Postmortem Human Samples Research Articles

Investigating 3-CMC metabolism: Insights from liver microsomes and postmortem biological matrix.

3-Chloromethcathinone (3-CMC) is a synthetic cathinone that has been identified as a new psychoactive substance (NPS) by the European Monitoring Centre for Drugs and Drug Addiction. Despite its increasing prevalence in the recreational drug market since 2014, scientific literature on 3-CMC remains limited. This study employed a multi-step approach to investigate 3-CMC metabolism. First, an in-silico prediction was conducted to compile a list of potential metabolites. Then, in vitro assays were performed using human liver microsomes at two concentrations of 3-CMC. Samples were analyzed using an ultra-performance liquid chromatography system coupled with a high-resolution mass spectrometer. Chromatographic separation was obtained with an Acquity UPLC HSS C18 1.8 µm, 2.1 × 150 mm column on an Ultimate 3000 system chromatography coupled with a QExactivePlus mass spectrometer). Finally, data mining for metabolite identification was conducted using Compound Discoverer software. The combined in silico and in vitro approaches identified four primary metabolites of 3-CMC in HLM assays:1) hydroxylation of the aliphatic group to give M1 2) followed by reduction of the β-keto group, yielding M4; 3) N-demethylation, affording M2; and 4) Reduction of the β-keto group, yielding M3. Subsequent analysis of biological samples from two postmortem cases revealed that urine was the most informative matrix for detecting 3-CMC and its metabolites. The M3 metabolite, was identified as the third abundant metabolite in human liver microsome but was identified as the predominant metabolite in human postmortem samples. Identifying these key metabolites is crucial for improving the accuracy of forensic investigations and extending the detection window beyond the parent compound.

Oxidative/Nitrosative Stress, Apoptosis, and Redox Signaling: Key Players in Neurodegenerative Diseases.

Neurodegenerative diseases are significant health concerns that have a profound impact on the quality and duration of life for millions of individuals. These diseases are characterized by pathological changes in various brain regions, specific genetic mutations associated with the disease, deposits of abnormal proteins, and the degeneration of neurological cells. As neurodegenerative disorders vary in their epidemiological characteristics and vulnerability of neurons, treatment of these diseases is usually aimed at slowing disease progression. The heterogeneity of genetic and environmental factors involved in the process of neurodegeneration makes current treatment methods inadequate. However, the existence of common molecular mechanisms in the pathogenesis of these diseases may allow the development of new targeted therapeutic strategies. Oxidative and nitrosative stress damages membrane components by accumulating ROS and RNS and disrupting redox balance. This process results in the induction of apoptosis, which is important in the pathogenesis of neurodegenerative diseases through oxidative stress. Studies conducted using postmortem human samples, animal models, and cell cultures have demonstrated that oxidative stress, nitrosative stress, and apoptosis are crucial factors in the development of diseases such as Alzheimer's, Parkinson's, Multiple Sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. The excessive production of reactive oxygen and nitrogen species, elevated levels of free radicals, heightened mitochondrial stress, disturbances in energy metabolism, and the oxidation and nitrosylation of cellular macromolecules are recognized as triggers for neuronal cell death. Challenges in managing and treating neurodegenerative diseases require a better understanding of this field at the molecular level. Therefore, this review elaborates on the molecular mechanisms by which oxidative and nitrosative stress are involved in neuronal apoptosis.

Spatial transcriptomic analysis of adult hippocampal neurogenesis in the human brain.

Adult hippocampal neurogenesis has been extensively characterized in rodent models, but its existence in humans remains controversial. We sought to assess the phenomenon in postmortem human hippocampal samples by combining spatial transcriptomics and multiplexed fluorescent in situ hybridization. We computationally examined the spatial expression of various canonical neurogenesis markers in postmortem dentate gyrus (DG) sections from young and middle-aged sudden-death males. We conducted in situ assessment of markers expressed in neural stem cells, proliferative cells, and immature granule neurons in postmortem DG sections from infant, adolescent, and middle-aged males. We examined frozen DG tissue from infant (n = 1, age 2 yr), adolescent (n = 1, age 16 yr), young adult (n = 2, mean age 23.5 yr), and middle-aged (n = 2, mean age 42.5 yr) males, and frozen-fixed DG tissue from middle-aged males (n = 6, mean age 43.5 yr). We detected very few cells expressing neural stem cell and proliferative markers in the human DG from childhood to middle age. However, at all ages, we observed a substantial number of DG cells expressing the immature neuronal marker DCX. Most DCX + cells displayed an inhibitory phenotype, while the remainder were non-committed or excitatory in nature. The study was limited by small sample sizes and included samples only from males. Our findings indicate very low levels of hippocampal neurogenesis throughout life and the existence of a local reserve of plasticity in the adult human hippocampus. Overall, our study provides important insight into the distribution and phenotype of cells expressing neurogenesis markers in the adult human hippocampus.

