Rashmi K. Ambasta

Cross-functional E3 ligases Parkin and C-terminus Hsp70-interacting protein in neurodegenerative disorders

J. Neurochem. (2012) 120, 350–370.

Rotenone-induced parkinsonism elicits behavioral impairments and differential expression of parkin, heat shock proteins and caspases in the rat.

Rotenone is a pesticide that inhibits mitochondrial complex I activity, thus creating an environment of oxidative stress in the cell. Many studies have employed rotenone to generate an experimental animal model of Parkinsons disease (PD) that mimics and elicits PD-like symptoms, such as motor and cognitive decline. Cytoprotective proteins including parkin and heat shock proteins (HSPs) play major roles in slowing PD progression. Moreover, evidence suggests that mitochondrial dysfunction and oxidative stress-dependent apoptotic pathways contribute to dopaminergic neuron degeneration in PD. Here, rats were chronically exposed to rotenone to confirm that it causes a debilitating phenotype and various behavioral defects. We also performed histopathological examinations of nigrostriatal, cortical and cerebellar regions of rotenone-treated brain to elucidate a plausible neurodegenerative mechanism. The results of silver, tyrosine hydroxylase (TH), parkin, ubiquitin and caspase staining of brain tissue sections further validated our findings. The stress response is known to trigger HSP in response to pharmacological insult. These protective proteins help maintain cellular homeostasis and may be capable of rescuing cells from death. Therefore, we assessed the levels of different HSPs in the rotenone-treated animals. Collectively, our studies indicated the following findings in the striatum and substantia nigra following chronic rotenone exposure in an experimental PD model: (i) behavioral deficit that correlated with histopathological changes and down regulation of TH signaling, (ii) decreased levels of the cytoprotective proteins parkin, DJ1 and Hsp70 and robust expression of mitochondrial chaperone Hsp60 according to Western blot, (iii) increased immunoreactivity for caspase 9, caspase 3 and ubiquitin and decreased parkin immunoreactivity.

Nanoparticle mediated targeting of VEGFR and cancer stem cells for cancer therapy

Angiogenesis is a crucial process in tumor pathogenesis as it sustains malignant cells with nutrients and oxygen. It is well known that tumor cells secrete various growth factors, including VEGF, which triggers endothelial cells to form new capillaries. Prevention of expansion of new blood vessel networks results in reduced tumor size and metastasis. Production of VEGF is driven by hypoxia via transcriptional activation of the VEGF gene by HIF-1α.Tumours are now understood to contain different types of cells, and it is the cancer stem cells that retain the ability to drive the tumours growth. They are called cancer stem cells because, like stem cells present in normal tissues of the body, they can self-renew and differentiate. These cancer stem cells are responsible for the relapse of cancer as they are found to be resistant to conventional modes of cancer therapy like chemotherapy and radiation.In this review, a novel mode of treatment of cancer is proposed, which utilizes the twin nanoparticle to target endothelial cells in the niche of cancer stem cell. The nanoparticle discussed in this review, is a twin nanoparticle of iron coated with gold, which targets VEGF positive cell in the vicinity of cancer stem cell. In the twin nanoparticle, one particle will recognize cancer stem cell, and another conjugated nanoparticle will recognize VEGF positive cells, thereby inhibiting endothelial cells in the proximity of cancer stem cell. This novel strategy will inhibit angiogenesis near cancer stem cell hence new tumour cannot grow and old tumour will be unable to metastasize.

Quail Sulf1 Function Requires Asparagine-linked Glycosylation

The heparan sulfate endosulfatases Sulf1 and Sulf2 are cell-surface enzymes that control growth factor signaling through regulation of the 6-O-sulfation states of cell-surface and matrix heparan sulfate proteoglycans. Here, we report that quail Sulf1 (QSulf1) is an asparagine-linked glycosylated protein. Domain mapping studies in combination with a protein glycosylation prediction program identified multiple asparagine-linked glycosylation sites in the enzymatic and C-terminal domains. Glycosylation inhibitor studies revealed that glycosylation of QSulf1 is essential for its enzymatic activity, membrane targeting, and secretion. Furthermore, N-glycanase cleavage of asparagine-linked sites in native QSulf1 provided direct evidence that these N-linked glycosylation sites are specifically required for QSulf1 heparin binding and its 6-O-desulfation activity, revealing that N-linked glycosylation has a key role in the control of sulfatase enzymatic function.

