Corpora amylacea negatively correlate with hippocampal tau pathology in Alzheimer's disease.

Introduction: Severity and distribution of aggregated tau and neurofibrillary tangles (NFT) are strongly correlated with the clinical presentation of Alzheimer's disease (AD). Clearance of aggregated tau could decrease the rate of NFT formation and delay AD onset. Recent studies implicate corpora amylacea (CA) as a regulator of onset or accumulation of tau pathology. Normally, CA clear brain waste products by amassing cellular debris, which are then extruded into the cerebrospinal fluid to be phagocytosed. The proper functioning of CA may slow progression of AD-associated NFT pathology, and this relationship may be influenced by amount and distribution of phospho-tau (pTau) produced, age, sex, and genetic risk. Objective: The goal of this study was to determine if CA size and number are associated with hippocampal location and local pTau severity while accounting for variations in age, sex, and genetic risk. Methods: Postmortem brain hippocampal tissue sections from 40 AD and 38 unaffected donors were immunohistochemically stained with AT8 (pTau) and counter stained with periodic acid Schiff (PAS). Stained sections of the CA1 and CA3 regions of the hippocampus were analyzed. The percent area occupied (%AO) of CA, pTau, and NFT was calculated. Pairwise comparisons and regression modeling were used to analyze the influence of age, pTau %AO, and genetic risk on %AO by CA in each region, separately in donors with AD and unaffected donors. Results: CA %AO was significantly higher in the CA3 region compared to CA1 in both groups. A significant negative correlation of CA %AO with both pTau %AO and neurofibrillary tangle %AO in the CA3 region of AD brain donors was found. Regression analysis in the CA3 region revealed a significant negative association between CA with both pTau and age. Conclusion: We found an increase of CA in the CA3 region, compared to CA1 region, in AD and unaffected donors. This may suggest that the CA3 region is a hub for waste removal. Additionally, the negative correlation between %AO by CA and NFT in the CA3 region of the hippocampus in donors with AD suggests CA could play a role in AD pathologic progression by influencing tau clearance.

Association of region-specific hippocampal reduction of neurogranin with inflammasome proteins in post mortem brains of Alzheimer's disease.

INTRODUCTION: Neurogranin (Ng) is considered a biomarker for synaptic dysfunction in Alzheimer's disease (AD). In contrast, the inflammasome complex has been shown to exacerbate AD pathology. METHODS: We investigated the protein expression, morphological differences of Ng, and correlated Ng to hyperphosphorylated tau in the post mortem brains of 17 AD cases and 17 age- and sex-matched controls. In addition, we correlated the Ng expression with two different epitopes of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). RESULTS: We show a reduction of Ng immunopositive neurons and morphological differences in AD compared to controls. Ng immunostaining was negatively correlated with neurofibrillary tangles, humanized anti-ASC (IC100) positive neurons and anti-ASC positive microglia, in AD. DISCUSSION: The finding of a negative correlation between Ng and ASC speck protein expression in post mortem brains of AD suggests that the activation of inflammasome/ASC speck pathway may play an important role in synaptic degeneration in AD. Highlights: We show the role that neurogranin plays on post-synaptic signaling in specific hippocampal regions.We demonstrate that there could be clinical implications of using neurogranin as a biomarker for dementia.We describe the loss of plasticity and neuronal scaffolding proteins in the present of AD pathology.We show the response of neuroinflammation when tau proteins phosphorylate in hippocampal neurons.We show that there is a potential therapeutic target for the inflammasome, and future studies may show that IC100, a humanized monoclonal antibody directed against ASC, may slow the progression of neurodegeneration.

Cell-type-specific CAG repeat expansions and toxicity of mutant Huntingtin in human striatum and cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships among CAG expansions, death of specific cell types and molecular events associated with these processes are not established. Here, we used fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise at mHTT in striatal medium spiny neurons (MSNs), cholinergic interneurons and cerebellar Purkinje neurons, and at mutant ATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1. Our data support a model in which CAG expansions are necessary but may not be sufficient for cell death and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Selective vulnerability of layer 5a corticostriatal neurons in Huntington's disease.

The properties of the cell types that are selectively vulnerable in Huntington's disease (HD) cortex, the nature of somatic CAG expansions of mHTT in these cells, and their importance in CNS circuitry have not been delineated. Here, we employed serial fluorescence-activated nuclear sorting (sFANS), deep molecular profiling, and single-nucleus RNA sequencing (snRNA-seq) of motor-cortex samples from thirteen predominantly early stage, clinically diagnosed HD donors and selected samples from cingulate, visual, insular, and prefrontal cortices to demonstrate loss of layer 5a pyramidal neurons in HD. Extensive mHTT CAG expansions occur in vulnerable layer 5a pyramidal cells, and in Betz cells, layers 6a and 6b neurons that are resilient in HD. Retrograde tracing experiments in macaque brains identify layer 5a neurons as corticostriatal pyramidal cells. We propose that enhanced somatic mHTT CAG expansion and altered synaptic function act together to cause corticostriatal disconnection and selective neuronal vulnerability in HD cerebral cortex.