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.

Transcriptomic characterization of human lateral septum neurons reveals conserved and divergent marker genes across species.

The lateral septum (LS) is a midline, subcortical structure, which regulates social behaviors that are frequently impaired in neurodevelopmental disorders including schizophrenia and autism spectrum disorder. Mouse studies have identified neuronal populations within the LS that express a variety of molecular markers, including vasopressin receptor, oxytocin receptor, and corticotropin releasing hormone receptor, which control specific facets of social behavior. Despite its critical role in regulating social behavior and notable gene expression patterns, comprehensive molecular profiling of the human LS has not been performed. Here, we conducted single nucleus RNA-sequencing (snRNA-seq) to generate the first transcriptomic profiles of the human LS using postmortem human brain tissue samples from 3 neurotypical donors. Our analysis identified 5 transcriptionally distinct neuronal cell types within the human LS that are enriched for TRPC4, the gene encoding Trp-related protein 4. Differential expression analysis revealed a distinct LS neuronal cell type that is enriched for OPRM1, the gene encoding the μ-opioid receptor. Leveraging recently generated mouse LS snRNA-seq datasets, we conducted a cross-species analysis. Our results demonstrate that TRPC4 enrichment in the LS is highly conserved between human and mouse, while FREM2, which encodes FRAS1 related extracellular matrix protein 2, is enriched only in the human LS. Together, these results highlight transcriptional heterogeneity of the human LS, and identify robust marker genes for the human LS.

Paraquat Poisoning: Insights from Autopsy, Histology, and Liquid Chromatography with Tandem Mass Spectrometry in Multidisciplinary Forensic Toxicology Practice.

The herbicide paraquat (PQ) is responsible for a significant number of fatalities resulting from self-poisoning. Nevertheless, only a limited number of comprehensive studies focusing on fatal PQ poisoning, which include examination of autopsy findings, histopathology, and quantitative analysis of post-mortem samples, have been published. This study aimed to evaluate autopsy findings, histopathology, and quantitative analysis of PQ in post-mortem human serum samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a simple, sensitive, and specific method. Autopsies were performed on all deaths due to PQ poisoning, and serum samples were sent to the toxicology laboratory for chemical analysis. The method was successfully applied to seven human serum samples, and the results indicate its reliability for detecting PQ. The study reports fatal serum PQ levels ranging from 0.5 to 372.0 µg/mL. The comprehensive data presented in this study can be useful for further research and practical applications.

Proteomic evidence of depression-associated astrocytic dysfunction in the human male olfactory bulb

The olfactory bulb (OB), a major structure of the limbic system, has been understudied in human investigations of psychopathologies such as depression. To explore more directly the molecular features of the OB in depression, a global comparative proteome analysis was carried out with human post-mortem OB samples from 11 males having suffered from depression and 12 healthy controls. We identified 188 differentially abundant proteins (with adjusted p < 0.05) between depressed cases and controls. Gene ontology and gene enrichment analyses suggested that these proteins are involved in biological processes including the complement and coagulation cascades. Cell type enrichment analysis displayed a significant reduction in several canonical astrocytic proteins in OBs from depressed patients. Furthermore, using RNA-fluorescence in-situ hybridization, we observed a decrease in the percentage of ALDH1L1+ cells expressing canonical astrocytic markers including ALDOC, NFIA, GJA1 (connexin 43) and SLC1A3 (EAAT1). These results are consistent with previous reports of downregulated astrocytic marker expression in other brain regions in depressed patients. We also conducted a comparative phosphoproteomic analysis of OB samples and found a dysregulation of proteins involved in neuronal and astrocytic functions. To determine whether OB astrocytic abnormalities is specific to humans, we also performed proteomics on the OB of socially defeated male mice, a commonly used model of depression. Cell-type specific analysis revealed that in socially defeated animals, the most striking OB protein alterations were associated with oligodendrocyte-lineage cells rather than with astrocytes, highlighting an important species difference. Overall, this study further highlights cerebral astrocytic abnormalities as a consistent feature of depression in humans.

