Postmortem Human Tissue Research Articles

Integrating gene expression and imaging data across Visium capture areas with visiumStitched.

Visium is a widely-used spatially-resolved transcriptomics assay available from 10x Genomics. Standard Visium capture areas (6.5mm by 6.5mm) limit the survey of larger tissue structures, but combining overlapping images and associated gene expression data allow for more complex study designs. Current software can handle nested or partial image overlaps, but is designed for merging up to two capture areas, and cannot account for some technical scenarios related to capture area alignment. We generated Visium data from a postmortem human tissue sample such that two capture areas were partially overlapping and a third one was adjacent. We developed the R/Bioconductor package visiumStitched, which facilitates stitching the images together with Fiji (ImageJ), and constructing SpatialExperiment R objects with the stitched images and gene expression data. visiumStitched constructs an artificial hexagonal array grid which allows seamless downstream analyses such as spatially-aware clustering without discarding data from overlapping spots. Data stitched with visiumStitched can then be interactively visualized with spatialLIBD. visiumStitched provides a simple, but flexible framework to handle various multi-capture area study design scenarios. Specifically, it resolves a data processing step without disrupting analysis workflows and without discarding data from overlapping spots. visiumStitched relies on affine transformations by Fiji, which have limitations and are less accurate when aligning against an atlas or other situations. visiumStitched provides an easy-to-use solution which expands possibilities for designing multi-capture area study designs.

Abstract 4145859: Clonal Hematopoiesis of Indeterminate Potential Increases Mortality Risk in Coronary Artery Disease

Background: Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with an increased incidence of common late-onset diseases including myocardial infarction. Research Question: Here we examined the impact of CHIP on mortality in patients with coronary artery disease (CAD) and further explored potential underlying mechanisms. Methods: CAD patients were examined for CHIP-defining mutations using deep DNA sequencing. Propensity score matching was used to compare CHIP CAD with non-CHIP CAD patients. Survival was analyzed using Kaplan-Meier curves and Cox proportional hazard models. Functional aspects were investigated at the protein and RNA level in cardiovascular relevant human postmortem tissue from the Munich cardIovaScular StudIes biObaNk (MISSION), in CAD patients from an independent cohort and in in-vitro macrophage models. Results: Within 7,933 CAD patients screened by deep-DNA-sequencing with a 13 gene panel, 2,002 carried at least one CHIP defining mutation with a variant allele frequency ≥2%. After 1:1 matching with non-CHIP carriers we revealed a significantly elevated 3-year mortality in the group of CHIP carriers [HR 1.48 (CI 95% 1.23-1.78); p<0.0001)] as well as in carriers of mutations in one out of seven individual genes – ASXL1 , DNMT3A , PPM1D , SF3B1 , SRSF2 , TET2 , and U2AF1 . In human coronary atherosclerotic plaque we visualized CHIP-mutations within leukocytes. Further, within coronary artery plaque of deceased TET2 mutation carriers (n=26 vs. n=13 controls) we identified by proteomic profiling an upregulation of inflammatory and metabolic pathways, including enhanced lipid metabolism. In an independent cohort of CAD patients undergoing coronary bypass surgery, RNA sequencing data from monocyte-derived macrophages revealed in TET2 CHIP carriers associations with CAD complexity. TET2 -mutated macrophages studied in-vitro revealed an increased uptake of low-density lipoprotein (LDL) and oxidized LDL-cholesterol besides a known upregulation of inflammatory pathways. Conclusion: In summary, this study emphasizes CHIP as a significant prognostic factor in CAD patients, associated with increased complexity of CAD and altered gene expression within human coronary artery plaques. Mechanistically, TET2 mutations modify the behavior of CHIP-affected macrophages, leading to heightened inflammation and increased uptake of LDL-cholesterol.

In vivo characterization of ACE2 expression in Sprague-Dawley rats and cultured primary brain pericytes highlights the utility of Rattus norvegicus in the study of COVID-19 brain pathophysiology.

