Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

Polygenic Parkinson's Disease Genetic Risk Score as Risk Modifier of Parkinsonism in Gaucher Disease.

BACKGROUND: Biallelic pathogenic variants in GBA1 are the cause of Gaucher disease (GD) type 1 (GD1), a lysosomal storage disorder resulting from deficient glucocerebrosidase. Heterozygous GBA1 variants are also a common genetic risk factor for Parkinson's disease (PD). GD manifests with considerable clinical heterogeneity and is also associated with an increased risk for PD. OBJECTIVE: The objective of this study was to investigate the contribution of PD risk variants to risk for PD in patients with GD1. METHODS: We studied 225 patients with GD1, including 199 without PD and 26 with PD. All cases were genotyped, and the genetic data were imputed using common pipelines. RESULTS: On average, patients with GD1 with PD have a significantly higher PD genetic risk score than those without PD (P = 0.021). CONCLUSIONS: Our results indicate that variants included in the PD genetic risk score were more frequent in patients with GD1 who developed PD, suggesting that common risk variants may affect underlying biological pathways. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Association between self-reported adherence to a low-sodium diet and dietary habits related to sodium intake in heart failure patients.

BACKGROUND: Sodium restriction is the primary dietary therapy in heart failure (HF); however, assessing sodium intake is challenging to clinicians, who commonly rely on patients' self-report of following a low-sodium diet to determine adherence. It is important to further explore the utility of self-reported adherence to a low-sodium diet in patients with HF. OBJECTIVES: The objective of this study was to evaluate the association between patients' self-reported adherence to a low-sodium diet and dietary habits related to sodium intake in patients with chronic HF. METHODS: Patients with HF seen in a tertiary care Heart Function Clinic and who have been taught on a low-sodium diet with a target of less than 2300 mg/d were included. Self-perception of compliance and dietary habits related to sodium intake was evaluated by using a dietary questionnaire. Patients were divided into 3 groups according to self-reported adherence to a low-sodium diet: never, sometimes, and always. RESULTS: Overall, 237 patients (median age, 66 years, 72.6% men) were included. Compared with the other 2 groups, patients who stated always following a low-sodium diet were less likely to use salt in cooking or at the table. However, 4.2% of the patients in the always group reported eating canned or package soups every day. Moreover, the highest proportion of patients eating fast foods 1 to 3 times a week was found among those in the sometimes group (22.9%) compared with the never (9.1%) and always (6.7%) groups (P = .002). Importantly, the rest of the food items did not show any significant differences between self-reported adherence groups. CONCLUSION: Self-report of adherence to a low-sodium diet is not reliable among patients with HF, who associate the idea of following a low-sodium diet mainly with not using salt for cooking or at the table but not with reducing frequency of intake of high-sodium processed foods.

Kynurenic acid inflammatory signaling expands in primates and impairs prefrontal cortical cognition.

Cognitive deficits from dorsolateral prefrontal cortex (dlPFC) dysfunction are common in neuroinflammatory disorders, including long-COVID, schizophrenia and Alzheimer's disease, and have been correlated with kynurenine inflammatory signaling. Kynurenine is further metabolized to kynurenic acid (KYNA) in brain, where it blocks NMDA and α7-nicotinic receptors (nic-α7Rs). These receptors are essential for neurotransmission in dlPFC, suggesting that KYNA may cause higher cognitive deficits in these disorders. The current study found that KYNA and its synthetic enzyme, KAT II, have greatly expanded expression in primate dlPFC in both glia and neurons. Local application of KYNA onto dlPFC neurons markedly reduced the delay-related firing needed for working memory via actions at NMDA and nic-α7Rs, while inhibition of KAT II enhanced neuronal firing in aged macaques. Systemic administration of agents that reduce KYNA production similarly improved cognitive performance in aged monkeys, suggesting a therapeutic avenue for the treatment of cognitive deficits in neuroinflammatory disorders.

Key Roles of CACNA1C/Cav1.2 and CALB1/Calbindin in Prefrontal Neurons Altered in Cognitive Disorders.

Importance: The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. Objective: To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. Design, Setting, and Participants: The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Main Outcomes and Measures: Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Results: Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by ß1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or ß1-adrenoceptor antagonist protected working memory from stress. Conclusions and Relevance: The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

Glutamate Metabotropic Receptor Type 3 (mGlu3) Localization in the Rat Prelimbic Medial Prefrontal Cortex.

