Genotype-Based Housing as a Potential Confounder in Studies Using Transgenic Mouse Models-Insight from the A53T Mouse Model of Parkinson's Disease

Abstract

Background/Objectives: Environmental factors, including the differences in genotype-based housing (GbH), can act as confounding variables in studies using transgenic mouse models, potentially influencing experimental outcomes and limiting their reproducibility and translational value. Despite the widespread use of transgenic models in preclinical studies, the extent to which housing conditions can affect the behavioral and molecular parameters of interest remains poorly understood. This study aims to investigate how different GbH conditions influence visuo-spatial memory and gene expression in the A53T mouse model (JAX006823) of Parkinson's disease (PD) during the pre-motor phase. Methods: A53T+ transgenic male mice and their non-transgenic littermates (A53T-) were housed in either mixed-genotype (MGH) or single-genotype (SGH) environments from postnatal day (PND) 30, with C57BL/6J mice serving as the controls. A behavioral assessment using the Novel Object Recognition and Object Location Tests was conducted at PND 180, followed by a qPCR analysis of Iba1, Gfapα, Bdnf, Tnfα, Il-1β, and Il-6 expression in the medial prefrontal cortex and the hippocampus. Results: The variations in GbH influenced behavior and mRNA expression differently in the A53T+ and A53T- animals. Specifically, the A53T- mice in SGH environments displayed behavioral and molecular profiles similar to the C57BL/6J controls, while the same was not evident in the MGH environments. In the A53T+ mice, the mRNA expression of Iba1, Gfapα, Bdnf, and Tnfα was sensitive to variations in GbH, while memory impairment was not. Conclusions: This study highlights the importance of considering environmental factors in studies using transgenic animal models. The obtained data suggests that GbH can influence the parameters of interest in preclinical research, implicating the need for the optimization of future study designs.

Keywords: A53T; Parkinson’s disease; genotype-based housing; memory impairment; reproducibility; transgenic animal models; translation.

Figure 1

Animal weaning and segregation into SGH and MGH. The colony of A53T mice was maintained by backcrossing transgenic males to C57BL/6J females. A53T+ and A53T− animals were weaned in the same cage, until PND 29 ± 2 when they were segregated into SGH or MGH. C57BL/6J animals were kept in SGH as additional controls. Mice were housed in stable social groups of four per cage.

Figure 2

Graphical representation of the procedure. All animals were habituated to the testing arena from experimental day 1−4; on experimental day 5 after a 10 min habituation period to the testing arena, NORT and OLT were performed to assess short-term memory. On experimental day 10, the mPFC and HPP of animals were extracted and stored at −80 °C for subsequent qRT-PCR analysis of the expression of relevant inflammatory and neurotrophic markers (i.e., Iba1, Gfapα, Il-6, Il-1β, Tnfα, and Bdnf).

Figure 3

Expression of human SNCA transgene and endogenous mouse Snca in HPP of adult C57BL/6J, A53T−, and A53T+ male mice. (A) Normalized expression counts of human SNCA and mouse Snca across different groups shown as boxplots with overlaid data points. (B) Paired dot plot showing the expression of SNCA and Snca within individual samples. Points are connected by lines to indicate sample pairing. (C) Boxplot representing the fold change between SNCA and Snca expression (SNCA/Snca ratio) in group samples, indicating the relative expression level of the inserted transgene compared with the endogenous gene. The data are expressed as mean ± SD, with individual data plots (visible as black dots) along the column bars. Statistical analysis of differential gene expression was performed using DESeq2, applying the Wald tests with Benjamini–Hochberg correction for multiple comparisons. Differences in SNCA/Snca expression ratios were evaluated using the Wilcoxon rank-sum test. All tests were two-sided, and a significance threshold of p < 0.05 was used (n = 5 animals per group). * p < 0.05 vs. C57BL/6J group. # vs. A53T−.

Figure 4

The behavior of adult C57BL/6J, A53T−, and A53T+ male mice in the Novel Object Recognition Test (NORT) and Novel Object Location Test (OLT). The discrimination index (DI) based on the number of approaches (DIna) and exploration time (DIet) in the NORT (A,B) and in OLT (C,D) are presented for the C57BL/6J, A53T−, and A53T+ mice kept in SGH and MGH. The data are expressed as mean ± SD, with individual data plots (visible as black symbols) along the column bars (n = 8 animals per group). * p < 0.05 vs. C57BL/6J group. # vs. A53T−. The AC inside the bars indicates that the DI was significantly above chance (DI > 0.5). The dotted line symbolically separates the bar representing the performance of C57BL/6J mice from the bars representing the performance of JAX006823 strain in a given test.

Figure 5

The expression profiles of Iba1, Gfapα, Bdnf, Tnfα, Il-1β, and Il-6 genes in the HPP of adult C57BL/6J, A53T−, and A53T+ male mice. Relative expression of Iba1 (A), Gfapα (B), Bdnf (C), Tnfα (D), Il-1β (E), and Il-6 (F) genes in C57BL6/J, A53T−, and A53T+ mice kept in SGH and MGH are displayed as a percentage of change from C57BL/6J mice. Gapdh was used as the reference gene. The data are expressed as mean ± SD, with individual data plots (visible as black symbols) along the column bars (n = 8 animals per group). * p < 0.05 vs. C57BL/6J group. # vs. A53T−, and & vs. the same genotype in different housing conditions. The dotted line symbolically separates the bars representing the measures obtained from C57BL/6J mice from the bars representing measures obtained from JAX006823 strain.

Figure 6

The expression profile of Iba1, Gfapα, Bdnf, Tnfα, Il-1β, and Il-6 genes in the mPFC of adult C57BL/6J, A53T−, and A53T+ male mice. Relative expression of Iba1 (A), Gfapα (B), Bdnf (C), Tnfα (D), Il-1β (E), and Il-6 (F) genes in C57BL/6J mice, A53T−, and A53T+ mice kept in SGH and MGH are displayed as a percentage of change. Gapdh was used as the reference gene. The data are expressed as mean ± SD, with individual data plots (visible as black symbols) along the column bars (n = 8 animals per group). * p < 0.05 vs. C57BL/6J group. The dotted line symbolically separates the bars representing the measures obtained from C57BL/6J mice from the bars representing measures obtained from JAX006823 strain.