Tau Phosphorylation, Molecular Chaperones, and Ubiquitin E3 Ligase: Clinical Relevance in Alzheimer's Disease

Alzheimers disease (AD) is characterized by dementia, cognitive disabilities, and tauopathy. Tau is a microtubule associated protein that helps maintain the neuronal network. While phosphorylation of tau protein causes disruption of the microtubular network, dephosphorylation allows reconstitution of the microtubule network. Several kinases, e.g., MARK, MAPK, and protein kinase C, are known to hyperphosphorylate tau, leading to disruption of the microtubular network and formation of neurofibrillary tangles (NFTs), which are further glycosylated, glycated, and have lipid peroxide adducts that impair the neuronal transport system and affect memory formation and retention. Moreover, intracerebral administration of amyloid-β oligomers causes hyperphosphorylation of tau, but whether it is involved in the formation of NFTs is still unclear. Further, amyloid burden activates AMP-activated protein kinase that increases phosphorylation of tau at position Ser262/Ser356 and Ser396. Several phosphatases are present at low levels in AD brains indicating that their down regulation results in abnormal hyperphosphorylation of tau. However, evidence strengthens a possible link between tau phosphorylation and molecular chaperone mediated tau metabolism for the clearance of toxic tau accumulation and has a crucial role in tauopathy. Furthermore, accumulation of phosphorylated tau protein and the possibility of removing the toxic phosphorylated tau protein from the milieu indicates that the chaperone interacts with phosphorylated tau and promotes its degradation. For instance, Hsp90 and cdc37 regulate tau stability and phosphorylation dynamics whereas Hsp27 is able to modulate neuronal plasticity, while 14-3-3 is involved in the interaction of tau with small HSPs. Hsp70 ATPase acts as a modulator in AD therapeutics while Hsc70 rapidly engages tau after microtubular destabilization. Herein, we highlight the various causes of tauopathy and HSP-E3 ligase mediated therapeutics in AD.

Sesamol and naringenin reverse the effect of rotenone-induced PD rat model.

In the previous report (Sonia Angeline et al., 2012), we showed an altered expression of protective proteins in rotenone-induced Parkinsons disease (PD)-like rat model. This model exhibited a marked attenuation in the expression of parkin, C terminus Hsp70 interacting protein (CHIP) and PARK 7 protein (DJ1) while enhanced levels of caspases and ubiquitin were seen. Herein, we confirmed the neuroprotective role of sesamol and naringenin individually on rotenone-induced rodent model of PD. Rotenone administration was given for 11days to generate the PD model (Sonia Angeline et al., 2012). From 11th day onward individual doses of sesamol (15mg/kg) and naringenin (10mg/kg) drugs were given orally to the rotenone PD rat model for 10 consecutive days. The impact of drugs markedly improved the motor skills, body weight, expression of parkin, DJ1, tyrosine hydroxylase and CHIP compared to the group treated with rotenone alone in the striatum and substantia nigra. These results were correlated with the reduction in caspase and ubiquitin levels by immunostaining and immunoblotting. Moreover, improved morphology and survivability of neurons were seen upon sesamol and naringenin treatment in the same rat PD model. Further we confirmed the efficacy of neuroprotective biomolecule administration on muscle from the above PD model and observed the restoration in muscle morphology, elevated level of parkin, DJ1, differential expression of heat shock proteins and reduced cell death. To conclude, for the first time we are demonstrating the comprehensive role of sesamol and naringenin (rotenone-induced PD model) in neuro and myoprotection that would have great clinical significance.

Naringenin and quercetin reverse the effect of hypobaric hypoxia and elicit neuroprotective response in the murine model

Exposure to high-altitude results in hypobaric hypoxia which is considered as an acute physiological stress. This condition often leads to high-altitude illnesses such as high-altitude cerebral edema, high altitude pulmonary edema and hypoxic muscle weakness. Hypoxic injuries can be prevented by either preconditioning with cobalt chloride or treatment with drugs. The aim of current investigation was to evaluate the effect of naringenin (NGEN) and quercetin (QUR) against behavioral impairment and neuronal damage in hypoxia induced murine model. An oral administration of NGEN or QUR (10mg/kg each) was given to the animal prior to every hypoxic treatment. Behavioral changes were evaluated along with the hypoxia exposure for all the groups. After hypoxia exposure and drug administration, the mice were euthanized; brains were harvested and stored for further analysis. Expressions of hypoxia induced proteins were ensured by Western blotting. Our results demonstrate expression of hypoxia inducible factor 1α (HIF1α), vascular endothelial growth factor (VEGF), active caspase 3 and ubiquitin levels were significantly reduced upon drug treatment. However, expressions of chaperones (Hsp70, Hsp90 and C-terminus Hsp70 interacting protein) were moderately changed. We established our findings based on behavioral test, hematoxylin and eosin as well as amino-cupric silver stainings. In addition, the protective nature of these drugs was corroborated with immunoblot and immunofluorescence results, where we confirmed the down regulation of caspase 3 and ubiquitinated proteins. To conclude, treatment with NGEN and QUR alone substantially ameliorated hypoxia induced brain dysfunction and acts like a neuroprotectant.