A high number of COVID-19 patients report ongoing neurological impairments including headache, fatigue and memory impairments. Our understanding of COVID-19 disease mechanisms in the brain is limited and relies on post-mortem human tissues, in vitro studies in various cell lines (both human and animal) as well as preclinical studies in a variety of species. Notably the use of rats in the study of COVID-19 has been scarce in part due to early reports of low infectivity of the original Wuhan strain in mice and rats. Evidence has shown that subsequent strains that have mutated from the original strain and are capable of infection in rats. Here we present an immunohistological characterization of ACE2 expression in the rat brain perivascular region. We found ACE2 to be expressed in pericytes but not endothelial cells or astrocytes in the perivascular space. We further examined the uptake of Omicron variants 1.1.529 and BA.2 receptor binding domains (RBD) of the SARS-CoV2 spike protein in primary brain pericytes derived from rats. We demonstrate that rat primary brain pericytes are susceptible to SARS-CoV2 spike protein uptake and induce functional changes in pericytes associated with a reduction in tight junction protein expression. These data provide evidence that rat primary cell responses to SARS-CoV2 infection are consistent with reports of infectivity in other species (transgenic mice expressing hACE2, ferrets, hamsters) and supports the use of this model organism with a long history of use in the study of disease which should be leveraged for study of COVID-19 in the brain.

Divergent and Convergent TMEM106B Pathology in Murine Models of Neurodegeneration and Human Disease.

TMEM106B is a lysosomal/late endosome protein that is a potent genetic modifier of multiple neurodegenerative diseases as well as general aging. Recently, TMEM106B was shown to form insoluble aggregates in postmortem human brain tissue, drawing attention to TMEM106B pathology and the potential role of TMEM106B aggregation in disease. In the context of neurodegenerative diseases, TMEM106B has been studied in vivo using animal models of neurodegeneration, but these studies rely on overexpression or knockdown approaches. To date, endogenous TMEM106B pathology and its relationship to known canonical pathology in animal models has not been reported. Here, we analyze histological patterns of TMEM106B in murine models of C9ORF72 -related amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD), SOD1-related ALS, and tauopathy and compare these to postmortem human tissue from patients with C9-ALS/FTD, Alzheimer's disease (AD), and AD with limbic-predominant age-related TDP-43 encephalopathy (AD/LATE). We show that there are significant differences between TMEM106B pathology in mouse models and human patient tissue. Importantly, we also identified convergent evidence from both murine models and human patients that links TMEM106B pathology to TDP-43 nuclear clearance specifically in C9-ALS. Similarly, we find a relationship at the cellular level between TMEM106B pathology and phosphorylated Tau burden in Alzheimer's disease. By characterizing endogenous TMEM106B pathology in both mice and human postmortem tissue, our work reveals considerations that must be taken into account when analyzing data from in vivo mouse studies and elucidates new insights supporting the involvement of TMEM106B in the pathogenesis and progression of multiple neurodegenerative diseases.

Vulnerability of neurofilament-expressing neurons in frontotemporal dementia

Frontotemporal dementia (FTD) is an umbrella term for several early onset dementias, that are caused by frontotemporal lobar degeneration (FTLD), which involves the atrophy of the frontal and temporal lobes of the brain. Neuron loss in the frontal and temporal lobes is a characteristic feature of FTLD, however the selective vulnerability of different neuronal populations in this group of diseases is not fully understood. Neurofilament-expressing neurons have been shown to be selectively vulnerable in other neurodegenerative diseases, including Alzheimer's disease and amyotrophic lateral sclerosis, therefore we sought to investigate whether this neuronal population is vulnerable in FTLD. We also examined whether neuronal sub-type vulnerability differed between FTLD with TDP-43 inclusions (FTLD-TDP) and FTLD with tau inclusions (FTLD-Tau). Post-mortem human tissue from the superior frontal gyrus (SFG) of FTLD-TDP (n = 15), FTLD-Tau (n = 8) and aged Control cases (n = 6) was immunolabelled using antibodies against non-phosphorylated neurofilaments (SMI32 antibody), calretinin and NeuN, to explore neuronal cell loss. The presence of non-phosphorylated neurofilament immunolabelling in axons of the SFG white matter was also quantified as a measure of axon pathology, as axonal neurofilaments are normally phosphorylated. We demonstrate the selective loss of neurofilament-expressing neurons in both FTLD-TDP and FTLD-Tau cases compared to aged Controls. We also show that non-phosphorylated neurofilament axonal pathology in the SFG white matter was associated with increasing age, but not FTLD. This data suggests neurofilament-expressing neurons are vulnerable in both FTLD-TDP and FTLD-Tau.

Analysis of tetrahydroisoquinolines formed after simultaneous consumption of ethanol and amphetamine or its derivatives by LC-MS/MS in human blood, brain, and liver tissue.

