Role of the prolyl isomerase Pin1 in the pathogenesis of Parkinson\u27s disease and neuroprotection by novel targeted compounds in pre-clinical animal models of the disease

Parkinson\u27s disease (PD) is a chronic and progressive neurodegenerative disorder named by the French neurologist Jean-Martin Charot, after the British physician James Parkinson who first described the disease as Shaking Palsy . Pathologically, PD is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), a marked reduction of dopamine in the striatum, the presence of ubiquitin and α-synuclein positive cytoplasmic inclusions known as Lewy bodies, and depigmentation of the locus cereleus. The prevailing theory regarding processes that are likely to account for progressive degeneration of the dopaminergic neurons in the nigrostriatal axis involves on mitochondrial dysfunction, oxidative stress, excitotoxicity and neuroinflammation. Of those, neuroinflammation and oxidative stress have gained the most focus recently. Research over the last decade has provided extensive evidence that the sustained microglial and astroglial neuroinflammatory responses cause progressive and delayed dopaminergic neurodegeneration. However, the mechanisms by which glial cells activation and subsequent inflammation lead to dopaminergic neurodegeneration remain poorly understood. Development of effective therapeutic approaches to halt the disease progression of PD is of paramount importance. My objective in this Ph.D. thesis work was to characterize important signaling molecules activated in neuroinflammation mediated neurodegenerative pathways as well as characterze of novel compounds in a pre-clinical mouse model of PD. Protein interacting with never in mitosis - A (Pin1) is a peptidyl-prolyl isomerase, that specifically recognizes phosphorylated serine or threonine residues immediately preceding proline (pSer/Thr-Pro) in a subset of proteins in which it isomerizes the cis/trans conformation of the peptide bond. High expression levels of Pin1 in terminally differentiated and post mitotic neurons suggest that it plays an important role in neurons, except in cell cycle regulation and proliferation. Recently, Pin1 overexpression was shown to facilitate formation of &alpha-synuclein inclusions in a cellular model of &alpha-synuclein aggregation. Pin1 was also localized in Lewy bodies in PD patients. But the level, activity, and role of Pin1 in the pathogenesis of PD are incompletely known. We hypothesized that Pin1 is differentially activated in neuronal and glial cells of the nigral dopaminergic system, and that it regulates NF-κB-mediated sustained neuroinflammatory processes in cell culture and in an animal model of Parkinson\u27s disease. Herein, we demonstrate for the first time that there is a dopaminergic neuron specific upregulation of Pin1 in human postmortem PD brain sections as well as in cell culture and animal models. We observed a rapid increase in Pin1 expression in both the 1-methyl-4phenyl pyridinium (MPP+)-treated cell culture and in 1-methyl-4-phenyl-1-2-3-6-tetrahydroptridine MPTP treated mice. Also, Pin1 acts as an important pro-apoptotic factor in the selective degeneration of dopaminergic neurons. Importantly, pharmacological inhibition of Pin1 attenuates MPTP-induced Pin1 expression in vitro and in vivo and protects against MPTP-induced neurodegeneration in the nigrostriatal axis. We also demonstrate for the first time that, microglia and astrocytes express Pin1 and that there is a strong association between Pin1 and NF-κB p65 in BV2 microglial cells. Recent studies demonstrated that the promoter regions of proinflammatory molecules contain the DNA binding site for NF-κB. We have shown that Pin1 and NF-κB p65 inhibition by Juglone leads to attenuation of glial cells activation and subsequent reduction of proinflammatory reactions in cell culture and in an animal model of PD. While characterizing the role of Pin1 in the pathogenesis of PD, we also tested the efficacy of novel compounds for protection of dopaminergic neurons in a mouse model of PD. We demonstrate that the novel compound diapocynin, a metabolite of apocynin, blocks MPTP-induced activation of microglial and astroglial cells, thus inhibiting the inflammatory and oxidative stress processes in MPTP-treated mice. Diapocynin also protects the nigrostriatum against MPTP toxicity. The final chapter of this work characterizes the anti-inflammatory and neuroprotective properties of mito-apocynin, a mitochondria targeted compound in the MPTP mouse model of PD. Mitoapocynin prevents the behavioral imapariments and dopamine loss caused by MPTP-induced toxicity. We have shown that mito-apocynin protects the nigrostriatum by attenuating glial cell mediated neuroinflammation and oxidative stress. Collectively, the research described herein characterizes the novel and important roles of Pin1 in the neuroinflammation and pathophysiology of Parkinson\u27s disease, as well as establishes the efficacy of novel compounds in protection of the nigrostriatum in a pre-clinical mouse model of Parkinson\u27s disease

