Alzheimer’s-like signaling in brains of COVID-19 patients

Authors: Steve Reiken,Leah Sittenfeld,Haikel Dridi,Yang Liu,Xiaoping Liu,Andrew R. Marks First published: 03 February 2022  https://doi.org/10.1002/alz.12558

Abstract

Introduction

The mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood.

Methods

Brain lysates from control and COVID-19 patients were analyzed for oxidative stress and inflammatory signaling pathway markers, and measurements of Alzheimer’s disease (AD)-linked signaling biochemistry. Post-translational modifications of the ryanodine receptor/calcium (Ca2+) release channels (RyR) on the endoplasmic reticuli (ER), known to be linked to AD, were also measured by co-immunoprecipitation/immunoblotting of the brain lysates.

Results

We provide evidence linking SARS-CoV-2 infection to activation of TGF-β signaling and oxidative overload. The neuropathological pathways causing tau hyperphosphorylation typically associated with AD were also shown to be activated in COVID-19 patients. RyR2 in COVID-19 brains demonstrated a “leaky” phenotype, which can promote cognitive and behavioral defects.

Discussion

COVID-19 neuropathology includes AD-like features and leaky RyR2 channels could be a therapeutic target for amelioration of some cognitive defects associated with SARS-CoV-2 infection and long COVID.

1 NARRATIVE

1.1 Contextual background

Patients suffering from COVID-19 exhibit multi-system organ failure involving not only pulmonary1 but also cardiovascular,2 neural,3 and other systems. The pleiotropy and complexity of the organ system failures both complicate the care of COVID-19 patients and contribute, to a great extent, to the morbidity and mortality of the pandemic.4 Severe COVID-19 most commonly manifests as viral pneumonia-induced acute respiratory distress syndrome (ARDS).5 Respiratory failure results from severe inflammation in the lungs, which arises when SARS-CoV-2 infects lung cells. Cardiac manifestations are multifactorial and include hypoxia, hypotension, enhanced inflammatory status, angiotensin-converting enzyme 2 (ACE2) receptor downregulation, endogenous catecholamine adrenergic activation, and direct viral-induced myocardial damage.67 Moreover, patients with underlying cardiovascular disease or comorbidities, including congestive heart failure, hypertension, diabetes, and pulmonary diseases, are more susceptible to infection by SARS-CoV-2, with higher mortality.67

In addition to respiratory and cardiac manifestations, it has been reported that approximately one-third of patients with COVID-19 develop neurological symptoms, including headache, disturbed consciousness, and paresthesias.8 Brain tissue edema, stroke, neuronal degeneration, and neuronal encephalitis have also been reported.2810 In a recent study, diffuse neural inflammatory markers were found in >80% of COVID-19 patient brains, processes which could contribute to the observed neurological symptoms.11 Furthermore, another pair of frequent symptoms of infection by SARS-CoV-2 are hyposmia and hypogeusia, the loss of the ability to smell and taste, respectively.3 Interestingly, hyposmia has been reported in early-stage Alzheimer’s disease (AD),3 and AD type II astrocytosis has been observed in neuropathology studies of COVID-19 patients.10

Systemic failure in COVID-19 patients is likely due to SARS-CoV-2 invasion via the ACE2 receptor,9 which is highly expressed in pericytes of human heart8 and epithelial cells of the respiratory tract,12 kidney, intestine, and blood vessels. ACE2 is also expressed in the brain, especially in the respiratory center and hypothalamus in the brain stem, the thermal center, and cortex,13 which renders these tissues more vulnerable to viral invasion, although it remains uncertain whether SARS-CoV-2 virus directly infects neurons in the brain.14 The primary consequences of SARS-CoV-2 infection are inflammatory responses and oxidative stress in multiple organs and tissues.1517 Recently it has been shown that the high neutrophil-to-lymphocyte ratio observed in critically ill patients with COVID-19 is associated with excessive levels of reactive oxygen species (ROS) and ROS-induced tissue damage, contributing to COVID-19 disease severity.15

Recent studies have reported an inverse relationship between ACE2 and transforming growth factor-β (TGF-β). In cancer models, decreased levels of ACE2 correlated with increased levels of TGF-β.18 In the context of SARS-CoV-2 infection, downregulation of ACE2 has been observed, leading to increased fibrosis formation, as well as upregulation of TGF-β and other inflammatory pathways.19 Moreover, patients with severe COVID-19 symptoms had higher blood serum TGF-β concentrations than those with mild symptoms,20 thus further implicating the role of TGF-β and warranting further investigation.

Interestingly, reduced angiotensin/ACE2 activity has been associated with tau hyperphosphorylation and increased amyloid beta (Aβ) pathology in animal models of AD.2122 The link between reduced ACE2 activity and increased TGF-β and tau signaling in the context of SARS-CoV-2 infection needs further exploration.

Our laboratory has shown that stress-induced ryanodine receptor (RyR)/intracellular calcium release channel post-translational modifications, including oxidation and protein kinase A (PKA) hyperphosphorylation related to activation of the sympathetic nervous system and the resulting hyper-adrenergic state, deplete the channel stabilizing protein (calstabin) from the channel complex, destabilizing the closed state of the channel and causing RyR channels to leak Ca2+ out of the endoplasmic/sarcoplasmic reticulum (ER/SR) in multiple diseases.2329 Increased TGF-β activity can lead to RyR modification and leaky channels,30 and SR Ca2+ leak can cause mitochondrial Ca2+ overload and dysfunction.29 Increased TGF-β activity31 and mitochondrial dysfunction32 are also associated with SARS-CoV-2 infection.

Here we show that SARS-CoV-2 infection is associated with adrenergic and oxidative stress and activation of the TGF-β signaling pathway in the brains of patients who have succumbed to COVID-19. One consequence of this hyper-adrenergic and oxidative state is the development of tau pathology normally associated with AD. In this article, we investigate potential biochemical pathways linked to tau hyperphosphorylation. Based on recent evidence that has linked tau pathology to Ca2+ dysregulation associated with leaky RyR channels in the brain,333 we investigated RyR2 biochemistry and function in COVID-19 patient brains.

RESEARCH-IN-CONTEXT

  1. Systematic review: The authors reviewed the literature using PubMed. While the mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood, there have been recent reports studying SARS-CoV-2 infection and brain biochemistry and neuropathology. These relevant citations are appropriately cited.
  2. Interpretation: Our findings link the inflammatory response to SARS-CoV-2 infection with the neuropathological pathways causing tau hyperphosphorylation typically associated with Alzheimer’s disease (AD). Furthermore, our data indicate a role for leaky ryanodine receptor 2 (RyR2) in the pathophysiology of SARS-CoV-2 infection.
  3. Future directions: The article proposes that the alteration of cellular calcium dynamics due to leaky RyR2 in COVID-19 brains is associated with the activation of neuropathological pathways that are also found in the brains of AD patients. Both the cortex and cerebellum of SARS-CoV-2–infected patients exhibited a reduced expression of the Ca2+ buffering protein calbindin. Decreased calbindin could render these tissues more vulnerable to cytosolic Ca2+ overload. Ex vivo treatment of the COVID-19 brain using a Rycal drug (ARM210) that targets RyR2 channels prevented intracellular Ca2+ leak in patient samples. Future experiments will explore calcium channels as a potential therapeutic target for the neurological complications associated with COVID-19.

