Molecular Aspects of Dopaminergic Neurodegeneration: Gene-Environment Interaction in Parkin Dysfunction

Parkinson’s disease (PD) is a common neurodegenerative movement disorder that is characterized pathologically by a progressive loss of midbrain dopaminergic neurons and by protein inclusions, designated Lewy bodies and Lewy neurites. PD is one of the most common neurodegenerative diseases, affecting almost 1% of the population over 60 years old. Although the symptoms and neuropathology of PD have been well characterized, the underlying mechanisms and causes of the disease are still not clear. Genetic mutations can provide important clues to disease mechanism, but most PD cases are sporadic rather than familial; environmental factors have long been suspected to contribute to the disease. Although more than 90% of PD cases occur sporadically and are thought to be due, in part, to oxidative stress and mitochondrial dysfunction, the study of genetic mutations has provided great insight into the molecular mechanisms of PD. Furthermore, rotenone, a widely used pesticide, and paraquat and maneb cause a syndrome in rats and mice that mimics, both behaviorally and neurologically, the symptoms of PD. In the current review, we will discuss various aspects of gene-environment interaction that lead to progressive dopaminergic neurodegenration, mainly focusing on our current finding based on stress-mediated parkin dysfunction

Neuroprotective efficacy of a new brain-penetrating C-Abl inhibitor in a murine Parkinson's disease model.

Experimental evidence suggests that oxidative and nitrative mechanisms account for much of the dopaminergic neuronal injury in Parkinson's disease (PD). The ubiquitously expressed non-receptor tyrosine kinase c-Abl is activated by oxidative stress and thus, may play a role in redox-mediated neurodegeneration. Recently, we reported that c-Abl is activated in PD and that a c-Abl inhibitor mitigated neuronal damage in a PD animal model, suggesting a novel neuroprotective therapeutic approach. In the studies presented here, we evaluated the efficacy of a potent and clinically relevant second-generation irreversible Abl kinase inhibitor, INNO-406, as a therapeutic agent for PD. Our studies reveal that INNO-406 is capable of preventing the progression of dopaminergic neuronal damage in a toxin-induced C57 mouse model of PD. Using bovine brain microvessel endothelium as an in vitro blood-brain barrier (BBB) model, we detected rapid and significant transfer of INNO-406. Additionally, pharmacokinetic analyses demonstrated significant nanomolar concentrations of INNO-406 in brain in the presence or absence of MPTP administration, however, INNO-406 did not alter the brain levels of MPP+ in MPTP-treated mice. Finally, we showed that 10 mg/kg of INNO-406 given to C57 mice for one week before MPTP treatment (4×20 mg/kg i.p., every 2 h) and then for one week after MPTP treatment decreased the loss of dopamine in the striatum by 45% and the loss of TH+ neurons in substantia nigra pars compacts by 40%. This treatment regimen also abrogated activation of c-Abl, tyrosine phosphorylation of the Abl substrate and E3-ubiquitin ligase parkin, and accumulation of the toxic parkin substrate AIMP2. We propose that compounds of the INNO-406 class of Abl inhibitors will be useful new neuroprotective drugs for the treatment of PD-like pathology in preclinical systems that should be easily translated to the clinic

BBMEC Trans-Endothelial Electrical Resistance (TEER).

<p>The data is presented as mean ± SD, n = 3.</p

Directional Apparent Permeability Comparison of Drug Analogues.

<p>Where, dQ/dt is the flux across the cell monolayers, A is the surface area of the membrane and C₀ is the initial concentration of drug. The data is presented as mean ± SD, and * considered statistically different directionally p<0.05, n = 3.</p

Directional transport of Imatinib (A) and INNO-406 (B) across BBMEC monolayers.

<p>The directional transport of two drug analogues Imatinib (A) and INNO-406 (B) were determined in BBMEC. The apical-to-basolateral transport (A→B) (open circles) and basolateral-to-apical (B→A)(closed circles). The data are presented as percent transported as a function of time (means ± SD, n = 3). * Considered statistically significant (p<0.05).</p

INNO-406 prevents dopaminergic depletion and c-Abl mediated parkin phosphorylation in mice striatum.

<p>(A) INNO-406 prevents MPTP-induced depletion of striatal dopamine and its metabolites DOPAC and HVA in adult male C57BL/J6 mice. Animals received INNO-406 (10 mg/kg, i.p.) as a single daily injection for 7 days before MPTP injection. On day 7 mice were treated with 4×20 mg/kg, i.p. of MPTP at 2 h interval. INNO-406 injection was continued for one more week after the last injection of MPTP. * Considered statistically significant from control (p<0.05). ** Considered statistically significant from MPTP (p<0.05). (B) Parkin is tyrosine phosphorylated in the striatal lysates of MPTP-treated mice. Animals received INNO-406 (10 mg/kg, i.p.) as a single daily injection for 7 days before MPTP injection. On day 7 mice were treated with 4×20 mg/kg, i.p. of MPTP at 2 h interval. INNO-406 injection was continued for one more week after the last injection of MPTP. Samples were immunoprecipitated with anti-parkin antibody and were immunoblotted for anti-<i>p</i>-tyrosine, anti-parkin or anti-c-Abl. A 10% input samples were immunoblotted for anti-<i>p</i>-c-Abl, anti-parkin, anti-AIMP2, anti-c-Abl and ant-Actin. A pretreatment with INNO-406, a c-Abl kinase inhibitor, blocks oxidative stress-mediated tyrosine phosphorykation of parkin, activation of c-Abl and accumulation of toxic substrate, AIMP2, in the striatum of MPTP-treated mice. The photomicrograph shown is a representative of three repeats from samples pooled from 3 different animals. The optical density quantification data as normalized amounts is presented. * Considered statistically significant from control (p<0.05). ** Considered statistically significant from MPTP (p<0.05). (C) Cytotoxicity plotted as a percentage of control as measured by LDH in SH-SY5Y cells treated with MPP⁺ (500 µM). Some samples were incubated with 0.5 or 1.0 µM INNO-406 for 6 h before MPP⁺ treatment. *p<0.05. Differences among means were analyzed using one-way analysis of variance (ANOVA). All experiments were repeated at least three times and representative examples are presented.</p

The c-Abl inhibitor INNO-406 protects against MPTP-induced loss of DA neurons.

<p>INNO-406 (10 mg/kg/i.p for one week prior to MPTP, 4×20 mg, and one week after MPTP treatment) protects (A–D) substantia nigra DA neurons and (E–H) striatal DA terminals from MPTP-induced toxicity. Pre-treatment of mice with INNO-406 leads to preservation of TH+ positive staining cells within the substantia nigra pars compacta and DA terminals in the striatum compared to MPTP treatment alone. Plots of quantitative stereological data showing the protective effect of INNO-406 in SNpc and striatum has been shown. * Considered statistically significant from control (p<0.05). ** Considered statistically significant from MPTP (p<0.05). SNpc = Substantia Nigra <i>pars compacta</i>.</p