Name: gene-environment interaction models to unmask susceptibility mechanisms in parkinson's disease
Uploaded: 2014-01-07T13:45:00
Description: Watch this Scientific Journal Video about Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease at JoVE.com

This work was funded by the National Institutes of Health NIGMS 056062.

Note: All animal procedures and animal care methods should be approved by the institution&#8217;s Institutional Animal Care and Usage Committee (IACUC). Study described here was performed in accordance to the guidelines established by SRI International&#8217;s IACUC. 
1. Acquisition and maintenance of LOX-deficient mice
<ol>
	<li>Purchase 5-LOX-deficient or 12/15-LOX-deficient mice and respective strain and sex-matched controls at age 7-8...

The design of this gene-environment interaction study allowed us to gain new information regarding the dual nature of the 5-LOX isozyme in the nigrostriatal pathway.&#160;By performing HPLC to measure striatal monoamines after saline or MPTP treatment in transgenics lacking the 5-LOX isozyme and their wildtype littermates, we were able to note that its deficiency appears to be protective under toxic conditions (Figure 1), but under normal conditions, lack of the enzyme reduces striatal dopamine levels an...

Use of gene-environment interaction models provides an approach to mimic risk factors that likely influence idiopathic Parkinson&#8217;s disease (PD) and affords an opportunity to discern mechanistic insights that are unlikely to be elucidated by use of a genetic or toxicant system alone1,2. Here we illustrate this point and describe application of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of nigrostriatal degeneration3&#160;to better understand the selectivity of lipoxygenase (LOX) isozyme activity on neuroinflammation and toxicity4. While a role for LOX isozymes has been widely evaluated in peripheral disorders5,6 as well as CNS disease including stroke7 and Alzheimer&#8217;s disease8,9, the role of the family of isozymes in nigrostriatal function and degeneration related to PD is not well understood and warrants study. The MPTP neurotoxin demonstrates preferential degeneration of the nigrostriatal pathway and recapitulates the striatal dopamine depletion and nigral dopaminergic cell loss that underlie motoric impairments in PD patients10. While this model does not reproduce the full cadre of nonmotor and motor PD behaviors and frank &#945;-synuclein-positive Lewy body pathology, it has been useful to elucidate novel mechanistic targets that contribute to nigrostriatal damage and for early-stage translational testing as it is the best characterized noninvasive model available to reliably produce nigral cell death accompanied by striatal dopamine loss11-15. Wide use of the MPTP mouse, with paradigms ranging from acute, subacute to chronic16-18, has allowed for standardization of dosing to result in mild to severe nigrostriatal damage19,20&#160;with activation of different mechanisms of toxicity depending on the treatment regimen18,21,22. Consequently, this permits a &#8216;window of lesioning&#8217; to be targeted that may result in enhanced or reduced nigrostriatal injury depending on the therapeutic agent or transgenic model utilized23-25.
Also essential for translational and discovery biology studies are the techniques used to assess damage and the evidence such methods provide.&#160;For the MPTP mouse model, established metrics to evaluate lesioning are measurement of markers of striatal dopaminergic tone, including dopamine and its metabolites by HPLC, and Western blot analysis of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and indicators of degenerative events such as glial activation using Western blot analysis and immunohistochemistry4.&#160;Although these are classical neurochemical, biochemical, and histological procedures, the techniques provide critical and reproducible readouts on the extent of damage within the nigrostriatal dopaminergic pathway, indicate mechanisms of toxicity, and have proven to be valuable tools in understanding degenerative events in PD.

