Spatial epigenome-transcriptome co-profiling of mammalian tissues.

Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context1-5. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.

Spatial profiling of chromatin accessibility in mouse and human tissues.

Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2-5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.

Brain and blood transcriptome profiles delineate common genetic pathways across suicidal ideation and suicide.

Human genetic studies indicate that suicidal ideation and behavior are both heritable. Most studies have examined associations between aberrant gene expression and suicide behavior, but behavior risk is linked to the severity of suicidal ideation. Through a gene network approach, this study investigates how gene co-expression patterns are associated with suicidal ideation and severity using RNA-seq data in peripheral blood from 46 live participants with elevated suicidal ideation and 46 with no ideation. Associations with the presence of suicidal ideation were found within 18 co-expressed modules (p < 0.05), as well as in 3 co-expressed modules associated with suicidal ideation severity (p < 0.05, not explained by severity of depression). Suicidal ideation presence and severity-related gene modules with enrichment of genes involved in defense against microbial infection, inflammation, and adaptive immune response were identified and investigated using RNA-seq data from postmortem brain that revealed gene expression differences with moderate effect sizes in suicide decedents vs. non-suicides in white matter, but not gray matter. Findings support a role of brain and peripheral blood inflammation in suicide risk, showing that suicidal ideation presence and severity are associated with an inflammatory signature detectable in blood and brain, indicating a biological continuity between ideation and suicidal behavior that may underlie a common heritability.

A Stress Protein-Based Suicide Prediction Score and Relationship to Reported Early-Life Adversity and Recent Life Stress.

BACKGROUND: The hypothalamic-pituitary-adrenal (HPA) axis is a major stress response system, and excessive HPA responses can impact major depressive disorder and suicide. We examined relationships between reported early-life adversity (ELA), recent-life stress (RLS), suicide, and corticotropin-releasing hormone (CRH), CRH binding protein, FK506-binding protein (FKBP5), glucocorticoid receptor (GR), and brain-derived neurotrophic factor (BDNF) in postmortem human prefrontal cortex (BA9), and anterior cingulate cortex (BA24). METHODS: Thirteen quadruplets, matched for sex, age, and postmortem interval and consisting of suicide decedents and healthy controls, were divided equally into those with and without ELA. ELA, RLS, and psychiatric diagnoses were determined by psychological autopsy. Protein levels were determined by western blots. RESULTS: There were no suicide- or ELA-related differences in CRH, CRH binding protein, GR, or FKBP5 in BA9 or BA24 and no interaction between suicide and ELA (P > .05). For BDNF, there was an interaction between suicide and ELA in BA24; suicides without ELA had less BDNF than controls without ELA, and controls with ELA had less BDNF than controls without ELA. CRH in BA9 and FKBP5 in anterior cingulate cortex correlated negatively with RLS. Least Absolute Shrinkage and Selection Operator logistic regression with cross-validation found combining BDNF, GR, and FKBP5 BA24 levels predicted suicide, but ELA did not contribute. A calculated "suicide risk score" using these measures had 71% sensitivity and 71% specificity. CONCLUSION: A dysregulated HPA axis is related to suicide but not with ELA. RLS was related to select HPA axis proteins in specific brain regions. BDNF appears to be dysregulated in a region-specific way with ELA and suicide.

COVID-19 induces CNS cytokine expression and loss of hippocampal neurogenesis.

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with acute and postacute cognitive and neuropsychiatric symptoms including impaired memory, concentration, attention, sleep and affect. Mechanisms underlying these brain symptoms remain understudied. Here we report that SARS-CoV-2-infected hamsters exhibit a lack of viral neuroinvasion despite aberrant blood-brain barrier permeability. Hamsters and patients deceased from coronavirus disease 2019 (COVID-19) also exhibit microglial activation and expression of interleukin (IL)-1ß and IL-6, especially within the hippocampus and the medulla oblongata, when compared with non-COVID control hamsters and humans who died from other infections, cardiovascular disease, uraemia or trauma. In the hippocampal dentate gyrus of both COVID-19 hamsters and humans, we observed fewer neuroblasts and immature neurons. Protracted inflammation, blood-brain barrier disruption and microglia activation may result in altered neurotransmission, neurogenesis and neuronal damage, explaining neuropsychiatric presentations of COVID-19. The involvement of the hippocampus may explain learning, memory and executive dysfunctions in COVID-19 patients.

