Venugopalan D. Nair

p53 Mediates Nontranscriptional Cell Death in Dopaminergic Cells in Response to Proteasome Inhibition

Proteasome dysfunction has been demonstrated in Parkinson disease (PD), and proteasome inhibitors have been shown to induce degeneration of dopaminergic neurons in vitro and in vivo. The mechanism whereby proteasome dysfunction leads to dopaminergic cell death, however, is unknown. In this study, we show that proteasome inhibition in both PC12 cells and dopaminergic neurons derived from embryonic stem cells is associated with mitochondrial membrane permeabilization, activation of caspase-3, and nuclear changes consistent with apoptosis. Prior to the emergence of apoptotic features, we found that proteasome inhibition induced increased levels of phosphorylated p53. Inhibition of p53 by pifithrin-α or by RNA interference prevented mitochondrial membrane permeabilization and cytotoxicity. There was no increase in p53 mRNA in proteasome-inhibited cells, suggesting that p53 was increased in a transcription-independent manner. Further, there was no increase in Puma or Bax mRNA and p53 co-immunoprecipitated with Bcl-xL and Mdm2. These findings suggest that p53 mediates cell death by way of a direct mitochondrial effect in this model. We also observed increased levels of phosphorylated p53 in dopamine neurons of the substantia nigra pars compacta of mice following systemic administration of a proteasome inhibitor. These changes preceded degeneration of dopaminergic neurons. Increased phosphorylated p53 was also demonstrated in the substantia nigra pars compacta of post-mortem PD brains. These results suggest that abnormalities in p53 signaling play a role in dopaminergic cell death induced by proteasome inhibition and may be relevant to neurodegeneration in PD.

Differential Modulation of Akt/Glycogen Synthase Kinase-3β Pathway Regulates Apoptotic and Cytoprotective Signaling Responses

We have previously reported that specific dopamine agonists mediate protection against apoptosis induced by oxidative stress by activating the D2 receptor-coupled phosphoinositide 3-kinase (PI-3K)/Akt pathway. In the present study we examined the downstream effectors of PI-3K/Akt signaling and their role in cell death after oxidative stress and protection provided by ropinirole, a D2 receptor agonist in PC12 cells and primary cultures of dopamine neurons. Ropinirole treatment was associated with rapid translocation and phosphorylation of the PI-3K substrate Akt and phosphorylation of Akt substrates. One of these Akt downstream substrates was identified as the pro-apoptotic factor glycogen synthase kinase-3β (GSK-3β). Ropinirole-induced protection was associated with phosphorylation of GSK-3β (inactivation). In contrast, inhibition of PI-3K blocked the phosphorylation of Akt and GSK-3β (activation) and prevented the protection mediated by ropinirole. Suppression of Akt with specific short hairpin RNA in normal PC12 cells caused cell death, which was associated with reduced phosphorylation of GSK-3β and reduced levels of β-catenin, a transcriptional activator that is regulated by GSK-3β. Knock-out of GSK-3β expression with a short hairpin RNA alone was itself sufficient to cause cell death. We further demonstrated that oxidative stress induced by hydrogen peroxide (H2O2) dephosphorylates Akt and GSK-3β, increases GSK-3β activity, and promotes an interaction with β-catenin and its degradation. Inhibition of GSK-3β activity by inhibitor VIII protects cells from H2O2 similar to ropinirole. These results indicate that GSK-3β downstream of Akt plays a critical role in cell death and survival in these models.

A genomic and clinical prognostic index for hepatitis C-related early-stage cirrhosis that predicts clinical deterioration

Objective The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. Design We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100 000/mm3), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). Results In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child–Turcotte–Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). Conclusions A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.

Interaction of NMDA and dopamine D2L receptors in human neuroblastoma SH-SY5Y cells.

Abstract: To understand the mechanism of interaction of the dopamine D2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH‐SY5Y cells with the human dopamine D2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK‐801 and (±)‐3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP) on human neuroblastoma SH‐SY5Y cells expressing human dopamine D2L receptors resulted in a significant increase in the density of D2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by ∼50% by the blockade of NMDA with MK‐801. These results suggest a possible interaction of NMDA and dopamine D2L receptors in neuroblastoma SH‐SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.

Ontogenic development of dopamine D4 receptor in rat brain

The postnatal development of rat brain dopamine D4 receptor gene expression was studied in animals 1 day to 1 year old, using the polymerase chain reaction technique. The level of expression of the D4 mRNA was appreciable at birth (day 1), increased to maximum at day 3, and showed declines at day 28. D4 mRNA expression remained unchanged at the ages of 6 months to 1 year. The mRNA expression at day 1 is about 50% of that observed on day 3, and declines approximately 50% by day 28. In contrast, the dopamine D2 mRNA expression was maximum at day 28, which is consistent with the reported studies.

Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells

ABSTRACT The histone demethylase LSD1, a component of the CoREST (corepressor for element 1-silencing transcription factor) corepressor complex, plays an important role in the downregulation of gene expression during development. However, the activities of LSD1 in mediating short-time-scale gene expression changes have not been well understood. To reveal the mechanisms underlying these two distinct functions of LSD1, we performed genome-wide mapping and cellular localization studies of LSD1 and its dimethylated histone 3 lysine 4 (substrate H3K4me2) in mouse embryonic stem cells (ES cells). Our results showed an extensive overlap between the LSD1 and H3K4me2 genomic regions and a correlation between the genomic levels of LSD1/H3K4me2 and gene expression, including many highly expressed ES cell genes. LSD1 is recruited to the chromatin of cells in the G1/S/G2 phases and is displaced from the chromatin of M-phase cells, suggesting that LSD1 or H3K4me2 alternatively occupies LSD1 genomic regions during cell cycle progression. LSD1 knockdown by RNA interference or its displacement from the chromatin by antineoplastic agents caused an increase in the levels of a subset of LSD1 target genes. Taken together, these results suggest that cell cycle-dependent association and dissociation of LSD1 with chromatin mediates short-time-scale gene expression changes during embryonic stem cell cycle progression.