<table border="0" cellpadding="0" cellspacing="0" width="1227">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:481px;">1-Methyl-4-phenyl-1,2,3,6-tetra-hydropyridine hydrochloride (MPTP-HCL)</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:121px;">M0896</td>
			<td style="width:351px;">for PD modeling</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% Formaldehyde (paraformaldehyde) solution, phosphate-buffered (PFA)</td>
			<td>American MasterTech Scientific</td>
			<td>BUP0157</td>
			<td>for immersion fixation</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid ACS reagent, 70% (PCA)</td>
			<td>Sigma-Aldrich</td>
			<td>244252</td>
			<td>for HPLC acid extraction</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminotetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E1644</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphatase inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P5726</td>
			<td>for tissue homogenization</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td>for Lowry protein assay</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose, molecular biology, &#8805;99.5% (GC)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>for cryoprotection</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline, powder, pH 7.4 (for 0.01 M PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BCA Protein Assay Kit</td>
			<td>Pierce/Thermo</td>
			<td>23225</td>
			<td>for protein determination</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex 12% Tris-Glycine Mini Gels 1.0 mm, 12-well</td>
			<td>Invitrogen/Life Technologies</td>
			<td>EC60052BOX</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NuPAGE LDS Sample Buffer (4x)</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0007</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex Sharp Prestained Protein Standard&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>LC5800</td>
			<td>protein ladder</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate, electrophoresis, 98.5% (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td>for SDS-PAGE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methyl Alcohol, Anhydrous, Reagent&#160;</td>
			<td>American MasterTech Scientific</td>
			<td>SPM1057C</td>
			<td>methanol for transfer</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride (NaCl), ACS reagent</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>saline and buffers</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nonfat dry milk powder</td>
			<td>Carnation</td>
			<td>n/a</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ponceau S solution in 5% acetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P7170</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Tyrosine Hydroxylase (TH), sheep polyclonal</td>
			<td>Chemicon/Millipore</td>
			<td>AB1542</td>
			<td>for immunofluorescence&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Tyrosine Hydroxylase (TH), rabbit polyclonal</td>
			<td>Pel-Freez Biologicals</td>
			<td>P40101-0</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-&#946; Actin, rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>A2066</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Glial Fibrillary Acidic Protein (GFAP), rabbit polyclonal</td>
			<td>Chemicon/Millipore</td>
			<td>AB5804</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-Glial Fibrillary Acidic Protein (GFAP), mouse monoclonal</td>
			<td>Covance Inc.</td>
			<td>SMI-22R</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated&#160;</td>
			<td>Fisher Scientific</td>
			<td>31462</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">goat anti-sheep, peroxidase conjugated</td>
			<td>Pierce/Thermo</td>
			<td>31480</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">goat anti-mouse, peroxidase conjugated</td>
			<td>Pierce/Thermo</td>
			<td>31430</td>
			<td>for immunofluorescence</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Pierce/Thermo</td>
			<td>34078</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CL-XPosure Film 7 in x 9.5 in&#160;</td>
			<td>Pierce/Thermo</td>
			<td>34089</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Restore Western Blot Stripping Buffer&#160;</td>
			<td>Pierce/Thermo</td>
			<td>21059</td>
			<td>for immunoblotting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citric acid monohydrate, ACS reagent, &#8805;99.0%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Normal Donkey Serum</td>
			<td>Millipore</td>
			<td>S30-100ML</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Polyvinylpyrrolidone (PVP)</td>
			<td>Sigma-Aldrich</td>
			<td>P5288</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Bovine Serum Albumin (BSA), lyophilized</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-01</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Donkey anti-Rabbit IgG, Alexa Fluor 568-labeled&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A10042</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Anti-Sheep IgG (H+L), FITC&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>713-095-147</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASHIELD Hard-Set Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1500</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Goat Serum</td>
			<td>Millipore</td>
			<td>S26-100ML</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASTAIN ABC Kit (Rabbit IgG )&#160;</td>
			<td>Vector Laboratories</td>
			<td>PK-4001</td>
