snhg11, a new candidate contributing to neurogenesis, plasticity, and memory deficits in Down syndrome.

Down syndrome (DS) stands as the prevalent genetic cause of intellectual disability, yet comprehensive understanding of its cellular and molecular underpinnings remains limited. In this study, we explore the cellular landscape of the hippocampus in a DS mouse model, the Ts65Dn, through single-nuclei transcriptional profiling. Our findings demonstrate that trisomy manifests as a highly specific modification of the transcriptome within distinct cell types. Remarkably, we observed a significant shift in the transcriptomic profile of granule cells in the dentate gyrus (DG) associated with trisomy. We identified the downregulation of a specific small nucleolar RNA host gene, Snhg11, as the primary driver behind this observed shift in the trisomic DG. Notably, reduced levels of Snhg11 in this region were also observed in a distinct DS mouse model, the Dp(16)1Yey, as well as in human postmortem brain tissue, indicating its relevance in Down syndrome. To elucidate the function of this long non-coding RNA (lncRNA), we knocked down Snhg11 in the DG of wild-type mice. Intriguingly, this intervention alone was sufficient to impair synaptic plasticity and adult neurogenesis, resembling the cognitive phenotypes associated with trisomy in the hippocampus. Our study uncovers the functional role of Snhg11 in the DG and underscores the significance of this lncRNA in intellectual disability. Furthermore, our findings highlight the importance of DG in the memory deficits observed in Down syndrome.

Neuroinflammatory disease signatures in SPG11-related hereditary spastic paraplegia patients

Biallelic loss of SPG11 function constitutes the most frequent cause of complicated autosomal recessive hereditary spastic paraplegia (HSP) with thin corpus callosum, resulting in progressive multisystem neurodegeneration. While the impact of neuroinflammation is an emerging and potentially treatable aspect in neurodegenerative diseases and leukodystrophies, the role of immune cells in SPG11–HSP patients is unknown. Here, we performed a comprehensive immunological characterization of SPG11–HSP, including examination of three human postmortem brain donations, immunophenotyping of patients’ peripheral blood cells and patient-specific induced pluripotent stem cell-derived microglia-like cells (iMGL). We delineate a previously unknown role of innate immunity in SPG11–HSP. Neuropathological analysis of SPG11–HSP patient brain tissue revealed profound microgliosis in areas of neurodegeneration, downregulation of homeostatic microglial markers and cell-intrinsic accumulation of lipids and lipofuscin in IBA1+ cells. In a larger cohort of SPG11–HSP patients, the ratio of peripheral classical and intermediate monocytes was increased, along with increased serum levels of IL-6 that correlated with disease severity. Stimulation of patient-specific iMGLs with IFNγ led to increased phagocytic activity compared to control iMGL as well as increased upregulation and release of proinflammatory cytokines and chemokines, such as CXCL10. On a molecular basis, we identified increased STAT1 phosphorylation as mechanism connecting IFNγ-mediated immune hyperactivation and SPG11 loss of function. STAT1 expression was increased both in human postmortem brain tissue and in an Spg11–/– mouse model. Application of an STAT1 inhibitor decreased CXCL10 production in SPG11 iMGL and rescued their toxic effect on SPG11 neurons. Our data establish neuroinflammation as a novel disease mechanism in SPG11–HSP patients and constitute the first description of myeloid cell/ microglia activation in human SPG11–HSP. IFNγ/ STAT1-mediated neurotoxic effects of hyperreactive microglia upon SPG11 loss of function indicate that immunomodulation strategies may slow down disease progression.

Spatial Neurolipidomics at the Single Amyloid-β Plaque Level in Postmortem Human Alzheimer's Disease Brain.

Lipid dysregulations have been critically implicated in Alzheimer's disease (AD) pathology. Chemical analysis of amyloid-β (Aβ) plaque pathology in transgenic AD mouse models has demonstrated alterations in the microenvironment in the direct proximity of Aβ plaque pathology. In mouse studies, differences in lipid patterns linked to structural polymorphism among Aβ pathology, such as diffuse, immature, and mature fibrillary aggregates, have also been reported. To date, no comprehensive analysis of neuronal lipid microenvironment changes in human AD tissue has been performed. Here, for the first time, we leverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) through a high-speed and spatial resolution commercial time-of-light instrument, as well as a high-mass-resolution in-house-developed orbitrap system to characterize the lipid microenvironment in postmortem human brain tissue from AD patients carrying Presenilin 1 mutations (PSEN1) that lead to familial forms of AD (fAD). Interrogation of the spatially resolved MSI data on a single Aβ plaque allowed us to verify nearly 40 sphingolipid and phospholipid species from diverse subclasses being enriched and depleted, in relation to the Aβ deposits. This included monosialo-gangliosides (GM), ceramide monohexosides (HexCer), ceramide-1-phosphates (CerP), ceramide phosphoethanolamine conjugates (PE-Cer), sulfatides (ST), as well as phosphatidylinositols (PI), phosphatidylethanolamines (PE), and phosphatidic acid (PA) species (including Lyso-forms). Indeed, many of the sphingolipid species overlap with the species previously seen in transgenic AD mouse models. Interestingly, in comparison to the animal studies, we observed an increased level of localization of PE and PI species containing arachidonic acid (AA). These findings are highly relevant, demonstrating for the first time Aβ plaque pathology-related alteration in the lipid microenvironment in humans. They provide a basis for the development of potential lipid biomarkers for AD characterization and insight into human-specific molecular pathway alterations.