Single-cell atlas of ABCA7 loss-of-function reveals impaired neuronal respiration via choline-dependent lipid imbalances.

Loss-of-function (LoF) variants in the lipid transporter ABCA7 significantly increase the risk of Alzheimer's disease (odds ratio ∼2), yet the pathogenic mechanisms and the neural cell types affected by these variants remain largely unknown. Here, we performed single-nuclear RNA sequencing of 36 human post-mortem samples from the prefrontal cortex of 12 ABCA7 LoF carriers and 24 matched non-carrier control individuals. ABCA7 LoF was associated with gene expression changes in all major cell types. Excitatory neurons, which expressed the highest levels of ABCA7, showed transcriptional changes related to lipid metabolism, mitochondrial function, cell cycle-related pathways, and synaptic signaling. ABCA7 LoF-associated transcriptional changes in neurons were similarly perturbed in carriers of the common AD missense variant ABCA7 p.Ala1527Gly (n = 240 controls, 135 carriers), indicating that findings from our study may extend to large portions of the at-risk population. Consistent with ABCA7's function as a lipid exporter, lipidomic analysis of isogenic iPSC-derived neurons (iNs) revealed profound intracellular triglyceride accumulation in ABCA7 LoF, which was accompanied by a relative decrease in phosphatidylcholine abundance. Metabolomic and biochemical analyses of iNs further indicated that ABCA7 LoF was associated with disrupted mitochondrial bioenergetics that suggested impaired lipid breakdown by uncoupled respiration. Treatment of ABCA7 LoF iNs with CDP-choline (a rate-limiting precursor of phosphatidylcholine synthesis) reduced triglyceride accumulation and restored mitochondrial function, indicating that ABCA7 LoF-induced phosphatidylcholine dyshomeostasis may directly disrupt mitochondrial metabolism of lipids. Treatment with CDP-choline also rescued intracellular amyloid β -42 levels in ABCA7 LoF iNs, further suggesting a link between ABCA7 LoF metabolic disruptions in neurons and AD pathology. This study provides a detailed transcriptomic atlas of ABCA7 LoF in the human brain and mechanistically links ABCA7 LoF-induced lipid perturbations to neuronal energy dyshomeostasis. In line with a growing body of evidence, our study highlights the central role of lipid metabolism in the etiology of Alzheimer's disease.

The Role of Alpha-Synuclein in Synucleinopathy: Impact on Lipid Regulation at Mitochondria-ER Membranes.

The protein alpha-synuclein (αSyn) plays a critical role in the pathogenesis of synucleinopathy, which includes Parkinson's disease and multiple system atrophy, and mounting evidence suggests that lipid dyshomeostasis is a critical phenotype in these neurodegenerative conditions. Previously, we identified that αSyn localizes to mitochondria-associated endoplasmic reticulum membranes (MAMs), temporary functional domains containing proteins that regulate lipid metabolism, including the de novo synthesis of phosphatidylserine. In the present study, we have analyzed the lipid composition of postmortem human samples, focusing on the substantia nigra pars compacta of Parkinson's disease and controls, as well as three less affected brain regions of Parkinson's donors. To further assess synucleinopathy-related lipidome alterations, similar analyses were performed on the striatum of multiple system atrophy cases. Our data show region-and disease-specific changes in the levels of lipid species. Specifically, our data revealed alterations in the levels of specific phosphatidylserine species in brain areas most affected in Parkinson's disease. Some of these alterations, albeit to a lesser degree, are also observed multiples system atrophy. Using induced pluripotent stem cell-derived neurons, we show that αSyn contributes to regulating phosphatidylserine metabolism at MAM domains, and that αSyn dosage parallels the perturbation in phosphatidylserine levels. Our results support the notion that αSyn pathophysiology is linked to the dysregulation of lipid homeostasis, which may contribute to the vulnerability of specific brain regions in synucleinopathy. These findings have significant therapeutic implications.