1.2 Study conclusions and disease implications

Our results indicate that SARS-CoV-2 infection activates inflammatory signaling and oxidative stress pathways resulting in hyperphosphorylation of tau, but normal amyloid precursor protein (APP) processing in COVID-19 patient cortex and cerebellum. There was reduced calbindin expression in both cortex and cerebellum rendering both tissues vulnerable to Ca2+-mediated pathology. Moreover, COVID-19 cortex and cerebellum exhibited RyR Ca2+ release channels with the biochemical signature of ‘‘leaky’’ channels and increased activity consistent with pathological intracellular Ca2+ leak. RyR2 were oxidized, associated with increased NADPH oxidase 2 (NOX2), and were PKA hyperphosphorylated on serine 2808, both of which cause loss of the stabilizing subunit calstabin2 from the channel complex promoting leaky RyR2 channels in COVID-19 patient brains. Furthermore, ex vivo treatment of COVID-19 patient brain samples with the Rycal drug ARM210, which is currently undergoing clinical testing at the National Institutes of Health for RyR1-myopathy (ClinicalTrials.gov Identifier: NCT04141670), fixed the channel leak. Thus, our experiments demonstrate that SARS-CoV-2 infection activates biochemical pathways linked to the tau pathology associated with AD and that leaky RyR Ca2+ channels may be a potential therapeutic target for the neurological complications associated with COVID-19.

The molecular basis of how SARS-CoV-2 infection results in ‘‘long COVID’’ is not well understood, and questions regarding the role of defective Ca2+ signaling in the brain in COVID-19 remain unanswered. A recent comprehensive molecular investigation revealed extensive inflammation and degeneration in the brains of patients that died from COVID-19,34 including in patients with no reported neurological symptoms. These authors also reported overlap between marker genes of AD and genes that are upregulated in COVID-19 infection, consistent with the findings of increased tau pathophysiology reported in the present study. We propose a potential mechanism that may contribute to the neurological complications caused by SARS-CoV-2: defective intracellular Ca2+ regulation and activation of AD-like neuropathology.

TGF-β belongs to a family of cytokines involved in the formation of cellular fibrosis by promoting epithelial-to-mesenchymal transition, fibroblast proliferation, and differentiation.35 TGF-β activation has been shown to induce fibrosis in the lungs and other organs by activation of the SMAD-dependent pathway. We have previously reported that TGF-β/SMAD3 activation leads to NOX2/4 translocation to the cytosol and its association with RyR channels, promoting oxidization of the channels and depletion of the stabilizing subunit calstabin in skeletal muscle and in heart.2830 Alteration of Ca2+ signaling may be particularly crucial in COVID-19-infected patients with cardiovascular/neurological diseases due, in part, to the multifactorial RyR2 remodeling after the cytokine storm, increased TGF-β activation, and increased oxidative stress. Moreover, SARS-CoV-2–infected patients exhibited a hyperadrenergic state. The elevated expression of glutamate carboxypeptidase 2 (GCPII) in COVID-19 brains reported in the present study could also contribute directly to increased PKA signaling of RyR2 by reducing PKA inhibition via metabotropic glutamate receptor 3 (mGluR3).36 Hyperphosphorylation of RyR2 channels can promote pathological remodeling of the channel and exacerbate defective Ca2+ regulation in these tissues. The increased Ca2+/cAMP/PKA signaling could also open nearby K+ channels which could potentially weaken synaptic connectivity, reduce neuronal firing,36 and could activate Ca2+ dependent enzymes.

Interestingly, both the cortex and cerebellum of SARS-CoV–2-infected patients exhibited a reduced expression of the Ca2+ buffering protein calbindin. Decreased calbindin could render these tissues more vulnerable to the cytosolic Ca2+ overload. This finding is in accordance with previous studies showing reduced calbindin expression levels in Purkinje cells and the CA2 hippocampal region of AD patients3739 and in cortical pyramidal cells of aged individuals with tau pathology.3340 In contrast to the findings in the brains of COVID-19 patients in the present study, calbindin was not reduced in the cerebellum of AD patients, possibly protecting these cells from AD pathology.3941

Leaky RyR channels, leading to increased mitochondrial Ca2+ overload and ROS production and oxidative stress, have been shown to contribute to the development of tau pathology associated with AD.3232933 Recent studies of the effects of COVID-19 on the central nervous system have found memory deficits and biological markers similar to those seen in AD patients.4243 Our data demonstrate increased activity of enzymes responsible for phosphorylating tau (pAMPK, pGSK3β), as well as increased phosphorylation at multiple sites on tau in COVID-19 patient brains. The tau phosphorylation observed in these samples exhibited some differences from what is typically observed in AD, occurring in younger patients and in areas of the brain, specifically the cerebellum, that usually do not demonstrate tau pathology in AD patients. Taken together, these data suggest a potential contributing mechanism to the development of tau pathology in COVID-19 patients involving oxidative overload-driven RyR2 channel dysfunction. Furthermore, we propose that these pathological changes could be a significant contributing factor to the neurological manifestations of COVID-19 and in particular the “brain fog” associated with long COVID, and represent a potential therapeutic target for ameliorating these symptoms. For example, tau pathology in the cerebellum could explain the recent finding that 74% of hospitalized COVID-19 patients experienced coordination deficits.44 The data presented also raise the possibility that prior COVID-19 infection could be a potential risk factor for developing AD in the future.

The present study was limited to the use of existing autopsy brain tissues at the Columbia University Biobank from SARS-CoV-2–infected patients. The number of subjects is small and information on their cognitive function as well as their brain histopathology and levels of Aβ in cerebrospinal fluid and plasma are lacking. Furthermore, we did not have access to a suitable animal model of SARS-CoV-2 infection in which to test whether the observed biochemical changes in COVID-19 brains and potential cognitive and behavioral deficits associated with the brain fog of long COVID could be reversed or attenuated by therapeutic interventions. The design of future studies should include larger numbers of subjects that are age- and sex-matched. The cognitive function of SARS-CoV-2–infected patients who presented cognitive symptoms should be assessed and regularly monitored. Moreover, it is important to know whether the observed neuropathological signaling is unique to SARS-CoV-2 infection or are common to all other viral infections. Previous studies have reported cognitive impairment in Middle East respiratory syndrome45 as well as Ebola4647 patients. Retrospective studies comparing the incidence and the magnitude of cognitive impairments caused by these different viral infections would improve our understanding of these neurological complications of viral infections.