Validated assays for the quantification of C9orf72 human pathology

A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to the C9orf72 locus in what has been a traditionally hard to sequence genomic region. This is of particular value to sizing and phasing the repeat expansion and determining changes to the gene locus after gene editing. We developed ddPCR assays to quantify two major C9orf72 transcript variants, which we validated by selective excision of their distinct transcriptional start sites. Using validated knock-out human iPSC lines, we validated 4 commercially available antibodies (of 9 tested) that were specific for C9orf72 protein quantification by Western blot, but none were specific for immunocytochemistry. We tested 15 combinations of antibodies against dipeptide repeat proteins (DPRs) across 66 concentrations using MSD immunoassay, and found two (against poly-GA and poly-GP) that yielded a 1.5-fold or greater signal increase in patient iPSC-motor neurons compared to knock-out control, and validated them in human postmortem and transgenic mouse brain tissue. Our validated DNA, RNA and protein assays are applicable to discovery research as well as clinical trials.

18F]MK6240 and [18F]PI2620 autoradiography on postmortem human brain tissue in AD

AbstractBackgroundTo compare the post mortem uptake of [18F]MK6240 and [18F]PI2620 in the brain hemisphere, of A‐T‐ (CN) and A+T+ tissueMethodAutoradiography (ARG) imaging using [18F]MK6240 and [18F]PI2620 was used to assess uptake of CN and AD tissues. One study used sections of whole‐hemisphere of one CN tissue and one AD tissue. The uptake value of [18F]MK6240 was calculated from a regions of interest (ROI) drawn manually from the area of cortex gray matter (GM), cortex white matter (WM), and background (BG).Other study was 24 tissues from the prefrontal cortex (Pfc) (12CN, 12AD), hippocampus (Hip) (12 CN, 12 AD) and cerebellum (Cer) (12 CN, 12 AD) were evaluated with [18F]MK6240, and [18F]PI2620. The uptake value of [18F]MK6240 and [18F]PI2620 was calculated from ROI drawn manually from the area of pfc GM, hip GM and cer GM.ResultThe ARG showed greater [18F]MK6240 uptake in AD than in CN tissue. There were significant differences between CN and AD tissue in terms of cortex gray GM distribution and cortex WM, but no significant differences in BG. These results also, there is no correlation between the specific activity of [18F]MK6240 and the uptake value in CN and AD tissues. In a second experiment, based on our findings there was a significant difference between CN and AD groups when assessed by [18F]MK6240 as well as [18F]PI2620 in the pfc_GM and hip_GM. Furthermore, we found no difference between AD and CN tissues when assessed by [18F]MK6240 and [18F]PI2620 in cerebellum GM. Ratio values Pfc/Cer and Hip/Cer assessed by [18F]MK6240 as well as there were significant differences between CN and AD. [18F]MK6240 and [18F]PI2620 were highly correlated in AD but not in CN brain tissues.ConclusionWe also found that [18F]PI2620 and [18F]MK6240 have similar brain uptake in post‐mortem tissue.

Spatial Transcriptomics of Human Postmortem Brain Tissue Uncovers Molecular Pathways Associated with Amyloid‐Beta Plaques

AbstractBackgroundAlzheimer’s disease (AD) pathologies include extracellular amyloid‐beta (A‐beta) plaques, intracellular neurofibrillary tangles, neuronal cell loss, and chronic glial inflammation. Recent studies profiling gene expression in human postmortem brain tissue have uncovered cell‐type‐specific changes in AD. However, these studies did not provide simultaneous, direct measurements of AD pathologies in the native microenvironment.MethodTo this end, we applied the 10X Genomics Visium platform to post‐mortem dorsolateral prefrontal cortex (DLPFC) from 20 ROSMAP participants with or without AD dementia. In total, we obtained spatial transcriptomic (ST) data for ∼260,000 tissue microdomains (“spots” comprised of ∼1‐10 cells).ResultWe clustered spots and annotated clusters according to expression of genes with known enrichment in anatomical layers (i.e. cortical layers 1‐6, meninges, and white matter). We deconvoluted the cell type(s) comprising each spot using cell2location, and found significant changes in the abundance of specific cell types in AD. Using a two‐stage pseudobulk regression approach, we identified hundreds of genes whose spatial expression is significantly associated with nearby A‐beta plaques and/or astrocyte reactivity (GFAP). A‐beta‐associated genes are enriched for diverse biological processes, including translation, metabolism, inflammation, gliosis, and immune cell infiltration. We also detected genes that distinguish A‐beta plaques surrounded by reactive glial nets. We validated the spatial expression pattern of a subset of plaque‐associated genes by RNAScope.ConclusionIn summary, our study reveals genes and pathways that shape molecular states in the spatial context of AD pathology. Our data represent a comprehensive application of ST technology in AD brains, providing a valuable resource for the research community.