TDP-43 and Alzheimer's Disease Pathology in the Brain of a Harbor Porpoise Exposed to the Cyanobacterial Toxin BMAA.

Cetaceans are well-regarded as sentinels for toxin exposure. Emerging studies suggest that cetaceans can also develop neuropathological changes associated with neurodegenerative disease. The occurrence of neuropathology makes cetaceans an ideal species for examining the impact of marine toxins on the brain across the lifespan. Here, we describe TAR DNA-binding protein 43 (TDP-43) proteinopathy and Alzheimer's disease (AD) neuropathological changes in a beached harbor porpoise (Phocoena phocoena) that was exposed to a toxin produced by cyanobacteria called ß-N-methylamino-L-alanine (BMAA). We found pathogenic TDP-43 cytoplasmic inclusions in neurons throughout the cerebral cortex, midbrain and brainstem. P62/sequestosome-1, responsible for the autophagy of misfolded proteins, was observed in the amygdala, hippocampus and frontal cortex. Genes implicated in AD and TDP-43 neuropathology such as APP and TARDBP were expressed in the brain. AD neuropathological changes such as amyloid-ß plaques, neurofibrillary tangles, granulovacuolar degeneration and Hirano bodies were present in the hippocampus. These findings further support the development of progressive neurodegenerative disease in cetaceans and a potential causative link to cyanobacterial toxins. Climate change, nutrient pollution and industrial waste are increasing the frequency of harmful cyanobacterial blooms. Cyanotoxins like BMAA that are associated with neurodegenerative disease pose an increasing public health risk.

Molecular profiling of human substantia nigra identifies diverse neuron types associated with vulnerability in Parkinson's disease.

Parkinson's disease (PD) is characterized pathologically by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Whether cell types beyond DA neurons in the SN show vulnerability in PD remains unclear. Through transcriptomic profiling of 315,867 high-quality single nuclei in the SN from individuals with and without PD, we identified cell clusters representing various neuron types, glia, endothelial cells, pericytes, fibroblasts, and T cells and investigated cell type-dependent alterations in gene expression in PD. Notably, a unique neuron cluster marked by the expression of RIT2, a PD risk gene, also displayed vulnerability in PD. We validated RIT2-enriched neurons in midbrain organoids and the mouse SN. Our results demonstrated distinct transcriptomic signatures of the RIT2-enriched neurons in the human SN and implicated reduced RIT2 expression in the pathogenesis of PD. Our study sheds light on the diversity of cell types, including DA neurons, in the SN and the complexity of molecular and cellular changes associated with PD pathogenesis.

Hypoxia and hypoxia-inducible factors in Kaposi sarcoma-associated herpesvirus infection and disease pathogenesis.

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma and several other tumors and hyperproliferative diseases seen predominantly in human immunodeficiency virus-infected and other immunocompromised persons. There is an increasing body of evidence showing that hypoxia and hypoxia-inducible factors (HIFs) play important roles in the biology of KSHV and in the pathogenesis of KSHV-induced diseases. Hypoxia and HIFs can induce lytic activation of KSHV and KSHV can in turn lead to a hypoxic-like state in infected cells. In this review, we describe the complex interactions between KSHV biology, the cellular responses to hypoxia, and the pathogenesis of KSHV-induced diseases. We also describe how interference with HIFs can lead to decreased tumor growth and/or death of infected cells and KSHV-induced tumors. Finally, we show how these observations may lead to novel strategies for the treatment of KSHV-induced diseases.

Identifying cancer-associated leukocyte profiles using high-resolution flow cytometry screening and machine learning.

Background: Machine learning (ML) is a valuable tool with the potential to aid clinical decision making. Adoption of ML to this end requires data that reliably correlates with the clinical outcome of interest; the advantage of ML is that it can model these correlations from complex multiparameter data sets that can be difficult to interpret conventionally. While currently available clinical data can be used in ML for this purpose, there exists the potential to discover new "biomarkers" that will enhance the effectiveness of ML in clinical decision making. Since the interaction of the immune system and cancer is a hallmark of tumor establishment and progression, one potential area for cancer biomarker discovery is through the investigation of cancer-related immune cell signatures. Hence, we hypothesize that blood immune cell signatures can act as a biomarker for cancer progression. Methods: To probe this, we have developed and tested a multiparameter cell-surface marker screening pipeline, using flow cytometry to obtain high-resolution systemic leukocyte population profiles that correlate with detection and characterization of several cancers in murine syngeneic tumor models. Results: We discovered a signature of several blood leukocyte subsets, the most notable of which were monocyte subsets, that could be used to train CATboost ML models to predict the presence and type of cancer present in the animals. Conclusions: Our findings highlight the potential utility of a screening approach to identify robust leukocyte biomarkers for cancer detection and characterization. This pipeline can easily be adapted to screen for cancer specific leukocyte markers from the blood of cancer patient.