The effects of the simultaneous consumption of amphetamine or amphetamine derivatives and alcohol have not yet been adequately clarified, particularly concerning potential condensation products resulting from the endogenous reaction between these substances and their metabolites (e.g., acetaldehyde, a metabolite of ethanol). In this study, we developed an LC-MS/MS method employing liquid-liquid extraction for the qualitative detection of some relevant condensation products belonging to the class of tetrahydroisoquinolines and their derivatives in human blood, brain, and liver samples. This includes the analysis of the substrates amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, as well as the condensation products 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, 1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline, and N-methyl-1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline. Therefore, the reference standards of the mentioned tetrahydroisoquinolines were synthesized in advance and the method was validated with regard to the question of the qualitative detection of these compounds. The validation parameters included selectivity, specificity, limit of detection, lower limit of quantification, recovery, matrix effects, and stability for blood, brain, and liver samples. Following the analysis of human blood and post-mortem tissue samples, evidence of the condensation product 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline originating from the interaction between amphetamine and acetaldehyde was identified in two liver samples. On the contrary, no evidence of this or other tetrahydroisoquinolines was found in the remaining tissue and serum samples.

Sex-specific Concordance of Striatal Transcriptional Signatures of Opioid Addiction in Human and Rodent Brains.

Opioid use disorder (OUD) has emerged as a severe, ongoing public health emergency. Current, frontline addiction treatment strategies fail to produce lasting abstinence in most users. This underscores the lasting effects of chronic opioid exposure and emphasizes the need to understand the molecular mechanisms of drug seeking and taking, but also how those alterations persist through acute and protracted withdrawal. Here, we used RNA sequencing in post-mortem human tissue from males (n=10) and females (n=10) with OUD and age and sex-matched comparison subjects. We compared molecular alterations in the nucleus accumbens (NAc) and dorsolateral prefrontal cortex (DLPFC) between humans with OUD and rodent models across distinct stages of opioid use and withdrawal (acute and prolonged) using differential gene expression and network-based approaches. We found that the molecular signature in the NAc of females with OUD mirrored effects seen in the NAc of female mice at all stages of exposure. Conversely, males with OUD showed strong overlap in expression profile with rats in acute withdrawal. Co-expression networks involved in post-transcriptional modification of RNA and epigenetic modification of chromatin state. This study provides fundamental insight into the converging molecular pathways altered by opioids across species. Further, this work helps to disentangle which alterations observed in humans with OUD are driven by acute drug exposure and which alterations are consequences of chronic exposure.

Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study

Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury (DAI) and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal swellings, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals with confirmed DAI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.

Unique Pathology in the Locus Coeruleus of Individuals with Down Syndrome.

Down syndrome (DS) is one of the most commonly occurring chromosomal conditions. Most individuals with DS develop Alzheimer's disease (AD) by 50 years of age. Recent evidence suggests that AD pathology in the locus coeruleus (LC) is an early event in sporadic AD. It is likely that the widespread axonal network of LC neurons contributes to the spread of tau pathology in the AD brain, although this has not been investigated in DS-AD. The main purpose of this study was to profile AD pathology and neuroinflammation in the LC, comparing AD and DS-AD in postmortem human tissues. We utilized immunofluorescence and semi-quantitative analyses of pTau (4 different forms), amyloid-β (Aβ), glial, and neuronal markers in the LC across 36 cases (control, DS-AD, and AD) to compare the different pathological profiles. Oligomeric tau was highly elevated in DS-AD cases compared to LOAD or EOAD cases. The distribution of staining for pT231 was elevated in DS-AD and EOAD compared to the LOAD group. The DS-AD group exhibited increased Aβ immunostaining compared to AD cases. The number of tau-bearing neurons was also significantly different between the EOAD and DS-AD cases compared to the LOAD cases. While inflammation, pTau, and Aβ are all involved in AD pathology, their contribution to disease progression may differ depending on the diagnosis. Our results suggest that DS-AD and EOAD may be more similar in pathology than LOAD. Our study highlights unique avenues to further our understanding of the mechanisms governing AD neuropathology.

