Astroglial transcriptome dysregulation in early disease of an ALS mutant SOD1 mouse model

Figures & data

Figure 1. Heatmaps from early- and mid-symptomatic G93A SOD1 astroglia show dramatic gene dysregulation. (A) Early onset P60 control vs mutant SOD1 astroglia display differential gene expression, (B) Mid onset P90 control vs mutant SOD1 astroglia display differential gene expression. A total of four mice were used for each time point and all probes being illustrated.

Figure 2. Disease astroglia display a strong trend in genes differentially expressed during disease progression. (A) Correlation plot shows genes most correlated being upregulated and downregulated in control vs mutant SOD1 at both time points. (B) Venn diagram displays each time point of isolated astroglia and time points combined to illustrate genes unique to each and genes showing similar trend of differentiation. A fold change of 1.5 and p values of .05 was used to allow for stringent parameters.

Table 1. Top differentially enriched genes in pre-clinical P60 ALS spinal cord astroglia.

Gene	FC (Control vs SOD1)	Function
KCNK1	5.872	Potassium channel on astroglia that can heterodimerize
PIK3C2B	4.672	Phosphoinositide 3-kinase family member involved in cell signaling
SDC3	4.538	Syndecan proteoglycan family member playing a role in cell shape organization
PLP1	4.123	Transmembrane proteolipid protein that is the predominant component of myelin
FAM107A	4.074	Expression in cells suppresses cell growth, potential involvement in tumor development
SPRY2	−5.437	Sprouty family protein essential for the inhibition of receptor tyrosine kinase signaling proteins
SLC41A1	−4.702	Na+/Mg2+ exchanger important in magnesium homeostasis
LUC7L3	−4.689	Pre-mRNA splicing factor found in the nucleus
DLG4	−4.658	Localizes to NMDA and potassium channel clusters; may act as a multimeric scaffold at postsynaptic sites
RHOA	−4.388	Member of the Rho family of small GTPases

Microarray results were subjected to two-way ANOVA and statistical analyses. Top candidates were selected based on their enrichment in the age-matched ALDH1L1-eGFP astroglia from mutant SOD1 as compared to control astroglia. Functional annotation was obtained from GeneCards.org. A complete list of enriched genes can be found in Supplemental Table 2.

Table 2. Top differentially enriched genes in P90 disease onset ALS spinal cord astroglia.

Gene	FC (Control vs SOD1)	Function
SPARCL1	12.424	Calcium ion binding
MALAT1	11.496	Gene results in two products: a mascRNA and a molecular scaffold in the nucleus for ribonucleoprotein complexes.
HSD11B1	6.860	Enzyme responsible for converting cortisol to cortisone and the reverse reaction.
MARCKS	6.541	Acts as an actin crosslinking protein and substrate for protein kinase C
GJA1	6.310	Component of gap junctions and member of the connexin family.
CTSS	−17.732	Lysosomal proteinase
RPS8	−6.448	Ribosomal protein, component of subunit 40s
Tmsb4x	−6.215	Actin sequestering protein involved in cell proliferation, migration, and differentiation
B2M	−6.194	Serum protein associated with MHC Class I heavy chain
SSR3	−5.895	Subunit of the signal sequence receptor of the endoplasmic reticulum (ER) involved in protein translocation across the ER membrane

Microarray results were subjected to two-way ANOVA and statistical analyses. Top candidates were selected based on their enrichment in the age-matched ALDH1L1-eGFP astroglia from mutant SOD1 as compared to control astroglia. Functional annotation was obtained from GeneCards.org. A complete list of enriched genes can be found in Supplemental Table 3.

Table 3. Top differentially regulated pathways in the P60 ALDH1L1-eGFP astroglia from mutant spinal cord compared to the control SOD1 G93A spinal cord.

Pathway	p Values	SOD1 vs Control
PI3K/AKT signaling	1.98E-05	Down in mutant
P70S6K signaling	1.74E-04	Down in mutant
Calcium transport I	1.44E-03	Up in mutant
Glutamate receptor signaling	2.36E-03	Up in mutant

Microarray results were subjected to two-way ANOVA and statistical analyses. Post-analyses candidate genes were subjected to Ingenuity Pathway Analysis. Top pathways differentially affected are listed in the table.

Table 4. Top differentially regulated pathways in the P90 ALDH1L1-eGFP astroglia from mutant spinal cord compared to the control SOD1 G93A spinal cord.

Pathway	p Values	SOD1 vs Control
Tight junction signaling	1.20E-04	Down in Mutant
Oxidative phosphorylation	4.90E-04	Down in Mutant
EIF2 signaling	8.54E-08	Up in Mutant
mTOR signaling	3.76E-08	Up in Mutant

Microarray results were subjected to two-way ANOVA and statistical analyses. Post-analyses candidate genes were subjected to ingenuity pathway analysis. Top pathways differentially affected are listed in the table.

Figure 3. Cellular compartmentalization shows unique preference at early- and mid-disease onset. (A, B) P60 and P90 showed that the highest amount of hits to be differentially regulated compared to control resided in the cytoplasm and nucleus, respectively. Stringent parameters of fold change of 1.5 and p values of 0.05 was used for this analyses and ingenuity pathway analyses for identification of comparts of top hits.

Figure 4. STRING map analysis to determine interacting proteins for highly enriched markers. (A,B) Kcnk1 and Spry2 have several established interacting proteins with some not detected (n.d.) and others detected in our microarray results. Detected proteins are represented by fold-change value (FC ±), control vs mutant SOD1.