Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

Human Brainstem and Cerebellum Atlas: Chemoarchitecture and Cytoarchitecture Paired to MRI.

Lesion localization is the basis for understanding neurologic disease, which is predicated on neuroanatomical knowledge carefully cataloged from histology and imaging atlases. However, it is often difficult to correlate clinical images of brainstem injury obtained by MRI scans with the details of human brainstem neuroanatomy represented in atlases, which are mostly based on cytoarchitecture using Nissl stain or a single histochemical stain, and usually do not include the cerebellum. Here, we report a high-resolution (200 µm) 7T MRI of a cadaveric male human brainstem and cerebellum paired with detailed, coregistered histology (at 2 µm single-cell resolution) of the immunohistochemically stained cholinergic, serotonergic, and catecholaminergic (dopaminergic, noradrenergic, and adrenergic) neurons, in relationship to each other and to the cerebellum. These immunohistochemical findings provide novel insights into the spatial relationships of brainstem cell types and nuclei, including subpopulations of melanin and TH+ neurons, and allows for more informed structural annotation of cell groups. Moreover, the coregistered MRI-paired histology helps validate imaging findings. This is useful for interpreting both scans and histology, and to understand the cell types affected by lesions. Our detailed chemoarchitecture and cytoarchitecture with corresponding high-resolution MRI builds on previous atlases of the human brainstem and cerebellum, and makes precise identification of brainstem and cerebellar cell groups involved in clinical lesions accessible for both laboratory scientists and clinicians alike.SIGNIFICANCE STATEMENT Clinicians and neuroscientists frequently use cross-sectional anatomy of the human brainstem from MRI scans for both clinical and laboratory investigations, but they must rely on brain atlases to neuroanatomical structures. Such atlases generally lack both detail of brainstem chemical cell types, and the cerebellum, which provides an important spatial reference. Our current atlas maps the distribution of key brainstem cell types (cholinergic, serotonergic, and catecholaminergic neurons) in relationship to each other and the cerebellum, and pairs this histology with 7T MR images from the identical brain. This atlas allows correlation of the chemoarchitecture with corresponding MRI, and makes the identification of cell groups that are often discussed, but rarely identifiable on MRI scan, accessible to clinicians and clinical researchers.

Succinate prodrugs as treatment for acute metabolic crisis during fluoroacetate intoxication in the rat.

Sodium fluoroacetate (FA) is a metabolic poison that systemically inhibits the tricarboxylic acid (TCA) cycle, causing energy deficiency and ultimately multi-organ failure. It poses a significant threat to society because of its high toxicity, potential use as a chemical weapon and lack of effective antidotal therapy. In this study, we investigated cell-permeable succinate prodrugs as potential treatment for acute FA intoxication. We hypothesized that succinate prodrugs would bypass FA-induced mitochondrial dysfunction, provide metabolic support, and prevent metabolic crisis during acute FA intoxication. To test this hypothesis, rats were exposed to FA (0.75 mg/kg) and treated with the succinate prodrug candidate NV354. Treatment efficacy was evaluated based on cardiac and cerebral mitochondrial respiration, mitochondrial content, metabolic profiles and tissue pathology. In the heart, FA increased concentrations of the TCA metabolite citrate (+ 4.2-fold, p < 0.01) and lowered ATP levels (- 1.9-fold, p < 0.001), confirming the inhibition of the TCA cycle by FA. High-resolution respirometry of cardiac mitochondria further revealed an impairment of mitochondrial complex V (CV)-linked metabolism, as evident by a reduced phosphorylation system control ratio (- 41%, p < 0.05). The inhibition of CV-linked metabolism is a novel mechanism of FA cardiac toxicity, which has implications for drug development and which NV354 was unable to counteract at the given dose. In the brain, FA induced the accumulation of ß-hydroxybutyrate (+ 1.4-fold, p < 0.05) and the reduction of mitochondrial complex I (CI)-linked oxidative phosphorylation (OXPHOSCI) (- 20%, p < 0.01), the latter of which was successfully alleviated by NV354. This promising effect of NV354 warrants further investigations to determine its potential neuroprotective effects.