Mitigation of synaptic and memory impairments via F-actin stabilization in Alzheimer’s disease

BackgroundSynaptic dysfunction, characterized by synapse loss and structural alterations, emerges as a prominent correlate of cognitive decline in Alzheimer’s disease (AD). Actin cytoskeleton, which serves as the structural backbone of synaptic architecture, is observed to be lost from synapses in AD. Actin cytoskeleton loss compromises synaptic integrity, affecting glutamatergic receptor levels, neurotransmission, and synaptic strength. Understanding these molecular changes is crucial for developing interventions targeting synaptic dysfunction, potentially mitigating cognitive decline in AD.MethodsIn this study, we investigated the synaptic actin interactome using mass spectrometry in a mouse model of AD, APP/PS1. Our objective was to explore how alterations in synaptic actin dynamics, particularly the interaction between PSD-95 and actin, contribute to synaptic and cognitive impairment in AD. To assess the impact of restoring F-actin levels on synaptic and cognitive functions in APP/PS1 mice, we administered F-actin stabilizing agent, jasplakinolide. Behavioral deficits in the mice were evaluated using the contextual fear conditioning paradigm. We utilized primary neuronal cultures to study the synaptic levels of AMPA and NMDA receptors and the dynamics of PSD-95 actin association. Furthermore, we analyzed postmortem brain tissue samples from subjects with no cognitive impairment (NCI), mild cognitive impairment (MCI), and Alzheimer’s dementia (AD) to determine the association between PSD-95 and actin.ResultsWe found a significant reduction in PSD-95-actin association in synaptosomes from middle-aged APP/PS1 mice compared to wild-type (WT) mice. Treatment with jasplakinolide, an actin stabilizer, reversed deficits in memory recall, restored PSD-95-actin association, and increased synaptic F-actin levels in APP/PS1 mice. Additionally, actin stabilization led to elevated synaptic levels of AMPA and NMDA receptors, enhanced dendritic spine density, suggesting improved neurotransmission and synaptic strength in primary cortical neurons from APP/PS1 mice. Furthermore, analysis of postmortem human tissue with NCI, MCI and AD subjects revealed disrupted PSD-95-actin interactions, underscoring the clinical relevance of our preclinical studies.ConclusionOur study elucidates disrupted PSD-95 actin interactions across different models, highlighting potential therapeutic targets for AD. Stabilizing F-actin restores synaptic integrity and ameliorates cognitive deficits in APP/PS1 mice, suggesting that targeting synaptic actin regulation could be a promising therapeutic strategy to mitigate cognitive decline in AD.

Pathophysiological processes underlying hidden hearing loss revealed in Kcnt1/2 double knockout mice.

Presbycusis is a prevalent condition in older adults characterized by the progressive loss of hearing due to age-related changes in the cochlea, the auditory portion of the inner ear. Many adults also struggle with understanding speech in noise despite having normal auditory thresholds, a condition termed "hidden" hearing loss because it evades standard audiological assessments. Examination of animal models and postmortem human tissue suggests that hidden hearing loss is also associated with age-related changes in the cochlea and may, therefore, precede overt age-related hearing loss. Nevertheless, the pathological mechanisms underlying hidden hearing loss are not understood, which hinders the development of diagnostic biomarkers and effective treatments for age-related hearing loss. To fill these gaps in knowledge, we leveraged a combination of tools, including transcriptomic profiling and morphological and functional assessments, to identify these processes and examine the transition from hidden to overt hearing loss. As a novel approach, we took advantage of a recently characterized model of hidden hearing loss: Kcnt1/2 double knockout mice. Using this model, we find that even before observable morphological pathology, hidden hearing loss is associated with significant alteration in several processes, notably proteostasis, in the cochlear sensorineural structures, and increased susceptibility to overt hearing loss in response to noise exposure and aging. Our findings provide the first insight into the pathophysiology associated with the earliest and, therefore, most treatable stages of hearing loss and provide critical insight directing future investigation of pharmaceutical strategies to slow and possibly prevent overt age-related hearing loss.

Hippocampal Availability of the α7 Nicotinic Acetylcholine Receptor in Recent-Onset Psychosis