2 CONSOLIDATED RESULTS AND STUDY DESIGN

There were increased markers of oxidative stress (glutathione disulfide [GSSG]/ glutathione [GSH]) in the cortex (mesial temporal lobe) and cerebellum (cerebellar cortex, lateral hemisphere) of COVID-19 tissue. Kynurenic acid, a marker of inflammation, was increased in COVID-19 cortex and cerebellum brain lysates compared to controls, is in accordance with recent studies showing a positive correlation between kynurenic acid and cytokines and chemokine levels in COVID-19 patients.4850

To determine whether SARS-CoV-2 infection also increases tissue TGF-β activity, we measured SMAD3 phosphorylation, a downstream signal of TGF-β, in control and COVID-19 tissue lysates. Phosphorylated SMAD3 (pSMAD3) levels were increased in COVID-19 cortex and cerebellum brain lysates compared to controls, indicating that SARS-CoV-2 infection increased TGF-β signaling in these tissues. Interestingly, brain tissues from COVID-19 patients exhibited activation of the TGF-β pathway, despite the absence of the detectable (by immunohistochemistry and polymerase chain reaction, data not shown) virus in these tissues. These results suggest that the TGF-β pathway is activated systemically by SARS-CoV-2, resulting in its upregulation in the brain, as well as other organs. In addition to oxidative stress, COVID-19 brain tissues also demonstrated increased PKA and calmodulin-dependent protein kinase II association domain (CaMKII) activity, most likely associated with increased adrenergic stimulation. Both PKA and CaMKII phosphorylation of tau have been reported in tauopathies.5152

The hallmarks of AD brain neuropathology are the formation of Aβ plaques from abnormal APP processing by BACE1, as well as tau ‘‘tangles’’ caused by tau hyperphosphorylation.53 Brain lysates from COVID-19 patients’ autopsies demonstrated normal BACE1 and APP levels compared to controls. The patients analyzed in the present study were grouped by age (young ≤ 58 years old, old ≥ 66 years old) to account for normal, age-dependent changes in APP and tau pathology. Abnormal APP processing was only observed in brain lysates from patients diagnosed with AD. However, AMPK and GSK3β phosphorylation were increased in both the cortex and cerebellum in COVID-19 brains. Activation of these kinases in SARS-CoV-2–infected brains leads to a hyperphosphorylation of tau consistent with AD tau pathology in the cortex. COVID-19 brain lysates from older patients showed increased tau phosphorylation at S199, S202, S214, S262, and S356. Lysates from younger COVID-19 patients showed increased tau phosphorylation at S214, S262, and S356, but not at S199 and S202, demonstrating increased tau phosphorylation in both young and old individuals and suggesting a tau pathology similar to AD in COVID-19–affected patients. Interestingly, both young and old patient brains demonstrated increased tau phosphorylation in the cerebellum, which is not typical of AD.

RyR channels may be oxidized due to the activation of the TGF-β signaling pathway.30 NOX2 binding to RyR2 causes oxidation of the channel, which activates the channel, manifested as an increased open probability that can be assayed using 3[H]ryanodine binding.54 When the oxidization of the channel is at pathological levels, there is destabilization of the closed state of the channel, resulting in spontaneous Ca2+ release or leak.2730 To determine the effect of the increased TGF-β signaling associated with SARS-CoV-2 infection on NOX2/RyR2 interaction, RyR2 and NOX2 were co-immunoprecipitated from brain lysates of COVID-19 patients and controls. NOX2 associated with RyR2 in brain tissues from SARS-CoV-2–infected individuals were increased compared to controls.

Given the increased oxidative stress and increased NOX2 binding to RyR2 seen in COVID-19 brains, RyR2 post-translational modifications were investigated. Immunoprecipitated RyR2 from brain lysates demonstrated increased oxidation, PKA phosphorylation on serine 2808, and depletion of the stabilizing protein subunit calstabin2 in SARS-CoV-2–infected tissues compared to controls. This biochemical remodeling of the channel is known as the ‘‘biochemical signature’’ of leaky RyR2235556 that is associated with destabilization of the closed state of the channel. This leads to SR/ER Ca2+ leak, which contributes to the pathophysiology of a number of diseases including AD.232426305557 RyR channel activity was determined by binding of 3[H]ryanodine, which binds only to the open state of the channel. RyR2 was immunoprecipitated from tissue lysates and ryanodine binding was measured at both 150 nM and 20 μM free Ca2+. RyR2 channels from SARS-CoV-2–infected brain tissue demonstrated abnormally high activity (increased ryanodine binding) compared to channels from control tissues at physiologically resting conditions (150 nM free Ca2+), when channels should be closed. Interestingly, cortex and cerebellum of SARS-CoV-2–infected patients also exhibited a reduced expression of the Ca2+ binding protein calbindin. Calbindin is typically not reduced in the cerebellum of AD patients, possibly providing some protection against AD pathology. The low calbindin levels in the cerebellum of COVID-19 brains could contribute to the observed tau pathology in this brain region. An additional atypical finding in the COVID-19 brains studied in this investigation is an increased level of GCPII. This could contribute to the observed RyR PKA phosphorylation by increasing cAMP and inhibiting the metabotropic glutamate receptor type 3.36

3 DETAILED METHODS AND RESULTS

3.1 Methods

3.1.1 Human samples

De-identified human heart, lung, and brain tissue were obtained from the COVID BioBank at Columbia University. The cortex samples were from the mesial temporal lobe and the cerebellum samples were from the cerebellar cortex, lateral hemisphere. The Columbia University BioBank functions under standard operating procedures, quality assurance, and quality control for sample collection and maintenance. Age- and sex-matched controls exhibited absence of neurological disorders and cardiovascular or pulmonary diseases. Sex, age, and pathology of patients are listed in Table 1.TABLE 1. Sex, age, and pathology of COVID-19 patients

Patient NumberSexAgePathology
1Male57Acute hypoxic-ischemic injury in the hippocampus, pons, and cerebellum.
2Female38Hypoxic ischemic encephalopathy, severe, global.
3Male58Hypoxic/ischemic injury, global, widespread astrogliosis/microgliosis.
4Male84Dementia. Beta-amyloid plaques are noted in cortex and cerebellum.
5Female80Severe hypoxic ischemic encephalopathy, severe. Global astrogliosis and microgliosis. Mild Alzheimer-type pathology.
6Female74Acute hypoxic-ischemic encephalopathy, global, moderate to severe. Arteriolosclerosis, mild. Metabolic gliosis, moderate
7Male66Left frontal subacute hemorrhagic infarct. Multifocal subacute infarcts in pons and left cerebral peduncle. Global astrogliosis and microgliosis (see microscopic description). Alzheimer’s pathology.
8Female76Hypoxic ischemic encephalopathy, moderate. Alzheimer’s pathology. Atherosclerosis, moderate. Arteriolosclerosis, moderate
9Male72Hypoxic/ischemic injury, acute to subacute, involving hippocampus, medulla and cerebellum. Mild atherosclerosis. Mild arteriolosclerosis
10Male71Hypoxic-ischemic encephalopathy, acute, global, mild to moderate. Diffuse Lewy body disease, neocortical type, consistent with Parkinson disease dementia. Atherosclerosis, severe. Arteriolosclerosis, mild.

Lysate preparation and Western blots

Tissues (50 mg) were isotonically lysed using a Dounce homogenizer in 0.25 ml of 10 mM Tris maleate (pH 7.0) buffer with protease inhibitors (Complete inhibitors from Roche). Samples were centrifuged at 8000 × g for 20 minutes and the protein concentrations of the supernatants were determined by Bradford assay. To determine protein levels in tissue lysates, tissue proteins (20 μg) were separated by 4% to 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots were developed using the following antibodies: pSMAD3 (Abcam, 1:1000), SMAD3 (Abcam, 1:1000), AMPK (Abcam, 1:1000), tau (Thermo Fisher, 1:1000), pTauS199 (Thermo Fisher, 1:1000), pTauS202/T205 (Abcam, 1:1000), pTauS262 (Abcam, 1:1000), GSK3β (Abcam, 1:1000), pGSK3βS9 (Abcam, 1:1000), pGSK3βT216 (Abcam, 1:1000), APP (Abcam, 1:1000), BACE1 (Abcam, 1:1000), GAPDH (Santa Cruz Biotech, 1:1000), CTF-β (Santa Cruz Biotechnology, Inc., 1:1000), Calbindin (Abcam, 1:1000), and GCPII (Thermo Fisher, 1:4000).