Characteristics of Bay of Bengal monsoon depressions in the 21st Century

We show that 21st century increase in radiative forcing does not significantly impact the frequency of South Asian summer monsoon depressions (MDs) or their trajectories in the Coupled Model Intercomparison Project Phase 5 general circulation models (GCMs). A significant relationship exists between the climatological occurrences of MDs and the strength of the background upper (lower) tropospheric meridional (zonal) winds and tropospheric moisture in the core genesis region of MDs. Likewise, there is a strong relationship between the strength of the meridional tropospheric temperature gradient in the GCMs and the trajectories of MDs over land. While monsoon dynamics progressively weakens in the future, atmospheric moisture exhibits a strong increase, limiting the impact of changes in dynamics on the frequency of MDs. Moreover, the weakening of meridional tropospheric temperature gradient in the future is substantially weaker than its inherent underestimation in the GCMs. Our results also indicate that future increases in the extreme wet events are dominated by nondepression day occurrences, which may render the monsoon extremes less predictable in the future

Mito-Apocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage, and Progressive Neurodegeneration in MitoPark Transgenic Mice

Aims: Parkinson\u27s disease (PD) is a neurodegenerative disorder characterized by progressive motor deficits and degeneration of dopaminergic neurons. Caused by a number of genetic and environmental factors, mitochondrial dysfunction and oxidative stress play a role in neurodegeneration in PD. By selectively knocking out mitochondrial transcription factor A (TFAM) in dopaminergic neurons, the transgenic MitoPark mice recapitulate many signature features of the disease, including progressive motor deficits, neuronal loss, and protein inclusions. In the present study, we evaluated the neuroprotective efficacy of a novel mitochondrially targeted antioxidant, Mito-apocynin, in MitoPark mice and cell culture models of neuroinflammation and mitochondrial dysfunction. Results: Oral administration of Mito-apocynin (10 mg/kg, thrice a week) showed excellent central nervous system bioavailability and significantly improved locomotor activity and coordination in MitoPark mice. Importantly, Mito-apocynin also partially attenuated severe nigrostriatal degeneration in MitoPark mice. Mechanistic studies revealed that Mito-apo improves mitochondrial function and inhibits NOX2 activation, oxidative damage, and neuroinflammation. Innovation: The properties of Mito-apocynin identified in the MitoPark transgenic mouse model strongly support potential clinical applications for Mito-apocynin as a viable neuroprotective and anti-neuroinflammatory drug for treating PD when compared to conventional therapeutic approaches. Conclusion: Collectively, our data demonstrate, for the first time, that a novel orally active apocynin derivative improves behavioral, inflammatory, and neurodegenerative processes in a severe progressive dopaminergic neurodegenerative model of PD. Antioxid. Redox Signal. 27, 1048–1066

Sodium Phenylbutyrate Controls Neuroinflammatory and Antioxidant Activities and Protects Dopaminergic Neurons in Mouse Models of Parkinson’s Disease

Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21rac, but not p21ras, attenuated the production of ROS from activated microglia. Inhibition of both p21ras and p21rac activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21ras and p21rac in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. Oral administration of NaPB reduced nigral activation of p21ras and p21rac, protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders

Role of the prolyl isomerase Pin1 in the pathogenesis of Parkinson's disease and neuroprotection by novel targeted compounds in pre-clinical animal models of the disease

