Protein Kinases - High Yield Targets for Cancer and Dementia Drug Discovery: Protein kinase treatment targets.

Cellular protein kinases are involved in diverse normal cellular functions. Many types of dysregulation of protein kinases are the molecular basis for development of common cancers and neurodegenerative diseases. More than 80 small-molecule protein kinase inhibitors now are available and FDA-approved for successful treatment of cancers and neurodegenerative diseases. Newly designed protein kinase inhibitors and related forms of therapy based on a greater understanding of molecular mechanisms have diminished the appearance of disease resistance to protein kinase inhibitors and other side-effects. These advances will further promote the success of protein kinase inhibitors in treatment of common cancers, Alzheimer's disease and other neurodegenerative conditions.

Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs.

Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins ß-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and ß-III-tubulin range from 30% to 63%, in contrast to 0.8%-3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.

Human Stem Cells in Regenerative Medicine.

Modern medicine now has the capacity to improve therapy for many human diseases by introducing adult somatic stem cells that can repair or replace defective or damaged tissues. However, the area is still in an early phase of development, so all new applications must be carefully designed for maximal safety as well as effectiveness.

Th2 cells in rapid immune responses and protective avoidance reactions.

Type 2 helper cells (Th2 cells) differentiate from CD4 helper T cells under the influence of IL-4 and conventional or monocyte-derived CD11b+ dendritic cells. Th2 cells are capable of generating IL-4, IL-5, and IL-13, as well as evoking immunoglobulin class-switch to IgE. Three types of rapid immune responses are Th2 cell-dependent: (1) mast cell-IgE mediated allergic reactions, (2) Th2 cell-derived cytokine-mediated reactions that complement allergic reactions and protect the host from toxins, xenobiotics, environmental irritants, and helminthic parasites, and (3) IgE-stimulated mast cell-derived cysteinyl-leukotriene mediated avoidance of toxins. The contributions of Th2 cell-derived cytokines to eosinophilia (IL-5), IgE class-switch, and epithelial barrier activation, mucous secretion, and metaplasia (IL-4 and IL-13) in asthma, allergic rhinitis with polyps and atopic dermatitis have led to anti-cytokine monoclonal antibody treatments. Anti-IL-5 neutralizing monoclonal antibody in asthma and anti-IL-4/IL-13 receptor neutralizing monoclonal antibody in asthma and atopic dermatitis are proven successful therapies in appropriately selected patients who are not sufficiently improved by conventional treatments.

An emerging spectrum of therapeutic targets for Alzheimer's disease.

Development of effective preventative and therapeutic measures for Alzheimer's disease has been unsuccessful because of the long and subtly symptomatic preclinical period, difficulties obtaining tissue and biochemical data from living patients, and the many complex underlying pathogenic processes. Recent applications of sensitive specific bioimaging techniques, analyses of RNAs and proteins of neural cell-derived extracellular vesicles in blood, and sophisticated genetic procedures in cellular and rodent models have yielded hopeful new therapeutic targets. These newer targets are described here in relation to their neural cellular location, potential genetic modifications and possible pharmacological approaches.

Neuron-derived extracellular vesicles in blood reveal effects of exercise in Alzheimer's disease.

BACKGROUND: Neuron-derived extracellular vesicles (NDEVs) in blood may be used to derive biomarkers for the effects of exercise in Alzheimer's disease (AD). For this purpose, we studied changes in neuroprotective proteins proBDNF, BDNF, and humanin in plasma NDEVs from patients with mild to moderate AD participating in the randomized controlled trial (RCT) of exercise ADEX. METHODS: proBDNF, BDNF, and humanin were quantified in NDEVs immunocaptured from the plasma of 95 ADEX participants, randomized into exercise and control groups, and collected at baseline and 16 weeks. Exploratorily, we also quantified NDEV levels of putative exerkines known to respond to exercise in peripheral tissues. RESULTS: NDEV levels of proBDNF, BDNF, and humanin increased in the exercise group, especially in APOE ε4 carriers, but remained unchanged in the control group. Inter-correlations between NDEV biomarkers observed at baseline were maintained after exercise. NDEV levels of putative exerkines remained unchanged. CONCLUSIONS: Findings suggest that the cognitive benefits of exercise could be mediated by the upregulation of neuroprotective factors in NDEVs. Additionally, our results indicate that AD subjects carrying APOE ε4 are more responsive to the neuroprotective effects of physical activity. Unchanged NDEV levels of putative exerkines after physical activity imply that exercise engages different pathways in neurons and peripheral tissues. Future studies should aim to expand upon the effects of exercise duration, intensity, and type in NDEVs from patients with early AD and additional neurodegenerative disorders. TRIAL REGISTRATION: The Effect of Physical Exercise in Alzheimer Patients (ADEX) was registered in ClinicalTrials.gov on April 30, 2012 with the identifier NCT01681602.

