Cholesterol contributes to dopamine-neuronal loss in MPTP mouse model of Parkinson's disease: Involvement of mitochondrial dysfunctions and oxidative stress.

Hypercholesterolemia is a known contributor to the pathogenesis of Alzheimer's disease while its role in the occurrence of Parkinson's disease (PD) is only conjecture and far from conclusive. Altered antioxidant homeostasis and mitochondrial functions are the key mechanisms in loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain in PD. Hypercholesterolemia is reported to cause oxidative stress and mitochondrial dysfunctions in the cortex and hippocampus regions of the brain in rodents. However, the impact of hypercholesterolemia on the midbrain dopaminergic neurons in animal models of PD remains elusive. We tested the hypothesis that hypercholesterolemia in MPTP model of PD would potentiate dopaminergic neuron loss in SN by disrupting mitochondrial functions and antioxidant homeostasis. It is evident from the present study that hypercholesterolemia in naïve animals caused dopamine neuronal loss in SN with subsequent reduction in striatal dopamine levels producing motor impairment. Moreover, in the MPTP model of PD, hypercholesterolemia exacerbated MPTP-induced reduction of striatal dopamine as well as dopaminergic neurons in SN with motor behavioral depreciation. Activity of mitochondrial complexes, mainly complex-I and III, was impaired severely in the nigrostriatal pathway of hypercholesterolemic animals treated with MPTP. Hypercholesterolemia caused oxidative stress in the nigrostriatal pathway with increased generation of hydroxyl radicals and enhanced activity of antioxidant enzymes, which were further aggravated in the hypercholesterolemic mice with Parkinsonism. In conclusion, our findings provide evidence of increased vulnerability of the midbrain dopaminergic neurons in PD with hypercholesterolemia

Effect of hypercholesterolemia on hydroxyl radical (•OH) generation in (A,C,E) striatum and (B,D,F) substantia nigra regions of brain of Parkinsonian mice.

<p>Animals were injected with salicylic acid (100 mg/kg) and sacrificed two hours post injection on the last day of treatment. 2,3- and 2,5-dihydroxy benzoic acid (DHBA; •OH adducts of salicylate) formed were measured from homogenates of NCP and SN by employing a sensitive HPLC-ECD method. Data are expressed as pmol/mg tissue and represented as mean ± S.E.M. *p ≤ 0.05 as compared with control and #p ≤ 0.05 as compared with MPTP alone treated group (n = 5).</p

Effect of hypercholesterolemia on Parkinsonian motor behavior.

<p>In the last week of the 14 week treatment period, all the groups of animals were tested for (A) akinesia (B) catalepsy and (C) Swim test. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 6).</p

Effect of hypercholesterolemia on striatal dopamine level in Parkinsonian mice.

<p>Mice were sacrificed by decapitation on the seventh day following the first dose of MPTP. Striatal dopamine content was analyzed by HPLC-ECD system. Hypercholesterolemia exaggerates striatal dopamine depletion in Parkinsonian mice. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 6).</p

Effects of hypercholesterolemia on tyrosine hydroxylase (TH)-immunoreactivity in striatum (NCP) and TH-positive nigral (SN) neurons in MPTP-treated mice.

<p>Representative NCP (A-D) and SN (E-H) photographs from CS, HCD, MPTP and HCD+MPTP (left to right) groups showing TH-immunoreactivity. Quantification of relative density of TH-immunostaining in NCP (I) and neuronal count in SN (J) analyzed using ImageJ software. Hypercholesterolemia aggravates nigral TH-positive neuronal loss in MPTP-treated mice. Results are expressed as mean ± SEM. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 5).</p

Effect of hypercholesterolemia on nigrostriatal mitochondrial complex-III activity in Parkinsonian mice.

<p>(A-D) representative striatal (NCP) sections and (E-H) substantia nigral (SN) sections were processed for mitochondrial complex-III activity by employing histoenzymology. The marked region in the photographs (E-H) represents substantia nigra pars compacta (SNpc) region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171285#pone.0171285.ref059" target="_blank">59</a>]. Optical density of serial sections of (I) NCP and (J) SN was analyzed using ImageJ software. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 4).</p

Effect of hypercholesterolemia on nigrostriatal reduced glutathione (GSH) level in Parkinsonian mice.

<p>(A) Striatum and (B) substantia nigra regions of brain were used for estimating GSH levels by employing a sensitive HPLC-ECD system. Data are expressed as nmol/mg tissue and represented as mean ± S.E.M. *p ≤ 0.05 as compared with control and #p ≤ 0.05 as compared with MPTP alone treated group (n = 5).</p

Schematic representation of the experimental paradigm.

<p>[Abbreviations: DA, dopamine; •OH, hydroxyl radical; GSH, reduced glutathione; SOD, superoxide dismutase; CAT, catalase; TH-IR, tyrosine hydroxylase-immunoreactivity].</p

Effect of high cholesterol diet on (A) body weight, (B) serum total cholesterol, and cholesterol level in (C-D) liver and (F-G) brain (striatum).

<p>Mice were fed with high cholesterol diet (HCD) or standard diet (control, CS) for 14 weeks. Body weight (in gram) was measured two weeks apart from the start of treatment till 14^th weeks. On the last day of diet, blood was collected by cardio-punctured method and serum total cholesterol was estimated by enzymatic method. Accumulation of cholesterol in liver and striatum region of brain was estimated from fixed tissues by Schultz’s method. Optical density of characteristic greenish-blue colour for cholesterol in liver (E) and striatum (H) was measured using ImageJ software. The results given are mean ± S.E.M. *p ≤0.05 as compared to CS (n = 8).</p