Genetic mechanisms for impaired synaptic plasticity in schizophrenia revealed by computational modeling.

Schizophrenia phenotypes are suggestive of impaired cortical plasticity in the disease, but the mechanisms of these deficits are unknown. Genomic association studies have implicated a large number of genes that regulate neuromodulation and plasticity, indicating that the plasticity deficits have a genetic origin. Here, we used biochemically detailed computational modeling of postsynaptic plasticity to investigate how schizophrenia-associated genes regulate long-term potentiation (LTP) and depression (LTD). We combined our model with data from postmortem RNA expression studies (CommonMind gene-expression datasets) to assess the consequences of altered expression of plasticity-regulating genes for the amplitude of LTP and LTD. Our results show that the expression alterations observed post mortem, especially those in the anterior cingulate cortex, lead to impaired protein kinase A (PKA)-pathway-mediated LTP in synapses containing GluR1 receptors. We validated these findings using a genotyped electroencephalogram (EEG) dataset where polygenic risk scores for synaptic and ion channel-encoding genes as well as modulation of visual evoked potentials were determined for 286 healthy controls. Our results provide a possible genetic mechanism for plasticity impairments in schizophrenia, which can lead to improved understanding and, ultimately, treatment of the disorder.

Schizophrenia risk-associated SNPs affect expression of microRNA 137 host gene: a postmortem study.

Common variants in the MicroRNA 137 host gene MIR137HG and its adjacent gene DPYD have been associated with schizophrenia risk and the latest Psychiatric Genomics Consortium (PGC). Genome-Wide Association Study on schizophrenia has confirmed and extended these findings. To elucidate the association of schizophrenia risk-associated SNPs in this genomic region, we examined the expression of both mature and immature transcripts of the miR-137 host gene (MIR137HG) in the dorsolateral prefrontal cortex (DLPFC) and subgenual anterior cingulate cortex (sgACC) of postmortem brain samples of donors with schizophrenia and psychiatrically-unaffected controls using qPCR and RNA-Seq approaches. No differential expression of miR-137, MIR137HG, or its transcripts was observed. Two schizophrenia risk-associated SNPs identified in the PGC study, rs11165917 (DLPFC: P = 2.0e-16; sgACC: P = 6.4e-10) and rs4274102 (DLPFC: P = 0.036; sgACC: P = 0.002), were associated with expression of the MIR137HG long non-coding RNA transcript MIR137HG-203 (ENST00000602672.2) in individuals of European ancestry. Carriers of the minor (risk) allele of rs11165917 had significantly lower expression of MIR137HG-203 compared with those carrying the major allele. However, we were unable to validate this result by short-read sequencing of RNA extracted from DLPFC or sgACC tissue. This finding suggests that immature transcripts of MIR137HG may contribute to genetic risk for schizophrenia.

Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

Multiple system atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.

Increased number of excitatory synapsis and decreased number of inhibitory synapsis in the prefrontal cortex in autism.

Previous studies in autism spectrum disorder demonstrated an increased number of excitatory pyramidal cells and a decreased number of inhibitory parvalbumin+ chandelier interneurons in the prefrontal cortex of postmortem brains. How these changes in cellular composition affect the overall abundance of excitatory and inhibitory synapses in the cortex is not known. Herein, we quantified the number of excitatory and inhibitory synapses in the prefrontal cortex of 10 postmortem autism spectrum disorder brains and 10 control cases. To identify excitatory synapses, we used VGlut1 as a marker of the presynaptic component and postsynaptic density protein-95 as marker of the postsynaptic component. To identify inhibitory synapses, we used the vesicular gamma-aminobutyric acid transporter as a marker of the presynaptic component and gephyrin as a marker of the postsynaptic component. We used Puncta Analyzer to quantify the number of co-localized pre- and postsynaptic synaptic components in each area of interest. We found an increase in the number of excitatory synapses in upper cortical layers and a decrease in inhibitory synapses in all cortical layers in autism spectrum disorder brains compared with control cases. The alteration in the number of excitatory and inhibitory synapses could lead to neuronal dysfunction and disturbed network connectivity in the prefrontal cortex in autism spectrum disorder.

A framework to improve the alignment of individual cytoarchitectonic maps of the Julich-Brain atlas using cortical folding landmarks.

