View Project lobsi-affy-mouse-193446

Project Summary
Status:	Public
Publications:	1 Published
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Mouse	Array Design Detail	true
Organ/Tissue Type:	spinal cord	Experiment Detail	true
Organ Region:	lumbar ventral horn	Sample Detail	false
Cell Type:	DRG, dorsal root ganglia	Hybridization Detail	false
Study Type:	time_series_design	Measurement Detail	false
Disease/Condition:	ALS
Replicates:	3
Expected Samples:	27

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Investigator Contact Detail
Name	Christian S Lobsiger
Street Address:	UCSD, 9500 Gilman Drive
	CMM-E, Room 3072
City, State/Province:	La Jolla , CA
Zip/Postal Code:	92093-0670
Country:	United States
Work Phone:	858 534 7899
Fax:	858 534 7659
E-mail:	clobsige@ucsd.edu

Proposal Detail

Grant:

3R01NS027036-17S1

Status:

Public

Service Type:

Hybrization through Analysis

IACUC:

S00225

IACUC date:

2000-11-01

Study Relevance:

Missense mutations in the gene for the ubiquitously expressed superoxide dismutase-1 (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (ALS), the most common adult onset motor neuron disease in humans killing selectively large motor neurons. Mice and rats overexpressing mutant SOD1 develop an adult onset neurodegenerative disease with hindlimb-paralysis and subsequent death similar to the human condition. In order to analyze the effects of mutant SOD1 expression onto the most affected cell-type in ALS, a small subpopulation of spinal cord cells, we propose to use laser microdissection to isolate mouse lumbar motor neurons and to assess the changes onto the mRNA expression profile using Affymetrix GeneChips compared to control animals. While two studies applying a genomic approach on the ALS mouse models used the entire spinal cord, contributions of changes to motor neurons were masked by the inflammatory effects of mutant SOD1 and the much larger population of non-motor neuronal cells. What is therefore needed is a cell-type specific expression profile that could reveal dysregulations in the transcriptome of the affected motor neurons.

Hypothesis:

Both, mice deleted in wildtype SOD1 and mice overexpressing mutant human SOD1 clearly established that the toxic property of mutant SOD1 is based upon a gain-of-toxic function phenomenon rather than a loss-of-function effect, since the SOD1 knock-out mice are overall normal. Furthermore, mice overexpressing wildtype SOD1 are healthy and serve as ideal control animals. Therefore, one of the most important question in ALS is to determine what slowly acting mechanism, or build-up of toxic products is responsible for ultimately killing more than 50-70% of the large lumbar spinal cord motor neurons. We hypothezise that the toxic property of mutant SOD1 must already induce changes onto the transcriptome of the most at-risk cell-type, the large lumbar spinal cord motor neuron, very early on in the presymptomatic phase of the disease course, when the animal is still without any paralysis signs and moving normally around the cage.

Specific Aim:

In order to find and analyze disease relevant gene expression profile changes in the mutant vs. the wildtype overexpressing SOD1 mice, we propose to use two different mutant SOD1 lines. Both lines develop the same ALS-like motor neuron disease, however, line 1 (with a G37R mutation) retains the full activity of the SOD1 enzyme while line 2 (with a G85R mutation) has almost no SOD1 enzymatic activity. We believe that by using these two most extreme variants of ALS-inducing mutant SOD1 forms, candidate genes that will be similarly dysregulated in both mutant lines will have great potential to be a disease relevant hit. We will collect from both mutant SOD1 mouse lines lumbar motor neurons from three presymptomatic timepoints, equally distributed during their adult life and before obvious hindlimb-weakness or paralysis signs and compare them to two control lines, negative littermates and wildtype SOD1 overexpressing mice. Furthermore, to determine if their are already mutant SOD1-induced changes since the formation of the affected cells, we will also isolate embryonic spinal cord motor neurons and compare them between mutant SOD1 and wildtype SOD1 overexpressing animals.

Experimental Procedure and Design:

We will choose 3 timepoints before phenotypic disease-onset that is at 22 weeks for the SOD1G37R line (L42) and at 11 months for the SOD1G85R line (L148). The timepoints are 8, 15 and 18 weeks for SOD1G37R line and 4, 7.5 and 9 months for the SOD1G85R line all compared to age-matched control animals (wildtype SOD1 overexpressing mice (L76) for the SOD1G37R line (high mutant SOD1 levels) and negative littermates for the SOD1G85R line (low mutant SOD1 levels)). Using the Leica lasermicrodissection system on fresh frozen spinal cord sections, we process a single spinal cord sample in 3 days resulting in approximately 4000 ventral horn motor neurons collected from 150 consecutive 20 um sections of the most affected lumbar spinal cord region (L4-L6). To visualize the motor neurons, we have established a fast Nissel-staining protocol that is optimized for speed to preserve the integrity of the RNA. Before lasermicrodissection slides containing 5 spinal cord sections are dessicated for 1 hour and samples are collected directly into RNA-preserving lysis-buffer. Using Stratagene's NanoPrep RNA-extraction columns a yield of around 150 ng of total RNA was obtained (Ribogreen-quantification), that is enough for both the 2-round linear RNA amplification and for future candidate confirmation studies using real-time PCR analysis. We are using the Affymetrix Two-Cycle 3'-Amplification Kit, starting with 100 ng of total RNA and will hybridize on Affymetrix Mouse 430 2.0 GeneChips. Embryonic motor neurons are isolated from e16 mutant SOD1G93A overexpressing rat embryos and compared to wildtype SOD1 overexpressing embryos using a metrazimide gradient followed by a p75-antibody purification in combination with magnetic beads. Cells from one litter of embryos were directly collected into lysis-buffer, yielding at least 200-500 ng of total RNA, enough for one Rat 230 2.0 GeneChip (2-round amplification using 100 ng total RNA).

