Deficiency of the Endocytic ProteinHip1Leads to DecreasedGdpd3expression, Low Phosphocholine, and Kypholordosis

Abstract

47Deficiency of huntingtin interacting protein 1 (Hip1) results in degenerative phenotypes. 48Here we generated a Hip1 deficiency allele where a floxed transcriptional stop-cassette 49 and a human HIP1 cDNA were knocked-in to intron 1 of mouse Hip1 locus. CMV-Cre-50 mediated germline excision of the stop-cassette resulted in expression of HIP1 and rescue 51 of the Hip1 knockout phenotype. Mx1-Cre-mediated excision led to HIP1 expression in 52 spleen, kidney and liver, and also rescued the phenotype. In contrast, GFAP-Cre-53 mediated HIP1 expression in brain did not rescue the phenotype. Metabolomics and 54 microarrays of several Hip1 knockout tissues identified low phosphocholine (PC) levels 55 and low Glycerophosphodiester Phosphodiesterase Domain Containing 3 (Gdpd3) 56 expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of 57 choline, a precursor of PC, Gdpd3 downregulation could lead to the low PC levels. To 58 test if Gdpd3 contributes to the Hip1 deficiency phenotype, we generated Gdpd3 59 knockout mice. Double knockout of Gdpd3 and Hip1 worsened the Hip1 phenotype. This 60 suggests that Gdpd3 compensates for Hip1 loss. More detailed knowledge of how Hip1 61 deficiency leads to low PC will improve our understanding of HIP1 in choline 62 metabolism in normal and disease states. 63 64 65 amplified from pcDNA3-hHip1 plasmid. The IRES (~0.6 kb) and eGFP (~0.9 kb) 128 sequences were amplified from pIRES2-AcGFP1 and pEGFP-N1 plasmid, respectively. 129The final vector was obtained by standard molecular cloning. Aside from the 130 homology arms, the final vector also contains a Lox-Stop-Lox cassette (~1.5 kb), mHip1 131 partial intron1+ partial exon 2, hHip1 cDNA, IRES, EGFP + polyA, Frt sequences flanking 132 the Neo expression cassette (the neo cassette was used for positive selection of the 133 electroporated ES cells), and a DTA expression cassette (for negative selection of the ES 134