Analyses of ryanodine receptor complex

Tissue lysates (0.1 mg) were treated with buffer or 10 μM Rycal (ARM210) at 4°C. RyR2 was immunoprecipitated from 0.1 mg lung, heart, and brain using an anti-RyR2 specific antibody (2 μg) in 0.5 ml of a modified radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.2, 0.9% NaCl, 5.0 mm NaF, 1.0 mm Na3VO4, 1% Triton X-100, and protease inhibitors; RIPA) overnight at 4°C. RyR2-specific antibody was an affinity-purified polyclonal rabbit antibody using the peptide CKPEFNNHKDYAQEK corresponding to amino acids 1367–1380 of mouse RyR2 with a cysteine residue added to the amino terminus. The immune complexes were incubated with protein A-Sepharose beads (Sigma) at 4°C for 1 hour, and the beads were washed three times with RIPA. The immunoprecipitates were size-fractionated on SDS-PAGE gels (4%–20% for RyR2, calstabin2, and NOX2) and transferred onto nitrocellulose membranes for 1 hour at 200 mA. Immunoblots were developed using the following primary antibodies: anti-RyR2 (Affinity BioReagents, 1:2500), anti-phospho-RyR-Ser(pS)-2808 (Affinity BioReagents 1:1000), anti- calstabin2 (FKBP12 C-19, Santa Cruz Biotechnology, Inc., 1:2500), and anti-NOX2 (Abcam, 1:1000). To determine channel oxidation, the carbonyl groups in the protein side chains were derivatized to DNP by reaction with 2,4-dinitrophenylhydrazine. The DNP signal associated with RyR2 was determined using a specific anti-DNP antibody according to the manufacturer using an Odyssey system (LI-COR Biosciences) with infrared-labeled anti-mouse and anti-rabbit immunoglobulin G (IgG; 1:5000) secondary antibodies.

Ryanodine binding

RyR2 was immunoprecipitated from 1.5 mg of tissue lysate using an anti-RyR2 specific antibody (25 μg) in 1.0 ml of a modified RIPA buffer overnight at 4°C. The immune complexes were incubated with protein A-Sepharose beads (Sigma) at 4°C for 1 hour, and the beads were washed three times with RIPA buffer, followed by two washes with ryanodine binding buffer (10 mM Tris-HCl, pH 6.8, 1 M NaCl, 1% CHAPS, 5 mg/ml phosphatidylcholine, and protease inhibitors). Immunoprecipitates were incubated in 0.2 ml of binding buffer containing 20 nM [3H] ryanodine and either of 150 nM and 20 μm free Ca2+ for 1 hour at 37°C. Samples were diluted with 1 ml of ice-cold washing buffer (25 mm Hepes, pH 7.1, 0.25 m KCl) and filtered through Whatman GF/B membrane filters pre-soaked with 1% polyethyleneimine in washing buffer. Filters were washed three times with 5 ml of washing buffer. The radioactivity remaining on the filters is determined by liquid scintillation counting to obtain bound [3H] ryanodine. Nonspecific binding was determined in the presence of 1000-fold excess of non-labeled ryanodine.

GSSG/GSH ratio measurement and SMAD3 phosphorylation

Approximately 20 mg of tissue suspended in 200 μL of ice-cold phosphate-buffered saline/0.5% NP-40, pH6.0 was used for lysis. Tissue was homogenized with a Dounce homogenizer with 10 to 15 passes. Samples were centrifuged at 8000 × g for 15 minutes at 4°C to remove any insoluble material. Supernatant was transferred to a clean tube. Deproteinizing of the samples was accomplished by adding 1 volume ice-cold 100% (w/v) trichloroacetic acid (TCA) into five volumes of sample and vortexing briefly to mix well. After incubating for 5 minutes on ice, samples were centrifuged at 12,000 × g for 5 minutes at 4°C and the supernatant was transferred to a fresh tube. The samples were neutralized by adding NaHCO3 to the supernatant and vortexing briefly. Samples were centrifuged at 13,000 × g for 15 minutes at 4°C and supernatant was collected. Samples were then deproteinized, neutralized, TCA was removed, and they were ready to use in the assay. The GSSG/GSH was determined using a ratio detection assay kit (Abcam, ab138881). Briefly, in two separate assay reactions, GSH (reduced) was measured directly with a GSH standard and Total GSH (GSH + GSSG) was measured by using a GSSG standard. A 96-well plate was set up with 50 μL duplicate samples and standards with known concentrations of GSH and GSSG. A Thiol green indicator was added, and the plate was incubated for 60 minutes at room temperature (RT). Fluorescence at Ex/Em = 490/520 nm was measured with a fluorescence microplate reader and the GSSG/GSH for samples were determined comparing fluorescence signal of samples with known standards.

Kynurenic acid assay

Kynurenic acid (KYNA) concentration in brain lysates was determined using an enzyme-linked immunosorbent assay (ELISA) kit for KYNA (ImmuSmol). Briefly, samples (50 μl) were added to a microtiter plate designed to extract the KCNA from the samples. An acylation reagent was added for 90 minutes at 37°C to derivatize the samples. After derivatization, 50 μl of the prepared standards and 100 μl samples were pipetted into the appropriate wells of the KYNA microtiter plate. KYNA Antiserum was added to all wells and the plate was incubated overnight at 4°C. After washing the plate four times, the enzyme conjugate was added to each well. The plate was incubated for 30 minutes at RT on a shaker at 500 rpm. The enzyme substrate was added to all wells and the plate was incubated for 20 minutes at RT. Stop solution was added to each well. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

PKA activity assay

PKA activity in brain lysates was determined using a PKA activity kit (Thermo Fisher, EIAPKA). Briefly, samples were added to a microtiter plate containing an immobilized PKA substrate that is phosphorylated by PKA in the presence of ATP. After incubating the samples with ATP at RT for 2 hours, the plate was incubated with the phospho-PKA substrate antibody for 60 minutes. After washing the plate with wash buffer, goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate was added to each well. The plate was aspirated, washed, and TMB substrate was added to each well, which was then incubated for 30 minutes at RT. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

CaMKII activity assay

CaMKII activity in brain lysates was determined using the CycLex CaM kinase II Assay Kit (MBL International). Briefly, samples were added to a microtiter plate containing an immobilized CaMKII substrate that is phosphorylated by CaMKII in the presence of Mg2+ and ATP. After incubating the samples in kinase buffer containing Mg2+ and ATP at RT for 1 hour, the plate was washed and incubated with the HRP conjugated anti-phospho-CaMKII substrate antibody for 60 minutes. The plate was aspirated, washed, and TMB substrate was added to each well, which was then incubated for 30 minutes at RT. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

Statistics

Group data are presented as mean ± standard deviation. Statistical comparisons between the two groups were determined using an unpaired t-test. Values of P < .05 were considered statistically significant. All statistical analyses were performed with GraphPad Prism 8.0.