Prediction of Post-Acute-Sequelae of COVID-19 by Cargo Protein Biomarkers of Blood Total Extracellular Vesicles in Acute COVID-19.

BACKGROUND: SARS-CoV-2 invades mitochondria of infected cells resulting in disordered metabolism, mitophagy, and abnormal levels of mitochondrial proteins in extracellular vesicles. Blood extracellular vesicle SARS-CoV-2 proteins and mitochondrial proteins were quantified in COVID-19 to assess possible roles as biomarkers. METHODS: Total extracellular vesicles were precipitated from blood of age- and gender-matched participants with no infection (n=10), acute COVID-19 (n=16), post-acute sequelae of COVID-19 (PASC or long COVID) (n=30), or post-acute COVID without PASC (n=8) and their extracted proteins quantified by enzyme-linked immunosorbent assays (ELISAs). RESULTS: Total extracellular vesicle levels of S1 (receptor-binding domain [RBD]) protein were significantly higher in acute infections than in uninfected controls, post-acute infection without PASC, and PASC. Total extracellular vesicle levels of nucleocapsid (N) protein were significantly higher in PASC than in uninfected controls, acute infections, and post-acute infection without PASC. Neither acute levels of S1(RBD) or N proteins predicted progression to PASC. Levels of neither SARS-CoV-2 protein in established PASC correlated with neuropsychiatric manifestations. Significant decreases in total extracellular vesicle levels of the mitochondrial proteins MOTS-c, VDAC-1, and humanin, and elevations of levels of SARM-1 were observed in acutely infected patients who would develop PASC. Significant decreases in total extracellular vesicle levels of MOTS-c and humanin, but not VDAC-1, and elevations of total extracellular vesicle levels of SARM-1 were characteristic of PASC patients with neuropsychiatric manifestations. CONCLUSIONS: Total extracellular vesicle levels of SARS-CoV-2 proteins in COVID-19 indicate intracellular presence of SARS-CoV-2. Abnormal total extracellular vesicles levels of mitochondrial proteins in acute infections predict a high risk of PASC and later in established PASC are indicative of neuropsychiatric manifestations.

Abnormal levels of mitochondrial Ca2+ channel proteins in plasma neuron-derived extracellular vesicles of early schizophrenia.

Structural alterations or quantitative abnormalities of some mitochondrial ion channels and exchangers are associated with altered neuronal functions and increased susceptibility to mental illness. Here we have assessed levels of functionally prominent mitochondrial calcium ion channel proteins in plasma neuron-derived extracellular vesicles (NDEVs) of living patients with first episodes of psychosis (FP) and matched controls (Cs). NDEVs were enriched with an established method of precipitation and immunoabsorption by anti-human CD171 neural adhesion protein (L1CAM) antibody and extracted proteins quantified with ELISAs. CD81 exosome marker-normalized NDEV levels of leucine zipper EF-hand containing transmembrane 1 protein (LETM1), transient receptor potential cation channel subfamily M, member 4 (TRPM4), and solute carrier family 8 member B1 (SLC24A6) or mitochondrial Na+ /Ca2+ exchanger (NCLX) were significantly lower for FP patients (n = 10) than Cs (n = 10), whereas NDEV levels of voltage-dependent L-type calcium channel subunit α-1C (CACNA-1C) were significantly higher for FP patients than Cs. Abnormal structures or mitochondrial levels of LETM1, NCLX, and CACNA-1C have been linked through analyses of individual proteins, genome-wide association studies, and whole exome protein-coding sequence studies to neurodevelopmental disorders, mental retardation, schizophrenia, and major depressive diseases. A greater understanding of the altered calcium homeostasis in schizophrenia, that is attributable to underlying mitochondrial calcium channel abnormalities, will lead to improved diagnosis and treatment.

SARS-CoV-2 and Mitochondrial Proteins in Neural-Derived Exosomes of COVID-19.