The genome-wide DNA methylation profiles among neurosurgery patients with and without post-operative delirium.

AIMS: There is no previous study demonstrating the differences of genome-wide DNA methylation (DNAm) profiles between patients with and without postoperative delirium (POD). We aimed to discover epigenetic (DNAm) markers that are associated with POD in blood obtained from patients before and after neurosurgery. METHODS: Pre- and post-surgical blood DNA samples from 37 patients, including 10 POD cases, were analyzed using the Illumina EPIC array genome-wide platform. We examined DNAm differences in blood from patients with and without POD. Enrichment analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes terms were also conducted. RESULTS: When POD cases were tested for DNAm change before and after surgery, enrichment analyses showed many relevant signals with statistical significance in immune response related-pathways and inflammatory cytokine related-pathways such as "cellular response to cytokine stimulus", "regulation of immune system process", "regulation of cell activation", and "regulation of cytokine production". Furthermore, after excluding the potential effect of common factors related to surgery and anesthesia between POD cases and non-POD controls, the enrichment analyses showed significant signals such as "immune response" and "T cell activation", which are same pathways previously identified from an independent non-surgical inpatient cohort. CONCLUSIONS: Our first genome-wide DNAm investigation of POD showed promising signals related to immune response, inflammatory response and other relevant signals considered to be associated with delirium pathophysiology. Our data supports the hypothesis that epigenetics play an important role in the pathophysiological mechanism of delirium and suggest the potential usefulness of an epigenetics-based biomarker of POD.

The evolution of microtubule associated proteins - a reference proteomic perspective.

Microtubule associated proteins (MAPs), defined as proteins that bind microtubules but are not molecular motors or severing enzymes, play a key role in regulating microtubule stability in neurons. Existing studies of the evolutionary relationships between these proteins are limited to genomic data from a small number of species. We therefore used a large collection of publicly available reference-quality eukaryotic proteomes to carry out a phylogenetic analysis of microtubule associated proteins in both vertebrates and invertebrates. Complete or near-complete reference quality proteomes were obtained from Uniprot. Microtubule associated proteins were identified using InterProtScan, aligned using MUSCLE and then phylogenetic trees constructed using the WAG algorithm. We identified 889 proteins with tubulin binding domains, of which 663 were in eukaryotes, including 168 vertebrates and 64 invertebrates. The vertebrate proteins separated into three families, resembling human MAP 2, MAP4 and MAPT, respectively, while invertebrate MAPs clustered separately. We found significant variation in number of microtubule associated proteins and number of microtubule binding domains between taxa, with fish and mollusks having an unexpectedly high number of MAPs and binding domains, respectively. Our findings represent a novel analysis of the evolution of microtubule associated proteins based on publicly available proteomics data sets. We were able to confirm the phylogeny of MAPs identified based on more limited genomic analyses, and in addition, derived several novel insights on the structure and function of MAPs.

Genome-wide association study and functional validation implicates JADE1 in tauopathy.

Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-ß (Aß) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aß toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n = 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (SLC24A4, MS4A6A, HS3ST1) and progressive supranuclear palsy (MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration.

Identification of HnRNPC as a novel Tau exon 10 splicing factor using RNA antisense purification mass spectrometry.