Alzheimer’s disease rewires gene coexpression networks coupling different brain regions

Connectome studies have shown how Alzheimer’s disease (AD) disrupts functional and structural connectivity among brain regions. But the molecular basis of such disruptions is less studied, with most genomic/transcriptomic studies performing within-brain-region analyses. To inspect how AD rewires the correlation structure among genes in different brain regions, we performed an Inter-brain-region Differential Correlation (Inter-DC) analysis of RNA-seq data from Mount Sinai Brain Bank on four brain regions (frontal pole, superior temporal gyrus, parahippocampal gyrus and inferior frontal gyrus, comprising 264 AD and 372 control human post-mortem samples). An Inter-DC network was assembled from all pairs of genes across two brain regions that gained (or lost) correlation strength in the AD group relative to controls at FDR 1%. The differentially correlated (DC) genes in this network complemented known differentially expressed genes in AD, and likely reflects cell-intrinsic changes since we adjusted for cell compositional effects. Each brain region used a distinctive set of DC genes when coupling with other regions, with parahippocampal gyrus showing the most rewiring, consistent with its known vulnerability to AD. The Inter-DC network revealed master dysregulation hubs in AD (at genes ZKSCAN1, SLC5A3, RCC1, IL17RB, PLK4, etc.), inter-region gene modules enriched for known AD pathways (synaptic signaling, endocytosis, etc.), and candidate signaling molecules that could mediate region-region communication. The Inter-DC network generated in this study is a valuable resource of gene pairs, pathways and signaling molecules whose inter-brain-region functional coupling is disrupted in AD, thereby offering a new perspective of AD etiology.

SNORA69 is up-regulated in the lateral habenula of individuals with major depressive disorder

Major depressive disorder (MDD) is a complex and potentially debilitating illness whose etiology and pathology remains unclear. Non-coding RNAs have been implicated in MDD, where they display differential expression in the brain and the periphery. In this study, we quantified small nucleolar RNA (snoRNA) expression by small RNA sequencing in the lateral habenula (LHb) of individuals with MDD (n = 15) and psychiatrically-healthy controls (n = 15). We uncovered five snoRNAs that exhibited differential expression between MDD and controls (FDR < 0.01). Specifically, SNORA69 showed increased expression in MDD and was technically validated via RT-qPCR. We further investigated the expression of Snora69 in the LHb and peripheral blood of an unpredicted chronic mild stress (UCMS) mouse model of depression. Snora69 was specifically up-regulated in mice that underwent the UCMS paradigm. SNORA69 is known to guide pseudouridylation onto 5.8S and 18S rRNAs. We quantified the relative abundance of pseudouridines on 5.8S and 18S rRNA in human post-mortem LHb samples and found increased abundance of pseudouridines in the MDD group. Overall, our findings indicate the importance of brain snoRNAs in the pathology of MDD. Future studies characterizing SNORA69’s role in MDD pathology is warranted.

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS

TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, the analysis of deeply phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 pathology and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic accumulation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic accumulation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS.

Identification of gene fusions associated with amyotrophic lateral sclerosis.

Genetics is an important risk factor for amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons. Recent findings demonstrate that in addition to specific genetic mutations, structural variants caused by genetic instability can also play a causative role in ALS. Genomic instability can lead to deletions, duplications, insertions, inversions, and translocations in the genome, and these changes can sometimes lead to fusion of distinct genes into a single transcript. Gene fusion events have been studied extensively in cancer; however, they have not been thoroughly investigated in ALS. The aim of this study was to determine whether gene fusions are present in ALS. Gene fusions were identified using STAR Fusion v1.10.0 software in bulk RNA-Seq data from human postmortem samples from publicly available data sets from Target ALS and the New York Genome Center ALS Consortium. We report the presence of gene fusion events in several brain regions as well as in spinal cord samples in ALS. Although most gene fusions were intra-chromosomal events between neighboring genes and present in both ALS and control samples, there was a significantly greater number of unique gene fusions in ALS compared to controls. Lastly, we identified specific gene fusions with a significant burden in ALS, that were absent from both control samples and known cancer gene fusion databases. Collectively, our findings reveal an enrichment of gene fusions in ALS and suggest that these events may be an additional genetic cause linked to ALS pathogenesis.

Maximum likelihood estimation of renal transporter ontogeny profiles for pediatric PBPK modeling.