OBJECTIVE: As SARS-CoV-2 is known to invade neural cell mitochondria, a plasma system for quantifying central nervous system proteins in living humans was used to investigate neuropathogenic mechanisms of long-COVID-19. METHODS: SARS-CoV-2 proteins and mitochondrial proteins (MPs) in enriched plasma neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) were quantified in resolved acute COVID-19 without post-acute sequelae of SARS-CoV-2 (PASC), PASC without neuropsychiatric manifestations (NP), PASC with NP and healthy controls. RESULTS: NDEV and ADEV mean levels of SARS-CoV-2 S1 and nucleocapsid (N) proteins were higher in all PASC sub-groups than controls, but only N levels were higher in PASC with than without NP. Exosome marker CD81-normalized NDEV mean levels of subunit 6 of MP respiratory chain complex I and subunit 10 of complex III, and neuroprotective MPs Humanin and mitochondrial open-reading frame of the 12S rRNA-c (MOTS-c) all were decreased significantly in PASC with NP but not in PASC without NP relative to controls. NDEV levels of MPs voltage-dependent anion-selective channel protein 1 (VDAC1) and N-methyl-D-aspartate receptor 1 (NMDAR1) were decreased in PASC without and with NP, whereas those of calcium channel MPs mitochondrial calcium uniporter (MCU), sodium/calcium exchanger (NCLX) and leucine zipper EF-hand containing transmembrane 1 protein (LETM1) were decreased only in PASC with NP. ADEV levels of MCU and NCLX only were increased in PASC without and with NP. INTERPRETATION: Abnormal NDEV and ADEV levels of SARS-CoV-2 N and S1 protein and MPs correlate with NP and may be biomarkers for long-COVID prognostics and therapeutic trials. ANN NEUROL 2022;91:772-781.

Mitochondrial Electron Transport Chain Protein Abnormalities Detected in Plasma Extracellular Vesicles in Alzheimer's Disease.

Mitochondria provide energy to neurons through oxidative phosphorylation and eliminate Reactive Oxygen Species (ROS) through Superoxide Dismutase 1 (SOD1). Dysfunctional mitochondria, manifesting decreased activity of electron transport chain (ETC) complexes and high ROS levels, are involved in Alzheimer's disease (AD) pathogenesis. We hypothesized that neuronal mitochondrial dysfunction in AD is reflected in ETC and SOD1 levels and activity in plasma neuron-derived extracellular vesicles (NDEVs). We immunoprecipitated NDEVs targeting neuronal marker L1CAM from two cohorts: one including 22 individuals with early AD and 29 control subjects; and another including 14 individuals with early AD and 14 control subjects. In the first cohort, we measured levels of complexes I, III, IV, ATP synthase, and SOD1; in the second cohort, we measured levels and catalytic activity of complexes IV and ATP synthase. AD individuals had lower levels of complexes I (p < 0.0001), III (p < 0.0001), IV (p = 0.0061), and V (p < 0.0001), and SOD1 (p < 0.0001) compared to controls. AD individuals also had lower levels of catalytic activity of complex IV (p = 0.0214) and ATP synthase (p < 0.0001). NDEVs confirm quantitative and functional abnormalities in ECT complexes and SOD1 previously observed in AD models and during autopsy, opening the way for using them as biomarkers for mitochondrial dysfunction in AD.

Abnormal levels of mitochondrial proteins in plasma neuronal extracellular vesicles in major depressive disorder.

To characterize neuronal mitochondrial abnormalities in major depressive disorder (MDD), functional mitochondrial proteins (MPs) extracted from enriched plasma neuron-derived extracellular vesicles (NDEVs) of MDD participants (n = 20) were quantified before and after eight weeks of treatment with a selective serotonin reuptake inhibitor (SSRI). Pretreatment baseline NDEV levels of the transcriptional type 2 nuclear respiratory factor (NRF2) which controls mitochondrial biogenesis and many anti-oxidant gene responses, regulators of diverse neuronal mitochondrial functions cyclophilin D (CYPD) and mitofusin-2 (MFN2), leucine zipper EF-hand containing transmembrane 1 protein (LETM1) component of a calcium channel/calcium channel enhancer, mitochondrial tethering proteins syntaphilin (SNPH) and myosin VI (MY06), inner membrane electron transport complexes I (subunit 6) and III (subunit 10), the penultimate enzyme of nicotinamide adenine dinucleotide (NAD) generation nicotinamide mononucleotide adenylytransferase 2 (NMNAT2), and neuronal mitochondrial metabolic regulatory and protective factors humanin and mitochondrial open-reading frame of the 12S rRNA-c (MOTS-c) all were significantly lower than those of NDEVs from matched controls (n = 10), whereas those of pro-neurodegenerative NADase Sterile Alpha and TIR motif-containing protein 1 (SARM1) were higher. The baseline NDEV levels of transcription factor A mitochondrial (TFAM) and the transcriptional master-regulator of mitochondrial biogenesis PPAR γ coactivator-1α (PGC-1α) showed no differences between MDD participants and controls. Several of these potential biomarker proteins showed substantially different changes in untreated MDD than those we reported in untreated first-episode psychosis. NDEV levels of MPs of all functional classes, except complex I-6, NRF2 and PGC-1α were normalized in MDD participants who responded to SSRI therapy (n = 10) but not in those who failed to respond (n = 10) by psychiatric evaluation. If larger studies validate NDEV MP abnormalities, they may become useful biomarkers and identify new drug targets.