Alternative splicing in Tau exon 10 generates 3 R- and 4 R-Tau proteoforms, which have equal abundance in healthy adult human brain. Aberrant alternative splicing in Tau exon 10 leads to distortion of the balanced 3 R- and 4 R-Tau expression levels, which is a causal factor to trigger toxic Tau aggregation, neuron dysfunction and patient death in a group of neurodegenerative diseases known as tauopathies. Hence, identification of regulators upstream of the Tau exon 10 splicing events are crucial to understanding pathogenic mechanisms driving tauopathies. In this study, we used RNA Antisense Purification with Mass Spectrometry (RAP-MS) analysis to identify RNA-binding proteins (RBPs) that interact with the Tau pre-mRNA near exon 10. Among the newly identified RBP candidates, we show that knockdown of hnRNPC induces Tau exon 10 skipping whereas overexpression of hnRNPC promotes Tau exon 10 inclusion. In addition, we show that hnRNPC interacts with the poly-uridine (U-tract) sequences in introns 9 and 10 of Tau pre-mRNA. Mutation of these U-tract motifs abolished binding of hnRNPC with Tau pre-mRNA fragment and blocked its impact on Tau exon 10 inclusion. These findings indicate that hnRNPC binds and utilizes these U-tract motifs located in introns 9 and 10 of Tau pre-mRNA to promote Tau exon 10 inclusion. Intriguingly, high hnRNPC expression level is associated with progressive supranuclear palsy (PSP), a sporadic tauopathy with pathological accumulation of Tau species that contain exon 10, which suggests a putative therapeutic role of hnRNPC for PSP treatment. [Figure: see text].

Assessment of Polychlorinated Biphenyls and Their Hydroxylated Metabolites in Postmortem Human Brain Samples: Age and Brain Region Differences.

Exposure to polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) has been implicated in neurodevelopmental disorders. However, the distribution of PCBs and OH-PCBs in the human brain has not been characterized. This study investigated the age-, sex-, and brain region-specific distribution of all 209 PCBs using gaschromatography-tandem mass spectrometry (GC-MS/MS) in neonatal (N = 7) and adult (N = 7) postmortem brain samples. OH-PCB analyses were performed by GC-MS/MS (as methylated derivatives) and, in a subset of samples, by nontarget liquid chromatography high-resolution mass spectrometry (Nt-LCMS). Fourteen higher chlorinated PCB congeners were observed with a detection frequency >50%. Six lower chlorinated PCBs were detected with a detection frequency >10%. Higher chlorinated PCBs were observed with higher levels in samples from adult versus younger donors. PCB congener profiles from adult donors showed more similarities across brain regions and donors than younger donors. We also assess the potential neurotoxicity of the PCB residues in the human brain with neurotoxic equivalency (NEQ) approaches. The median ΣNEQs, calculated for the PCB homologues, were 40-fold higher in older versus younger donors. Importantly, lower chlorinated PCBs made considerable contributions to the neurotoxic potential of PCB residues in some donors. OH-PCBs were identified for the first time in a small number of human brain samples by GC-MS/MS and Nt-LCMS analyses, and all contained four or fewer chlorine.

Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains.

Microtubule-associated protein tau, an integral component of neurofibrillary tangles, interacts with a variety of signaling molecules. Previously, our laboratory reported that nerve growth factor (NGF)-induced MAPK activation in a PC12-derived cell line was potentiated by tau, with phosphorylation at T231 being required. Therefore, we sought to identify a signaling molecule involved in the NGF-induced Ras-MAPK pathway that interacted with phospho-T231-tau. Here, we report that the protein tyrosine phosphatase SHP2 (also known as PTPN11) interacted with tau, with phospho-T231 significantly enhancing the interaction. By using proximity ligation assays, we found that endogenous tau-SHP2 complexes were present in neuronal cells, where the number of tau-SHP2 complexes significantly increased when the cells were treated with NGF, with phosphorylation at T231 being required for the increase. The interaction did not require microtubule association, and an association between tau and activated SHP2 was also found. Tau-SHP2 complexes were also found in both primary mouse hippocampal cultures and adult mouse brain. Finally, SHP2 levels were upregulated in samples from patients with mild and severe Alzheimer's disease (AD), and the level of tau-SHP2 complexes were increased in AD patient samples. These findings strongly suggest a role for the tau-SHP2 interaction in NGF-stimulated neuronal development and in AD.This article has an associated First Person interview with the first author of the paper.

Adenosine arrests breast cancer cell motility by A3 receptor stimulation.

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.

