Cell cycle activation in p21 dependent pathway: An alternative mechanism of organophosphate induced dopaminergic neurodegeneration.

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G0/G1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells.

Dichlorvos-induced cell cycle arrest and DNA damage repair activation in primary rat microglial cells.

Dichlorvos, an organophosphate (OP), is known to cause oxidative stress in the central nervous system (CNS). Previously we have shown that dichlorvos treatment promoted the levels of proinflammatory molecules and ultimately induced apoptotic cell death in primary microglial cells. Here we studied the effect of dichlorvos on crucial cell cycle regulatory proteins and the DNA damage sensor ataxia-telangiectasia mutated (ATM). We found a significant increase in p53 and its downstream target, p21, levels in dichlorvos-treated microglial cells compared with control cells. Moreover, dichlorvos exposure promoted the levels of different cell cycle regulatory proteins. These results along with flow cytometry results suggested that primary microglial cells were arrested at G1 and G2/M phase after dichlorvos exposure. We have shown in a previous study that dichlorvos can induce DNA damage in microglia; here we found that microglial cells also tried to repair this damage by inducing a DNA repair enzyme, i.e., ATM. We observed a significant increase in the levels of ATM after dichlorvos treatment compared with control.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases.

Dichlorvos exposure results in activation induced apoptotic cell death in primary rat microglia.

Dichlorvos [2,2-dichlorovinyl dimethyl phosphate] is one of the most common in-use organophosphate (OP) in developing nations. Previous studies from our lab have shown chronic Dichlorvos exposure leads to neuronal cell death in rats. However, the extent of damage caused by Dichlorvos to other cells of the central nervous system (CNS) is still not clear. Microglial cells are the primary threat sensors of CNS which become activated in many pathological conditions. Activation of microglial cells results in reactive microgliosis, manifested by increased cellular damage in the affected regions. Using rat primary microglial cultures, here we show that Dichlorvos exposure can activate and induce apoptotic cell death in microglia. We observed significant up-regulation of pro-inflammatory molecules like nitric oxide, TNF-α, and IL-1ß when microglia were treated with Dichlorvos (10 µM). Significant up-regulation of CD11b, microglial specific activation marker, was also observed after 24 h of Dichlorvos treatment. The activated microglial cells eventually undergo cell death after 48 h of Dichlorvos treatment. The DNA fragmentation pattern of Dichlorvos treated microglia along with increased expression of Bax in mitochondria, cytochrome c release from mitochondria, and caspase-3 activation led us to assume that microglia were undergoing apoptosis. Thus, the present study showed that Dichlorvos can induce microglial activation and ultimately apoptotic cell death. These findings gave new perspective to the current knowledge of Dichlorvos (OPs) mediated CNS damage and presents microglial activation as a potential therapeutic target for preventing the OP induced neuronal damage.

Neurobehavioral impairments, generation of oxidative stress and release of pro-apoptotic factors after chronic exposure to sulphur mustard in mouse brain.

Recent global events have focused attention on the potential threat of international and domestic chemical terrorism, as well as the possibility of chemical warfare proliferation. Sulphur mustard (SM) is one of the potent chemical warfare agents (CWA), which initiates a cascade of events that converge on the redox mechanisms common to brain injury. The present study was designed to examine the effects of chronic SM exposure on neurobehavioral impairments, mitochondrial oxidative stress in male Swiss Albino mice and its role in inducing apoptotic neuronal cell death. The animals were divided into four groups (control, low, medium and high dose) of 5 animals each. Exposure to SM was given percutaneously daily for 12 weeks. The results demonstrated impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests in a dose dependent manner. There was a significant increase in lipid peroxidation and protein carbonyl content whereas, decrease in the activity of manganese superoxide dismutase (MnSOD), glutathione reductase and glutathione peroxidase suggesting impaired antioxidant defense system. Immunoblotting of cytochrome c, Bcl-2, Bax and activation of caspase-3 suggest induction of apoptosis in a dose dependent manner. Finally, increased p53 expression suggests that it may target the mitochondrial pathway for inducing apoptosis in response to DNA damage signals. In conclusion, chronic SM exposure may have the potential to generate oxidative stress which may trigger the release of cytochrome c as well as caspase-3 activation in neurons leading to cell death by apoptosis in a dose dependent manner which may in the end be responsible for the disruption of cognitive functions in mice.

Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway.

The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits - ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases.

4-Hydroxy TEMPO attenuates dichlorvos induced microglial activation and apoptosis.