The segregation of the cortical mantle into cytoarchitectonic areas provides a structural basis for the specialization of different brain regions. In vivo neuroimaging experiments can be linked to this postmortem cytoarchitectonic parcellation via Julich-Brain. This atlas embeds probabilistic maps that account for inter-individual variability in the localization of cytoarchitectonic areas in the reference spaces targeted by spatial normalization. We built a framework to improve the alignment of architectural areas across brains using cortical folding landmarks. This framework, initially designed for in vivo imaging, was adapted to postmortem histological data. We applied this to the first 14 brains used to establish the Julich-Brain atlas to infer a refined atlas with more focal probabilistic maps. The improvement achieved is significant in the primary regions and some of the associative areas. This framework also provides a tool for exploring the relationship between cortical folding patterns and cytoarchitectonic areas in different cortical regions to establish new landmarks in the remainder of the cortex.

Parvalbumin interneurons in human ventromedial prefrontal cortex: a comprehensive post-mortem study of myelination and perineuronal nets in neurotypical individuals and depressed suicides with and without a history of child abuse.

Cortical parvalbumin interneurons (PV+) are major regulators of excitatory/inhibitory information processing, and their maturation is associated with the opening of developmental critical periods (CP). Recent studies reveal that cortical PV+ axons are myelinated, and that myelination along with perineuronal net (PNN) maturation around PV+ cells is associated with the closures of CP. Although PV+ interneurons are susceptible to early-life stress, their relationship between their myelination and PNN coverage remains unexplored. This study compared the fine features of PV+ interneurons in well-characterized human post-mortem ventromedial prefrontal cortex samples (n = 31) from depressed suicides with or without a history of child abuse (CA) and matched controls. In healthy controls, 81% of all sampled PV+ interneurons displayed a myelinated axon, while a subset (66%) of these cells also displayed a PNN, proposing a relationship between both attributes. Intriguingly, a 3-fold increase in the proportion of unmyelinated PV+ interneurons with a PNN was observed in CA victims, along with greater PV-immunofluorescence intensity in myelinated PV+ cells with a PNN. This study, which is the first to provide normative data on myelination and PNNs around PV+ interneurons in human neocortex, sheds further light on the cellular and molecular consequences of early-life adversity on cortical PV+ interneurons.

Disentangling transcriptomic heterogeneity within the human subgenual anterior cingulate cortex.

The subgenual anterior cingulate cortex (sgACC) is a critical site for understanding the neural correlates of affect and emotion. While the activity of the sgACC is functionally homogenous, it is comprised of multiple Brodmann Areas (BAs) that possess different cytoarchitectures. In some sgACC BAs, Layer 5 is sublaminated into L5a and L5b which has implications for its projection targets. To understand how the transcriptional profile differs between the BAs, layers, and sublayers of human sgACC, we collected layer strips using laser capture microdissection followed by RNA sequencing. We found no significant differences in transcript expression in these specific cortical layers between BAs within the sgACC. In contrast, we identified striking differences between Layers 3 and 5a or 5b that were concordant across sgACC BAs. We found that sublayers 5a and 5b were transcriptionally similar. Pathway analyses of L3 and L5 revealed overlapping biological processes related to synaptic function. However, L3 was enriched for pathways related to cell-to-cell junction and dendritic spines whereas L5 was enriched for pathways related to brain development and presynaptic function, indicating potential functional differences across layers. Our study provides important insight into normative transcriptional features of the sgACC.

Lecanemab demonstrates highly selective binding to Aß protofibrils isolated from Alzheimer's disease brains.

