Post-treatment with apocynin at a lower dose regulates the UPR branch of eIF2α and XBP-1 pathways after stroke.

Stroke leads to disturbance in the physiology of the ER (Endoplasmic Reticulum) that triggers UPR (Unfolded Protein Response) pathways aimed to compensate neuronal cell damage. However, sustained UPR causes stressful conditions in the ER lumen forming abnormal protein aggregates. Stroke-induced oxidative stress also amalgamates with UPR to safeguard and ensure the proper functioning of brain cells. Thus we tested the effect of apocynin (a potent antioxidant) post-treatment in experimental stroke on the outcome of ER stress and UPR branch pathways. We administered a low dose of apocynin at 1 mg/kg (intraperitoneal) to adult Sprague-Dawley rats subjected to Middle Cerebral Artery Occlusion (MCAO) for two-time points. The first dose immediately after re-establishing the blood flow and another at 6 h of reperfusion. Apocynin post-treatment significantly reduced ROS (Reactive Oxygen Species) generation at an early reperfusion time point of 4 h. It preserved neuronal morphology, dendritic spine density, reduced protein aggregation, and brain damage after 24 h of reperfusion. Apocynin post-treatment regulates the two UPR branch pathways in our experimental paradigm. 1) Down-regulation of eIF2α (Eukaryotic Initiation Factor 2α) phosphorylation, and CHOP (C/EBP homologous protein) 2) by reducing the XBP-1 (X-Box binding Protein-1) mRNA splicing downstream to PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α (Inositol Requiring Enzyme 1alpha) UPR pathways, respectively. Bioinformatics prediction showed that apocynin has binding sites for PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α proteins. The amino acid residues interacting with apocynin were Cys891 and Gln889 (for PERK), and the amino acids Ser726, Arg722, and Ala719 (for IRE1α) lying within their activation loop. Overall, these studies indicate that apocynin post-treatment might regulate ER stress/UPR pathways and minimize stroke brain damage, thus having implications for developing newer strategies for stroke treatment.

Optimisation of chloroquine phosphate loaded nanostructured lipid carriers using Box-Behnken design and its antimalarial efficacy.

Chloroquine was once the most widely used antimalarial for nearly eight decades for its safety, efficiency, stability, low cost and finally for its less toxic nature. But its use and efficacy got slowly decreased with the increase of chloroquine resistant strains of Plasmodium species throughout the world. Lipid based nanodrug delivery systems have been very popular in the recent times as they are very less toxic, have drug targeting capabilities and also reduces the dosing frequency by increasing efficacy of the drug. In the present research work, response surface methodology was employed to optimise chloroquine phosphate (CQ) loaded nanostructured lipid carriers (NLCs) using a modified double emulsion technique. The optimised CQ loaded NLC showed a particle size of 66.50 ± 1.21 nm, PDI of 0.210 ± 0.016, ZP of +38.4 ± 1.44 and EE of 78.2 ± 1.2%, respectively. The in vitro and in vivo antimalarial studies of CQ loaded NLCs showed an enhanced antimalarial efficacy of the nanoformulation with a better suppression of parasitemia and with an increased efficacy of more than 23% in comparison to pure drug. This study demonstrated that by loading a drug into an NLCs system can help in overcoming the problems associated with the present antimalarials available.

Design, characterization and antimalarial efficacy of PEGylated galactosylated nano lipid carriers of primaquine phosphate.

This study was aimed to design and optimize primaquine phosphate (PQ) loaded nanostructured lipid carriers (NLCs) using response surface methodology. The optimized NLCs were evaluated for various physical and morphological characterizations. The in vitro studies for drug release showed that PQ loaded NLCs had a sustained release up to 72 h and the stability studies confirmed that the PQ-NLCs were stable for 90 d at 4 °C and 25 °C. In vitro erythrocyte toxicity revealed that PQ-NLCs were less toxic than the pure drug. In vitro parasite growth inhibition assay showed an IC50 value of 71.11 ± 6.47 ng/ml for the 3D7 Plasmodium falciparum (CQ sensitive) strain and 263.86 ± 5.68 ng/ml for RKL9 P. falciparum (CQ resistant) strain for the PQ-NLCs. Enhanced parasitaemia suppression of 99.46% at 2 mg/kg/d, a better suppression of parasitaemia of about 28% more than pure drug and a higher survivality rate of 66.66% even after the 35th day was observed for the PQ loaded NLCs. Also from the comparative fluorescent imaging study, it was clearly observed that accumulation of PQ-NLCs in the liver was more that of the pure drug. These results clearly indicated that the limitations of antimalarial drug PQ can be overcomed by loading it into the NLCs.