An optimized integrated functional genomics toolkit identifies novel role of SORL1 in microglia

AbstractBackgroundRecent genome wide association studies implicated microglia in the pathophysiology of neurodegeneration; however, the translation of these genetic discoveries is hindered by the current lack of a robust human microglia in vitro model system and standardized protocols for functional genomics assessment. Though microglia differentiated from human induced pluripotent stem cells (iMG) could be a promising cellular model, the available differentiation protocols are variable, lengthy and give inefficient, mixed yields, limiting our ability to utilize modern gene editing tools (e.g., CRISPR‐Cas9) in this model for elucidating the functional consequences of disease relevant genes and variants.MethodsIn order to streamline microglia functional genomics, we optimized a highly efficient protocol to differentiate human induced pluripotent stem cells into iMG in two easy steps, offering a significant improvement in purity and time requirement for iMG production. We validated this iMG model system in a robust way through a comprehensive single cell RNA‐sequencing (scRNA‐Seq) study, mass spectrometry‐based proteomics and microglial functional assays. In addition, the iMG differentiation protocol includes sequential iMG harvesting and cell freezing steps, that will allow the researchers to scale up the production of iMG for high‐throughput assays. Furthermore, the toolkit also includes a drug inducible CRISPR‐ON/OFF approach in our newly established microglia model system that enables the investigation of gene function in a temporal fashion. All the data generated as part of the comprehensive validation is publicly available in the form of a searchable online platform (https://jasonchungngo.shinyapps.io/IPSC-derived-Microglia/).ResultsWe used this newly developed integrated microglia functional genomics toolkit, along with human primary microglia freshly purified from dorsolateral prefrontal cortex (DLPFC), to investigate the thus far unidentified role of SORL1, an AD associated gene, in microglia. By combining state‐of‐the art (e.g. single cell RNA‐Seq, proteomics and focused CRISPR‐Cas9 screen) and standard (immunohistochemistry on postmortem human aged brain tissues) approaches we established the molecular mechanisms by which SORL1 shapes the functional phenotypes of human microglia. Most interestingly, we have found divergent roles for SORL1 in microglia when compared to neurons.ConclusionsOur findings highlight the importance of cell type specific functional genomics of susceptibility genes in AD.

Explainable AI and its instantiation in Computational Pathology for a better understanding of Alzheimer’s disease

AbstractBackgroundEXplainable Artificial Intelligence (XAI) has an essential role to accelerate the AI adoption by empowering biomedical experts and catalyzing modern research through the emergence of a new generation of virtual actors, enabling traceable knowledge‐based quantification, as a guided qualification support. XAI instantiation is shown through our study, hypothesizing that rapid Alzheimer’s Disease (rAD) brains display subtle histological changes that would be undercovered by high‐throughput automated microscopic analysis. We study the topography and morphology of tau lesions’ aggregates to better understand the morphological substratum of Alzheimer’s Disease (AD) heterogeneity.MethodTo address this question at a large scale, we designed, tested and implemented a software for automatic segmentation, annotation and quantitation of brain lesions in histopathological whole slide images (WSI). A dataset of 15 whole slide images of postmortem human brain tissue is used in this study. These are fully annotated by neuropathologists yielding a set of more than 30,000 annotated plaques and tangle objects. A deep learning pipeline consisting of an attention‐UNet model with explainability features is trained for segmentation of tau aggregates in WSIs. The attention maps corresponding to segmentation predictions from the model are used by pathologists as a comparable reference to assist their decision making process.ResultThe DL‐based WSI analysis framework allows detecting a majority of the tau aggregates and refining tau aggregate boundaries with higher accuracy compared to manual annotations. Besides, with such a DL model based tau identification, the pathologist needs to annotate only a few samples for the DL model training. Overall, DL assisted analysis of tau aggregates could ease the effort of pathologists and facilitate analysis of hundreds of WSIs which are vital in AD research areas such as identifying different AD forms and AD patient stratification.ConclusionXAI is not only providing traceability by opening the way to an effective adoption, but also allows optimizing the ML/DL design, by keeping high performances. The green footprint is, therefore, considered, as well as the GDPR (European General Data Protection Regulation), towards Responsible Artificial Intelligence.

Exploratory analysis of 3000 proteins in postmortem human brain tissue lysates using proximity extension assay

Exploratory analysis of 3000 proteins in postmortem human brain tissue lysates using proximity extension assay