Studies using human postmortem tissue and imaging with positron emission tomography (PET) support a low hippocampal availability of the α7 nicotinic acetylcholine receptor (α7-nAChR) in psychotic conditions, particularly in schizophrenia or schizoaffective disorder (nonaffective psychosis). If validated further, the finding may have implications for clinical diagnosis and treatment. To test for lower availability of the α7-nAChR in the hippocampus of individuals with recent-onset psychosis compared with healthy control individuals and its association with lower cognitive performance or higher psychotic symptom burden within recent-onset psychosis. In this cross-sectional study, healthy individuals without history of psychosis and patients within 10 years of a first onset of psychotic disorder were recruited from the greater Baltimore, Maryland, and Washington, DC, area. Fluorine 18-labeled ASEM ([18F] ASEM) PET data were acquired from participants enrolled between March 1, 2014, and July 31, 2023, from an academic research institution. Data acquired between March 1, 2014, and January 31, 2018 (n = 26), were published as a pilot study and were combined with new data acquired between January 1, 2019, and July 31, 2023 (n = 33). Regional [18F]ASEM total distribution volume (VT) that measures α7-nAChR availability, global cognition composite score, and total scores on the Scale for the Assessment of Positive Symptoms and the Scale for the Assessment of Negative Symptoms. A total of 59 participants (30 women [51%]; mean [SD] age, 25.5 [5.2] years), including 35 with recent-onset psychosis and 24 healthy controls, completed the study. In age-adjusted analyses, lower hippocampal [18F]ASEM VT was found in individuals with recent-onset psychosis (mean [SE], 17.87 [0.60]) compared with healthy controls (mean [SE], 19.82 [0.73]) (P = .04). In addition, [18F]ASEM VT was lower in individuals with nonaffective psychosis (mean [SE], 16.30 [0.83]) compared with healthy controls (P = .006) or those with affective psychosis (mean [SE], 19.34 [0.80]) (P = .03). Across recent-onset psychosis and after controlling for age, lower hippocampal [18F]ASEM VT was associated with more positive (r = -0.44; P = .009) but not negative symptoms, and higher hippocampal VT was associated with better global cognition composite score (r = 0.38; P = .03). In this cross-sectional study of individuals with recent-onset psychosis compared with healthy controls, a lower hippocampal α7-nAChR availability was found in recent-onset psychosis, and its availability was lower in those with nonaffective vs affective psychosis. Further study of the association between low availability of the α7-nAChR and recent-onset psychosis is warranted toward informing diagnostic or therapeutic strategies related to these findings.

The neocortical infrastructure for language involves region-specific patterns of laminar gene expression

The language network of the human brain has core components in the inferior frontal cortex and superior/middle temporal cortex, with left-hemisphere dominance in most people. Functional specialization and interconnectivity of these neocortical regions is likely to be reflected in their molecular and cellular profiles. Excitatory connections between cortical regions arise and innervate according to layer-specific patterns. Here, we generated a gene expression dataset from human postmortem cortical tissue samples from core language network regions, using spatial transcriptomics to discriminate gene expression across cortical layers. Integration of these data with existing single-cell expression data identified 56 genes that showed differences in laminar expression profiles between the frontal and temporal language cortex together with upregulation in layer II/III and/or layer V/VI excitatory neurons. Based on data from large-scale genome-wide screening in the population, DNA variants within these 56 genes showed set-level associations with interindividual variation in structural connectivity between the left-hemisphere frontal and temporal language cortex, and with the brain-related disorders dyslexia and schizophrenia which often involve affected language. These findings identify region-specific patterns of laminar gene expression as a feature of the brain's language network.

Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study.

Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons following brain injury in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal segments, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals that sustained a TBI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.

Novel Monoclonal Antibody Specific toward Amyloid-β Binds to a Unique Epitope within the N-Terminal Region.

Amyloid-β (Aβ) deposition throughout the neuroaxis is a classical hallmark of several neurodegenerative diseases, most notably Alzheimer's disease (AD). Aβ peptides of varied length and diverse structural conformations are deposited within the parenchyma and vasculature in the brains of individuals with AD. Neuropathologically, Aβ pathology can be assessed using antibodies to label and characterize their features, which in turn leads to a more extensive understanding of the pathological process. In the present study, we generated a novel monoclonal antibody, which we found to be specific for the N-terminal region of Aβ. This antibody reacted to amyloid precursor protein expressed in cultured cells and labels Aβ plaques and cerebral amyloid angiopathy in brain tissue from a mouse model of amyloidosis as well as post-mortem brain tissue from patients diagnosed with AD. This highly specific novel antibody will serve as a unique tool for future studies investigating Aβ deposition in novel mouse models and cross-sectional studies using post-mortem human tissue.

Integrating gene expression and imaging data across Visium capture areas with visiumStitched.