3.2 Results

3.2.1 Oxidative stress and TGF-β, PKA, and CaMKII activation

Oxidative stress levels were determined in brain tissues (cortex, cerebellum) from COVID-19 patient autopsy tissues and controls by measuring the ratio of GSSG to GSH by an ELISA kit. COVID-19 patients exhibited significant oxidative stress with a 3.8- and 3.2-fold increase in GSSG/GSH ratios in cortex (Ctx) and cerebellum (CB) compared to controls, respectively (Figure 1A). High circulating levels of kynurenine have been reported in COVID-19.4850 However, the expression of KYNA in COVID-19 brain tissue has not been examined. Levels in the Ctx and CB were measured using an ELISA kit. COVID-19 brains had a significant increase in the Ctx and CB compared to controls (Figure 1A). An additional marker of tissue inflammation is increased cytokine expression. SMAD3 phosphorylation, a downstream signal of TGF-β, was increased in COVID-19 Ctx and CB tissue lysates compared to controls (Figure 1B and 1C). Increased adrenergic activation in the brain of patients infected with SARS-CoV-2 was also demonstrated by measuring PKA activity in the Ctx and CB and CaMKII activity was increased as well (Figure 1D).

Details are in the caption following the image
FIGURE 1Open in figure viewerIncreased oxidative stress, inflammatory and adrenergic signaling in brains of COVID-19 patients. A, Bar graph depicting the glutathione disulfide (GSSG)/ glutathione (GSH) ratio and kynurenic acid (KYNA) enzyme-linked immunsorbent assay signal from control (n = 6) and COVID-19 (n = 6) tissue lysates. CB, cerebellum; Ctx, cortex. Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19. B, Western blots showing phospho-SMAD3 and total SMAD3 from control (n = 4) and COVID-19 (n = 7) brain lysates. C, Bar graphs depicting quantification of pSMAD3/SMAD3 from Western blot signals in B. D, Calmodulin-dependent protein kinase II association domain (CaMKII) and protein kinase A (PKA) activity of brain tissue lysates. Data are mean ± SD. *P < .05 control versus COVID-19

Activation of AD-linked signaling

Both PKA and CaMKII have been directly implicated in the increased phosphorylation of tau associated with AD.5152 Because COVID-19 brain lysates had increased PKA and CaMKII activity, AD-linked biochemistry was evaluated in the COVID-19 brain lysates. Normal APP processing was observed in COVID-19 brain lysates as demonstrated by normal BACE1 and APP levels compared to controls (Figure 2A and B). Abnormal APP processing was only observed in brain lysates from patients diagnosed with AD (see Table 1 for patient details). However, phosphorylation/activation of AMPK and GSK3β was observed in SARS-CoV-2–infected patient brain lysates. Activation of these kinases along with the activation of PKA and CaMKII (Figure 1) leads to a hyperphosphorylation of tau at multiple residues (Figure 2C and D). Tau hyperphosphorylation in the cerebellum is not typical of AD pathology. The CB tau pathology demonstrated in COVID-19 warrants further investigation.

Details are in the caption following the image
FIGURE 2Open in figure viewerHyperphosphorylation of tau but normal amyloid precursor protein (APP) processing in COVID-19 brains. A, Brain (CB, cerebellum; Ctx, cortex) lysates were separated by 4% to 20% polyacrylamide gel electrophoresis. Immunoblots were developed for pAMPK, AMPK, GSK3β, pGSK3β (T216), APP, BACE1, and GAPDH loading control. The numbers (1–10) above immunoblots refer to patient numbers listed in Table 1. B, Bar graphs showing quantification of pAMPK, pGSK3β, APP/GAPDH, and BACE1/GAPDH from Western blots in (A). Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19; **P < .05 CB versus Ctx; #P < .05 COVID (Young) versus COVID (Old). C, Immunoblots of brain lysates showing total tau and tau phosphorylation on residues S199, S202/T205, S214, S262, and S356. D, Bar graphs showing quantification phosphorylated tau at the residues shown on Western blots in (C). Data are mean ± SD. *P < .05 control versus COVID-19; **P < .05 CB versus Ctx; #P < .05 COVID (Young) versus COVID (Old)

RyR2 channel oxidation and leak

RyR2 biochemistry was investigated to determine whether RyR2 in COVID-19 brain tissues demonstrated a “leaky” phenotype. Increased NOX2/RyR2 binding was shown in Ctx and CB lysates from SARS-CoV-2–infected individuals compared to controls using co-immunoprecipitation (Figure 3A and B). In addition, RyR2 from SARS-CoV-2–infected brains had increased oxidation, increased serine 2808 PKA phosphorylation, and depletion of the stabilizing protein subunit calstabin2 compared to controls (Figure 3A and B). RyR channels exhibiting these characteristics can be inappropriately activated at low cytosolic Ca2+ concentrations resulting in a pathological ER/SR Ca2+ leak. 3[H]Ryanodine binding to immunoprecipitated RyR2 was measured at both 150 nM and 20 μM free Ca2+. Because ryanodine binds only to the open state of the channel under these conditions, 3[H]Ryanodine binding may be used as a surrogate measure of channel open probability. The total amount of RyR immunoprecipitated was the same for control and COVID-19 samples (data not shown). Increased RyR2 channel activity at resting conditions (150 nM free Ca2+) was observed in COVID-19 channels compared to controls (Figure 3C). Under these conditions, RyR channels should be closed. Rebinding of calstabin2 to RyR2, using a Rycal, has been shown to reduce SR/ER Ca2+ leak, despite the persistence of the channel remodeling. Indeed, calstabin2 binding to RyR2 was increased when COVID-19 patient brain tissue lysates were treated ex vivo with the Rycal drug ARM210 (Figure 3A and B). Abnormal RyR2 activity observed at resting Ca2+ concentration was also decreased by Rycal treatment (Figure 3C).

Details are in the caption following the image
FIGURE 3Open in figure viewerDysregulation of calcium-handling proteins in COVID-19 brains. A, Western blots depicting ryanodine receptor 2 (RyR2) oxidation, protein kinase A (PKA) phosphorylation, and calstabin2 or NADPH oxidase 2 (NOX2) bound to the channel from brain (CB, cerebellum; Ctx, cortex) lysates. B, Bar graphs quantifying DNP/RyR2, pS2808/RyR2, and calstabin2 and NOX2 bound to the channel from the Western blots. Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19; # P < .05 COVID-19 versus COVID-19+ARM210. C, 3[H]ryanodine binding from immunoprecipitated RyR2. Bar graphs show ryanodine binding at 150 nM Ca2+ as a percent of maximum binding (Ca2+ = 20 μM). Data are mean ± SD. *P < .05 control versus COVID-19; #P < .05 COVID-19 versus COVID-19+ARM210. D, Western blots showing the levels of glutamate carboxypeptidase 2 (GCPII), calbindin, and GAPDH loading control in brain (Ctx, CB). E, Bar graphs quantifying GCPII/GAPDH and calbindin/GAPDH from the western blots. Data are mean ± SD. *P < .05 control versus COVID-19

An interesting finding concerning the tau phosphorylation in brain lysates from SARS-CoV-2 patients was the increase of phosphorylation at multiple sites in the cerebellum. This is atypical of AD. One potential mechanism to explain this finding is the significantly decreased levels of calbindin expressed in COVID-19 cerebellum (Figure 3D3E). The decreased cerebellar calbindin levels could make this area of the brain more susceptible to Ca2+-induced activation of enzymes upstream of tau phosphorylation. Moreover, increased GCPII expression was observed in COVID-19 cortex and cerebellar lysates (Figure 3D3E), which would reduce mGluR3 inhibition of PKA signaling and could contribute to the PKA hyperphosphorylation of RyR2.