			<td>for IHC; 10 &#181;l each of solutions A and B per 1 ml PBS (per instructions )</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">DAB Peroxidase Substrate Kit, 3,3&#8217;-diaminobenzidine</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>for IHC; per 5 ml cold ddH2O, add 2 drops buffer stock solution, 2 drops DAB, and 1 drop H2O2 (H2O2 is added immediately before use)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrogen peroxide, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>216763</td>
			<td>for quench step in IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-Iba1</td>
			<td>Biocare Medicals</td>
			<td>CP290A</td>
			<td>for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:481px;">Cresyl Violet Solution, Regular Strength&#160;</td>
			<td>FD Neurotechnologies</td>
			<td>PS102-01&#160;</td>
			<td>counterstain for Iba1 IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">95% Ethanol, reagent alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R8382</td>
			<td>dehydration for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Absolute ethanol</td>
			<td>Mallinckrodt&#160;</td>
			<td>7019-10</td>
			<td>dehydration for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>destaining for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td>clearing agent for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPX Mountant</td>
			<td>Sigma-Aldrich</td>
			<td>06522</td>
			<td>mounting medium for DAB IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O.C.T. Compound - Frozen Section Embedding Medium&#160;</td>
			<td>American MasterTech Scientific</td>
			<td>EMOCTCS</td>
			<td>embeddium medium for cryostat cutting</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium permanganate</td>
			<td>Sigma-Aldrich</td>
			<td>223468</td>
			<td>to decontaminate DAB solution</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dopamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8502</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,4-Dihydroxyphenylacetic acid (DOPAC)</td>
			<td>Sigma-Aldrich</td>
			<td>850217</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Homovanillic acid (HVA)</td>
			<td>Sigma-Aldrich</td>
			<td>H1252</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid (PCA) - 70%</td>
			<td>Sigma-Aldrich</td>
			<td>244252</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dihydrogen phosphate monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>71504</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Octanesulfonic acid sodium salt (OSA)</td>
			<td>Sigma-Aldrich</td>
			<td>O8380</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E1644</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>EMD</td>
			<td>AX0145-1</td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC-grade distilled deionized water (ddH2O)</td>
			<td>Millipore</td>
			<td></td>
			<td>for HPLC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.22 &#181;m GSTF membrane</td>
			<td>Millipore</td>
			<td></td>
			<td>for filtration</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning Netwells</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3477</td>
			<td>polystyrene insert with polyester mesh bottom, for IHC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic cell disrupter (Soniprep 150)</td>
			<td>MSE</td>
			<td>MSE.41371.274</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5414R</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ESA MD-150 reverse-phase column&#160;</td>
			<td>ESA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Pump (Ultimate 3000)</td>
			<td>Dionex</td>
			<td>ISO-3100BM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Autosampler (Ultimate 3000)</td>
			<td>Dionex</td>
			<td>WPS-3000TSL</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electrochemical detector</td>
			<td>ESA</td>
			<td>Coulochem III</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Guard Cell</td>
			<td>ESA</td>
			<td>5020</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analytical Cell</td>
			<td>ESA</td>
			<td>5011A</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromeleon software</td>
			<td>Dionex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse E400</td>
			<td>Nikon</td>
			<td>E400</td>
			<td>light/fluorescent microscope</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable mouse cage</td>
			<td>Ancare</td>
			<td>N10HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfilter top</td>
			<td>Ancare</td>
			<td>N10MBT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-LOX- deficient mice</td>
			<td>The Jackson Laboratory</td>
			<td>004155</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12/15-LOX-deficient mice</td>
			<td>The Jackson Laboratory</td>
			<td>002778</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

gene-environment interaction models to unmask susceptibility mechanisms in parkinson's disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

This toxin exposure paradigm can produce a significant and detectable 20% striatal dopamine depletion in MPTP- vs. saline-injected animals. It is important to note that different lots of MPTP may yield slightly more or less lesioning; thus, for better precision, a preliminary experiment in wildtype mice is recommended prior to use in transgenics when a new lot of neurotoxin is utilized.&#160;The use of mild-to-moderate lesioning allows for impact of the transgene to be observed; a severe lesion may produce a &#39;floor e...

Watch this Scientific Journal Video about Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease at JoVE.com

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

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