Association of BDNF Val66Met Polymorphism and Brain BDNF Levels with Major Depression and Suicide.

Background: Brain-derived neurotrophic factor is implicated in the pathophysiology of major depressive disorder and suicide. Both are partly caused by early life adversity, which reduces brain-derived neurotrophic factor protein levels. This study examines the association of brain-derived neurotrophic factor Val66Met polymorphism and brain brain-derived neurotrophic factor levels with depression and suicide. We hypothesized that both major depressive disorder and early life adversity would be associated with the Met allele and lower brain brain-derived neurotrophic factor levels. Such an association would be consistent with low brain-derived neurotrophic factor mediating the effect of early life adversity on adulthood suicide and major depressive disorder. Methods: Brain-derived neurotrophic factor Val66Met polymorphism was genotyped in postmortem brains of 37 suicide decedents and 53 nonsuicides. Additionally, brain-derived neurotrophic factor protein levels were determined by Western blot in dorsolateral prefrontal cortex (Brodmann area 9), anterior cingulate cortex (Brodmann area 24), caudal brainstem, and rostral brainstem. The relationships between these measures and major depressive disorder, death by suicide, and reported early life adversity were examined. Results: Subjects with the Met allele had an increased risk for depression. Depressed patients also have lower brain-derived neurotrophic factor levels in anterior cingulate cortex and caudal brainstem compared with nondepressed subjects. No effect of history of suicide death or early life adversity was observed with genotype, but lower brain-derived neurotrophic factor levels in the anterior cingulate cortex were found in subjects who had been exposed to early life adversity and/or died by suicide compared with nonsuicide decedents and no reported early life adversity. Conclusions: This study provides further evidence implicating low brain brain-derived neurotrophic factor and the brain-derived neurotrophic factor Met allele in major depression risk. Future studies should seek to determine how altered brain-derived neurotrophic factor expression contributes to depression and suicide.

GLUCOCORTICOID RECEPTOR-RELATED GENES: GENOTYPE AND BRAIN GENE EXPRESSION RELATIONSHIPS TO SUICIDE AND MAJOR DEPRESSIVE DISORDER.

INTRODUCTION: We tested the relationship between genotype, gene expression and suicidal behavior and major depressive disorder (MDD) in live subjects and postmortem samples for three genes, associated with the hypothalamic-pituitary-adrenal axis, suicidal behavior, and MDD; FK506-binding protein 5 (FKBP5), Spindle and kinetochore-associated protein 2 (SKA2), and Glucocorticoid Receptor (NR3C1). MATERIALS AND METHODS: Single-nucleotide polymorphisms (SNPs) and haplotypes were tested for association with suicidal behavior and MDD in a live (N = 277) and a postmortem sample (N = 209). RNA-seq was used to examine gene and isoform-level brain expression postmortem (Brodmann Area 9; N = 59). Expression quantitative trait loci (eQTL) relationships were examined using a public database (UK Brain Expression Consortium). RESULTS: We identified a haplotype within the FKBP5 gene, present in 47% of the live subjects, which was associated with increased risk of suicide attempt (OR = 1.58, t = 6.03, P = .014). Six SNPs on this gene, three SNPs on SKA2, and one near NR3C1 showed before-adjustment association with attempted suicide, and two SNPs of SKA2 with suicide death, but none stayed significant after adjustment for multiple testing. Only the SKA2 SNPs were related to expression in the prefrontal cortex (pFCTX). One NR3C1 transcript had lower expression in suicide relative to nonsuicide sudden death cases (b = -0.48, SE = 0.12, t = -4.02, adjusted P = .004). CONCLUSION: We have identified an association of FKBP5 haplotype with risk of suicide attempt and found an association between suicide and altered NR3C1 gene expression in the pFCTX. Our findings further implicate hypothalamic pituitary axis dysfunction in suicidal behavior.

A pilot integrative genomics study of GABA and glutamate neurotransmitter systems in suicide, suicidal behavior, and major depressive disorder.