Model for the role for leaky RyR2 in the pathophysiology of SARS-CoV-2 infection

Our data indicate a role for leaky RyR2 in the pathophysiology of SARS-CoV-2 infection (Figure 4). In addition to the brain of COVID-19 patients, we observed increased systemic oxidative stress and activation of the TGF-β signaling pathway in lung, and heart, which correlates with oxidation-driven biochemical remodeling of RyR2 (Figure 3 and S1 in supporting inormation). This RyR2 remodeling results in intracellular Ca2+ leak, which can play a role in heart failure progression, pulmonary insufficiency, as well as cognitive dysfunction.232628 The alteration of cellular Ca2+ dynamics has also been implicated in COVID-19 pathology.5859 Taken together, the present data suggest that leaky RyR2 may play a role in the long-term sequelae of COVID-19, including the “brain fog” associated with SARS-CoV-2 infection which could be a forme fruste of AD,60 and could predispose long COVID patients to developing AD later in life. Leaky RyR2 channels may be a therapeutic target for amelioration of some of the persistent cognitive deficits associated with long COVID.

Details are in the caption following the image
FIGURE 4Open in figure viewerSARS-CoV-2 infection results in leaky ryanodine receptor 2 (RyR2) that may contribute to cardiac, pulmonary, and cognitive dysfunction. SARS-CoV-2 infection targets cells via the angiotensin-converting enzyme 2 (ACE2) receptor, inducing inflammasome stress response/activation of stress signaling pathways. This results in increased transforming growth factor-β (TGF-β) signaling, which activates SMAD3 (pSMAD) and increases NADPH oxidase 2 (NOX2) expression and the amount of NOX2 associated with RyR2. Increased NOX2 activity at RyR2 oxidizes the channel, causing calstabin2 depletion from the channel macromolecular complex, destabilization of the closed state, and ER/SR calcium leak that is known to contribute to cardiac dysfunction,55 arrhythmias,61 pulmonary insufficiency,2325 and cognitive and behavioral abnormalities associated with neurodegenreation.2426 Decreased calbindin in COVID-19 may render brain more susceptible to tau pathology. Rycal drugs fix the RyR2 channel leak by restoring calstabin2 binding and stabilizing the channel closed state. Fixing leaky RyR2 may improve cardiac, pulmonary, and cognitive function in COVID-19.

A Case Of Shrunken Brains: How Covid-19 May Damage Brain Cells

Authors: William A. Haseltine Forbes March 22, 2022

Thanks to a new study from the UK we are now beginning to uncover the effects of SARS-CoV-2 infections in the brain. Comparing brain volume before and after individuals were exposed to SARS-CoV-2, this study documents significant cortical gray matter loss, equivalent to nearly 10 years of aging. Gweanaelle Douaud, the study’s first author and Professor at the University of Oxford, says that infected individuals display structural “differences over time above and beyond any potential baseline differences.” Most strikingly, individuals that experienced no or only mild symptoms with Covid-19 displayed specifically significant changes, but cortical damage seems to occur regardless of disease severity, age, sex, or vaccination status. It may be years before the long-term consequences of these structural differences are fully understood.

Douand et al had unique access to an extensive biomedical database, called the UK BioBank. The UK BioBank provided pre-pandemic brain scans from 785 individuals that were used as a baseline for normal size and structure relative to each participant. About three years later, in May 2021, the same population of participants returned for new brain scans. From the total cohort, 401 individuals were infected with Covid-19 between scans and the remaining served recruited as controls.

Between the first and second brain scans, individuals previously infected with Covid-19 experienced a 0.7% reduction in overall cortical gray matter on average, compared to the control group. To put that into perspective, people middle aged and beyond only lose 0.2% to 0.3% of volume per year.

Although it is clear that SARS-CoV-2 can damage the brain, how this damage occurs without directly infecting nerve cells remains a mystery. Current theories suggest there may be multiple factors contributing to these deficits. Structural abnormalities in the brain may in fact be secondary to infection occurring in other sites around the body, including the olfactory epithelium. Considering how close the nose is to the brain, this Oxford University Study considered whether losing the sense of smell may be linked to other neurological damage.

Loss of smell is a consistent clinical feature of Covid-19, with recent studies suggesting that 86% of individuals exposed to the virus may experience partial or complete loss of smell. A smaller percentage of people also develop additional neurological complications, including brain fog, fatigue, impaired cognitive function, and encephalography, characterized by impairments in brain structure and function. For some, these symptoms may continue to linger anywhere from a few months to more than a year after the initial infection. These effects, however, do not discriminate between mild and severe disease.

First, Douand et al. found reduced gray matter in several regions associated with olfaction, consistent with symptoms of prolonged loss of smell. Individuals exposed to Covid-19 experienced the most significant neural damage to the primary olfactory cortex, or piriform cortex. This structure receives input directly from olfactory receptors that pass through the olfactory bulb, making it the first neural target for processing and perceiving odors. Reduced cortical thickness was also observed in a connected region called the orbitofrontal cortex. Illustrated in Figure 1, the orbitofrontal cortex (OFC) receives inputs from the primary olfactory cortex and is commonly referred to as the secondary olfactory cortex.

Schematic-view-of-the-human-olfactory-system-The-primary-and-secondary-olfactory
Figure 1: Schematic view of the human olfactory system. The primary and secondary olfactory cortices … [+] FROM: “ A REVIEW ON THE NEURAL BASES OF EPISODIC ODOR MEMORY: FROM LABORATORY-BASED TO AUTOBIOGRAPHICAL APPROACHES” SAIVE ET AL. 2014.

Damage to these areas may be linked to loss of smell, but there is no evidence of causation. Instead, researchers speculate that the loss of volume to brain regions associated with olfaction may be partially attributed to widespread damage to the olfactory epithelium that disrupts neural pathways and impairs function. As with many other neural systems, pathways that are not being used over a long period of time cease to exist, a process neuroscientists often call “use it or lose it” that causes brain tissues to shrink.

Surprisingly, Douand et al. also found additional abnormalities in regions not normally associated with the sense of smell. In particular, they observed reduced gray matter volume in some regions of the limbic system, involving several structures important for producing behavioral and emotional responses. The largest differences, ranging from 0.2% to 2% reductions, were seen in the left parahippocampal gyrus and the entorhinal cortex. These regions play an important role in the hippocampal memory system, so gray matter loss could signify future memory impairments. Structures are shown below in Figure 2 for reference.