Recent advances in immunotherapeutic approaches to the treatment of Alzheimer's disease (AD) have increased the importance of understanding the exact binding preference of each amyloid-beta (Aß) antibody employed, since this determines both efficacy and risk for potentially serious adverse events known as amyloid-related imaging abnormalities. Lecanemab is a humanized IgG1 antibody that was developed to target the soluble Aß protofibril conformation. The present study prepared extracts of post mortem brain samples from AD patients and non-demented elderly controls, characterized the forms of Aß present, and investigated their interactions with lecanemab. Brain tissue samples were homogenized and extracted using tris-buffered saline. Aß levels and aggregation states in soluble and insoluble extracts, and in fractions prepared using size-exclusion chromatography or density gradient ultracentrifugation, were analyzed using combinations of immunoassay, immunoprecipitation (IP), and mass spectrometry. Lecanemab immunohistochemistry was also conducted in temporal cortex. The majority of temporal cortex Aß (98 %) was in the insoluble extract. Aß42 was the most abundant form present, particularly in AD subjects, and most soluble Aß42 was in soluble aggregated protofibrillar structures. Aß protofibril levels were much higher in AD subjects than in controls. Protofibrils captured by lecanemab-IP contained high levels of Aß42 and lecanemab bound to large, medium, and small Aß42 protofibrils in a concentration-dependent manner. Competitive IP showed that neither Aß40 monomers nor Aß40-enriched fibrils isolated from cerebral amyloid angiopathy reduced lecanemab's binding to Aß42 protofibrils. Immunohistochemistry showed that lecanemab bound readily to Aß plaques (diffuse and compact) and to intraneuronal Aß in AD temporal cortex. Taken together, these findings indicate that while lecanemab binds to Aß plaques, it preferentially targets soluble aggregated Aß protofibrils. These are largely composed of Aß42, and lecanemab binds less readily to the Aß40-enriched fibrils found in the cerebral vasculature. This is a promising binding profile because Aß42 protofibrils represent a key therapeutic target in AD, while a lack of binding to monomeric Aß and cerebral amyloid deposits should reduce peripheral antibody sequestration and minimize risk for adverse events.

iPSC-induced neurons with the V337M MAPT mutation are selectively vulnerable to caspase-mediated cleavage of tau and apoptotic cell death.

BACKGROUND: Tau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies. METHODS: To understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons. RESULTS: FTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases. CONCLUSIONS: Our results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions.

Brain lncRNA-mRNA co-expression regulatory networks and alcohol use disorder.

Prolonged alcohol consumption can disturb the expression of both coding and noncoding genes in the brain. These dysregulated genes may co-express in modules and interact within networks, consequently influencing the susceptibility to developing alcohol use disorder (AUD). In the present study, we performed an RNA-seq analysis of the expression of both long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) in 192 postmortem tissue samples collected from eight brain regions (amygdala, caudate nucleus, cerebellum, hippocampus, nucleus accumbens, prefrontal cortex, putamen, and ventral tegmental area) of 12 AUD and 12 control subjects of European ancestry. Applying the limma-voom method, we detected a total of 57 lncRNAs and 51 mRNAs exhibiting significant differential expression (Padj < 0.05 and fold-change ≥2) across at least one of the eight brain regions investigated. Machine learning analysis further confirmed the potential of these top genes in predicting AUD. Through Weighted Gene Co-expression Network Analysis (WGCNA), we identified distinct lncRNA-mRNA co-expression modules associated with AUD in each of the eight brain regions. Additionally, lncRNA-mRNA co-expression networks were constructed for each brain region using Cytoscape to reveal gene regulatory interactions implicated in AUD. Hub genes within these networks were found to be enriched in several key KEGG pathways, including Axon Guidance, MAPK Signaling, p53 Signaling, Adherens Junction, and Neurodegeneration. Our results underscore the significance of networks involving AUD-associated lncRNAs and mRNAs in modulating neuroplasticity in response to alcohol exposure. Further elucidating these molecular mechanisms holds promise for the development of targeted therapeutic interventions for AUD.

Post-mortem analysis of dolutegravir, tenofovir, lamivudine and efavirenz penetration in multiple CNS compartments.

BACKGROUND: Central nervous system (CNS) compartmentalization provides opportunity for HIV persistence and resistance development. Differences between cerebrospinal fluid (CSF) and cerebral matter regarding HIV persistence are well described. However, CSF is often used as surrogate for CNS drug exposure, and knowledge from solid brain tissue is rare. METHODS: Dolutegravir, tenofovir, lamivudine and efavirenz concentrations were measured across 13 CNS regions plus plasma in samples collected during autopsy in 49 Ugandan decedents. Median time from death to autopsy was 8 (IQR 5,15) hours. To evaluate postmortem redistribution, a time course study was performed in a mouse model. RESULTS: Regions with the highest penetration ratios were choroid plexus/arachnoid (dolutegravir and tenofovir), CSF (lamivudine), and cervical spinal cord/meninges (efavirenz); the lowest were corpus callosum (dolutegravir and tenofovir), frontal lobe (lamivudine), and parietal lobe (efavirenz). On average, brain concentrations were 84%, 87%, and 76% of CSF for dolutegravir, tenofovir, and lamivudine respectively. Postmortem redistribution was observed in the mouse model, with tenofovir and lamivudine concentration increased by 350% and efavirenz concentration decreased by 24% at 24-hours post-mortem. CONCLUSION: Analysis of postmortem tissue provides a unique opportunity to investigate CNS antiretroviral penetration. Regional differences were observed paving the way to identify mechanisms of viral compartmentalization and/or neurotoxicity.