Gamma-amino butyric acid (GABA) and glutamate are the major inhibitory and excitatory neurotransmitters in the mammalian central nervous system, respectively, and have been associated with suicidal behavior and major depressive disorder (MDD). We examined the relationship between genotype, brain transcriptome, and MDD/suicide for 24 genes involved in GABAergic and glutamatergic signaling. In part 1 of the study, 119 candidate SNPs in 24 genes (4 transporters, 4 enzymes, and 16 receptors) were tested for associations with MDD and suicidal behavior in 276 live participants (86 nonfatal suicide attempters with MDD and 190 non-attempters of whom 70% had MDD) and 209 postmortem cases (121 suicide deaths of whom 62% had MDD and 88 sudden death from other causes of whom 11% had MDD) using logistic regression adjusting for sex and age. In part 2, RNA-seq was used to assay isoform-level expression in dorsolateral prefrontal cortex of 59 postmortem samples (21 with MDD and suicide, 9 MDD without suicide, and 29 sudden death non-suicides and no psychiatric illness) using robust regression adjusting for sex, age, and RIN score. In part 3, SNPs with subthreshold (uncorrected) significance levels below 0.05 for an association with suicidal behavior and/or MDD in part 1 were tested for eQTL effects in prefrontal cortex using the Brain eQTL Almanac (www.braineac.org). No SNPs or transcripts were significant after adjustment for multiple comparisons. However, a protein coding transcript (ENST00000414552) of the GABA A receptor, gamma 2 (GABRG2) had lower brain expression postmortem in suicide (P = 0.01) and evidence for association with suicide death (P = 0.03) in a SNP that may be an eQTL in prefrontal cortex (rs424740, P = 0.02). These preliminary results implicate GABRG2 in suicide and warrant further investigation and replication in larger samples.

Postnatal day 2 to 11 constitutes a 5-HT-sensitive period impacting adult mPFC function.

Early-life serotonin [5-hydroxytryptamine (5-HT)] signaling modulates brain development, which impacts adult behavior, but 5-HT-sensitive periods, neural substrates, and behavioral consequences remain poorly understood. Here we identify the period ranging from postnatal day 2 (P2) to P11 as 5-HT sensitive, with 5-HT transporter (5-HTT) blockade increasing anxiety- and depression-like behavior, and impairing fear extinction learning and memory in adult mice. Concomitantly, P2-P11 5-HTT blockade causes dendritic hypotrophy and reduced excitability of infralimbic (IL) cortex pyramidal neurons that normally promote fear extinction. By contrast, the neighboring prelimbic (PL) pyramidal neurons, which normally inhibit fear extinction, become more excitable. Excitotoxic IL but not PL lesions in adult control mice reproduce the anxiety-related phenotypes. These findings suggest that increased 5-HT signaling during P2-P11 alters adult mPFC function to increase anxiety and impair fear extinction, and imply a differential role for IL and PL neurons in regulating affective behaviors. Together, our results support a developmental mechanism for the etiology and pathophysiology of affective disorders and fear-related behaviors.

Benzodiazepines and the potential trophic effect of antidepressants on dentate gyrus cells in mood disorders.

Modest antidepressant response rates of mood disorders (MD) encourage benzodiazepine (BZD) co-medication with debatable benefit. Adult hippocampal neurogenesis may underlie antidepressant responses, but diazepam co-administration impairs murine neuron maturation and survival in response to fluoxetine. We counted neural progenitor cells (NPCs), mitotic cells, and mature granule neurons post-mortem in dentate gyrus (DG) from subjects with: untreated Diagnostic and Statistical Manual of Mental Disorders (DSM) IV MD (n = 17); antidepressant-treated MD (MD*ADT, n = 10); benzodiazepine-antidepressant-treated MD (MD*ADT*BZD, n = 7); no psychopathology or treatment (controls, n = 18). MD*ADT*BZD had fewer granule neurons vs. MD*ADT in anterior DG and vs. controls in mid DG, and did not differ from untreated-MD in any DG subregion. MD*ADT had more granule neurons than untreated-MD in anterior and mid DG and comparable granule neuron number to controls in all dentate subregions. Untreated-MD had fewer granule neurons than controls in anterior and mid DG, and did not differ from any other group in posterior DG. MD*ADT*BZD had fewer NPCs vs. MD*ADT in mid DG. MD*ADT had more NPCs vs. untreated-MD and controls in anterior and mid DG. MD*ADT*BZD and MD*ADT had more mitotic cells in anterior DG vs. controls and untreated-MD. There were no between-group differences in mid DG in mitotic cells or in posterior DG for any cell type. Our results in mid-dentate, and to some degree anterior dentate, gyrus are consistent with murine findings that benzodiazepines counteract antidepressant-induced increases in neurogenesis by interfering with progenitor proliferation. We also confirmed, in this expanded sample, our previous finding of granule neuron deficit in untreated MD.