Visium is a widely-used spatially-resolved transcriptomics assay available from 10x Genomics. Standard Visium capture areas (6.5mm by 6.5mm) limit the survey of larger tissue structures, but combining overlapping images and associated gene expression data allow for more complex study designs. Current software can handle nested or partial image overlaps, but is designed for merging up to two capture areas, and cannot account for some technical scenarios related to capture area alignment. We generated Visium data from a postmortem human tissue sample such that two capture areas were partially overlapping and a third one was adjacent. We developed the R/Bioconductor package visiumStitched, which facilitates stitching the images together with Fiji (ImageJ), and constructing SpatialExperiment R objects with the stitched images and gene expression data. visiumStitched constructs an artificial hexagonal array grid which allows seamless downstream analyses such as spatially-aware clustering without discarding data from overlapping spots. Data stitched with visiumStitched can then be interactively visualized with spatialLIBD. visiumStitched provides a simple, but flexible framework to handle various multi-capture area study design scenarios. Specifically, it resolves a data processing step without disrupting analysis workflows and without discarding data from overlapping spots. visiumStiched relies on affine transformations by Fiji, which have limitations and are less accurate when aligning against an atlas or other situations. visiumStiched provides an easy-to-use solution which expands possibilities for designing multi-capture area study designs.

Molecular properties and diagnostic potential of monoclonal antibodies targeting cytotoxic α-synuclein oligomers

α-Synuclein (α-syn) accumulates as insoluble amyloid but also forms soluble α-syn oligomers (αSOs), thought to be even more cytotoxic than fibrils. To detect and block the unwanted activities of these αSOs, we have raised 30 monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs’ relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, while the commercial antibody MJF14 only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. Five selected mAbs identified α-syn-related pathologies like Lewy bodies (LBs) and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs or multiple system atrophy, although to different extents. Three mAbs were particularly useful for pathological evaluation of postmortem brain human tissue, including early stages of PD. Although there was no straightforward connection between the mAbs’ biophysical and immunohistochemical properties, it is encouraging that this comprehensive collection of mAbs able to recognize different aggregated α-syn species in vitro also holds diagnostic potential.

Complement Factor B Inhibition or Deletion Is Not Sufficient to Prevent Neurodegeneration in a Murine Model of Glaucoma.

Purpose: Activation of the classical complement pathway is thought to contribute to the development and progression of glaucoma. The role of alternative complement or amplification pathways in glaucoma is not well understood. We evaluated complement factor B (FB) expression in postmortem human ocular tissues with or without glaucoma and the effect of FB inhibition and deletion in a mouse ocular hypertensive model of glaucoma induced by photopolymerized hyaluronic acid glycidyl methacrylate (HAGM). Methods: Human CFB mRNA in human eyes was assessed by RNAscope and TaqMan. HAGM model was performed on C57BL6/J mice. The effect of FB in HAGM model was evaluated with an oral FB inhibitor and Cfb-/- mice. Complement mRNA and proteins in mouse eyes were assessed by TaqMan and western blot, respectively. Results: CFB mRNA in human glaucomatous macular neural retina and optic nerve head was upregulated. Cfb mRNA is also upregulated in the HAGM model. Oral FB inhibitor, ED-79-GX17, dosed daily at 200 mg/kg for 3 days after intraocular pressure (IOP) induction in wild-type mice showed complement inhibition in ocular tissues and significantly inhibited systemic complement levels. Daily dosing of ED-79-GX17 for 30 days or Cfb deletion was also unable to prevent retinal ganglion cell or axon loss 30 days after IOP induction in mice. Conclusion: The alternative complement component FB may not substantially contribute to RGC loss in the HAGM mouse glaucoma model despite upregulation of Cfb expression and activation of the alternative pathway. The relevance of these findings to human glaucoma remains to be determined.

Alterations to metabolic hormones in amyotrophic lateral sclerosis and frontotemporal dementia postmortem human tissue.

Metabolic changes are observed in patients with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although regulation of metabolic processes in the CNS is predominantly carried out within the hypothalamus, extra-hypothalamic CNS areas contain metabolic hormone receptors, including those for leptin (LEPR), insulin (INSR), and neuropeptide Y (NPY), indicating that they may play a role in biological processes underlying pathogenic disease processes. The status of these hormones within regions vulnerable in ALS/FTD is not well described. This study sought to determine whether the expression of these hormones and their receptors is altered in pathology-rich regions in cases of human FTD (superior frontal gyrus and insular cortex) and ALS (primary motor cortex and lumbar spinal cord) with TDP-43 pathology compared to matched healthy controls. LEPR mRNA was increased within the superior frontal gyrus of FTD cases and within primary motor cortex and lumbar spinal cord of ALS cases; INSR mRNA was increased in superior frontal gyrus and insular cortex of FTD cases. NPY protein was decreased in primary motor cortex and lumbar spinal cord of ALS cases. Our results demonstrate that metabolic hormones undergo complex alterations in ALS and FTD and suggest that these hormones could play critical roles in the pathogenesis of these diseases.

Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue

The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.