From flesh to bones: Multi-omics approaches in forensic science.

Recent advancements in omics techniques have revolutionised the study of biological systems, enabling the generation of high-throughput biomolecular data. These innovations have found diverse applications, ranging from personalised medicine to forensic sciences. While the investigation of multiple aspects of cells, tissues or entire organisms through the integration of various omics approaches (such as genomics, epigenomics, metagenomics, transcriptomics, proteomics and metabolomics) has already been established in fields like biomedicine and cancer biology, its full potential in forensic sciences remains only partially explored. In this review, we have presented a comprehensive overview of state-of-the-art analytical platforms employed in omics research, with specific emphasis on their application in the forensic field for the identification of the cadaver and the cause of death. Moreover, we have conducted a critical analysis of the computational integration of omics approaches, and highlighted the latest advancements in employing multi-omics techniques for forensic investigations.

A method to image brain tissue frozen at autopsy.

Magnetic Resonance Imaging (MRI) can provide the location and signal characteristics of pathological regions within a postmortem tissue block, thereby improving the efficiency of histopathological studies. However, such postmortem-MRI guided histopathological studies have so far only been performed on fixed samples as imaging tissue frozen at the time of extraction, while preserving its integrity, is significantly more challenging. Here we describe the development of cold-postmortem-MRI, which can preserve tissue integrity and help target techniques such as transcriptomics. As a first step, RNA integrity number (RIN) was used to determine the rate of tissue biomolecular degradation in mouse brains placed at various temperatures between -20 °C and +20 °C for up to 24 h. Then, human tissue frozen at the time of autopsy was immersed in 2-methylbutane, sealed in a bio-safe tissue chamber, and cooled in the MRI using a recirculating chiller to determine MRI signal characteristics. The optimal imaging temperature, which did not show significant RIN deterioration for over 12 h, at the same time giving robust MRI signal and contrast between brain tissue types was deemed to be -7 °C. Finally, MRI was performed on human tissue blocks at this optimal imaging temperatures using a magnetization-prepared rapid gradient echo (MPRAGE, isotropic resolution between 0.3-0.4 mm) revealing good gray-white matter contrast and revealing subpial, subcortical, and deep white matter lesions. RINs measured before and after imaging revealed no significant changes (n = 3, p = 0.18, paired t-test). In addition to improving efficiency of downstream processes, imaging tissue at sub-zero temperatures may also improve our understanding of compartment specificity of MRI signal.

Scaling of smaller pyramidal neuron size and lower energy production in schizophrenia.

BACKGROUND: Dorsolateral prefrontal cortex (DLPFC) dysfunction in schizophrenia appears to reflect alterations in layer 3 pyramidal neurons (L3PNs), including smaller cell bodies and lower expression of mitochondrial energy production genes. However, prior somal size studies used biased strategies for identifying L3PNs, and somal size and levels of energy production markers have not been assessed in individual L3PNs. STUDY DESIGN: We combined fluorescent in situ hybridization (FISH) of vesicular glutamate transporter 1 (VGLUT1) mRNA and immunohistochemical-labeling of NeuN to determine if the cytoplasmic distribution of VGLUT1 mRNA permits the unbiased identification and somal size quantification of L3PNs. Dual-label FISH for VGLUT1 mRNA and cytochrome C oxidase subunit 4I1 (COX4I1) mRNA, a marker of energy production, was used to assess somal size and COX4I1 transcript levels in individual DLPFC L3PNs from schizophrenia (12 males; 2 females) and unaffected comparison (13 males; 1 female) subjects. STUDY RESULTS: Measures of L3PN somal size with NeuN immunohistochemistry or VGLUT1 mRNA provided nearly identical results (ICC = 0.96, p < 0.0001). Mean somal size of VGLUT1-identified L3PNs was 8.7% smaller (p = 0.004) and mean COX4I1 mRNA levels per L3PN were 16.7% lower (p = 0.01) in schizophrenia. These measures were correlated across individual L3PNs in both subject groups (rrm = 0.81-0.86). CONCLUSIONS: This preliminary study presents a novel method for combining unbiased neuronal identification with quantitative assessments of somal size and mRNA levels. We replicated findings of smaller somal size and lower COX4I1 mRNA levels in DLPFC L3PNs in schizophrenia. The normal scaling of COX4I1 mRNA levels with somal size in schizophrenia suggests that lower markers of energy production are secondary to L3PN morphological alterations in the illness.