Late-stage borreliosis and substance abuse.

Background: Infectious diseases can contribute to substance abuse. Here, a fatal case of borreliosis and substance abuse is reported. This patient had a history of multiple tick bites and increasing multisystem symptoms, yet diagnosis and treatment were delayed. He experimented with multiple substances including phencyclidine (PCP), an N-methyl-d-aspartate (NMDA) receptor antagonist that opposes NMDA agonism caused by Borrelia infection. During PCP withdrawal, he committed one homicide, two assaults, and suicide. Methods: Brain tissue was obtained from autopsy and stained for microglial activation and quinolinic acid (QA). Immunoflouresence (IFA) and fluorescence in situ hybridization (FISH) were used to identify the presence of pathogens in autopsy tissue. Results: Autopsy tissue evaluation demonstrated Borrelia in the pancreas by IFA and heart by IFA and FISH. Activated microglia and QA were found in the brain, indicating neuroinflammation. It is postulated that PCP withdrawal may exacerbate symptoms produced by Borrelia-induced biochemical imbalances in the brain. This combination may have greatly increased his acute homicidal and suicidal risk. Patient databases also demonstrated the risk of homicide or suicide in patients diagnosed with borreliosis and confirmed multiple symptoms in these patients, including chronic pain, anxiety, and anhedonia. Conclusions: Late-stage borreliosis is associated with multiple symptoms that may contribute to an increased risk of substance abuse and addictive disorders. More effective diagnosis and treatment of borreliosis, and attention to substance abuse potential may help reduce associated morbidity and mortality in patients with borreliosis, particularly in endemic areas.

A Human Brain Map of Mitochondrial Respiratory Capacity and Diversity.

Mitochondrial oxidative phosphorylation (OxPhos) powers brain activity1,2, and mitochondrial defects are linked to neurodegenerative and neuropsychiatric disorders3,4, underscoring the need to define the brain's molecular energetic landscape5-10. To bridge the cognitive neuroscience and cell biology scale gap, we developed a physical voxelization approach to partition a frozen human coronal hemisphere section into 703 voxels comparable to neuroimaging resolution (3×3×3 mm). In each cortical and subcortical brain voxel, we profiled mitochondrial phenotypes including OxPhos enzyme activities, mitochondrial DNA and volume density, and mitochondria-specific respiratory capacity. We show that the human brain contains a diversity of mitochondrial phenotypes driven by both topology and cell types. Compared to white matter, grey matter contains >50% more mitochondria. We show that the more abundant grey matter mitochondria also are biochemically optimized for energy transformation, particularly among recently evolved cortical brain regions. Scaling these data to the whole brain, we created a backward linear regression model integrating several neuroimaging modalities11, thereby generating a brain-wide map of mitochondrial distribution and specialization that predicts mitochondrial characteristics in an independent brain region of the same donor brain. This new approach and the resulting MitoBrainMap of mitochondrial phenotypes provide a foundation for exploring the molecular energetic landscape that enables normal brain functions, relating it to neuroimaging data, and defining the subcellular basis for regionalized brain processes relevant to neuropsychiatric and neurodegenerative disorders.

A Human Brain Map of Mitochondrial Respiratory Capacity and Diversity.

