Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)‐binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. ...In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation‐contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2), l‐type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life‐threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild‐type CaM protein in the presence of Ca2+. Finally, Ca2+‐binding studies indicates that all CaM mutations display reduced CaM Ca2+‐binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.
Calmodulin (CaM) is a universal calcium (Ca2+)‐binding messenger that regulates many vital cellular events. In cardiac muscle, CaM associates with ryanodine receptor 2 (RyR2) and regulates ...excitation–contraction coupling. Mutations in human genes CALM1, CALM2, and CALM3 have been associated with life‐threatening heart disorders, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia. A novel de novo LQTS‐associated missense CaM mutation (E105A) was recently identified in a 6‐year‐old boy, who experienced an aborted first episode of cardiac arrest. Herein, we report the first molecular characterization of the CaM E105A mutation. Expression of the CaM E105A mutant in zebrafish embryos resulted in cardiac arrhythmia and increased heart rate, suggestive of ventricular tachycardia. In vitro biophysical and biochemical analysis revealed that E105A confers a deleterious effect on protein stability and a reduced Ca2+‐binding affinity due to loss of cooperativity. Finally, the CaM E105A mutation resulted in reduced CaM–RyR2 interaction and defective modulation of ryanodine binding. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function by a complex mechanism involving alterations in both CaM–Ca2+ and CaM–RyR2 interactions.
A novel de novo long QT syndrome–associated missense calmodulin (CaM) mutation E105A was recently identified in a 6‐year‐old boy, who experienced an aborted first episode of cardiac arrest. Our study reports the first in vivo and in vitro molecular characterization of the CaM E105A mutation. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function in zebrafish by a complex mechanism involving alterations in both CaM–Ca2+ and CaM–ryanodine receptor 2 interactions.
Despite decades of intensive research, to date, there is no accepted diagnosis for Parkinson's disease (PD) based on biochemical analysis of blood or CSF. However, neurodegeneration in the brains of ...PD patients begins several years before the manifestation of the clinical symptoms, pointing to serious flaw/limitations in this approach.
To explore the potential use of alpha-synuclein (α-syn) species as candidate biomarkers for PD, we generated specific antibodies directed against wide array of α-syn species, namely total-, oligomeric- and phosphorylated-Ser129-α-syn (t-, o- and p-S129-α-syn). Next we sought to employ our antibodies to develop highly specific ELISA assays to quantify α-syn species in biological samples. Finally we verified the usefulness of our assays in CSF samples from 46 PD patients and 48 age-matched healthy controls. We also assessed the discriminating power of combining multiple CSF α-syn species with classical Alzheimer's disease biomarkers. The combination of CSF o-/t-α-syn, p-S129-α-syn and p-tau provided the best fitting predictive model for discriminating PD patients from controls. Moreover, CSF o-α-syn levels correlated significantly with the severity of PD motor symptoms (r = -0.37).
Our new ELISA assays can serve as research tools to address the unmet need for reliable CSF biomarkers for PD and related disorders.
Abstract Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of ...compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no β-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.
Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca
) oscillations that induce egg activation and early embryonic ...development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca
release via the inositol 1,4,5-trisphosphate (InsP
) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca
oscillations within mammalian eggs. Moreover, studies describing PLCζ "knockout" mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca
oscillations and egg activation in mammals, while also discussing the nature of a potential "alternative" sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.
Neuropathic pain is considered to be pathological in nature and has been shown to involve, at least partially, dysregulated inflammatory processes. It is a severe chronic disease that can develop ...following lesions to the central nervous system or to peripheral nerves. The peripheral nerve damage can be caused by either diseases such as diabetes, or by trauma. A common underlying mechanism of neuropathic pain is the presence of inflammation at the site of the damaged or affected nerve(s). This inflammatory response, especially when unresolved, initiates and maintains a cascade of events resulting in the activation of innate immune cells at the site of tissue injury. The release of inflammatory mediators such as cytokines, neurotrophic factors, and chemokines initiates local actions and can result in a more generalized immune response. The resultant neuroinflammatory environment can cause activation of glial cells, which can release, in an uncontrolled manner, more of these mediators and exasperate the situation, thus having a prominent role in nociception. The neuropathic pain pathophysiology is complex and includes peripheral and central neuronal alterations as well as neuro-immune interactions, which become more prominent during inflammatory reactions. This report focuses on how targeting inflammatory mediators may result in novel therapeutic approaches to neuropathic pain management.