Replicate Description:

We are using biological quadruplets samples (4) for every timepoint with all the mouse lines on a pure C57Bl/6 background, analyzing only females. All the breeding to obtain the needed animals were setup and aged under identical conditions at approx. the same time and the 4 animals are from on or two different litters. Fresh frozen spinal cords are stored in TissueTek at -80C for max. 6 months before use. We can isolate enough lasermicrodissected motor neurons from one single spinal cord (4000 cells) to obtain sufficient total RNA (150 ng) for one GeneChip (2-round amplification). Amplifications are performed in parallel for all the 8 samples per timepoint, so are the hybridizations. For the embryonic rat motor neurons biological quadruplet samples (4) were done, with 4 individual preparations consisting of both the mutant SOD1 and the wildtype SOD1 motor neurons isolated on the same day. Rats are from an outbred Sprague-Dawley strain and for both the mice and the rats the levels of overexpression between the mutant and the wildtype SOD1 are similar.

Replicate Types:

biological_replicate

Experimental Factors:

Conditions that are tested in the experiment. At least one is required. Experimental factors are the independent variables in the experiment.

Factor Name	Description	Factor Category
temporal changes during the disease between wildtype and mutant	For mice: Analyze the mutant SOD1 ind...	disease_state

Project Samples

Samples associated with this project.

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Name	Description	Bio-Source	Extracts
SOD1-G37R-8w-Rep1	mutant SOD1 - biological re...	mutant hSOD1 8w G37R	1
SOD1-G37R-8w-Rep2	mutant SOD1 - biological re...	mutant hSOD1 8w G37R	1
SOD1-G37R-8w-Rep3	mutant SOD1 - biological re...	mutant hSOD1 8w G37R	1
SOD1-WT-8w-Rep1	wildtype SOD1 - biological ...	wildtype hSOD1 8w	1
SOD1-WT-8w-Rep2	wildtype SOD1 - biological ...	wildtype hSOD1 8w	1
SOD1-G85R-7.5m-Rep1	mutant SOD1 - biological re...	mutant hSOD1 7.5m G85R	1
SOD1-WT-8w-Rep3	wildtype SOD1 - biological ...	wildtype hSOD1 8w	1
SOD1-G37R-15w-Rep1	mutant SOD1 - biological re...	mutant hSOD1 15w G37R	1
SOD1-G37R-15w-Rep2	mutant SOD1 - biological re...	mutant hSOD1 15w G37R	1
SOD1-G85R-7.5m-Rep2	mutant SOD1 - biological re...	mutant hSOD1 7.5m G85R	1
SOD1-G37R-15w-Rep3	mutant SOD1 - biological re...	mutant hSOD1 15w G37R	1
SOD1-G85R-7.5m-Rep3	mutant SOD1 - biological re...	mutant hSOD1 7.5m G85R	1
SOD1-G37R-15w-Rep4	mutant SOD1 - biological re...	mutant hSOD1 15w G37R	1
SOD1-WT-15w-Rep1	wildtype SOD1 - biological ...	wildtype hSOD1 15w	1
SOD1-G85R-7.5m-Rep4	mutant SOD1 - biological re...	mutant hSOD1 7.5m G85R	1
neg-control-7.5m-Rep1	negative littermate to muta...	negative control 7.5m	1
SOD1-WT-15w-Rep2	wildtype SOD1 - biological ...	wildtype hSOD1 15w	1
neg-control-7.5m-Rep2	negative littermate to muta...	negative control 7.5m	1
SOD1-WT-15w-Rep3	wildtype SOD1 - biological ...	wildtype hSOD1 15w	1
neg-control-7.5m-Rep3	negative littermate to muta...	negative control 7.5m	1
neg-control-7.5m-Rep4	negative littermate to muta...	negative control 7.5m	1
SOD1-G85R-9m-Rep1	mutant SOD1 - biological re...	mutant hSOD1 9m G85R	1
SOD1-G85R-9m-Rep2	mutant SOD1 - biological re...	mutant hSOD1 9m G85R	1
SOD1-G85R-9m-Rep3	mutant SOD1 - biological re...	mutant hSOD1 9m G85R	1
neg-control-9m-Rep1	negative littermate to muta...	negative control 9m	1
neg-control-9m-Rep2	negative littermate to muta...	negative control 9m	1
neg-control-9m-Rep3	negative littermate to muta...	negative control 9m	1

Project Hybridizations

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Project Reference Files

File Name	Size
NINDS-LM-EP105-9Mon-G85RMT-aM430A.CEL	11.79 Mb
NINDS-LM-EP105-9Mon-G85RMT-aM430A.CHP	13.86 Mb
NINDS-LM-EP105-9Mon-G85RMT-aM430A.EXP	1.04 Kb
NINDS-LM-EP105-9Mon-G85RMT-aM430B.CEL	11.69 Mb
NINDS-LM-EP105-9Mon-G85RMT-aM430B.CHP	13.79 Mb
NINDS-LM-EP105-9Mon-G85RMT-aM430B.EXP	1.04 Kb
NINDS-LM-EP64-7.5Mon-G85RMut-aM430A.CHP	288.92 Kb
NINDS-LM-EP91-9Mon-G85RWT-aM430A.CEL	11.78 Mb
NINDS-LM-EP91-9Mon-G85RWT-aM430A.CHP	13.86 Mb
NINDS-LM-EP91-9Mon-G85RWT-aM430A.DAT	19.0 Mb
NINDS-LM-EP91-9Mon-G85RWT-aM430A.EXP	489.0 bytes

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