Mitochondrial oxidative phosphorylation (OxPhos) powers brain activity1,2, and mitochondrial defects are linked to neurodegenerative and neuropsychiatric disorders3,4, underscoring the need to define the brain's molecular energetic landscape5-10. To bridge the cognitive neuroscience and cell biology scale gap, we developed a physical voxelization approach to partition a frozen human coronal hemisphere section into 703 voxels comparable to neuroimaging resolution (3×3×3 mm). In each cortical and subcortical brain voxel, we profiled mitochondrial phenotypes including OxPhos enzyme activities, mitochondrial DNA and volume density, and mitochondria-specific respiratory capacity. We show that the human brain contains a diversity of mitochondrial phenotypes driven by both topology and cell types. Compared to white matter, grey matter contains >50% more mitochondria. We show that the more abundant grey matter mitochondria also are biochemically optimized for energy transformation, particularly among recently evolved cortical brain regions. Scaling these data to the whole brain, we created a backward linear regression model integrating several neuroimaging modalities11, thereby generating a brain-wide map of mitochondrial distribution and specialization that predicts mitochondrial characteristics in an independent brain region of the same donor brain. This new approach and the resulting MitoBrainMap of mitochondrial phenotypes provide a foundation for exploring the molecular energetic landscape that enables normal brain functions, relating it to neuroimaging data, and defining the subcellular basis for regionalized brain processes relevant to neuropsychiatric and neurodegenerative disorders.

Mitochondrial abnormalities in temporal lobe of autistic brain.

Autism spectrum disorder (ASD) consists of a group of complex developmental disabilities characterized by impaired social interactions, deficits in communication and repetitive behavior. Multiple lines of evidence implicate mitochondrial dysfunction in ASD. In postmortem BA21 temporal cortex, a region that exhibits synaptic pathology in ASD, we found that compared to controls, ASD patients exhibited altered protein levels of mitochondria respiratory chain protein complexes, decreased Complex I and IV activities, decreased mitochondrial antioxidant enzyme SOD2, and greater oxidative DNA damage. Mitochondrial membrane mass was higher in ASD brain, as indicated by higher protein levels of mitochondrial membrane proteins Tom20, Tim23 and porin. No differences were observed in either mitochondrial DNA or levels of the mitochondrial gene transcription factor TFAM or cofactor PGC1α, indicating that a mechanism other than alterations in mitochondrial genome or mitochondrial biogenesis underlies these mitochondrial abnormalities. We further identified higher levels of the mitochondrial fission proteins (Fis1 and Drp1) and decreased levels of the fusion proteins (Mfn1, Mfn2 and Opa1) in ASD patients, indicating altered mitochondrial dynamics in ASD brain. Many of these changes were evident in cortical pyramidal neurons, and were observed in ASD children but were less pronounced or absent in adult patients. Together, these findings provide evidence that mitochondrial function and intracellular redox status are compromised in pyramidal neurons in ASD brain and that mitochondrial dysfunction occurs during early childhood when ASD symptoms appear.

Functional Architecture of Brain and Blood Transcriptome Delineate Biological Continuity Between Suicidal Ideation and Suicide.

Human genetic studies indicate that suicidal ideation and behavior are both heritable. Most studies have examined associations between aberrant gene expression and suicide behavior, but behavior risk is linked to severity of suicidal ideation. Through a gene network approach, this study investigates how gene co-expression patterns are associated with suicidal ideation and severity using RNA-seq data in peripheral blood from 46 live participants with elevated suicidal ideation and 46 with no ideation. Associations with presence and severity of suicidal ideation were found within 18 and 3 co-expressed modules respectively (p < 0.05), not explained by severity of depression. Suicidal ideation presence and severity-related gene modules with enrichment of genes involved in defense against microbial infection, inflammation, and adaptive immune response were identified, and tested using RNA-seq data from postmortem brain that revealed gene expression differences in suicide decedents vs. non-suicides in white matter, but not gray matter. Findings support a role of brain and peripheral blood inflammation in suicide risk, showing that suicidal ideation presence and severity is associated with an inflammatory signature detectable in blood and brain, indicating a biological continuity between ideation and suicidal behavior that may underlie a common heritability.

Altered adult neurogenesis and gliogenesis in patients with mesial temporal lobe epilepsy.