Elevated complement mediator levels in endothelial-derived plasma exosomes implicate endothelial innate inflammation in diminished brain function of aging humans.

We test the hypothesis that endothelial cells adopt an inflammatory phenotype in functionally intact aged human subjects with radiographic evidence of white matter hyperintensity (WMH) suggestive of small cerebrovascular disease. Components of all three complement effector pathways and regulatory proteins were quantified in extracts of plasma endothelial-derived exosomes (EDE) of 11 subjects (age 70-82) with and 15 without evidence of WMH on MRI. Group differences and associations with plasma markers of immune activation (IL6, ICAM1), cognition and neuroimaging were calculated via regression modelling. EDE complement factors within the alternative and classical pathways were found to be higher and regulatory proteins lower in subjects with WMH. EDE levels of some complement components demonstrated significant associations with cognitive slowing and elevated systolic blood pressure. The inhibitor of the membrane attack complex, CD46, showed a significant positive association with cerebral grey matter volume. Plasma inflammatory markers, IL6 and ICAM1, were positively associated with EDE levels of several complement components. These findings provide the first in vivo evidence of the association of endothelial cell inflammation with white matter disease, age-associated cognitive changes, and brain degeneration in functionally normal older individuals. Future endothelial biomarker development may permit recognition of early or preclinical stages of vascular contributions to cognitive impairment and dementia.

Neuronal and Astrocytic Extracellular Vesicle Biomarkers in Blood Reflect Brain Pathology in Mouse Models of Alzheimer's Disease.

Circulating neuronal extracellular vesicles (NEVs) of Alzheimer's disease (AD) patients show high Tau and ß-amyloid (Aß) levels, whereas their astrocytic EVs (AEVs) contain high complement levels. To validate EV proteins as AD biomarkers, we immunocaptured NEVs and AEVs from plasma collected from fifteen wild type (WT), four 2xTg-AD, nine 5xFAD, and fifteen 3xTg-AD mice and assessed biomarker relationships with brain tissue levels. NEVs from 3xTg-AD mice had higher total Tau (p = 0.03) and p181-Tau (p = 0.0004) compared to WT mice. There were moderately strong correlations between biomarkers in NEVs and cerebral cortex and hippocampus (total Tau: cortex, r = 0.4, p = 0.009; p181-Tau: cortex, r = 0.7, p < 0.0001; hippocampus, r = 0.6, p < 0.0001). NEVs from 5xFAD compared to other mice had higher Aß42 (p < 0.005). NEV Aß42 had moderately strong correlations with Aß42 in cortex (r = 0.6, p = 0.001) and hippocampus (r = 0.7, p < 0.0001). AEV C1q was elevated in 3xTg-AD compared to WT mice (p = 0.005); AEV C1q had moderate-strong correlations with C1q in cortex (r = 0.9, p < 0.0001) and hippocampus (r = 0.7, p < 0.0001). Biomarkers in circulating NEVs and AEVs reflect their brain levels across multiple AD mouse models supporting their potential use as a "liquid biopsy" for neurological disorders.

Neural cell-derived plasma exosome protein abnormalities implicate mitochondrial impairment in first episodes of psychosis.

