Identification of glyoxalase-I as a protein marker in a mouse model of extremes in trait anxiety

Abstract

For >15 generations, CD1 mice have been selectively and bidirectionally bred for either high-anxiety-related behavior (HAB-M) or low-anxiety-related behavior (LAB-M) on the elevated plus-maze. Independent of gender, HAB-M were more anxious than LAB-M animals in a variety of additional tests, including those reflecting risk assessment behaviors and ultrasound vocalization, with unselected CD1 "normal" control (NAB-M) and cross-mated (CM-M) mice displaying intermediate behavioral scores in most cases. Furthermore, in both the forced-swim and tail-suspension tests, LAB-M animals showed lower scores of immobility than did HAB-M and NAB-M animals, indicative of a reduced depression-like behavior. Using proteomic and microarray analyses, glyoxalase-I was identified as a protein marker, which is consistently expressed to a higher extent in LAB-M than in HAB-M mice in several brain areas. The same phenotype-dependent difference was found in red blood cells with NAB-M and CM-M animals showing intermediate expression profiles of glyoxalase-I. Additional studies will examine whether glyoxalase-I has an impact beyond that of a biomarker to predict the genetic predisposition to anxiety- and depression-like behavior.

Figure 1.

EPM data (percentage of time open arms) of the parental male and female CD1 mice (8+SEM and range) and G1-G15 generations of male and female HAB-M and LAB-M mice. CD1 mice selected as controls independent of their performance (NAB-M) are shown for comparison (G11, G13-G15). Male animals are indicated by solid lines and filled rhombs and circles, and females are indicated by dotted lines and empty rhombs and circles. Male and female NAB-M animals are displayed as bigger and smaller crosses, respectively. Independent of gender, HAB-M and LAB-M animals differed significantly in their anxiety-related behavior (G4-G15), and NAB-M differed significantly from both HAB-M and LAB-M animals (n = 40-80 per line and generation; ^***p < 0.001).

Figure 2.

EPM behavior of male and female HAB-M, NAB-M, CM-M, and LAB-M animals. A, B, Percentage of time spent on the open arms (A) and latency to the first entry into an open arm (B). ^***p < 0.001 versus HAB-M; ^###p < 0.001 versus LAB-M; ⁺p < 0.05 versus NAB-M. Results represent data from G13-G14.

Figure 3.

Behavioral data of male and female HAB-M, NAB-M, and LAB-M animals in the dark-light avoidance test. A, B, Percentage of time spent in the lit compartment (A) and number of rearings (B). Number of rearings were measured as total within both the lit and dark compartments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus HAB-M; ^##p < 0.01, ^###p < 0.001 versus LAB-M. Results represent data from G16-G17.

Figure 4.

A, B, Immobility time of male and female HAB-M, NAB-M, and LAB-M mice in the tail-suspension test (A) and of HAB-M, NAB-M, CM-M, and LAB-M mice in the forced swim test (B). ^**p < 0.01, ^***p < 0.001 versus HAB-M; ^###p < 0.001 versus LAB-M. Results represent data from G14-G15.

Figure 5.

A-C, Correlation analyses of anxiety- and depression-like indices (A, percentage of time open-arms EPM vs immobility time in the tail-suspension test; B, percentage of time open-arms EPM vs floating time in the forced swim test) and between depression-like indices (C, immobility time in the tail-suspension test vs floating time in the forced swim test). Data from male and female animals of G14-G15 are shown.

Figure 6.

Representative 2D PAGE of amygdala protein extracts from a male HAB-M animal (A) and a male LAB-M animal (B). The difference in the expression of Glx1 is indicated by the arrow. The pH gradient of the first-dimension isoelectric focusing is indicated at the bottom of the gels. Results represent data from G12; a total of eight animals were analyzed for each group.

Figure 7.

Western blot analysis of red blood cell protein extracts from male HAB-M, NAB-M, CM-M, and LAB-M animals. Red cell extracts representing equal amounts of total protein (100 μg) were run on a 12% SDS gel and transferred to a PVDF membrane. Subsequently, the membrane was probed with an anti-Glx1-specific antibody and developed. The immunoreactive Glx1 protein bands are shown (A). The signal volumes of the bands obtained from Western blot analysis in A were quantified with a densitometer, and means + SEM of optical density for each animal group are shown (B). ^**p < 0.01, ^***p < 0.001 versus HAB-M; ^###p < 0.001 versus LAB-M. Results represent data from G14-G17.

Figure 8.

Validation of Glx1 as a biomarker of trait anxiety in inbred mouse strains differing in their EPM behavior (percentage of time open arms). Male BALB/c mice expressed more Glx1 than the more anxious male C57BL/6 mice (C57BL/6J), thus supporting the data presented in Figure 7. ^**p < 0.05, ^***p < 0.01 versus C57BL/6.

Figure 9.

Predictive validity of Glx1 as a biomarker of trait anxiety. Based on Glx1 expression in (unknown) blood samples, the HAB-M (H) versus LAB-M (L) phenotypes could unambiguously be identified in a blind manner. The results of 17 samples from Western blot analysis using an anti-Glx1-specific antibody are shown. Results represent data from G16-G17.

Figure 10.

Microarray analysis of Glx1 gene (Glo1) expression. Chromosomal origin of the Glx1 gene, location of the cDNA probes used in the microarrays, and Glx1 mRNA expression in the hypothalamic PVN of male LAB-M and HAB-M mice; n = 3 each; PVNs from 2 animals were pooled into one sample) are shown. As the fold regulation values are backtransformed from a logarithmic scale, the value for HAB-M is 1.0; all other values represent the fold gene regulation in LAB-M compared with HAB-M. The design is based on Ensembl (www.ensembl.org); data are from the National Center for Biotechnology Information (www.ncbi.n-lm.nih.gov). Results represent data from G18.