Sudden unexpected death in early childhood: general observations in a series of 151 cases: Part 1 of the investigations of the San Diego SUDC Research Project.

PURPOSE: The purpose of this study was to determine the major subcategories and clinicopathologic features of sudden unexpected death in young children in a large retrospective cohort, and to confirm the association of sudden unexplained death in children (abbreviated by us for unexplained deaths as SUDC) with hippocampal pathology and/or febrile seizures. METHODS: We undertook analysis of a retrospective cohort of 151 cases, of which 80% (121/151) were subclassified as SUDC, 11% (16/151) as explained, 7% (10/151) as undetermined, and 3% (4/151) as seizure-related. RESULTS: There were no significant differences between SUDC and explained cases in postnatal, gestational, or postconceptional age, frequency of preterm birth, gender, race, or organ weights. In contrast, 96.7% (117/121) of the SUDC group were discovered during a sleep period compared to 53.3% (8/15) of the explained group (p < 0.001), and 48.8% (59/121) of the SUDC cases had a personal and/or family history of febrile seizures compared to 6.7% (1/15) of the explained group (p < 0.001). Of the explained deaths, 56% (9/16) were subclassified as infection, 31% (5/16) cardiac, 6% (1/16) accidental, and 6% (1/16) metabolic. Two of the three cases specifically tested for cardiac channelopathies at autopsy based upon clinical indications had genetic variants in cardiac genes, one of uncertain significance. Bacterial cultures at autopsy typically revealed organisms interpreted as contaminants. Two of the four seizure-related deaths were witnessed, with two of the brains from these cases showing generalized malformations. Hippocampal anomalies, including a specific combination we termed hippocampal maldevelopment associated with sudden death, were found in almost 50% (40/83) of the SUDC and undetermined cases in which hippocampal sections were available. CONCLUSIONS: This study highlights the key role for the hippocampus, febrile seizures, and sleep in SUDC pathophysiology. It also demonstrates the role of known predisposing conditions such as cardiac channelopathies and infections in causing sudden unexpected death in childhood, and the need for improved ancillary testing and protective strategies in these cases, even when the cause of death is established at autopsy.

Hippocampal malformation associated with sudden death in early childhood: a neuropathologic study: Part 2 of the investigations of The San Diego SUDC Research Project.

PURPOSE: Sudden unexplained death in childhood (SUDC), while rare, accounts for an important fraction of unexpected deaths in children >1 year of age. Previously we reported an association between febrile seizures, hippocampal maldevelopment, and sudden, unexpected deaths in young children (1-6 years), termed "hippocampal maldevelopment associated with sudden death (HMASD)." Here, we characterize in greater detail the hippocampal pathology in a large cohort of cases (n = 42) of this entity, and attempt to define possible new entities responsible for sudden, unexplained death in young children without HMASD/febrile seizure phenotypes. METHODS: We performed comparative analysis on cases, which we classified in a cohort of 89 sudden and unexpected deaths as HMASD, explained deaths, SUDC with febrile seizure phenotype (SUDC-FS) but without hippocampal pathology, and SUDC (without hippocampal pathology or febrile seizure phenotype). RESULTS: The frequency of each subgroup was: HMASD 48% (40/83); SUDC 27% (22/83); SUDC-FS 18% (15/83); explained 7% (6/83). HMASD was characterized clinically by sudden, sleep-related death, term birth, and discovery in the prone position. Key morphologic features of HMASD were focal granule cell bilamination of the dentate gyrus with or without asymmetry and/or malrotation of the hippocampus, associated with significantly increased frequencies of 11 other developmental abnormalities. We identified no other distinct phenotype in the unexplained categories, except for an association of febrile seizures without hippocampal maldevelopment. CONCLUSIONS: HMASD is a distinct clinicopathologic entity characterized by a likely developmental failure of neuronal migration in the dentate gyrus. Future research is needed to determine the causal role of HMASD in sudden death in early childhood.

Significance analysis of prognostic signatures.