Metabotropic glutamate receptors type 3 (mGlu3, encoded by GRM3) are increasingly related to cognitive functioning, including the working memory operations of the prefrontal cortex (PFC). In rhesus monkeys, mGlu3 are most commonly expressed on glia (36%), but are also very prominent on layer III dendritic spines (23%) in the dorsolateral PFC (dlPFC) where they enhance working memory-related neuronal firing. In contrast, mGlu2 are predominately presynaptic in layer III of macaque dlPFC, indicating a pre- vs. post-synaptic dissociation by receptor subtype. The current study examined the cellular and subcellular localizations of mGlu3 in the rat prelimbic medial PFC (PL mPFC), a region needed for spatial working memory performance in rodents. Multiple label immunofluorescence demonstrated mGlu3 expression in neurons and astrocytes, with rare labeling in microglia. Immunoelectron microscopy of layers III and V found that the predominant location for mGlu3 was on axons (layer III: 35.9%; layer V: 44.1%), with labeling especially prominent within the intervaricose segments distant from axon terminals. mGlu3 were also found on glia (likely astrocytes), throughout the glial membrane (layer III: 28.2%; layer V: 29.5%). Importantly, mGlu3 could be seen on dendritic spines, especially in layer III (layer III: 15.6%; layer V: 8.2%), with minor labeling on dendrites. These data show that there are some similarities between mGlu3 expression in rat PL mPFC and macaque dlPFC, but the spine expression enriches and differentiates in the more recently evolved primate dlPFC.

Unusual Molecular Regulation of Dorsolateral Prefrontal Cortex Layer III Synapses Increases Vulnerability to Genetic and Environmental Insults in Schizophrenia.

Schizophrenia is associated with reduced numbers of spines and dendrites from layer III of the dorsolateral prefrontal cortex (dlPFC), the layer that houses the recurrent excitatory microcircuits that subserve working memory and abstract thought. Why are these synapses so vulnerable, while synapses in deeper or more superficial layers are little affected? This review describes the special molecular properties that govern layer III neurotransmission and neuromodulation in the primate dlPFC and how they may render these circuits particularly vulnerable to genetic and environmental insults. These properties include a reliance on NMDA receptor rather than AMPA receptor neurotransmission; cAMP (cyclic adenosine monophosphate) magnification of calcium signaling near the glutamatergic synapse of dendritic spines; and potassium channels opened by cAMP/PKA (protein kinase A) signaling that dynamically alter network strength, with built-in mechanisms to take dlPFC "offline" during stress. A variety of genetic and/or environmental insults can lead to the same phenotype of weakened layer III connectivity, in which mechanisms that normally strengthen connectivity are impaired and those that normally weaken connectivity are intensified. Inflammatory mechanisms, such as increased kynurenic acid and glutamate carboxypeptidase II expression, are especially detrimental to layer III dlPFC neurotransmission and modulation, mimicking genetic insults. The combination of genetic and inflammatory insults may cross the threshold into pathology.

Predominant Founder Effect among Recurrent Pathogenic Variants for an X-Linked Disorder.

For disorders with X-linked inheritance, variants may be transmitted through multiple generations of carrier females before an affected male is ascertained. Pathogenic RS1 variants exclusively cause X-linked retinoschisis (XLRS). While RS1 is constrained to variation, recurrent variants are frequently observed in unrelated probands. Here, we investigate recurrent pathogenic variants to determine the relative burden of mutational hotspot and founder allele events to this phenomenon. A cohort RS1 variant analysis and standardized classification, including variant enrichment in the XLRS cohort and in RS1 functional domains, were performed on 332 unrelated XLRS probands. A total of 108 unique RS1 variants were identified. A subset of 19 recurrently observed RS1 variants were evaluated in 190 probands by a haplotype analysis, using microsatellite and single nucleotide polymorphisms. Fourteen variants had at least two probands with common variant-specific haplotypes over ~1.95 centimorgans (cM) flanking RS1. Overall, 99/190 of reportedly unrelated probands had 25 distinct shared haplotypes. Examination of this XLRS cohort for common RS1 haplotypes indicates that the founder effect plays a significant role in this disorder, including variants in mutational hotspots. This improves the accuracy of clinical variant classification and may be generalizable to other X-linked disorders.

Chronic Stress Weakens Connectivity in the Prefrontal Cortex: Architectural and Molecular Changes.