The hippocampus is the most common seizure focus in people. In the hippocampus, aberrant neurogenesis plays a critical role in the initiation and progression of epilepsy in rodent models, but it is unknown whether this also holds true in humans. To address this question, we used immunofluorescence on control healthy hippocampus and surgical resections from mesial temporal lobe epilepsy (MTLE), plus neural stem-cell cultures and multi-electrode recordings of ex vivo hippocampal slices. We found that a longer duration of epilepsy is associated with a sharp decline in neuronal production and persistent numbers in astrogenesis. Further, immature neurons in MTLE are mostly inactive, and are not observed in cases with local epileptiform-like activity. However, immature astroglia are present in every MTLE case and their location and activity are dependent on epileptiform-like activity. Immature astroglia, rather than newborn neurons, therefore represent a potential target to continually modulate adult human neuronal hyperactivity.

Hemispheric comparisons of neuron density in the planum temporale of schizophrenia and nonpsychiatric brains.

Postmortem and in vivo studies of schizophrenia frequently reveal reduced cortical volume, but the underlying cellular abnormalities are incompletely defined. One influential hypothesis, especially investigated in Brodmann's area 9 of prefrontal cortex, is that the number of neurons is normal, and the volume change is caused by reduction of the surrounding neuropil. However, studies have differed on whether the cortex has the increased neuron density that is predicted by this hypothesis. In a recent study of bilateral planum temporale (PT), we reported smaller volume and width of the outer cortex (layers I-III), especially in the left hemisphere, among subjects with schizophrenia. In the present study, we measured neuron density and size in the same PT samples, and also in prefrontal area 9 of the same brains. In the PT, separate stereological measurements were made in layers II, IIIc, and VI, whereas area 9 was sampled in layer IIIb-c. In both cortical regions, there was no significant effect of schizophrenia on neuronal density or size. There was, nevertheless, a trend-level right>left hemispheric asymmetry of neuron density in the PT, which may partially explain the previously reported left>right asymmetry of cortical width. In schizophrenia, our findings suggest that closer packing of neurons may not always explain reduced cortical volume, and subtly decreased neuron number may be a contributing factor.

Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.

The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64-69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SIGNIFICANCE: Here we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE tissue analysis.

Detecting Borrelia Spirochetes: A Case Study With Validation Among Autopsy Specimens.

The complex etiology of neurodegenerative disease has prompted studies on multiple mechanisms including genetic predisposition, brain biochemistry, immunological responses, and microbial insult. In particular, Lyme disease is often associated with neurocognitive impairment with variable manifestations between patients. We sought to develop methods to reliably detect Borrelia burgdorferi, the spirochete bacteria responsible for Lyme disease, in autopsy specimens of patients with a history of neurocognitive disease. In this report, we describe the use of multiple molecular detection techniques for this pathogen and its application to a case study of a Lyme disease patient. The patient had a history of Lyme disease, was treated with antibiotics, and years later developed chronic symptoms including dementia. The patient's pathology and clinical case description was consistent with Lewy body dementia. B. burgdorferi was identified by PCR in several CNS tissues and by immunofluorescent staining in the spinal cord. These studies offer proof of the principle that persistent infection with the Lyme disease spirochete may have lingering consequences on the CNS.

COVID-19 induces neuroinflammation and loss of hippocampal neurogenesis.

Infection with the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is associated with onset of neurological and psychiatric symptoms during and after the acute phase of illness 1-4 . Acute SARS-CoV-2 disease (COVID-19) presents with deficits of memory, attention, movement coordination, and mood. The mechanisms of these central nervous system symptoms remain largely unknown.In an established hamster model of intranasal infection with SARS-CoV-2 5 , and patients deceased from COVID-19, we report a lack of viral neuroinvasion despite aberrant BBB permeability, microglial activation, and brain expression of interleukin (IL)-1ß and IL-6, especially within the hippocampus and the inferior olivary nucleus of the medulla, when compared with non-COVID control hamsters and humans who died from other infections, cardiovascular disease, uremia or trauma. In the hippocampus dentate gyrus of both COVID-19 hamsters and humans, fewer cells expressed doublecortin, a marker of neuroblasts and immature neurons.Despite absence of viral neurotropism, we find SARS-CoV-2-induced inflammation, and hypoxia in humans, affect brain regions essential for fine motor function, learning, memory, and emotional responses, and result in loss of adult hippocampal neurogenesis. Neuroinflammation could affect cognition and behaviour via disruption of brain vasculature integrity, neurotransmission, and neurogenesis, acute effects that may persist in COVID-19 survivors with long-COVID symptoms.