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder named by the French neurologist Jean-Martin Charot, after the British physician James Parkinson who first described the disease as "Shaking Palsy". Pathologically, PD is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), a marked reduction of dopamine in the striatum, the presence of ubiquitin and α-synuclein positive cytoplasmic inclusions known as Lewy bodies, and depigmentation of the locus cereleus. The prevailing theory regarding processes that are likely to account for progressive degeneration of the dopaminergic neurons in the nigrostriatal axis involves on mitochondrial dysfunction, oxidative stress, excitotoxicity and neuroinflammation. Of those, neuroinflammation and oxidative stress have gained the most focus recently. Research over the last decade has provided extensive evidence that the sustained microglial and astroglial neuroinflammatory responses cause progressive and delayed dopaminergic neurodegeneration. However, the mechanisms by which glial cells activation and subsequent inflammation lead to dopaminergic neurodegeneration remain poorly understood. Development of effective therapeutic approaches to halt the disease progression of PD is of paramount importance. My objective in this Ph.D. thesis work was to characterize important signaling molecules activated in neuroinflammation mediated neurodegenerative pathways as well as characterze of novel compounds in a pre-clinical mouse model of PD. Protein interacting with never in mitosis - A (Pin1) is a peptidyl-prolyl isomerase, that specifically recognizes phosphorylated serine or threonine residues immediately preceding proline (pSer/Thr-Pro) in a subset of proteins in which it isomerizes the cis/trans conformation of the peptide bond. High expression levels of Pin1 in terminally differentiated and post mitotic neurons suggest that it plays an important role in neurons, except in cell cycle regulation and proliferation. Recently, Pin1 overexpression was shown to facilitate formation of &alpha-synuclein inclusions in a cellular model of &alpha-synuclein aggregation. Pin1 was also localized in Lewy bodies in PD patients. But the level, activity, and role of Pin1 in the pathogenesis of PD are incompletely known. We hypothesized that Pin1 is differentially activated in neuronal and glial cells of the nigral dopaminergic system, and that it regulates NF-κB-mediated sustained neuroinflammatory processes in cell culture and in an animal model of Parkinson's disease. Herein, we demonstrate for the first time that there is a dopaminergic neuron specific upregulation of Pin1 in human postmortem PD brain sections as well as in cell culture and animal models. We observed a rapid increase in Pin1 expression in both the 1-methyl-4phenyl pyridinium (MPP+)-treated cell culture and in 1-methyl-4-phenyl-1-2-3-6-tetrahydroptridine MPTP treated mice. Also, Pin1 acts as an important pro-apoptotic factor in the selective degeneration of dopaminergic neurons. Importantly, pharmacological inhibition of Pin1 attenuates MPTP-induced Pin1 expression in vitro and in vivo and protects against MPTP-induced neurodegeneration in the nigrostriatal axis. We also demonstrate for the first time that, microglia and astrocytes express Pin1 and that there is a strong association between Pin1 and NF-κB p65 in BV2 microglial cells. Recent studies demonstrated that the promoter regions of proinflammatory molecules contain the DNA binding site for NF-κB. We have shown that Pin1 and NF-κB p65 inhibition by Juglone leads to attenuation of glial cells activation and subsequent reduction of proinflammatory reactions in cell culture and in an animal model of PD. While characterizing the role of Pin1 in the pathogenesis of PD, we also tested the efficacy of novel compounds for protection of dopaminergic neurons in a mouse model of PD. We demonstrate that the novel compound diapocynin, a metabolite of apocynin, blocks MPTP-induced activation of microglial and astroglial cells, thus inhibiting the inflammatory and oxidative stress processes in MPTP-treated mice. Diapocynin also protects the nigrostriatum against MPTP toxicity. The final chapter of this work characterizes the anti-inflammatory and neuroprotective properties of mito-apocynin, a mitochondria targeted compound in the MPTP mouse model of PD. Mitoapocynin prevents the behavioral imapariments and dopamine loss caused by MPTP-induced toxicity. We have shown that mito-apocynin protects the nigrostriatum by attenuating glial cell mediated neuroinflammation and oxidative stress. Collectively, the research described herein characterizes the novel and important roles of Pin1 in the neuroinflammation and pathophysiology of Parkinson's disease, as well as establishes the efficacy of novel compounds in protection of the nigrostriatum in a pre-clinical mouse model of Parkinson's disease.</p