Combinatorial Antitumor Effect of Naringenin and Curcumin Elicit Angioinhibitory Activities In Vivo

Curcumin has long been used as an antioxidative, antiinflammatory, and modulator of pathological angiogenesis, whereas naringenin is a well-known immunomodulator. In this report, we investigated the effect of curcumin and naringenin on the growth of Ehrlich ascites carcinoma tumor model. To achieve this, Swiss albino mice were implanted intraperitoneally with 1 × 106 Ehrlich ascites carcinoma cells followed by the administration of oral doses of naringenin and curcumin either individually (50 mg/kg body weight) or in combination (20 mg/kg body weight each). A marked reduction has been seen in the total number of cells (80%) and accumulation of ascetic fluid (55%) when these drugs were administered together. These drugs proved to be an effective angio-inhibitory compound and confirmed by different in vivo assay systems, viz. peritoneal/skin angiogenesis and chorioallantoic membrane assay. Antiangiogenic and antiproliferative effect of these compounds alone or in combination was further corroborated with immunoblot results where we confirmed the downregulation of vascular endothelial growth factor, Hif1α, heat shock protein 90, and p-Akt. Furthermore, treatment with naringenin and curcumin alone or in combination substantially improved hepatocellular architecture and no noticeable neoplastic lesions or cellular alteration were reported. These outcomes put forward a plausible clinical application of these diet-derived compounds, as both angioinhibitory and antitumor in association with conventional therapy.

Linking mitochondrial dysfunction, metabolic syndrome and stress signaling in Neurodegeneration.

Mounting evidence suggests a link between metabolic syndrome (MetS) such as diabetes, obesity, non-alcoholic fatty liver disease in the progression of Alzheimers disease (AD), Parkinsons disease (PD) and other neurodegenerative diseases (NDDs). For instance, accumulated Aβ oligomer is enhancing neuronal Ca2+ release and neural NO where increased NO level in the brain through post translational modification is modulating the level of insulin production. It has been further confirmed that irrespective of origin; brain insulin resistance triggers a cascade of the neurodegeneration phenomenon which can be aggravated by free reactive oxygen species burden, ER stress, metabolic dysfunction, neuorinflammation, reduced cell survival and altered lipid metabolism. Moreover, several studies confirmed that MetS and diabetic sharing common mechanisms in the progression of AD and NDDs where mitochondrial dynamics playing a critical role. Any mutation in mitochondrial DNA, exposure of environmental toxin, high-calorie intake, homeostasis imbalance, glucolipotoxicity is causative factors for mitochondrial dysfunction. These cumulative pleiotropic burdens in mitochondria leads to insulin resistance, increased ROS production; enhanced stress-related enzymes that is directly linked MetS and diabetes in neurodegeneration. Since, the linkup mechanism between mitochondrial dysfunction and disease phenomenon of both MetS and NDDs is quite intriguing, therefore, it is pertinent for the researchers to identify and implement the therapeutic interventions for targeting MetS and NDDs. Herein, we elucidated the pertinent role of MetS induced mitochondrial dysfunction in neurons and their consequences in NDDs. Further, therapeutic potential of well-known biomolecules and chaperones to target altered mitochondria has been comprehensively documented. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Ion Channels in Neurological Disorders

The convergent endeavors of the neuroscientist to establish a link between clinical neurology, genetics, loss of function of an important protein, and channelopathies behind neurological disorders are quite intriguing. Growing evidence reveals the impact of ion channels dysfunctioning in neurodegenerative disorders (NDDs). Many neurological/neuromuscular disorders, viz, Alzheimers disease, Parkinsons disease, Huntingtons disease, multiple sclerosis, amyotrophic lateral sclerosis, and age-related disorders are caused due to altered function or mutation in ion channels. To maintain cell homeostasis, ion channels are playing a crucial role which is a large transmembrane protein. Further, these channels are important as it determines the membrane potential and playing critically in the secretion of neurotransmitter. Behind NDDs, losses of pathological proteins and defective ion channels have been reported and are found to aggravate the disease symptoms. Moreover, ion channel dysfunctions are eliciting a range of symptoms, including memory loss, movement disabilities, neuromuscular sprains, and strokes. Since the possible mechanistic role played by aberrant ion channels, their receptor and associated factors in neurodegeneration remained elusive; therefore, it is a challenging task for the neuroscientist to implement the therapeutics for targeting NDDs. This chapter reviews the potential role of the ion channels in membrane physiology and brain homeostasis, where ion channels and their associated factors have been characterized with their functional consequences in neurological diseases. Moreover, mechanistic role of perturbed ion channels has been identified in various NDDs, and finally, ion channel modulators have been investigated for their therapeutic intervention in treating common NDDs.