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The hGFAP-driven Conditional TSPO Knockout Is Protective in a Mouse Model of Multiple Sclerosis

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The hGFAP-driven Conditional TSPO Knockout Is Protective in a Mouse Model of Multiple Sclerosis

Daniel J Daugherty et al. Sci Rep.

Abstract

The mitochondrial translocator protein (TSPO) has been implicated in CNS diseases. Here, we sought to determine the specific role of TSPO in experimental autoimmune encephalomyelitis (EAE), the most studied animal model of multiple sclerosis (MS). To fundamentally elucidate the functions of TSPO, we first developed a viable TSPO knockout mouse. A conditional TSPO knockout mouse was generated by utilizing the Cre-Lox system. We generated a TSPO floxed mouse, and then crossed this mouse with a Cre recombinase expressing mouse driven by the human glial fibrillary acidic protein (hGFAP) promoter. The resultant mouse was a neural linage line specific TSPO knockout. The loss of TSPO in the CNS did not result in overt developmental defects or phenotypes. The TSPO-/- mouse showed a decrease in GFAP expression, correlating with a decrease in astrogliosis in response to neural injury during EAE. This decrease in astrogliosis was also witnessed in the lessening of severity of EAE clinical scoring, indicating an in vivo functional role for TSPO in suppressing EAE. The TSPO-/- mouse could be a useful tool in better understanding the role of TSPO in CNS disease, and our results implicate TSPO as a potential therapeutic target in MS.

Figures

Figure 1
Figure 1. Generation of a conditional allele, via a sequence replacement strategy to knock-out the Tspo gene.
The construct contains loxP sites that flank exons 2 & 3, a 2.8 kb 5′ short arm of homology, a 7.3 kb 3′ long arm of homology, a Diphtheria Toxin A (DTA) cassette, and a Neomycin (Neo) cassette flanked by frt sites for selective deletion. The Neo element allows for positive selection in ES cells, while the DTA element permits negative selection in ES cells. After homologous recombination of our conditional knock-out construct, the PGK-Neo can be excised via Flp-e electroporation. The Tspo gene has normal expression until Cre-mediated deletion of exons 2 & 3. This recombination creates a frameshift mutation and a premature stop, rendering the Tspo gene inactive.
Figure 2
Figure 2. Cre and TSPO expression in the CNS in TSPO−/− mice.
Cre expression was observed in co-localization with GFAP + astrocytes and neural precursors in the brain (A) and spinal cord (B) in TSPO−/− mice. TSPOFlox/Flox mice showed normal expression of TSPO (C), and the expression was strongest in the central canal (B). TSPO−/− mice showed a marked decrease in TSPO expression (C), particularly evident around the central canal (B). Cre expression was seen in TSPO−/− mice, but not in TSPOFlox/Flox mice (B,C). TSPO expression was generally low in the normal mouse CNS (D). There was a marked increase in TSPO expression in response to EAE (D). TSPO expression was seen in cells of the non-neural linage, microglia (arrows) and endothelial cells (arrowheads), in the hGFAP-driven conditional TSPO−/− mice (E). CC, corpus callosum; LV, lateral ventricle; SVZ, subventricular zone. Scale bars: 20 μm for E; 50 μm for A and B; 100 μm for C and D.
Figure 3
Figure 3. Clinical scores of TSPOFlox/Flox and TSPO−/− mice during EAE.
Clinical scores during EAE were decreased in the TSPO−/− group compared to the TSPOFlox/Flox group. There was a significant difference in the groups starting at day 16 and continuing throughout the experiment (n = 8 animals per group).
Figure 4
Figure 4. GFAP expression in the CNS in normal mice and during EAE.
GFAP expression in normal TSPOFlox/Flox (A) and TSPO−/− (B) mice was seen throughout the CNS. GFAP expression was contained to the grey and white matter astrocytes and the cells of the central canal. There was no Cre expression seen in TSPOFlox/Flox (A) while Cre + cells were present in TSPO−/− mice (B). GFAP expression was increased in EAE mice (C). TSPOFlox/Flox mice showed an increase in GFAP and TSPO expression, in particular within the cells of the central canal. Central canal cells also co-localized with TSPO, along with astrocytes within the grey matter of the spinal cord. TSPO−/− mice retained lower GFAP expression upon induction of EAE, and TSPO expression was not seen as increased in GFAP expressing cells (D). Scale bars: 100 and 50 μm for (A,B) 100, 100, 50 and 50 μm for (C,D). Representative Western Blot shows increase in GFAP protein level in EAE mice with a reduction seen in TSPO−/− group (E). Quantitative analysis of GFAP protein shows dramatic increase in GFAP expression in TSPOFlox/Flox mice after EAE. Reduced GFAP expression is detected in TSPO−/− mice (F). n = 7; *p < 0.05; **p < 0.01.
Figure 5
Figure 5. mRNA expression during EAE of TSPOFlox/Flox and TSPO−/− mice.
There was a significant decrease in TSPO mRNA in the TSPO−/− mice compared to the TSPOFlox/Flox group. Significant decreases in mRNA levels of cytokines TNFα and CXCL10 were also seen. ***p < 0.001.

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