Altered thalamocortical tract trajectory growth with undisrupted thalamic parcellation pattern in human lissencephaly brain at mid-gestational stage.

Proper topographically organized neural connections between the thalamus and the cerebral cortex are mandatory for thalamus function. Thalamocortical (TC) fiber growth begins during the embryonic period and completes by the third trimester of gestation, so that human neonates at birth have a thalamus with a near-facsimile of adult functional parcellation. Whether congenital neocortical anomaly (e.g., lissencephaly) affects TC connection in humans is unknown. Here, via diffusion MRI fiber-tractography analysis of long-term formalin-fixed postmortem fetal brain diagnosed as lissencephaly in comparison with an age-matched normal one, we found similar topological patterns of thalamic subregions and of internal capsule parcellated by TC fibers. However, lissencephaly fetal brain showed white matter structural changes, including fewer/less organized TC fibers and optic radiations, and much less cortical plate invasion by TC fibers - particularly around the shallow central sulcus. Diffusion MRI fiber tractography of normal fetal brains at 15, 23, and 26 gestational weeks (GW) revealed dynamic volumetric change of each parcellated thalamic subregion, suggesting coupled developmental progress of the thalamus with the corresponding cortex. Moreover, from GW23 and GW26 normal fetal brains, TC endings in the cortical plate could be delineated to reflect cumulative progressive TC invasion of cortical plate. By contrast, lissencephaly brain showed a dramatic decrease in TC invasion of the cortical plate. Our study thus shows the feasibility of diffusion MRI fiber tractography in postmortem long-term formalin-fixed fetal brains to disclose the developmental progress of TC tracts coordinating with thalamic and neocortical growth both in normal and lissencephaly fetal brains at mid-gestational stage.

Diagnostic Protocols for Evaluating the Degradation Mechanisms in Gel-Polymer Lithium Batteries.

Gel polymer electrolytes (GPEs) present a promising alternative to standard liquid electrolytes (LE) for Lithium-ion Batteries (LIBs) and Lithium Metal Batteries bridging the advantages of both liquid and solid polymer electrolytes. However, their cycle life still lags behind that of standard LIBs, and their degradation mechanisms remain poorly understood. A significant challenge is the need for specific diagnostic protocols to systematically study the degradation mechanisms of GPE-based cells. Challenges include the separation of cell components and effective washing, as well as the study of the solid electrolyte interfaces, all complicated by the semi-solid nature of GPEs. This paper provides a brief review of existing literature and proposes a comprehensive set of diagnostic tools for dismantling and evaluating the degradation of GPE-based LIBs. Finally, these methods and recommendations are applied to LiNi0.5Mn1.5O4 (LNMO)-graphite cells, revealing electrolyte oxidation as a major source of cell degradation.

Stability of Ni-Fe-Layered Double Hydroxide Under Long-Term Operation in AEM Water Electrolysis.