Dysregulation of LIMK-1/cofilin-1 pathway: A possible basis for alteration of neuronal morphology in experimental cerebral malaria.

OBJECTIVE: Loss of cognition even after survival is the salient feature of cerebral malaria (CM). Currently, the fate of neuronal morphology is not studied at the ultrastructural level in CM. Recent studies suggest that maintenance of neuronal morphology and dendritic spine density (actin dynamics in particular) are essential for proper cognitive function. LIMK-1/cofilin-1 signaling pathway is known to be involved in the maintenance of actin dynamics through regulation of cofilin-1, and in executing learning and memory functions. METHODS: Using an experimental mouse model, we analyzed the behavioral parameters of asymptomatic mice with CM by performing a rapid murine coma and behavior scale experiment. We performed Golgi-Cox staining to assess neuronal morphology, dendritic spine density, and arborization in brain cortex subjected to Plasmodium berghei ANKA infection compared to asymptomatic, anemic, and control groups. We studied the neural gene expression pattern of LIMK-1, cofilin-1, and ß-actin in all the experimental groups by semiquantitative and quantitative polymerase chain reaction followed by immunoblotting and immunofluorescence. RESULTS: We observed significant loss of dendritic spine density, abnormal spine morphology, reduced dendritic arborization, and extensive dendritic varicosities in the cortical neurons of CM-infected brain. Furthermore, these observations correlated with diminished protein levels of LIMK-1, cofilin-1, phospho-cofilin-1, and ß-actin in the whole brain lysates as well as formation of actin-cofilin rods in the brain sections of symptomatic mice with CM. INTERPRETATION: Overall, our findings suggest that the altered neuronal morphology and dysregulation of LIMK-1/cofilin-1 pathway could affect the cognitive outcome after experimental CM. Therefore, this study could help to establish newer therapeutic strategies addressing long-term cognitive impairment after CM. Ann Neurol 2017;82:429-443.

Poly(ADP-ribose)polymerase-1 hyperactivation in neurodegenerative diseases: The death knell tolls for neurons.

Neurodegeneration is a salient feature of chronic refractory brain disorders like Alzheimer's, Parkinson's, Huntington's, amyotropic lateral sclerosis and acute conditions like cerebral ischemia/reperfusion etc. The pathological protein aggregates, mitochondrial mutations or ischemic insults typifying these disease conditions collude with and intensify existing oxidative stress and attendant mitochondrial dysfunction. Interlocking these mechanisms is poly(ADP-ribose) polymerase (PARP-1) hyperactivation that invokes a distinct form of neuronal cell death viz., 'parthanatos'. PARP-1, a typical 'moonlighting protein' by virtue of its ability to poly(ADP-ribosyl)ate a plethora of cellular proteins exerts diverse functions that impinge significantly on cellular processes. In addition, its interactions with various nuclear proteins like transcription factors and chromatin modifiers elicit varied transcriptional outcomes that wield pathological cellular responses. Further, emerging leitmotifs like mitochondrial and nucleolar PARPs and the novel aspects of gene expression regulation by PARP-1 and poly(ADP-ribosyl)ation can provide a holistic view of PARP-1's influence on cell vitality. In this review, we discuss the pathological underpinnings of PARP-1, with a special emphasis on mitochondrial dysfunction and cell death subroutines, in the realm of neurodegeneration. This would provide a deeper insight into the functions of PARP-1 in neurodegenerative conditions that would enable the development of more effective therapeutic strategies.