Blausen_0614_LimbicSystem
Figure 2: Illustration of the limbic system. The parahippocampal gyrus and hippocampus, which … [+] BRUCEBLAUS, WIKIPEDIA COMMONS

Are the differences seen in these limbic structures also linked to deficits in the olfactory system during Covid-19? Douand et al. argues that the parahippocampal gyrus, the orbitofrontal cortex and other parts of the limbic system are in some way connected to the olfactory cortex. Since sensory inputs, including those for olfaction, are transmitted and integrated all over the brain to guide a range of behavioral responses, damage to the olfactory epithelium may also have disastrous consequences on regions of the brain not exclusively involved in olfaction. More likely, however, these and other changes in brain structure may be a consequence of a robust immune response occurring all around the brain, albeit the mechanisms underlying inflammation-induced brain damage remain unclear.

Additional exploratory analyses found gray matter loss in the amygdala, the insula which borders the temporal cortex, and the front-most portion of the cingulate gyrus, known as the anterior cingulate gyrus. Interestingly, all these regions play a role in emotion processing and regulation. More research is needed to determine whether deficits in these brain regions may be linked to mood disorders associated with long-haul Covid-19, including depression and anxiety.

ApolloMed_2017_14_4_198_224728_f1 (1)
Figure 3:Reference locations for amygdala, anterior cingulate cortex, and insula. APOLLO MEDICINE 2020

It is important to note that not every individual infected with Covid-19 will experience a reduction in brain volume, while others will experience much greater losses. Those hospitalized with Covid-19, for example, had more widespread tissue damage and atrophy, compared to those not hospitalized for infection. Beyond hospitalized vs. non-hospitalized, there was limited data from this study showing how severity of infection may contribute to these effects.

Finally, Douand et al. asked whether these structural changes in the cerebral cortex are linked to new neurological symptoms following Covid-19 infection. Interestingly, they did not find any significant correlations. No correlation between structural changes and the prevalence of new neurological symptoms, however, does not mean that these changes will not impact brain function.

For a vast majority of people, the regenerative properties of the olfactory bulb restores the sense of smell within a few weeks or months. What about the rest of the brain? Damage to brain cells cannot be reversed. When tissues die, cerebrospinal fluid and other biomolecules fill the excess space to maintain the integrity of the brain. Perhaps, this explains why neurological complications associated with long-haul Covid-19 show little improvement over time. Years of additional research are needed before the consequences of losing so much gray matter are fully understood. Identifying these changes now will help us to better support and treat what will be a growing class of people with cognitive impairments.

Although the loss of smell is often one of the first symptoms of Covid-19 preceding any respiratory complications, the hypothesis that the SARS-CoV-2 damages the brain when it infects cells in the olfactory epithelium remains heavily-debated. Researchers do seem confident that the virus does not directly infect brain cells. If the nose is a window to the brain, it may be time to develop new vaccines that aim to close it off from the SARS-CoV-2 virus.

Acute inflammatory demyelinating polyneuropathy or Guillain-Barré syndrome associated with COVID-19: a case report

Journal of Medical Case Reports volume 15, Article number: 219 (2021) 

Abstract

Background

Coronavirus disease 2019 (COVID-19) is a global pandemic. The disease, typically characterized by bilateral pulmonary infiltrates and profound elevation of inflammatory markers, can range in severity from mild or asymptomatic illness to a lethal cytokine storm and respiratory failure. A number of recognized complications of COVID-19 infection are described in the literature. Common neurological complications include headache and anosmia. Guillain-Barré syndrome (GBS) is an uncommon complication described in isolated case reports. However, a causal relationship has yet to be established. This case report adds to the growing body of evidence that GBS is a potential COVID-19 complication.

Case presentation

A 70-year-old Caucasian woman with recently diagnosed COVID-19 infection presented to the emergency department with 4 days of gradually worsening ascending lower extremity weakness. Exam revealed bilateral lower extremity weakness, mute reflexes, and sensory loss. Soon after starting intravenous administration of immunoglobulin (IVIG), the patient developed respiratory distress, eventually requiring intubation. She remained intubated for the duration of her IVIG treatment. After five rounds of treatment, the patient was successfully extubated and transferred to acute rehab. Following 4 weeks of intense physical therapy, she was able to walk with assistance on room air.

Conclusion

At the present time, this is one of the few reports of acute inflammatory demyelinating polyneuropathy (AIDP) or GBS associated with COVID-19 in the United States. It is unclear whether a causal relationship exists given the nature of the syndrome. However, in light of the growing number of reported cases, physicians should be aware of this possible complication when evaluating COVID-19 patients.

For More Information: https://jmedicalcasereports.biomedcentral.com/articles/10.1186/s13256-021-02831-4

COVID-19 and emerging spinal cord complications: A systematic review

Authors: Ritwick Mondal,aShramana Deb,bGourav Shome,cUpasana Ganguly,aDurjoy Lahiri,a,d,⁎ and Julián Benito-Leóne,f,g,⁎⁎ Mult Scler Relat Disord. 2021 Jun; 51: 102917.Published online 2021 Mar 21. doi: 10.1016/j.msard.2021.102917

Abstract

Background

Spinal cord complications associated with coronavirus infectious disease of 2019 (COVID-19) are being widely reported. The purpose of this systematic review was to summarize so far available pieces of evidence documenting de novo novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) mediated spinal cord demyelinating diseases. Indeed, the spinal demyelinating disorders that have been reported in those patients who have suffered from COVID-19 rather than on the people already living with diagnosed or undiagnosed primary demyelinating disorders.

Methods

We used the existing PRISMA consensus statement. Data were collected from PubMed, NIH Litcovid, EMBASE and Cochrane library databases, as well as Pre-print servers (medRxiv, bioRxiv, and pre-preints.org), until September 10, 2020, using pre-specified searching strategies.

Results

The 21 selected articles were all case reports and included 11 (52%) men and 10 (48%) women. The mean age was of 46.7 ± 18.0. The neurological manifestations included weakness, sensory deficit, autonomic dysfunction and ataxia. In most cases, elevated cerebrospinal fluid protein as well as lymphocytic pleocytosis were found. SARS-CoV-2 was detected in five (24%) patients, meanwhile in 13 (62%) patients, the testing was negative. Testing was not performed in two cases and, in one, data were unavailable. Nearly half of the cases (N = 9) were associated with isolated long extensive transverse myelitis (LETM), whereas a combination of both LETM and patchy involvement was found in two. Only five patients had isolated short segment involvement and two patchy involvement. Furthermore, concomitant demyelination of both brain and spine was reported in six patients. Concerning the prognosis, most of the patients improved and the mortality rate was low (N = 2, <10%).

Conclusion

Spinal cord demyelination should be added to the plethora of immune mediated neurologic complications associated with COVID-19.

For More Information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7981271/

Coronavirus and the Nervous System

What is SARS-CoV-2 and COVID-19?

Coronaviruses are common causes of usually mild to moderate upper respiratory tract illnesses like the common cold, with symptoms that may include runny nose, fever, sore throat, cough, or a general feeling of being ill. However, a new coronavirus called Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) emerged and spread to cause the COVID-19 pandemic.

COVID-19, which means Coronavirus disease 2019, is an infectious disease that can affect people of all ages in many ways. It is most dangerous when the virus spreads from the upper respiratory tract into the lungs to cause viral pneumonia and lung damage leading to Acute Respiratory Distress Syndrome (ARDS). When severe, this impairs the body’s ability to maintain critical levels of oxygen in the blood stream—which can cause multiple body systems to fail and can be fatal.

