Targeted deletion of EPO and EPOR in pyramidal neurons attenuates hypoxia- hypoxia-induced motor learning and endurance

To pinpoint the importance of EPOR in pyramidal neurons for the enhanced motor learning and endurance, mice with targeted knockout of EPOR in pyramidal neurons were generated (NexCre::EPOR^fl/fl; as termed as EPOR-cKO). To generate this mouse model, the present work first validated whether the generated EPOR-cKO mice was indeed a functional knockout. Therefore, the female EPOR^fl/fl mice was cross-bred with male mice homozygous for the Cre-recombinase gene under the control of the adenovirus EllA-promoter, which was used as a deleter mice. EllA-regulated Cre-recombinase was expressed in pre-implantation embryos leading to site specific deletion of LoxP flanked (fl) sequence in all tissues including the germ cells. This cross breeding generated the first familial generation (F1) of pups consisting of one EPOR allele with wild-type sequence (EPOR^+/+) and one EPOR deleted allele (EPOR^+/del). Since the EPOR^+/del allele was present in the germ cells, a F2 cross resulted in pups possessing all possible genotypes (EPOR^+/+; EPOR^+/del and EPOR^del/del). Interbreeding of these first generation progenies resulted in efficient germ line transmission of the deletion to subsequent generations. The presence of deleted alleles were determined by PCR based genotyping using specific primers as listed in Methods (Fig. 49B). As global deletion of EPOR is known to be lethal by E13.5 (Lin et al., 1996; Wu et al., 1995), the global EPOR-cKO embryos at E12.5 were observed to be smaller and without vasculature due to the global knockout as compared to their WT littermates (Fig. 49A).

A global EPOR-cKO was observed at E12.5, thereby validating the functionality of the

DEBIA WAKHLOO 118 generated EPOR-cKO mice. After validation, the EPOR^fl/fl mice were cross-bred with a Cre-recombinase mouse under the control of Nex promoter. This cross breeding resulted in a conditional deletion of EPOR specifically in mature pyramidal neurons.

Since previously, an improved learning performance of WT mice on CRW was observed, this thesis considered a hypothesis whether this effect is attenuated with the conditional knockout of EPOR in pyramidal neurons. To achieve this, NexCre::EPOR-KO mice were exposed to CRW in normoxic or hypoxic conditions from P28-P48 followed by 1-week break (Fig. 49C; 49H).

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DEBIA WAKHLOO 120 A significant reduction in the number of neurons (normalized to their respective NR controls) in the CA1 of NexCre::EPOR-KO mice was quantified as compared to wild-type mice exposed to either normoxic (Fig. 49D) or hypoxic conditions (Fig. 49F).

Subsequently, lack of EPOR in pyramidal neurons resulted in a distinct and significant reduction of the learning curve slope over time in normoxic (Fig. 49E) as well as hypoxic conditions (Fig. 49G) compared to the respective WT controls. To further pinpoint that this enhanced learning is due to the EPO present within pyramidal neurons, NexCRE::EPO-KO mice were generated. These mice consisted of knockout of EPO specifically in pyramidal neurons. These mice along with control wild-type were subjected to endogenous hypoxia via complex running wheel. Similar to results observed in NexCre::EPOR-KO mice, significant defects in learning in NexCre::EPO-KO mice (Fig. 49I) were observed as compared to the wild-type mice. Together, these data suggests the importance of EPO and EPOR expression in pyramidal neurons to enhance learning in mice.

Figure 49. Voluntary running induced learning is mediated by EPOR. (A) Representative pictures of Wildtype and E2A-Cre global KO in e12.5 embryos. Scale bars: 25µm. (B) Genotyping results depicts the functional KO using specific primers. (C) Experimental schematic to determine the effect of endogenous hypoxia on learning. WT and NexCre::EPOR-KO mice (at P28) were exposed to NR or CRW under normoxia (21%O2) or hypoxia (12%O2) for 3 weeks. After 1 week of break, CRW mice were again exposed to voluntary CRW for 4h before being sacrificed at P55. (D; F) Quantification of Ctip2 positive neurons (normalized to their respective NR controls) present in the CA1 region of the hippocampus of runner (CRW) exposed to either normoxic (D) or hypoxic (F) condition. Data represents average number of cells ± SEM per mm². Quantification performed from 3-8 independent mice and p-values presented via unpaired Student’s t-test. (E; G) Percent of distance run per night (normalized to mean distance run over the first 3 nights) over 17 nights by WT and NexCre::EPOR-KO (in collaboration with Franziska Scharkowski) mice exposed to normoxia or hypoxia. Data presented as mean ± SEM. Quantification performed from 7-16 independent mice and p-values presented via 2-way repeated measures ANOVA.

(H) Experimental schematic to determine the effect of endogenous hypoxia on learning. WT and NexCre::EPO-KO mice (at P28) were exposed to CRW for 3 weeks. After 1 week of break, the mice were sacrificed at P55. (I) Percent of distance run per night (normalized to mean distance run over the first 3 nights) over 17 nights by WT and NexCre::EPO-KO mice. Data presented as mean ± SEM. Quantification performed from 5-7 independent mice and p-values presented via 2-way repeated measures ANOVA.

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Discussion

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5. Discussion.

Although several reports have provided individual facets of the brain EPO system, the overall view was lacking. This work contributes to the broader view by providing evidence of a novel role of EPO as a potent growth factor, which acts as a local mediator of neuronal adaptation, presumably in response to increased network activity. A new working model is proposed where cognitive challenge leads to physiological hypoxia which upregulates EPO expression in the pyramidal neurons. This induced endogenous hypoxia leads to enhanced dendritic spine growth and precursors to differentiate into neurons. This observation of substantial numbers of newly generated pyramidal neurons without proliferation may change our present concepts of adult hippocampal neurogenesis.