Anion exchange membrane water electrolysis (AEMWE) is an attractive method for green hydrogen production. It allows the use of non-platinum group metal catalysts and can achieve performance comparable to proton exchange membrane water electrolyzers due to recent technological advances. While current systems already show high performances with available materials, research gaps remain in understanding electrode durability and degradation behavior. In this study, the performance and degradation tracking of a Ni3Fe-LDH-based single-cell is implemented and investigated through the correlation of electrochemical data using chemical and physical characterization methods. A performance stability of 1000 h, with a degradation rate of 84 µV h-1 at 1 A cm-2 is achieved, presenting the Ni3Fe-LDH-based cell as a stable and cost-attractive AEMWE system. The results show that the conductivity of the formed Ni-Fe-phase is one key to obtaining high electrolyzer performance and that, despite Fe leaching, change in anion-conducting binder compound, and morphological changes inside the catalyst bulk, the Ni3Fe-LDH-based single-cells demonstrate high performance and durability. The work reveals the importance of longer stability tests and presents a holistic approach of electrochemical tracking and post-mortem analysis that offers a guideline for investigating electrode degradation behavior over extended measurement periods.

Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h.

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

Measles vaccination - An underestimated prevention measure: Analyzing a fatal case in Hildesheim, Germany.

Measles and rubella are targeted for elimination in the WHO region Europe. To reach the elimination goal, vaccination coverage of 95% must be achieved and sustained, the genotype information has to be provided for 80% of all outbreaks and transmission chains of a certain variant must not be detected for >12 months. The latter information is collected at Germany's National Reference Center Measles, Mumps, Rubella (NRC MMR). We describe here an outbreak of measles occurring in Hildesheim. The outbreak comprised 43 cases and lasted 14 weeks. Surprisingly, a high number of vaccination failures was observed since 11 cases had received two doses of the MMR vaccine and 4 additional cases were vaccinated once. A 33-year-old woman passed away during the outbreak. She was the mother of 5 children between 4 and 16 years of age. Two schoolchildren contracted measles and passed it on to the rest of the family. Due to delivery bottlenecks, the vaccination of the mother was delayed. She developed measles-like symptoms 3 days after vaccination and was found dead on the morning of day 8 after vaccination. A post-mortem examination was done to identify the cause of death. Moreover, molecular characterization of the virus was performed to analyze whether she was infected by the wildtype virus circulating at that time in Hildesheim or whether the vaccine may have been a concomitant and aggravating feature of her death. The result showed that the samples taken from her at the time of death and during necropsy contained the wildtype measles virus variant corresponding to MVs/Gir Somnath.IND/42.16 (WHO Seq-ID D8-4683) that fueled the Hildesheim outbreak and circulated in Germany from March 2018 to March 2020. The vaccine virus was not detected. Moreover, two aspects uncovered by the post-mortem examination were remarkable; the woman died from giant cell pneumonia, which is a complication seen in immune-suppressed individuals and she was actively using cannabis. THC is known to influence the immune system, but literature reports describing the effects are limited.

Changes in the locus coeruleus during the course of Alzheimer's disease and their relationship to cortical pathology.

AIMS: In Alzheimer's disease (AD), the locus coeruleus (LC) undergoes early and extensive neuronal loss, preceded by abnormal intracellular tau aggregation, decades before the onset of clinical disease. Neuromelanin-sensitive MRI has been proposed as a method to image these changes during life. Surprisingly, human post-mortem studies have not examined how changes in LC during the course of the disease relate to cerebral pathology following the loss of the LC projection to the cortex. METHODS: Immunohistochemistry was used to examine markers for 4G8 (pan-Aß) and AT8 (ptau), LC integrity (neuromelanin, dopamine ß-hydroxylase [DßH], tyrosine hydroxylase [TH]) and microglia (Iba1, CD68, HLA-DR) in the LC and related temporal lobe pathology of 59 post-mortem brains grouped by disease severity determined by Braak stage (0-II, III-IV and V-VI). The inflammatory environment was assessed using multiplex assays. RESULTS: Changes in the LC with increasing Braak stage included increased neuronal loss (p < 0.001) and microglial Iba1 (p = 0.005) together with a reduction in neuromelanin (p < 0.001), DßH (p = 0.002) and TH (p = 0.041). Interestingly in LC, increased ptau and loss of neuromelanin were detected from Braak stage III-IV (p = 0.001). At Braak stage V/VI, the inflammatory environment was different in the LC vs TL, highlighting the anatomical heterogeneity of the inflammatory response. CONCLUSIONS: Here, we report the first quantification of neuromelanin during the course of AD and its relationship to AD pathology and neuroinflammation in the TL. Our findings of neuromelanin loss early in AD and before the neuroinflammatory reaction support the use of neuromelanin-MRI as a sensitive technique to identify early changes in AD.