A major goal in translational cancer research is to identify biological signatures driving cancer progression and metastasis. A common technique applied in genomics research is to cluster patients using gene expression data from a candidate prognostic gene set, and if the resulting clusters show statistically significant outcome stratification, to associate the gene set with prognosis, suggesting its biological and clinical importance. Recent work has questioned the validity of this approach by showing in several breast cancer data sets that "random" gene sets tend to cluster patients into prognostically variable subgroups. This work suggests that new rigorous statistical methods are needed to identify biologically informative prognostic gene sets. To address this problem, we developed Significance Analysis of Prognostic Signatures (SAPS) which integrates standard prognostic tests with a new prognostic significance test based on stratifying patients into prognostic subtypes with random gene sets. SAPS ensures that a significant gene set is not only able to stratify patients into prognostically variable groups, but is also enriched for genes showing strong univariate associations with patient prognosis, and performs significantly better than random gene sets. We use SAPS to perform a large meta-analysis (the largest completed to date) of prognostic pathways in breast and ovarian cancer and their molecular subtypes. Our analyses show that only a small subset of the gene sets found statistically significant using standard measures achieve significance by SAPS. We identify new prognostic signatures in breast and ovarian cancer and their corresponding molecular subtypes, and we show that prognostic signatures in ER negative breast cancer are more similar to prognostic signatures in ovarian cancer than to prognostic signatures in ER positive breast cancer. SAPS is a powerful new method for deriving robust prognostic biological signatures from clinically annotated genomic datasets.

Repetitive head impacts induce neuronal loss and neuroinflammation in young athletes.

Repetitive head impacts (RHI) sustained from contact sports are the largest risk factor for chronic traumatic encephalopathy (CTE). Currently, CTE can only be diagnosed after death and the multicellular cascade of events that trigger initial hyperphosphorylated tau (p-tau) deposition remain unclear. Further, the symptoms endorsed by young individuals with early disease are not fully explained by the extent of p-tau deposition, severely hampering development of therapeutic interventions. Here, we show that RHI exposure associates with a multicellular response in young individuals (<51 years old) prior to the onset of CTE p-tau pathology that correlates with number of years of RHI exposure. Leveraging single nucleus RNA sequencing of tissue from 8 control, 9 RHI-exposed, and 11 low stage CTE individuals, we identify SPP1+ inflammatory microglia, angiogenic and inflamed endothelial cell profiles, reactive astrocytes, and altered synaptic gene expression in excitatory and inhibitory neurons in all individuals with exposure to RHI. Surprisingly, we also observe a significant loss of cortical sulcus layer 2/3 neurons in contact sport athletes compared to controls independent of p-tau pathology. These results provide robust evidence that multiple years of RHI exposure is sufficient to induce lasting cellular alterations that may underlie p-tau deposition and help explain the early clinical symptoms observed in young former contact sport athletes. Furthermore, these data identify specific cellular responses to repetitive head impacts that may direct future identification of diagnostic and therapeutic strategies for CTE.

Effect of dimethyl fumarate on mitochondrial metabolism in a pediatric porcine model of asphyxia-induced in-hospital cardiac arrest.

Neurological and cardiac injuries are significant contributors to morbidity and mortality following pediatric in-hospital cardiac arrest (IHCA). Preservation of mitochondrial function may be critical for reducing these injuries. Dimethyl fumarate (DMF) has shown potential to enhance mitochondrial content and reduce oxidative damage. To investigate the efficacy of DMF in mitigating mitochondrial injury in a pediatric porcine model of IHCA, toddler-aged piglets were subjected to asphyxia-induced CA, followed by ventricular fibrillation, high-quality cardiopulmonary resuscitation, and random assignment to receive either DMF (30 mg/kg) or placebo for four days. Sham animals underwent similar anesthesia protocols without CA. After four days, tissues were analyzed for mitochondrial markers. In the brain, untreated CA animals exhibited a reduced expression of proteins of the oxidative phosphorylation system (CI, CIV, CV) and decreased mitochondrial respiration (p < 0.001). Despite alterations in mitochondrial content and morphology in the myocardium, as assessed per transmission electron microscopy, mitochondrial function was unchanged. DMF treatment counteracted 25% of the proteomic changes induced by CA in the brain, and preserved mitochondrial structure in the myocardium. DMF demonstrates a potential therapeutic benefit in preserving mitochondrial integrity following asphyxia-induced IHCA. Further investigation is warranted to fully elucidate DMF's protective mechanisms and optimize its therapeutic application in post-arrest care.

