In light of the fact that in Chapter 4, I found that overexpressing Vps35 led to an exacerbation of the ALS-like phenotype in SOD1G93A-Tg mice, I then wondered what the effect of
deleting Vps35 might be in this mouse model of ALS. To address this question, I employed a Cre- lox-based conditional mouse Vps35 knockout line generated by the lab of Dr Scott Small, which they graciously shared with me for this work. This mouse was built with LoxP sites flanking exons 3-5 of Vps35. Cre-recombinase can therefore induce a deletion of these exons of Vps35, resulting in the failure of producing Vps35 protein. For their work in Alzheimer’s disease (AD), they have
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crossed these mutant mice with mice expressing Cre under the forebrain neuronal CamKIIα promoter. They found that the generated mice are viable and have a knockout of Vps35 in forebrain neurons as seen in IHC (Fig. 5.1A). Their Western blot of whole forebrain lysate also shows a decrease in Vps35 protein levels—albeit modest, probably due to a dilution effect of other cell types—as well as a secondary decrease in other retromer components (Fig. 5.1B, C). This decrease in levels of Vps26 and Vps29 secondary to the deletion of Vps35 replicates the previously seen effects of Vps35 knockdown in cultured neurons (Bhalla, Vetanovetz et al. 2012).
Figure 5.1 Successful Conditional Knockout of Vps35 in Neurons
Data and figure provided by the Small Lab. Mice with a heterozygous conditional knockout Vps35 gene were bred to express Cre under the forebrain neuronal CamKIIα promoter. IHC for Vps35 shows a lack of Vps35 expression in neurons of the hippocampus (A). A Western blot for Vps35 was run on hippocampal samples from these mice and controls. A representative blot is presented here (B) with quantification of 7 replicates per group (C). Results presented as mean ± SEM. n=7, **p<0.01, ***p<0.001 in unpaired Student t-test
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In Chapter 2, I found a retromer deficiency in both MNs and astrocytes of the SOD1G93A-
Tg mouse, indicating that the effects of retromer dynamics in this mouse may not be limited to a single cell type. Thus, I chose to study the effect of a complete heterozygous deletion of Vps35 on the disease progression of SOD1G93A-Tg mice, rather than knocking out Vps35 in any single cell type. To create a Vps35 heterozygote mouse from the floxed Vps35 line, I first crossed it with a mouse expressing Cre under the CMV promoter. This CMV-Cre gene induces recombination across cell types in the mouse, including, importantly, the gametes. This cross, thus, resulted in a mouse that was effectively a Vps35 heterozygote. I then crossed this resulting mouse with an NTg mouse to separate out the Vps35 null allele from the CMV-Cre transgene, resulting in a true Vps35 heterozygote with no other genetic alterations. Finally, the cross between the Vps35 heterozygous and the SOD1G93A-Tg mice resulted in the mice I ultimately used for my experiments in this Chapter. A visual schematic of the breeding paradigm for these mice is provided in Figure 5.2.
It is important to note here that all experiments done in the SOD1G93A-Tg mouse in
Chapters 2-4 have been performed on mice with the mixed B6/SJL background. However, the fact that the founder conditional Vps35 knockout mice produced by the lab of Dr Scott Small were produced in the B6 background necessitated for these experiments to be undertaken in SOD1G93A- Tg mice with the B6 background. These mice have been shown to have a similar, though slowed disease progression as compared to those with the B6/SJL background (Heiman-Patterson et al. 2005).
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Figure 5.2 Breeding Scheme for Vps35 Heterozygous SOD1G93A-Tg Mice
Mice homozygous for the conditional knockout floxed (fl) allele of Vps35 were bred with mice expressing Cre under the CMV promoter (CMV-Cre-Tg). Of the resulting mice, in those that expressed CMV-Cre transgene, Cre induced recombination of the floxed Vps35 allele, resulting in a tissue-wide heterozygous knockout. These were then crossed with a NTg mouse, resulting in, among other genotypes, a Vps35 heterozygous mouse with no transgene. This mouse was then bred with a SOD1G93A-Tg mouse, and those that expressed the SOD1G93A transgene were used in the
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Before, reporting the results for the cross between Vps35 conditional mutant and SOD1G93A-Tg mice, it is worth mentioning the effects that a widespread deletion of 1 or 2 alleles for Vps35 using a CMV-Cre mouse line might have on the lower motor neuron pathway. I was consistently unable to obtained viable Vps35-/- pups by genotyping more than 6 litters, consistent with the previous reports showing that abrogating Vps35 in mice is lethal (Wen et al. 2011). In contrast, I found that Vps35+/- pups were viable and were indistinguishable from their Vps35+/+
littermates in terms of their morphometry and development. More importantly, up to 1.5 years, Vps35+/- mice did not show any evidence of clinical ALS-like phenotype.
Figure 5.3 Hemizygous Vps35 deletion decreases retromer protein levels
A western blot was run comparing retromer protein expression levels between Vps35 heterozygous and Vps35 homozygous wild-type SOD1G93A-Tg mice at P120 (A) and quantified (B). Results presented as mean ± SEM. n=4,
**p<0.01, ***p<0.001 in unpaired Student t-test
Previous studies have shown that the single allele deletion of Vps35 in mice produced a robust decrease in the CNS in levels of expression of not only Vps35, but also of Vps26a (Tang, Erion, et al. 2015). I confirmed that this was still the case if the heterozygote mouse also expressed the SOD1G93A transgene via a Western blot of SCs from Vps35 wild-type SOD1G93A-Tg and Vps35
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heterozygote SOD1G93A-Tg mice at P120. Indeed, the Vps35 heterozygote mice displayed a robust
decrease in both Vps35 (Student t-test, p=0.0016) and Vps26a (Student t-test, p=0.00028) expression in the SC (Fig. 5.3). Of note, protein expression was not compared between Vps35+/- ;SOD1G93A-Tg and Vps35+/+;NTg mice. However, it can be deduced that the changes found in the present experiment compound with those found in Chapter 2 between SOD1G93A-Tg and NTg mice to produce an even greater retromer expression decrease.