Microglial cells have been implicated in various neurodegenerative diseases. Previous studies from our lab have shown that dichlorvos (an organophosphate) could induce Parkinson's like features in rats. Recently, we have shown that dichlorvos can induce microglial activation, and if not checked in time could ultimately induce neuronal apoptosis. However, this activation does not always pose a threat to the neurons. Activated microglia also secrete various neuronal growth factors, suggesting that they have beneficial roles in CNS repair. Therefore, it is essential to control their detrimental functions selectively. Here, we tried to find out how microglial cells behave when exposed to dichlorvos in either the presence or absence of potent nitric oxide scavenger and superoxide dismutase mimetic, 4-hydroxy TEMPO (4-HT). Wistar rat pups (1 day) were used to isolate and culture primary microglial cells. We found 4-HT pretreatment successfully attenuated the dichlorvos mediated microglial activation. Moreover, 4-HT pretreatment decreased the up-regulated levels of p53 and its downstream effector, p21. The expression of various cell cycle regulators such as Chk2, CDC25a, and cyclin A remained close to their basal levels when 4-HT pretreatment was given. DNA fragmentation analysis showed significant reduction in the DNA damage of 4-HT pretreated microglia as compared to dichlorvos treated cells. In addition to this, we found 4-HT pretreatment prevented the microglial cells from undergoing apoptotic cell death even after 48 h of dichlorvos exposure. Taken together, our results showed 4-HT pretreatment could successfully ameliorate the dichlorvos induced microglial cell damage.

Attenuation of dichlorvos-induced microglial activation and neuronal apoptosis by 4-hydroxy TEMPO.

The neurotoxic consequences of acute high-level as well as chronic low-level organophosphates exposure are associated with a range of abnormalities in nerve functions. Previously, we have shown that after 24 h of dichlorvos exposure, microglia become activated and secrete pro-inflammatory molecules like nitric oxide, tumour necrosis factor-α and interleukin-1ß. Here, we extended our findings and focused on the neuronal damage caused by dichlorvos via microglial activation. For this, neurons and microglia were isolated separately from 1-day-old Wistar rat pups. Microglia were treated with dichlorvos for 24 h and supernatant was collected (dichlorvos-induced conditioned medium, DCM). However, when 4-hydroxy TEMPO (4-HT) pretreatment was given, we observed significant attenuation of dichlorvos-induced microglial activation; we also collected the supernatant of this culture (4-HT + DCM, TDCM). Next, we checked the effects of DCM on neurons and found heavy loss in viability as evident from NF-H immunostaining and MTT results, whereas dichlorvos alone-treated neurons showed comparatively less damage. However, we observed significant increase in neuronal viability when cells were treated with TDCM. Semi-quantitative PCR and western blot results revealed significant increase in p53, Bax and cytochrome c levels along with caspase 3 activation after 24 h of DCM treatment. However, TDCM-treated neurons showed significant decrease in the expression of these pro-apoptotic molecules. Taken together, these findings suggest that 4-HT can significantly attenuate dichlorvos-induced microglial activation and prevent apoptotic neuronal cell death.

Quercetin protects against chronic aluminum-induced oxidative stress and ensuing biochemical, cholinergic, and neurobehavioral impairments in rats.

In this study, we investigated the protective effect of chronic quercetin (a natural flavanoid) administration against Al-induced cognitive impairments, oxidative damage, and cholinergic dysfunction in male Wistar rats. Al lactate (10 mg/kg b.wt./day) was administered intragastrically to rats which were pre-treated with quercetin (10 mg/kg b.wt./day, intragastrically) for 12 weeks. At the end of 6 or 12 weeks of the study, several behavioral parameters were carried out to evaluate cognitive functions. Further after 12 weeks of exposure, various biochemical tests and H&E staining were performed to assess the extent of oxidative damage and neurodegeneration, respectively. Al levels were also estimated in HC and CS regions of rat brain. Chronic administration of quercetin caused significant improvement in the muscle coordination, cognition, anxiety, locomotion, and initial exploratory patterns in Al-treated rats. Quercetin supplementation to Al-treated animals also reduced oxidative stress, decreased ROS production, increased MnSOD activity and glutathione levels with decreased lipid peroxidation and protein oxidation. It increased AChE activity and ATP levels in HC and CS regions of rat brain compared to Al-treated rats. Quercetin administration ameliorates Al-induced neurodegenerative changes in Al-treated rats as seen by H&E staining. Further with the help of atomic absorption spectrophotometer, we found that quercetin supplementation to Al-treated rats also decreases the accumulation of Al in the HC and CS regions of rat brain. Taken together the results of this study show that quercetin offers neuroprotection against Al-induced cognitive impairments, cholinergic dysfunction, and associated oxidative damage in rats.

CSF p-Tau levels in the prediction of Alzheimer's disease.