Estrogen receptor negative/progesterone receptor positive breast cancer is not a reproducible subtype.

INTRODUCTION: Estrogen receptor (ER) and progesterone receptor (PR) testing are performed in the evaluation of breast cancer. While the clinical utility of ER as a predictive biomarker to identify patients likely to benefit from hormonal therapy is well-established, the added value of PR is less well-defined. The primary goals of our study were to assess the distribution, inter-assay reproducibility, and prognostic significance of breast cancer subtypes defined by patterns of ER and PR expression. METHODS: We integrated gene expression microarray (GEM) and clinico-pathologic data from 20 published studies to determine the frequency (n = 4,111) and inter-assay reproducibility (n = 1,752) of ER/PR subtypes (ER+/PR+, ER+/PR-, ER-/PR-, ER-/PR+). To extend our findings, we utilized a cohort of patients from the Nurses' Health Study (NHS) with ER/PR data recorded in the medical record and assessed on tissue microarrays (n = 2,011). In both datasets, we assessed the association of ER and PR expression with survival. RESULTS: In a genome-wide analysis, progesterone receptor was among the least variable genes in ER- breast cancer. The ER-/PR+ subtype was rare (approximately 1 to 4%) and showed no significant reproducibility (Kappa = 0.02 and 0.06, in the GEM and NHS datasets, respectively). The vast majority of patients classified as ER-/PR+ in the medical record (97% and 94%, in the GEM and NHS datasets) were re-classified by a second method. In the GEM dataset (n = 2,731), progesterone receptor mRNA expression was associated with prognosis in ER+ breast cancer (adjusted P <0.001), but not in ER- breast cancer (adjusted P = 0.21). PR protein expression did not contribute significant prognostic information to multivariate models considering ER and other standard clinico-pathologic features in the GEM or NHS datasets. CONCLUSION: ER-/PR+ breast cancer is not a reproducible subtype. PR expression is not associated with prognosis in ER- breast cancer, and PR does not contribute significant independent prognostic information to multivariate models considering ER and other standard clinico-pathologic factors. Given that PR provides no clinically actionable information in ER+ breast cancer, these findings question the utility of routine PR testing in breast cancer.

Thioflavin S Staining and Amyloid Formation Are Unique to Mixed Tauopathies.

Tau phosphorylation, aggregation, and toxicity are the main drivers of neurodegeneration in multiple tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau. Although aggregation and amyloid formation are often assumed to be synonymous, the ability of tau aggregates in different diseases to form amyloids in vivo has not been systematically studied. We used the amyloid dye Thioflavin S to look at tau aggregates in mixed tauopathies such as AD and primary age-related tauopathy, as well as pure 3R or 4R tauopathies such as Pick's disease, progressive supranuclear palsy, and corticobasal degeneration. We found that aggregates of tau protein only form thioflavin-positive amyloids in mixed (3R/4R), but not pure (3R or 4R), tauopathies. Interestingly, neither astrocytic nor neuronal tau pathology was thioflavin-positive in pure tauopathies. As most current positron emission tomography tracers are based on thioflavin derivatives, this suggests that they may be more useful for differential diagnosis than the identification of a general tauopathy. Our findings also suggest that thioflavin staining may have utility as an alternative to traditional antibody staining for distinguishing between tau aggregates in patients with multiple pathologies and that the mechanisms for tau toxicity may differ between different tauopathies.

Determinants of astrocytic pathology in stem cell models of primary tauopathies.