Determinants of Skilled Delivery Assistance in a Rural Population: Findings from an HDSS Site of Rural West Bengal, India

Objective This study examines the determinants of utilisation of skilled birth attendants (SBAs) amongst 2886 rural women in the state of West Bengal, India, using data from a survey of 2012–2013 conducted by the Birbhum Health and Demographic Surveillance System. Method Multilevel logit regression models were estimated and qualitative investigations conducted to understand the determinants of utilisation of SBAs in rural West Bengal. Results Among women who delivered their last child during the 3 years preceding the survey, 69.1 % of deliveries were assisted by SBAs, while 30.9 % were home deliveries without any SBA assistance. Multivariate analysis revealed that apart from socio-demographic and economic factors (such as household affluence, women’s education, birth order, uptake of comprehensive ANC check-ups, advice regarding danger signs of pregnancy and household’s socio-religious affiliation), supply side factors, such as availability of skilled birth attendants in the village and all-weather roads, have significant effect on seeking skilled assistance. Our findings also show that unobserved factors at village level independently influence uptake of SBA-assisted delivery. Conclusions for Practice The present findings emphasise that both demand and supply side intervention strategies are essential prerequisites to enhance skilled birth attendance. Ample communication is observed at the individual level, but improving community level outreach and advocacy activities could generate further demand. SBAs can be better integrated by accommodating the socio-religious needs of local communities, such as providing female doctors and doctors with similar socio-religious backgrounds

Combination of self-report method and observational method in assessment of postoperative pain severity in 2 to 7 years of age group: A cross-sectional analytical study

Background: Postoperative pain management is based on assessment of severity of pain. Adult patients can express their pain accurately but difficulty occurs in paediatric population. Children between 2 and 7 years of age may give biased response to any scale of pain assessment as they belong to the preoperational stage of cognitive development. Objectives: To establish the agreement between two pain scale, namely Faces Pain Scale-Revised (FPS-R) and Face, Legs, Activity, Cry, Consolability scale (FLACC) regarding assessment of severity of postoperative pain and to find out true negative in terms of specificity of combination of scale for assessment of postoperative pain. Settings and Design: Postoperative recovery unit, cross-sectional analytical study. Materials and Methods: Four hours after short surgical procedure 95 children were assessed by two pain scale and by two observers simultaneously and data submitted to analyser. Statistical Analysis: IBM SPSS (Version 20.0). P < 0.05 was considered as statistically significant. Results: Combination of these two scales show high odds ratio (39%) and kappa coefficient (0.76) suggesting excellent agreement. Specificity of combination of these scales is very high (95.1%) than individual (FPS-R-17.85%, FLACC-2.2%). Spearman′s correlation coefficient (ρ) was computed to ascertain the correlation between two scales and a significant positive correlation was found (ρ = 0.727, P = 0.00). Conclusion: FPS-R and FLACC scale has excellent agreement to diagnose the severity of postoperative pain in 2-7 years of age group and combination of these two scales has high specificity to assess the severity of postoperative pain than individual

Role of Munc13-1 and Munc13-2 Proteins in Alcoholism, and its Potential Clinical Applications