Chronic exposure to uncontrollable stress causes loss of spines and dendrites in the prefrontal cortex (PFC), a recently evolved brain region that provides top-down regulation of thought, action, and emotion. PFC neurons generate top-down goals through recurrent excitatory connections on spines. This persistent firing is the foundation for higher cognition, including working memory, and abstract thought. However, exposure to acute uncontrollable stress drives high levels of catecholamine release in the PFC, which activates feedforward calcium-cAMP signaling pathways to open nearby potassium channels, rapidly weakening synaptic connectivity to reduce persistent firing. Chronic stress exposures can further exacerbate these signaling events leading to loss of spines and resulting in marked cognitive impairment. In this review, we discuss how stress signaling mechanisms can lead to spine loss, including changes to BDNF-mTORC1 signaling, calcium homeostasis, actin dynamics, and mitochondrial actions that engage glial removal of spines through inflammatory signaling. Stress signaling events may be amplified in PFC spines due to cAMP magnification of internal calcium release. As PFC dendritic spine loss is a feature of many cognitive disorders, understanding how stress affects the structure and function of the PFC will help to inform strategies for treatment and prevention.

The Role of Exosomes in Lysosomal Storage Disorders.

Exosomes, small membrane-bound organelles formed from endosomal membranes, represent a heterogenous source of biological and pathological biomarkers capturing the metabolic status of a cell. Exosomal cargo, including lipids, proteins, mRNAs, and miRNAs, can either act as inter-cellular messengers or are shuttled for autophagic/lysosomal degradation. Most cell types in the central nervous system (CNS) release exosomes, which serve as long and short distance communicators between neurons, astrocytes, oligodendrocytes, and microglia. Lysosomal storage disorders are diseases characterized by the accumulation of partially or undigested cellular waste. The exosomal content in these diseases is intrinsic to each individual disorder. Emerging research indicates that lysosomal dysfunction enhances exocytosis, and hence, in lysosomal disorders, exosomal secretion may play a role in disease pathogenesis. Furthermore, the unique properties of exosomes and their ability to carry cargo between adjacent cells and organs, and across the blood-brain barrier, make them attractive candidates for use as therapeutic delivery vehicles. Thus, understanding exosomal content and function may have utility in the treatment of specific lysosomal storage disorders. Since lysosomal dysfunction and the deficiency of at least one lysosomal enzyme, glucocerebrosidase, is associated with the development of parkinsonism, the study and use of exosomes may contribute to an improved understanding of Parkinson disease, potentially leading to new therapeutics.

Glutamate Carboxypeptidase II in Aging Rat Prefrontal Cortex Impairs Working Memory Performance.

Glutamate carboxypeptidase II (GCPII) expression in brain is increased by inflammation, and reduces NAAG (N-acetyl aspartyl glutamate) stimulation of mGluR3 signaling. Genetic insults in this signaling cascade are increasingly linked to cognitive disorders in humans, where increased GCPII and or decreased NAAG-mGluR3 are associated with impaired prefrontal cortical (PFC) activation and cognitive impairment. As aging is associated with increased inflammation and PFC cognitive deficits, the current study examined GCPII and mGluR3 expression in the aging rat medial PFC, and tested whether GCPII inhibition with 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) would improve working memory performance. We found that GCPII protein was expressed on astrocytes and some microglia as expected from previous studies, but was also prominently expressed on neurons, and showed increased levels with advancing age. Systemic administration of the GCPII inhibitor, 2-MPPA, improved working memory performance in young and aged rats, and also improved performance after local infusion into the medial PFC. As GCPII inhibitors are well-tolerated, they may provide an important new direction for treatment of cognitive disorders associated with aging and/or inflammation.

Design and Region-Specific Adaptation of the Dietary Intervention Used in the SODIUM-HF Trial: A Multicentre Study.

BACKGROUND: Restricting dietary sodium consumption has been considered a major component of self-care management in heart failure (HF); however, the evidence supporting this recommendation has not been conclusive. The Study of Dietary Intervention Under 100 MMOL in Heart Failure (SODIUM-HF) trial aims to assess the effects of dietary sodium reduction on clinical outcomes in a HF population using a pragmatic design to provide empirical evidence to guide dietary sodium intake recommendations in patients with chronic HF. METHODS: SODIUM-HF is a multicentre, open-label, blinded adjudicated endpoint, randomized controlled trial in ambulatory patients with chronic HF. This trial involves participants recruited from sites in Canada, Australia, New Zealand, Mexico, Colombia, and Chile, who are followed up to 24 months. Rationale and methods of the SODIUM-HF trial were published elsewhere. As an international pragmatic dietary trial, SODIUM-HF was designed to address several challenges, such as defining the most suitable intervention to account for country-specific variations in food intake and availability. In SODIUM-HF, we implemented the Nutrition-Care Model to provide a comprehensive intervention delivered directly to patients, focusing on modifying the nutrient composition of the diet (sodium restriction), using a personalized counselling and close follow-up. RESULTS: Available upon completion of the trial. CONCLUSIONS: This long-term dietary trial is one of the first in its type in the HF field. This article describes in detail the rationale and methods for the dietary intervention employed and the region-specific adaptation of the SODIUM-HF intervention, so that the learning and processes taken in this trial can be applied to future multicountry dietary clinical trials.