GRL-142 binds to and impairs HIV-1 integrase nuclear localization signal and potently suppresses highly INSTI-resistant HIV-1 variants.

Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIVKGD) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIVKGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC50 of 130 femtomolar. When cells were exposed to HIVKGD IN-containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS's putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142-bound HIVKGD's PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant-harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.

Mechanism and therapeutic implications of pomalidomide-induced immune surface marker upregulation in EBV-positive lymphomas.

Epstein-Barr virus (EBV) downregulates immune surface markers to avoid immune recognition. Pomalidomide (Pom) was previously shown to increase immune surface marker expression in EBV-infected tumor cells. We explored the mechanism by which Pom leads to these effects in EBV-infected cells. Pom increased B7-2/CD86 mRNA, protein, and surface expression in EBV-infected cells but this was virtually eliminated in EBV-infected cells made resistant to Pom-induced cytostatic effects. This indicates that Pom initiates the upregulation of these markers by interacting with its target, cereblon. Interestingly, Pom increased the proinflammatory cytokines IP-10 and MIP-1∝/ß in EBV infected cells, supporting a possible role for the phosphoinositide 3-kinase (PI3K)/AKT pathway in Pom's effects. Idelalisib, an inhibitor of the delta subunit of PI3 Kinase, blocked AKT-Ser phosphorylation and Pom-induced B7-2 surface expression. PU.1 is a downstream target for AKT that is expressed in EBV-infected cells. Pom treatment led to an increase in PU.1 binding to the B7-2 promoter based on ChIP analysis. Thus, our data indicates Pom acts through cereblon leading to degradation of Ikaros and activation of the PI3K/AKT/PU.1 pathway resulting in upregulation of B7-2 mRNA and protein expression. The increased immune recognition in addition to the increases in proinflammatory cytokines upon Pom treatment suggests Pom may be useful in the treatment of EBV-positive lymphomas.

Cell Type Specific CAG Repeat Expansions and Toxicity of Mutant Huntingtin in Human Striatum and Cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Selective Vulnerability of Layer 5a Corticostriatal Neurons in Huntington's Disease.

The properties of the cell types that are selectively vulnerable in Huntington's disease (HD) cortex, the nature of somatic CAG expansions of mHTT in these cells, and their importance in CNS circuitry have not been delineated. Here we employed serial fluorescence activated nuclear sorting (sFANS), deep molecular profiling, and single nucleus RNA sequencing (snRNAseq) to demonstrate that layer 5a pyramidal neurons are vulnerable in primary motor cortex and other cortical areas of HD donors. Extensive mHTT -CAG expansions occur in vulnerable layer 5a pyramidal cells, and in Betz cells, layer 6a, layer 6b neurons that are resilient in HD. Retrograde tracing experiments in macaque brains identify the vulnerable layer 5a neurons as corticostriatal pyramidal cells. We propose that enhanced somatic mHTT -CAG expansion and altered synaptic function act together to cause corticostriatal disconnection and selective neuronal vulnerability in the HD cerebral cortex.

Daratumumab induces cell-mediated cytotoxicity of primary effusion lymphoma and is active against refractory disease.

Primary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10-22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara's effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Detection of ß-N-methylamino-l-alanine in postmortem olfactory bulbs of Alzheimer's disease patients using UHPLC-MS/MS: An autopsy case-series study.

Introduction: Cyanobacterial blooms produce toxins that may become aerosolized, increasing health risks through inhalation exposures. Health related effects on the lower respiratory tract caused by these toxins are becoming better understood. However, nasal exposures to cyanotoxins remain understudied, especially for those with neurotoxic potential. Here, we present a case series study evaluating exposure to ß-N-methylamino-l-alanine (BMAA), a cyanobacterial toxin linked to neurodegenerative disease, in postmortem olfactory tissues of individuals with varying stages of Alzheimer's disease (AD). Methods: Olfactory bulb (Ob) tissues were collected during autopsies performed between 2014 and 2017 from six South Florida brain donors (ages 47-78) with residences less than 140 m from a freshwater body. A triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) method validated according to peer AOAC International guidelines was used to detect BMAA and two BMAA isomers: 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Quantitative PCR was performed on the contralateral Ob to evaluate the relative expression of genes related to proinflammatory cytokines (IL-6 & IL-18), apoptotic pathways (CASP1 & BCL2), and mitochondrial stress (IRF1 & PINK1). Immunohistochemistry was also performed on the adjacent olfactory tract (Ot) to evaluate co-occurring neuropathology with BMAA tissue concentration. Results: BMAA was detected in the Ob of all cases at a median concentration of 30.4 ng/g (Range

Regulation of Retroviral and SARS-CoV-2 Protease Dimerization and Activity through Reversible Oxidation.