What do we know about the effects of SARS-CoV-2 and COVID-19 on the nervous system?

Much of the research to date has focused on the acute infection and saving lives. These strategies have included preventing infection with vaccines, treating COVID-19 symptoms with medicines or antibodies, and reducing complications in infected individuals.

Research shows the many neurological symptoms of COVID-19 are likely a result of the body’s widespread immune response to infection rather than the virus directly infecting the brain or nervous system. In some people, the SARS-CoV-2 infection causes an overreactive response of the immune system which can also damage body systems. Changes in the immune system have been seen in studies of the cerebrospinal fluid, which bathes the brain, in people who have been infected by SARS-CoV-2. This includes the presence of antibodies—proteins made by the immune system to fight the virus—that may also react with the nervous system. Although still under intense investigation, there is no evidence of widespread viral infection in the brain. Scientists are still learning how the virus affects the brain and other organs in the long-term. Research is just beginning to focus on the role of autoimmune reactions and other changes that cause the set of symptoms that some people experience after their initial recovery. It is unknown if injury to the nervous system or other body organs cause lingering effects that will resolve over time, or whether COVID-19 infection sets up a more persistent or even chronic disorder.

What are the immediate (acute) effects of SARS-CoV-2 and COVID-19 on the brain?

Most people infected with SARS-CoV-2 virus will have no or mild to moderate symptoms associated with the brain or nervous system. However, most individuals hospitalized due to the virus do have symptoms related to the brain or nervous system, most commonly including muscle aches, headaches, dizziness, and altered taste and smell. Some people with COVID-19 either initially have, or develop in the hospital, a dramatic state of confusion called delirium. Although rare, COVID-19 can cause seizures or major strokes. Muscular weakness, nerve injury, and pain syndromes are common in people who require intensive care during infections. There are also very rare reports of conditions that develop after SARS-CoV-2 infection, as they sometimes do with other types of infections. These disorders of inflammation in the nervous system include Guillain-Barré syndrome (which affects nerves), transverse myelitis (which affects the spinal cord), and acute necrotizing leukoencephalopathy (which affects the brain).

Bleeding in the brain, weakened blood vessels, and blood clots in acute infection

The SARS-CoV-2 virus attaches to a specific molecule (called a receptor) on the surface of cells in the body. This molecule is concentrated in the lung cells but is also present on certain cells that line blood vessels in the body. The infection causes some arteries and veins—including those in the brain—to  become thin, weaken, and leak. Breaks in small blood vessels have caused bleeding in the brain (so-called microbleeds) in some people with COVID-19 infection. Studies in people who have died due to COVID-19 infection show leaky blood vessels in different areas of the brain that allow water and a host of other molecules as well as blood cells that are normally excluded from the brain to move from the blood stream into the brain. This leak, as well as the resulting inflammation around blood vessels, can cause multiple small areas of damage. COVID-19 also causes blood cells to clump and form clots in arteries and veins throughout the body. These blockages reduce or block the flow of blood, oxygen, and nutrients that cells need to function and can lead to a stroke or heart attack.

stroke is a sudden interruption of continuous blood flow to the brain. A stroke occurs either when a blood vessel in the brain becomes blocked or narrowed or when a blood vessel bursts and spills blood into the brain. Strokes can damage brain cells and cause permanent disability. The blood clots and vascular (relating to the veins, capillaries, and arteries in the body) damage from COVID-19 can cause strokes even in young healthy adults who do not have the common risk factors for stroke.

COVID-19 can cause blood clots in other parts of the body, too. A blood clot in or near the heart can cause a heart attack. A heart attack orInflammation in the heart, called myocarditis, can causeheart failure, and reduce the flow of blood to other parts of the body. A blood clot in the lungs can impair breathing and cause pain. Blood clots also can damage the kidneys and other organs.

Low levels of oxygen in the body (called hypoxia) can permanently damage the brain and other vital organs in the body. Some hospitalized individuals require artificial ventilation on respirators. To avoid chest movements that oppose use of the ventilator it may be necessary to temporarily “paralyze” the person and use anesthetic drugs to put the individual to sleep. Some individuals with severe hypoxia require artificial means of bringing oxygen into their blood stream, a technique called extra corporeal membrane oxygenation (ECMO). Hypoxia combined with these intensive care unit measure generally cause cognitive disorders that show slow recovery.

Diagnostic imaging of some people who have had COVID-19 show changes in the brain’s white matter that contains the long nerve fibers, or “wires,” over which information flows from one brain region to another. These changes may be due to a lack of oxygen in the brain, the inflammatory immune system response to the virus, injury to blood vessels, or leaky blood vessels. This “diffuse white matter disease” might contribute to cognitive difficulties in people with COVID-19. Diffuse white matter disease is not uncommon in individuals requiring intensive hospital care but it not clear if it also occurs in those with mild to moderate severity of COVID-19 illness.

For More Information: https://www.ninds.nih.gov/Current-Research/Coronavirus-and-NINDS/nervous-system

Pathological findings in organs and tissues of patients with COVID-19: A systematic review

Authors: Sasha Peiris 1 2Hector Mesa 3Agnes Aysola 4Juan Manivel 5Joao Toledo 1 2Marcio Borges-Sa 6Sylvain Aldighieri 1 2Ludovic Reveiz 2 7

Abstract

Background: Coronavirus disease (COVID-19) is the pandemic caused by SARS-CoV-2 that has caused more than 2.2 million deaths worldwide. We summarize the reported pathologic findings on biopsy and autopsy in patients with severe/fatal COVID-19 and documented the presence and/or effect of SARS-CoV-2 in all organs.

Methods and findings: A systematic search of the PubMed, Embase, MedRxiv, Lilacs and Epistemonikos databases from January to August 2020 for all case reports and case series that reported histopathologic findings of COVID-19 infection at autopsy or tissue biopsy was performed. 603 COVID-19 cases from 75 of 451 screened studies met inclusion criteria. The most common pathologic findings were lungs: diffuse alveolar damage (DAD) (92%) and superimposed acute bronchopneumonia (27%); liver: hepatitis (21%), heart: myocarditis (11.4%). Vasculitis was common only in skin biopsies (25%). Microthrombi were described in the placenta (57.9%), lung (38%), kidney (20%), Central Nervous System (CNS) (18%), and gastrointestinal (GI) tract (2%). Injury of endothelial cells was common in the lung (18%) and heart (4%). Hemodynamic changes such as necrosis due to hypoxia/hypoperfusion, edema and congestion were common in kidney (53%), liver (48%), CNS (31%) and GI tract (18%). SARS-CoV-2 viral particles were demonstrated within organ-specific cells in the trachea, lung, liver, large intestine, kidney, CNS either by electron microscopy, immunofluorescence, or immunohistochemistry. Additional tissues were positive by Polymerase Chain Reaction (PCR) tests only. The included studies were from numerous countries, some were not peer reviewed, and some studies were performed by subspecialists, resulting in variable and inconsistent reporting or over statement of the reported findings.

Conclusions: The main pathologic findings of severe/fatal COVID-19 infection are DAD, changes related to coagulopathy and/or hemodynamic compromise. In addition, according to the observed organ damage myocarditis may be associated with sequelae.

For More Information: https://pubmed.ncbi.nlm.nih.gov/33909679/