Amphetamine-induced dopaminergic toxicity:: a single dose animal model of Parkinson's disease

Parkinson's disease affects millions of people worldwide and is characterized by loss of dopaminergic neurons of the nigro-striatal pathway. Although many mechanisms have been postulated to account for the destruction of these cells, no clear cause has been elucidated. The hypothesis that oxidative stress plays an important role in dopamine depletion in Parkinson's disease was examined through use of amphetamine, a dopaminergic toxicant known to act through oxidative stress. First, a thorough characterization of a single high dose of amphetamine was completed as a new model of Parkinson's disease. Then, antioxidant and anti-inflammatory treatments were used to protect against amphetamine's neurotoxic effects. The antioxidant ascorbic acid was successful in attenuating amphetamine-induced dopamine depletion, while others tested, including Trolox and EGCG, did not attenuate dopaminergic toxicity. In addition, an end product of lipid peroxidation, malondialdehyde, was measured in response to amphetamine treatment and evaluated with the time course of amphetamine-induced dopamine depletion. Studies have shown increased levels of malondialdehyde in the blood and brains of Parkinson's patients. Finally, the behavior and sensitivity of mice with selective deletions of genes coding for GSTM1, PAK5, PAK6, or both PAK5 and PAK6 to amphetamine was examined. Multiple genes have been implicated in the etiology of Parkinson's disease, some of which may be associated with oxidative stress response, mitochondrial function, protein kinase function and/or neuronal survival mechanisms. A null mutation in GSTM1 has been associated with Parkinson's disease and plays a role as an antioxidant in the brain. Mice lacking the GSTM1 gene did not show an abnormal behavioral phenotype compared to controls and were not sensitive to amphetamine toxicity. The p21-activated kinases (PAKs) are highly expressed in the brain as well and have been implicated in several neurological disorders, including Parkinson's disease. Mice lacking one or more of the PAK genes showed motoric similarities to Parkinson's disease, although they were relatively resistant to amphetamine toxicity. Collectively, these experiments explored the role of oxidative stress, antioxidant function and related genetic components in a single dose, amphetamine animal model of Parkinson's disease.Ph.D.Includes bibliographical references (p. 253-266)

Functional deficits in PAK5, PAK6 and PAK5/PAK6 knockout mice.

The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory

Summary of results and conclusions.

<p>WT = wild type mice, <i>Pak5</i>  =  <i>Pak5</i> knockout mice, <i>Pak6</i>  =  <i>Pak6</i> knockout mice. DKO =  <i>Pak5/Pak6</i> double knockout mice.</p

Figure 5

<p><b>a. Total Elevated Plus crosses</b>. The PAK5 knockout mice made more crosses into any arm of the elevated plus maze compared to the WT mice, the PAK6 knockout mice, and the DKO mice. The double knockout mice made significantly less crosses compared to the WT, PAK5 and PAK6 knockout mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Open vs. Closed crosses.</b> The mice made significantly more crosses into the closed arms of the maze compared to the number of crosses they made into the open arms. (& = p<.05 compared to closed arms). WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>c. </b><b>Elevated plus maze.</b> There was no difference in number of fecal boli between the genotypes. Only the PAK5 knockout mice jumped off the apparatus. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Figure 2

<p><b>a. Total activity first run</b>. The DKO mice were significantly less active than the WT(a), PAK5(b) and PAK6(c) mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Activity over 30 minutes.</b> The double knockout mice were significantly less active than the wild type mice (a), the PAK5 knockout mice (b), and the PAK6 knockout mice (c). WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Resident/intruder paradigm.

<p>The PAK5, PAK6, and DKO mice initiated significantly less attacks on the intruder mice, compared with the WT mice. The PAK5 and DKO mice were attacked significantly more times by the intruder mice than the WT mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Figure 1

<p><b>a. Total Body Weight Change</b>. PAK6 knockout mice had the largest increase in weight, significantly different from WT (a  =  p<.05 compared to WT), PAK5 (b =  p<.05 compared to PAK5), and DKO (d =  p<.05 compared to DKO). WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Body weight.</b> PAK6 knockout mice were significantly heavier than PAK5(b) and DKO(d) by 5 months of age and significantly heavier than WT(a) from 6 months of age. The DKO and PAK5 knockout mice weighed significantly less than the WT (a) and PAK6(c =  p<.05 compared to PAK6) at 5 months of age, but weighed similarly to the WT after 6 months of age. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Figure 7

<p><b>a. Total touches to target and control cylinders</b>. The WT, PAK5 and PAK6 knockout mice made significantly more touches to the target cylinder compared to the control cylinder (* = p<.05 compared to control). The PAK6 knockout mice and the DKO mice made significantly less touches to the target cylinder compared to the number of touches the WT (a) and PAK5 knockout mice (b) made. The PAK6 knockout mice and the DKO mice made significantly less touches to the control cylinder compared to the WT mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Percent touches to the target cylinder.</b> The double knockout mice had a significantly lower percentage of contact with the target cylinder compared to the WT, PAK5, and PAK6 knockout mice. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Figure 9

<p><b>a. Acquisition phase for active avoidance</b>. The DKO and PAK5 knockout mice made significantly less avoidances compared to the PAK6 and WT mice by the end of the 5 days of testing. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5. <b>b. Retention phase for active avoidance.</b> The DKO mice made less avoidances compared to the WT, PAK5, and PAK6 knockout mice over the 8 days of testing. WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p

Latency to fall from the rotorod.

<p>The PAK5 knockout mice and the DKO mice fell from the rotorod faster than the wild type mice (a = p<.05 compared to WT) and the PAK6 mice (c = p<.05 compared to PAK6 knockout mice). WT n = 8. PAK5 n = 5. PAK6 n = 8. DKO n = 5.</p