Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection

Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box–binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity

Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in α2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBCVL). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic α- and β-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrinVL) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to α- and β-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrinN) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrinN. Although the presence of both N- and O-glycosylations was found both in spectrinN and spectrinVL, enhanced sialylation was predominantly induced in spectrinVL. Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of α- and β-spectrinVL confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrinVL showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrinN suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBCVL. The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrinVL as evidenced by the presence of an additional 60 kDa fragment, absent in spectrinN which possibly affects the biology of RBCVL linked to both severe distortion of erythrocyte development and impairment of erythrocyte membrane integrity and may provide an explanation for their sensitivity to hemolysis and anemia in VL patients

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (104–105M−1) and number of binding sites (106–107sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10μg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two disease