Astrocytic tau aggregates are seen in several primary and secondary tauopathies, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and chronic traumatic encephalopathy (CTE). In all of these diseases, astrocytic tau consists mostly of the longer (4R) tau isoform, even when adjacent neuronal aggregates consist of a mixture of 3- and 4R tau, as in CTE. Even the rare astrocytic tau aggregates seen in Pick's disease appear to contain both 3R and 4R tau. The reasons for this, and the mechanisms by which astrocytic tau aggregates form, remain unclear. We used a combination of RNA in situ hybridization and immunofluorescence in post-mortem human brain tissue, as well as tau uptake studies in human stem cell-derived astrocytes, to determine the origins of astrocytic tau in 4R tauopathies. We found no differences in tau mRNA expression between diseases or between tau positive and negative astrocytes within PSP. We then found that stem cell-derived astrocytes preferentially take up long isoform (4R) recombinant tau and that this uptake is impaired by induction of reactivity with inflammatory stimuli or nutritional stress. Astrocytes exposed to either 3R or 4R tau also showed downregulation of genes related to astrocyte differentiation. Our findings suggest that astrocytes preferentially take up neuronal 4R tau from the extracellular space, potentially explaining why 4R tau is the predominant isoform in astrocytic tau aggregates.

Determinants of Astrocytic Pathology in Stem Cell Models of Primary Tauopathies.

Astrocytic tau aggregates are seen in several primary and secondary tauopathies, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and chronic traumatic encephalopathy (CTE). In all cases, astrocytic tau consists exclusively of the longer (4R) tau isoform, even when adjacent neuronal aggregates consist of a mixture of 3- and 4R tau, as in CTE. The reasons for this and the mechanisms by which astrocytic tau aggregates form remain unclear. We used a combination of RNA in situ hybridization and immunofluorescence in post-mortem human brain tissue, as well as tau uptake studies in human stem cell-derived astrocytes, to determine the origins of astrocytic tau in 4R tauopathies. We found that astrocytes across tauopathies do not upregulate tau mRNA expression between diseases or between tau-positive and -negative astrocytes within PSP. We then found that stem cell-derived astrocytes preferentially take up long isoform (4R) labeled recombinant tau and that this uptake is impaired by induction of reactivity with inflammatory stimuli or nutritional stress. Astrocytes exposed to either 3R or 4R tau also showed downregulation of genes related to astrocyte differentiation. Our findings suggest that astrocytes preferentially take up neuronal 4R tau from the extracellular space, which potentially explains why astrocytic tau aggregates contain only 4R tau, and that tau uptake is impaired by decreased nutrient availability or neuroinflammation, both of which are common in the aging brain.

Alcohol inhibits sociability via serotonin inputs to the nucleus accumbens.

Social interaction is a core component of motivational behavior that is perturbed across multiple neuropsychiatric disorders, including alcohol use disorder (AUD). Positive social bonds are neuroprotective and enhance recovery from stress, so reduced social interaction in AUD may delay recovery and lead to alcohol relapse. We report that chronic intermittent ethanol (CIE) induces social avoidance in a sex-dependent manner and is associated with hyperactivity of serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN). While 5-HT DRN neurons are generally thought to enhance social behavior, recent evidence suggests that specific 5-HT pathways can be aversive. Using chemogenetic iDISCO, the nucleus accumbens (NAcc) was identified as one of 5 regions that were activated by 5-HT DRN stimulation. We then employed an array of molecular genetic tools in transgenic mice to show that 5-HT DRN inputs to NAcc dynorphin neurons drive social avoidance in male mice after CIE by activating 5-HT 2C receptors. NAcc dynorphin neurons also inhibit dopamine release during social interaction, reducing the motivational drive to engage with social partners. This study reveals that excessive serotonergic drive after chronic alcohol can promote social aversion by inhibiting accumbal dopamine release. Drugs that boost brain serotonin levels may be contraindicated for individuals with AUD.