The two hallmarks of Alzheimer's disease (AD) are neurofibrillary tangles and amyloid plaques. Neurofibrillary tangles are formed due to the hyperphosphorylation of tau protein. There is an urgent need to develop a reliable biomarker for the diagnosis of AD. Cerebrospinal fluid (CSF) is surrounding the brain and reflects the major neuropathological features in the AD brain. Diagnosis, disease progression and drug actions rely on the AD biomarkers. Mainly CSF tau and phosphorylated tau (p-Tau) have been observed to serve the purpose for early AD. Keeping in view the early appearance of p-Tau in CSF, we analyzed p-Tau levels in 23 AD, 23 Non AD type dementia (NAD), 23 Neurological control (NC) and 23 Healthy control (HC) North Indian patients. The levels of p-Tau were found to be increased in AD patients (67.87±18.05 pg/ml, SEM 3.76) compared with NAD (47.55±7.85 pg/ml, SEM 1.64), NC (34.42±4.51 pg/ml, SEM 0.94) and HC (27.09±7.18 pg/ml, SEM 1.50). The resulting sensitivity for AD with NAD was 80.27% whereas with respect to the NAD, NC and HC was 85.40%. Therefore elevated levels of p-Tau in AD can be exploited as a predictive biomarker in North Indian AD patients.

Cerebrospinal fluid profile of amyloid ß42 (Aß42), hTau and ubiquitin in North Indian Alzheimer's disease patients.

Alzheimer's disease (AD) is the most common form of dementia, and is characterized by the degeneration of neurons and their synapses, and a higher number of amyloid plaques and neurofibrillary tangles (NFTs) compared with that found in non-demented individuals. Amyloid-ß-peptides (Aß) are major components of amyloid plaques in AD brain whereas NFTs are composed of Tau and associated with ubiquitin. The aim of the present study was to analyze the levels of Aß42, hTau (total Tau) and ubiquitin in CSF of North Indian population. CSF Aß42, Tau and ubiquitin were measured in CSF of AD patients as well as controls using ELISA assays. Here we report low Aß42 levels in AD patients (324.24±76.38pg/ml) as compared to those in non-AD (NAD) (668.34±43.13pg/ml), neurological controls (NCs) (727.28±46.49pg/ml) and healthy controls (HCs) (976.47±124.46pg/ml). In contrast, hTau and ubiquitin levels were significantly high (568.65±48.89pg/ml and 36.82±4.34ng/ml, respectively) in AD patients compared to those in NAD, NC and HC. The hTau levels were 267.37±36.64pg/ml, 167.34±44.27pg/ml and 107.62±24.27pg/ml in NAD, NC and HC, respectively. Similarly, ubiquitin levels were 23.57±2.32ng/ml, 19.76±3.64ng/ml and 13.24±4.56ng/ml in NAD, NC and HC, respectively. In conclusion, low Aß42 and high Tau-ubiquitin levels were found in North Indian AD patients.

Protective efficacy of mitochondrial targeted antioxidant MitoQ against dichlorvos induced oxidative stress and cell death in rat brain.

Dichlorvos is a synthetic insecticide that belongs to the family of chemically related organophosphate (OP) pesticides. It can be released into the environment as a major degradation product of other OPs, such as trichlorfon, naled, and metrifonate. Dichlorvos exerts its toxic effects in humans and animals by inhibiting neural acetylcholinesterase. Chronic low-level exposure to dichlorvos has been shown to result in inhibition of the mitochondrial complex I and cytochrome oxidase in rat brain, resulting in generation of reactive oxygen species (ROS). Enhanced ROS production leads to disruption of cellular antioxidant defense systems and release of cytochrome c (cyt c) from mitochondria to cytosol resulting in apoptotic cell death. MitoQ is an antioxidant, selectively targeted to mitochondria and protects it from oxidative damage and has been shown to decrease mitochondrial damage in various animal models of oxidative stress. We hypothesized that if oxidative damage to mitochondria does play a significant role in dichlorvos induced neurodegeneration, then MitoQ should ameliorate neuronal apoptosis. Administration of MitoQ (100 µmol/kg body wt/day) reduced dichlorvos (6 mg/kg body wt/day) induced oxidative stress (decreased ROS production, increased MnSOD activity and glutathione levels) with decreased lipid peroxidation, protein and DNA oxidation. In addition, MitoQ also suppressed DNA fragmentation, cyt c release and caspase-3 activity in dichlorvos treated rats compared to the control group. Further electron microscopic studies revealed that MitoQ attenuates dichlorvos induced mitochondrial swelling, loss of cristae and chromatin condensation. These results indicate that MitoQ may be beneficial against OP (dichlorvos) induced neurodegeneration.