CONTEXTE: La restriction de l'apport en sodium alimentaire est considérée comme étant un élément important de l'auto-prise en charge de l'insuffisance cardiaque (IC); or, les données à l'appui de cette recommandation ne sont pas concluantes. L'étude SODIUM-HF ( S tudy o f D ietary I ntervention U nder 100 M MOL in H eart F ailure) vise à évaluer en contexte réel les effets de la réduction de l'apport en sodium alimentaire sur les résultats cliniques au sein d'une population de patients atteints d'IC, afin d'obtenir des données empiriques qui serviront à orienter les recommandations en matière d'apport en sodium alimentaire chez les patients atteints d'IC chronique. MÉTHODOLOGIE: L'étude SODIUM-HF est un essai multicentrique contrôlé avec répartition aléatoire et mené en mode ouvert auprès de patients ambulatoires atteints d'IC chronique, dont les paramètres sont évalués à l'insu. Les participants sont recrutés dans des centres situés au Canada, en Australie, en Nouvelle-Zélande, au Mexique, en Colombie et au Chili, et font l'objet d'un suivi pendant 24 mois. La raison d'être et la méthodologie de l'étude SODIUM-HF sont présentées ailleurs. Comme il s'agit d'une étude internationale sur l'alimentation menée en contexte réel, l'étude SODIUM-HF a été conçue de manière à résoudre plusieurs difficultés, dont la façon la plus appropriée de tenir compte des variations entre pays en matière d'apport alimentaire et de disponibilité des aliments. L'étude met en œuvre le processus de soins en nutrition, afin d'assurer directement auprès des patients une intervention exhaustive axée sur la modification de la composition en nutriments de l'alimentation (restriction de l'apport en sodium) et faisant appel à des conseils personnalisés et à un suivi étroit. RÉSULTATS: Les résultats seront publiés à la fin de l'étude. CONCLUSIONS: Cette étude de longue durée sur l'alimentation est l'une des premières en son genre dans le domaine de l'IC. Les auteurs décrivent en détail la raison d'être et la méthodologie des mesures d'intervention alimentaire employées ainsi que les adaptations propres à chaque région mises en œuvre dans le cadre de l'étude SODIUM-HF, afin que les apprentissages et les processus issus de l'étude puissent être utilisés dans d'autres études cliniques multinationales sur l'alimentation.

BIN1 localization is distinct from Tau tangles in Alzheimer's disease.

BIN1 is the second most significant Alzheimer's disease (AD) risk factor gene identified through genome-wide association studies. BIN1 is an adaptor protein that can bind to several proteins including c-Myc, clathrin, adaptor protein-2 and dynamin. BIN1 is widely expressed in the brain and peripheral tissue as ubiquitous and tissue-specific alternatively spliced isoforms that regulate membrane dynamics and endocytosis in multiple cell types. The function of BIN1 in the brain and the mechanism(s) by which AD-associated BIN1 alleles increase the risk for the disease are not known. BIN1 has been shown to interact with Tau and two studies reported a positive correlation between BIN1 expression and neurofibrillary tangle pathology in AD. However, an inverse correlation between BIN1 expression and Tau propagation has also been reported. Moreover, there have been conflicting reports on whether BIN1 is present in tangles. A recent study characterized predominant BIN1 expression in mature oligodendrocytes in the gray matter and the white matter in rodent, and the human brain. Here, we have examined BIN1 localization in the brains of patients with AD using immunohistochemistry and immunofluorescence techniques to analyze BIN1 cellular expression in relation to cellular markers and pathological lesions in AD. We report that BIN1 immunoreactivity in human AD is not associated with neurofibrillary tangles or senile plaques. Moreover, our results show that BIN1 is not expressed by resting and activated microglia, astrocytes, or macrophages in human AD. In accordance with a recent report, low-level de novo BIN1 expression can be observed in a subset of neurons in the AD brain. Further investigations are warranted to understand the complex cellular mechanisms underlying the observed correlation between BIN1 expression and the severity of tangle pathology in AD.