Optimal treatment of infants with many renally cleared drugs must account for maturational differences in renal transporter (RT) activity. Pediatric physiologically-based pharmacokinetic (PBPK) models may incorporate RT activity, but this requires ontogeny profiles for RT activity in children, especially neonates, to predict drug disposition. Therefore, RT expression measurements from human kidney postmortem cortical tissue samples were normalized to represent a fraction of mature RT activity. Using these data, maximum likelihood estimated the distributions of RT activity across the pediatric age spectrum, including preterm and term neonates. PBPK models of four RT substrates (acyclovir, ciprofloxacin, furosemide, and meropenem) were evaluated with and without ontogeny profiles using average fold error (AFE), absolute average fold error (AAFE), and proportion of observations within the 5-95% prediction interval. Novel maximum likelihood profiles estimated ontogeny distributions for the following RT: OAT1, OAT3, OCT2, P-gp, URAT1, BCRP, MATE1, MRP2, MRP4, and MATE-2 K. Profiles for OAT3, P-gp, and MATE1 improved infant furosemide and neonate meropenem PBPK model AFE from 0.08 to 0.70 and 0.53 to 1.34 and model AAFE from 12.08 to 1.44 and 2.09 to 1.36, respectively, and improved the percent of data within the 5-95% prediction interval from 48% to 98% for neonatal ciprofloxacin simulations, respectively. Even after accounting for other critical population-specific maturational differences, novel RT ontogeny profiles substantially improved neonatal PBPK model performance, providing validated estimates of maturational differences in RT activity for optimal dosing in children.

Abstract 19018: Unraveling the Regulatory Landscape of NKX2-5 in Human Left Atrial Tissue: Implications for Atrial Fibrillation

Cardiac transcription factors are emerging as crucial modulators of atrial fibrillation (AF), with rare and common variation implicated in AF. Mutations in NKX2-5 and more recently, NFATC1 were reported in families with autosomal dominant young-onset AF. Here, we explore the landscape of NKX2-5 and NFATC1 in human left atrium, using Chromatin Immunoprecipitation Sequencing (ChIP-seq), DAVID functional annotation and HOMER motif analyses. Post-mortem human left atrial tissue samples (n=5) were obtained from the National Disease Research Interchange (NDRI) within a recovery interval of &lt; 4 hours. Preserved microstructural integrity and normal staining patters for TNNT2 and vimentin were verified using confocal microscopy. ChIP-seq analysis revealed an average of 2.62k NKX2-5 binding peaks, with 118 genes bound in all samples. Functional analysis identified significant enrichment in gene ontology categories and pathways (Table 1), including adrenergic and PPAR signaling pathways known to participate in AF susceptibility. Using HOMER, we mapped 55 NFATC1 consensus binding sites to the NKX2-5 ChIP peaks. Two NFATC1 consensus sites localized to an NKX2-5 binding peak located upstream from MEF2A (Figure 1). Published HI-C data from human right atrium indicates the NFATC1 binding sites are adjacent to the promoter of MEF2A in 3D space, suggesting that NKX2-5 and NFATC1 co-regulate MEF2A. In murine atria, mef2A regulates genes involved in fibrosis and adhesion, processes that are crucial in the pathogenesis of atrial fibrillation. Our data, together with published literature, provide a framework for understanding transcriptional networks that may predispose to human AF.

Cyanide determination in postmortem blood samples using Headspace-Ion Mobility Spectrometry (HS-IMS)

Interest in facile and sensitive methods for cyanide detection is related to the extreme cyanide toxicity and its importance in forensic toxicology. In this research a novel application of Ion Mobility Spectrometry (IMS) was demonstrated for rapid determination of cyanide poisoning in postmortem toxicology. In addition, a simple method for the sample preparation was applied based on the analyte (cyanide anion) transfer as HCN to the headspace and direct injection to IMS. The method showed a linear dynamic range of 50–2000 µg/L with R2 > 0.99, a high sensitivity (LOD of 20.4 µg/L and LOQ of 68.1 µg/L). Good repeatability (intra- and inter-assays, CV < 15 %) and excellent extraction recovery (82 -94 %) were obtained. After validation, the method was applied in the analysis of postmortem human blood samples in forensic cases. In all samples, the cyanide was promptly quantified.

Emerging Landscape of Preclinical Models for Studying COVID-19 Neurologic Diseases.