Neuroprotective and other functional proteins of mitochondria were quantified in extracts of plasma neural-derived exosomes from ten first-episode psychosis (FP) patients and ten matched psychiatrically normal controls (ctls). Astrocyte-derived extracellular vesicles (ADEVs) and neuron-derived extracellular vesicles (NDEVs) were immunoabsorbed separately from physically precipitated plasma total EVs. Extracted mitochondrial ATP synthase was specifically immunofixed to plastic wells for quantification of catalytic activity based on conversion of NADH to NAD+ . Other extracted mitochondrial functional proteins were quantified by ELISAs. All protein levels were normalized with EV content of the CD81 exosome marker. FP patient ADEV level but not NDEV level of mitochondrial ATP synthase activity was significantly lower than that of ctls. FP patient ADEV and NDEV levels of the functionally critical mitochondrial proteins mitofusin 2 and cyclophilin D, but not of transcription factor A of mitochondria, and of the mitochondrial short open-reading frame neuroprotective and metabolic regulatory peptides humanin and MOTS-c were significantly lower than those of ctls. In contrast, FP patient NDEV, but not ADEV, level of the mitochondrial-tethering protein syntaphilin, but not of myosin VI, was significantly higher than that of ctls. The distinctively different neural levels of some mitochondrial proteins in FP patients than ctls now should be correlated with diverse clinical characteristics. Drugs that increase depressed levels of proteins and mimetics of deficient short open-reading frame peptides may be of therapeutic value in early phases of schizophrenia.

Altered Functional Mitochondrial Protein Levels in Plasma Neuron-Derived Extracellular Vesicles of Patients With Gadolinium Deposition.

The retention of the heavy metal, gadolinium, after a Gadolinium-Based Contrast Agent-assisted MRI may lead to a symptom cluster termed Gadolinium Deposition Disease. Little is known of the disorder's underlying pathophysiology, but a recent study reported abnormally elevated serum levels of pro-inflammatory cytokines compared to normal controls. As a calcium channel blocker in cellular plasma and mitochondrial membranes, gadolinium also interferes with mitochondrial function. We applied to sera from nine Gadolinium Deposition Disease and two Gadolinium Storage Condition patients newly developed methods allowing isolation of plasma neuron-derived extracellular vesicles that contain reproducibly quantifiable levels of mitochondrial proteins of all major classes. Patients' levels of five mitochondrial functional proteins were statistically significantly lower and of two significantly higher than the levels in normal controls. The patterns of differences between study patients and controls for mitochondrial dynamics and mitochondrial proteins encompassing neuronal energy generation, metabolic regulation, ion fluxes, and survival differed from those seen for patients with first episode psychosis and those with Major Depressive Disorder compared to their controls. These findings suggest that mitochondrial dysfunction due to retained gadolinium may play a role in causing Gadolinium Deposition Disease. Larger samples of both GDD and GSC patients are needed to allow not only testing the repeatability of our findings, but also investigation of relationships of specific mitochondrial protein deficiencies or excesses and concurrent cytokine, genetic, or other factors to GDD's neurological and cognitive symptoms. Studies of neuronal mitochondrial proteins as diagnostic markers or indicators of treatment effectiveness are also warranted.

Decreased mitochondrial electron transport proteins and increased complement mediators in plasma neural-derived exosomes of early psychosis.

Potentially neurotoxic systems involved in traumatic and degenerative diseases of the brain were assessed in acute psychosis. Astrocyte-derived exosomes (ADEs) and neuron-derived exosomes (NDEs) were immunoprecipitated from plasma of ten untreated first-episode psychotics (FPs) and ten matched normal controls (Cs). Neural mitochondrial electron transport and complement proteins were extracted, quantified by ELISAs and normalized with levels of CD81 exosome marker. Levels of subunits 1 and 6 of NADH-ubiquinone oxidoreductase (complex I) and subunit 10 of cytochrome b-c1 oxidase (complex III), but not of subunit 1 of cytochrome C oxidase (complex IV) or superoxide dismutase 1 (SOD1) were significantly lower in ADEs and NDEs of FPs than Cs. This dysregulated pattern of electron transport proteins is associated with increased generation of reactive oxygen species. ADE glial fibrillary acidic protein levels were significantly higher in FPs than Cs, indicating a higher percentage of inflammatory astrocytes in FPs. ADE levels of C3b opsonin were significantly higher and those of C5b-9 attack complex was marginally higher in FPs than Cs. A significantly lower ADE level of the C3 convertase inhibitor CD55 may explain the higher levels of C3 convertase-generated C3b. ADE levels of the neuroprotective protein leukemia inhibitory factor (LIF) were significantly lower in FPs than Cs, whereas levels of IL-6 were no different. Plasma neural exosome levels of electron transport and complement proteins may be useful in predicting FP and guiding therapy. SOD mimetics, C3 convertase inhibitors and LIF receptor agonists also may have therapeutic benefits in FP.