Total CSF α-synuclein (t-α-syn), phosphorylated α-syn (pS129-α-syn) and α-syn oligomers (o-α-syn) have been studied as candidate biomarkers for synucleinopathies, with suboptimal specificity and ...sensitivity in the differentiation from healthy controls. Studies of α-syn species in patients with other underlying pathologies are lacking. The aim of this study was to investigate possible alterations in CSF α-syn species in a cohort of patients with diverse underlying pathologies. A total of 135 patients were included, comprising Parkinson's disease (PD;
= 13), multiple system atrophy (MSA;
= 9), progressive supranuclear palsy (PSP;
= 13), corticobasal degeneration (CBD;
= 9), Alzheimer's disease (AD;
= 51), frontotemporal degeneration (FTD;
= 26) and vascular dementia patients (VD;
= 14). PD patients exhibited higher pS129-α-syn/α-syn ratios compared to FTD (
= 0.045), after exclusion of samples with CSF blood contamination. When comparing movement disorders (i.e., MSA vs. PD vs. PSP vs. CBD), MSA patients had lower
syn levels compared to CBD (
= 0.024). Patients with a synucleinopathy (PD and MSA) exhibited lower t-
syn levels (
= 0.002; cut-off value: ≤865 pg/mL; sensitivity: 95%, specificity: 69%) and higher
/t-
syn ratios (
= 0.020; cut-off value: ≥0.122; sensitivity: 71%, specificity: 77%) compared to patients with tauopathies (PSP and CBD). There are no significant α-syn species alterations in non-synucleinopathies.
: The thymic peptide thymulin is known for its immunomodulatory role. However, several recent reports have indicated that thymulin is capable of interacting directly and/or indirectly with the ...nervous system. One of the first lines of evidence of this interaction was obtained in a series of experiments showing the hyperalgesic actions of this peptide. We demonstrated that, at low doses (ng), local (intraplantar) or systemic (intraperitoneal) injections of thymulin resulted in hyperalgesia with an increase in proinflammatory mediators, and that this peptide could act directly on the afferent nerve terminals through prostaglandin‐E2 (PGE2)‐dependent mechanisms, thus forming a neuroimmune loop involving capsaicin‐sensitive primary afferent fibers. In further experiments, systemic injections of relatively high doses (1–25 μg) of thymulin or of an analogue peptide (PAT) deprived of hyperalgesic effect, have been shown to reduce the inflammatory pain and the upregulated levels of cytokines induced by endotoxin (ET) injection. In addition, PAT treatment appeared to alleviate the sickness behavior (motor behavior and fever) induced by systemic inflammation. These effects could be attributed, at least partly, to the downregulation of proinflammatory mediators. Furthermore, when compared with the effects of other anti‐inflammatory drugs, PAT exerted equal or even stronger analgesic effects, and at much lower concentrations. Subsequent experiments were designed to examine the effects of intracerebroventricular (i.c.v.) injections of thymulin on cerebral inflammation induced by i.c.v. injection of ET. Pretreatment with thymulin reduced, in a dose‐dependant manner, the ET‐induced hyperalgesia, and exerted differential effects on the upregulated levels of cytokines in different areas of the brain, suggesting a neuroprotective role for thymulin in the central nervous system (CNS). Preliminary results demonstrate that thymulin inhibits in the hippocampus the ET‐induced nuclear activation of NF‐κB, the transcription factor required for the expression of proinflammatory cytokines genes. Although the mechanism of action of these molecules is not totally elucidated, our results indicate a possible therapeutic use of thymulin or PAT as analgesic and anti‐inflammatory drugs.
•The prevalence of homocystinuria in Qatar is 1:1800, mainly due to a founder missense mutation p.R336C.•The cystathionine beta-synthase (CBS) R336C mutant was bacterially expressed, purified and its ...molecular properties were compared to CBS wild type (WT) recombinant protein.•Our data revealed that p.R336C mutation results in a dramatic reduction (∼86%) of CBS enzymatic activity.•Circular Dichroism experiments suggested that the p.R336C mutation does not significantly alter the secondary structure of the CBS protein.•CD spectra also revealed distinct differences in the thermal unfolding mechanisms of CBS WT and R336C mutant protein species.•Chemical denaturation experiments indicated that the WT CBS protein is thermodynamically more stable than the R336C mutant, suggesting a destabilizing effect of the p.R336C mutation.•This study provides mechanistic insight into the pathogenicity of the p.R336C mutation that leads to a severe homocystinuria phenotype.
PDF