COVID-19 (Coronavirus Disease 2019) is an infectious disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and has globally infected 768 million people and caused over 6 million deaths. COVID-19 primarily affects the respiratory system but increasing reports of neurologic symptoms associated with COVID-19 have been reported in the literature. The exact mechanism behind COVID-19 neurologic pathophysiology remains poorly understood due to difficulty quantifying clinical neurologic symptoms in humans and correlating them to findings in human post-mortem samples and animal models. Thus, robust preclinical experimental models for COVID-19 neurologic manifestations are urgently needed. Here, we review recent advances in in vitro, in vivo, and other models and technologies for studying COVID-19 including primary cell cultures, pluripotent stem cell-derived neurons and organoids, rodents, nonhuman primates, 3D bioprinting, artificial intelligence, and multiomics. We specifically focus our discussion on the contribution, recent advancements, and limitations these preclinical models have on furthering our understanding of COVID-19's neuropathic physiology. We also discuss these models' roles in the screening and development of therapeutics, vaccines, antiviral drugs, and herbal medicine, and on future opportunities for COVID-19 neurologic research and clinical management.

Early Alzheimer’s disease pathology in human cortex involves transient cell states

Cellular perturbations underlying Alzheimer's disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the early cortical amyloid response-were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with β-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

Development of a radiographic technique for porcine head ballistic research

IntroductionThe porcine model shows structural features comparable to that of humans and are routinely used within research, due to the ethical, legal, and practical use of post-mortem human samples. Methods for obtaining high quality and comparable reference data using standardised acquisition protocols are essential. MethodsThe decapitated heads of three adult white sows were subjected to radiographic imaging before and after cranial trauma (9 mm, Heckler and Koch MP5). Digital radiographs were generated using a Siemens MULTIX TOP system with an Agfa digital detector, with foam blocks and sandbags as ancillary equipment. An iterative approach was adopted by the authors to generate reproducible radiographic views from two perpendicular angles. Specimens were kept at 5 °C and wrapped in polythene bags to reduce the impact of putrefaction. ResultsStandardised head radiography technique was developed for superior-inferior and lateral views demonstrating porcine anatomy. Key parameters included: automatic exposure control for tube current (∼4 mAs), tube voltage of 73 kVp, 100 cm source to image receptor distance, and an anti-scatter grid. Slight variances in specimen morphology, developmental status, and soft tissue changes did not affect imaging outcomes. ConclusionThe technique and positioning proposed in this study allows for the acquisition of high quality and reproducible radiographic images for comparable ballistic research datasets. Specimen positioning and centring of the primary beam may be applied across porcine breeds, although individual radiographic parameters may differ according to equipment specifications and specimen size. Implications for practiceDevelopment of a reproducible radiographic technique of porcine heads in forensic and veterinary research.

The mitochondrial protein TSPO in Alzheimer’s disease: relation to the severity of AD pathology and the neuroinflammatory environment

The 18kD translocator protein (TSPO) is used as a positron emission tomography (PET) target to quantify neuroinflammation in patients. In Alzheimer’s disease (AD), the cerebellum is the pseudo-reference region for comparison with the cerebral cortex due to the absence of AD pathology and lower levels of TSPO. However, using the cerebellum as a pseudo-reference region is debated, with other brain regions suggested as more suitable. This paper aimed to establish the neuroinflammatory differences between the temporal cortex and cerebellar cortex, including TSPO expression. Using 60 human post-mortem samples encompassing the spectrum of Braak stages (I–VI), immunostaining for pan-Aβ, hyperphosphorylated (p)Tau, TSPO and microglial proteins Iba1, HLA–DR and MSR-A was performed in the temporal cortex and cerebellum. In the cerebellum, Aβ but not pTau, increased over the course of the disease, in contrast to the temporal cortex, where both proteins were significantly increased. TSPO increased in the temporal cortex, more than twofold in the later stages of AD compared to the early stages, but not in the cerebellum. Conversely, Iba1 increased in the cerebellum, but not in the temporal cortex. TSPO was associated with pTau in the temporal cortex, suggesting that TSPO positive microglia may be reacting to pTau itself and/or neurodegeneration at later stages of AD. Furthermore, the neuroinflammatory microenvironment was examined, using MesoScale Discovery assays, and IL15 only was significantly increased in the temporal cortex. Together this data suggests that the cerebellum maintains a more homeostatic environment compared to the temporal cortex, with a consistent TSPO expression, supporting its use as a pseudo-reference region for quantification in TSPO PET scans.