Clinicopathological indices to predict hepatocellular carcinoma molecular classification.

Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti‐HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification.

Molecular cloning, localization and characterization of a 40-kDa catecholamine-regulated protein

We have previously described catecholamine‐regulated proteins of molecular masses 47, 40 and 26 kDa (CRP47/40/26). In mammals, these proteins are detected only in brain and have been implicated as playing a role in dopaminergic neurotransmission. In this report, we have cloned the cDNA encoding CRP40 from bovine brain. Analysis of the predicted amino acid sequence revealed that the CRP40 product contains an hsp70 motif and shares homology with heat‐shock protein hsp70. Immunolocalization studies using mAbs to dopamine show that it colocalizes with CRP40 in the vesicles of dopaminergic neuroblastoma SH‐SY5Y cells. The constitutive expression of CRP40 was increased by exposure to heat shock similar to inducible heat‐shock protein hsp70 in SH‐SY5Y cells. Dopamine significantly modulated the levels of CRP40, whereas, the expression of hsp70 remained unchanged upon dopamine treatment of these cells. Moreover, CRP40 is able to prevent the thermal aggregation of luciferase in vitro, similar to hsp70, suggesting that CRP40 encodes a dopamine‐inducible protein with properties similar to heat‐shock proteins. The immunofluorescence analyses show that in SH‐SY5Y cells, CRP40 translocates to the nucleus during dopamine‐induced apoptosis. These results suggest that CRP40 could play a protective role against the harmful effects of catecholamine metabolites.

Modulation of brain catecholamine absorbing proteins by dopaminergic agents.

Catecholamine absorbing proteins (CATNAPs) are localized in the brain and thus far have no known biochemical and pharmacological characteristics consistent with other receptor proteins or metabolic enzymes in the central nervous system. The oxidative metabolism of catecholamines in the brain, especially the catabolism of dopamine and its conjugation with metabolic brain proteins, results in the production of highly toxic free radicals. Since such processes are implicated in the pathophysiology of various neurodegenerative diseases, including parkinsonism, and since CATNAPs bind catecholamines with high affinity, there is a need to further investigate if these novel proteins could play a protective role against these harmful catecholamine metabolites. In this study, we demonstrate the purification, pharmacological characterization and modulation of CATNAPs, as the first steps necessary to elucidate the function of these proteins in the brain. First, CATNAPs were identified from tissues using [3H]N-n-propylnorapomorphine (a specific dopamine receptor agonist) and [125I]6-hydroxy-5-iodo[N(N-2,4-dinitro-phenyl)- aminopropyl]1,2,3,4-tetrahydronaphthalene ([125I]DATN; a highly specific ligand synthesized in our laboratory). Three proteins, with molecular masses of 47, 40 and 26 kDa, were identified and purified, which allowed for the subsequent production of antibodies against each of these CATNAPs. The effects of in vivo chronic administration of several dopaminergic agents on CATNAPs were also examined by Western immunoblotting. L-3,4-Dihydroxyphenylalanine (L-DOPA) treatment in rats resulted in the increase of all of the three proteins, as compared to controls. Treatment in rats with the dopamine depleting agent, reserpine, produced a significant decrease in all of the three CATNAPs. In addition, the effects of direct administration of apomorphine, dopamine, epinephrine, isopropylnorepinephrine, norepinephrine, N-n-propylnorapomorphine and 6-hydroxydopamine on CATNAP levels in rats were examined. Interestingly, we observed an increase (as compared to control) of the 47, 40 and 26 kDa proteins in animals treated with dopamine, norepinephrine, N-n-propylnorapomorphine and apomorphine. In contrast, animals treated with 6-hydroxydopamine showed significant decreases in the levels of all three proteins. It is evident that as the concentration of catecholamines increases, there is a corresponding increase in the levels of CATNAPs in the brain. These results clearly demonstrate the pharmacological modulation of CATNAPs by dopaminergic agents and suggest their possible role in the cytoprotection against damage caused by free radicals generated by oxidative stress.

Modulation of agonist stimulated adenylyl cyclase and GTPase activity by l-pro-l-leu-glycinamide and its peptidomimetic analogue in rat striatal membranes

L-prolyl-L-leucyl-glycinamide (PLG), also known as melanocyte-stimulating hormone release inhibiting factor (MIF-1), is an endogenous brain tripeptide. Previous studies have shown that PLG, and its peptidomimetic analogues, render dopamine D2 receptors more responsive to agonists by maintaining the high-affinity binding state of the receptors. In the present study, we examined the effect PLG and its analogue 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetam ide (PAOPA) on dopamine-stimulated adenylyl cyclase and NPA-stimulated GTPase activity in rat striatal membranes. Dopamine-stimulated adenylyl cyclase activity was inhibited by both PLG and PAOPA in a dose-dependent manner, whereas R(-)-propylnorapomorphine (NPA)-stimulated low Km GTPase activity was significantly increased by 1 microM PLG or 1 nM PAOPA. These results suggest that PLG and PAOPA maintain the high affinity state of the D2 receptor by increasing GTP hydrolysis through stimulation of agonist-induced GTPase activity.