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets.

Single nucleus multi-omics identifies human cortical cell regulatory genome diversity.

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases.

Irradiation immunity interactions.

The immune system can influence cancer development by both impeding and/or facilitating tumour growth and spread. A better understanding of this complex relationship is fundamental to optimise current and future cancer therapeutic strategies. Although typically regarded as a localised and immunosuppressive anti-cancer treatment modality, radiation therapy has been associated with generating profound systemic effects beyond the intended target volume. These systemic effects are immune-driven suggesting radiation therapy can enhance anti-tumour immunosurveillance in some instances. In this review, we summarise how radiation therapy can positively and negatively affect local and systemic anti-tumour immune responses, how co-administration of immunotherapy with radiation therapy may help promote anti-tumour immunity, and how the use of immune biomarkers may help steer radiation therapy-immunotherapy personalisation to optimise clinical outcomes.

Machine learning predicts cancer subtypes and progression from blood immune signatures.

Clinical adoption of immune checkpoint inhibitors in cancer management has highlighted the interconnection between carcinogenesis and the immune system. Immune cells are integral to the tumour microenvironment and can influence the outcome of therapies. Better understanding of an individual's immune landscape may play an important role in treatment personalisation. Peripheral blood is a readily accessible source of information to study an individual's immune landscape compared to more complex and invasive tumour bioipsies, and may hold immense diagnostic and prognostic potential. Identifying the critical components of these immune signatures in peripheral blood presents an attractive alternative to tumour biopsy-based immune phenotyping strategies. We used two syngeneic solid tumour models, a 4T1 breast cancer model and a CT26 colorectal cancer model, in a longitudinal study of the peripheral blood immune landscape. Our strategy combined two highly accessible approaches, blood leukocyte immune phenotyping and plasma soluble immune factor characterisation, to identify distinguishing immune signatures of the CT26 and 4T1 tumour models using machine learning. Myeloid cells, specifically neutrophils and PD-L1-expressing myeloid cells, were found to correlate with tumour size in both the models. Elevated levels of G-CSF, IL-6 and CXCL13, and B cell counts were associated with 4T1 growth, whereas CCL17, CXCL10, total myeloid cells, CCL2, IL-10, CXCL1, and Ly6Cintermediate monocytes were associated with CT26 tumour development. Peripheral blood appears to be an accessible means to interrogate tumour-dependent changes to the host immune landscape, and to identify blood immune phenotypes for future treatment stratification.

BMAA, Methylmercury, and Mechanisms of Neurodegeneration in Dolphins: A Natural Model of Toxin Exposure.

Dolphins are well-regarded sentinels for toxin exposure and can bioaccumulate a cyanotoxin called ß-N-methylamino-l-alanine (BMAA) that has been linked to human neurodegenerative disease. The same dolphins also possessed hallmarks of Alzheimer's disease (AD), suggesting a possible association between toxin exposure and neuropathology. However, the mechanisms of neurodegeneration in dolphins and the impact cyanotoxins have on these processes are unknown. Here, we evaluate BMAA exposure by investigating transcription signatures using PCR for dolphin genes homologous to those implicated in AD and related dementias: APP, PSEN1, PSEN2, MAPT, GRN, TARDBP, and C9orf72. Immunohistochemistry and Sevier Münger silver staining were used to validate neuropathology. Methylmercury (MeHg), a synergistic neurotoxicant with BMAA, was also measured using PT-GC-AFS. We report that dolphins have up to a three-fold increase in gene transcription related to Aß+ plaques, neurofibrillary tangles, neuritic plaques, and TDP-43+ intracytoplasmic inclusions. The upregulation of gene transcription in our dolphin cohort paralleled increasing BMAA concentration. In addition, dolphins with BMAA exposures equivalent to those reported in AD patients displayed up to a 14-fold increase in AD-type neuropathology. MeHg was detected (0.16-0.41 µg/g) and toxicity associated with exposure was also observed in the brain. These results demonstrate that dolphins develop neuropathology associated with AD and exposure to BMAA and MeHg may augment these processes.