Munc13-1 and Munc13-2 are presynaptic proteins that are involved in the vesicular priming and subsequent release of glutamate neurotransmitter. Previous research has demonstrated some of the effects of ethanol in specific areas of the brain related to addiction, but the effects of ethanol on the expression of Munc13-1 and Munc13-2 and their glutamatergic effects in different brain regions is unknown. First, we performed immunocytochemistry (ICC) of Munc13-1 and Munc13-2 on ethanol-treated differentiated HT22 cells in-vitro and found that there was a significant change in the overall expression of Munc13-1 in a dose and time-dependent manner. Next, we harvested primary hippocampal neurons, treated them with ethanol in a dose and time-dependent manner, and then measured both Munc13-1 and Munc13-2 protein expression using Western-blot analysis. Here, our results showed that ethanol significantly upregulates the expression of both Munc13-1 and Munc13-2. We then tested the effects of ethanol on Munc13 proteins in-vivo on both wild-type (Wt) and heterozygous Munc13-1 knockout mice, using the drinking in the dark (DID) paradigm. In comparison to our previous ex-vivo data, these results showed that with alcohol exposure there was a significant increase in the expression of Munc13-1 in the hippocampus and cerebellum of Wt mice, but a decrease in the expression of Munc13-2 in the hippocampus, cerebellum and cortex. In the heterozygous Munc13-1 knockout mice, ethanol caused significant compensation for the loss of Munc13-1 in the hippocampus and cerebellum, and also an increase in Munc13-2 expression in the cerebellum and cortex. Since both Munc13-1 and Munc13-2 predominantly control glutamatergic synapses, their modulation by ethanol exposure could potentiate an increase in glutamate release from the pre-synaptic terminals of these regions and stimulate excitation of the Central Nervous System (CNS). This is analogous to the CNS excitation we see with chronic AUD patients experiencing alcohol withdrawal symptoms. By identifying these proteins as drug targets, we may be able to unfold a different mechanism of alleviating alcohol withdrawal symptoms in AUD patients, without the GABAergic adverse effects that usually present with the current mainstay treatment options.Pharmacological and Pharmaceutical Sciences, Department o

An epoxide hydrolase inhibitor reduces neuroinflammation in a mouse model of Alzheimer’s disease

Neuroinflammation has been increasingly recognized to play a critical role in Alzheimer's disease (AD). The epoxy fatty acids (EpFAs) are derivatives of the arachidonic acid metabolism pathway and have anti-inflammatory activities. However, their efficacy is limited because of their rapid hydrolysis by the soluble epoxide hydrolase (sEH). We report that sEH is predominantly expressed in astrocytes and is elevated in postmortem brain tissue from patients with AD and in the 5xFAD β amyloid mouse model of AD. The amount of sEH expressed in AD mouse brains correlated with a reduction in brain EpFA concentrations. Using a specific small-molecule sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), we report that TPPU treatment protected wild-type mice against LPS-induced inflammation in vivo. Long-term administration of TPPU to the 5xFAD mouse model via drinking water reversed microglia and astrocyte reactivity and immune pathway dysregulation. This was associated with reduced β amyloid pathology and improved synaptic integrity and cognitive function on two behavioral tests. TPPU treatment correlated with an increase in EpFA concentrations in the brains of 5xFAD mice, demonstrating brain penetration and target engagement of this small molecule. These findings support further investigation of TPPU as a potential therapeutic agent for the treatment of AD

NaPB inhibits the production of ROS in mouse microglial cells.

<p>Cells were treated with 500 µM NaPB for 6 h followed by stimulation with 1 µM MPP⁺. At 15 min of stimulation, the generation of ROS was monitored by carboxy-H₂DCFDA (A). At different intervals (measured in minutes), superoxide production was assayed in whole cells (B). Cells preincubated with 500 µM NaPB for 6 h were stimulated with LPS (1 µg/ml), TNF-α (50 ng/ml), IL-1β (20 ng/ml), gp120 (200 pg/ml), and fibrillar Aβ1-42 (1 µM). At 10 min of stimulation, superoxide production was assayed in whole cells (C). Results are mean <u>+</u> SD of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs stimuli. D) Cells were incubated with NaPB in the presence or absence of HMG-CoA, mevalonate, GGPP, FPP, cholesterol, and coenzyme Q. After 6 h of incubation, cells were stimulated with LPS for 10 min followed by assay of superoxide. Results are mean <u>+</u> SD of three different experiments. <i>^ap<0.001</i> vs control; <i>^bp<0.001</i> vs LPS; <i>^cp<0.001</i> vs LPS+NaPB.</p