Deletion of PREPl causes growth impairment and hypotonia in mice

Abstract

Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL(-/-)) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL(-/-) mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL(+/+)) counterparts. A righting assay revealed that PREPL(-/-) pups took significantly longer than PREPL(+/+) pups to right themselves when placed on their backs. This deficit indicates that PREPL(-/-) mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease.

Figure 1. Construct design and validation.

A) The genome region around PREPL's exon 11 was used to design a targeting vector for homologous recombination. This vector included a neomycin (NEO) cassette for positive selection, Frt sites flanking the NEO cassette, LoxP sites flanking exon 11, a HindIII restriction site for 5′-Southern blots, and an NdeI site for 3′-Southern blots. Homologous recombination in ES cells generated cells carrying recombinant genomic DNA, which according to standard nomenclature (Gene^tm#Labcode) is referred to as Prepl^tm1Sagh. These ES cells were then used to generate chimeric mice designated Prepl^+/tm1Sagh. B) The germ line transmission of the Prepl^tm1Sagh allele to the offspring from chimera-C57BL/6J crosses was confirmed by Southern blots of tail genomic DNA. Restriction digests of the genomic DNA with HindIII (5′) or NdeI (3′), followed by probing with a 5′ or 3′ specific probe generated a single band for Prepl^+/+ (left lane), whereas an additional, lower molecular weight band is generated in mice carrying the Prepl^tm1Sagh allele (right lane). C) Prepl^+/tm1Sagh were then crossed with mice expressing Flp recombinase to produce Prepl^+/tm1.1Sagh mice (middle) lacking the NEO cassette, and finally crossed with mice ubiquitously expressing Cre recombinase, resulting in Prepl^+/tm1.2Sagh mice (bottom) which lack exon 11. We refer to these Prepl^+/tm1.2Sagh mice as PREPL^+/− mice. D) Representative PCR genotyping results for PREPL^+/+, PREPL^−/− and PREPL^+/− mice. Using this strategy, one can easily distinguish these three genotypes as well as other possible genotypes, such as the Prepl^tm1.1Sagh allele.

Figure 2. Homozygous PREPL^−/− mice lack PREPL and have diminished Prepl mRNA levels.

A) Western blot for PREPL in the spinal cords of PREPL^+/+ and PREPL^−/− mice. Recombinant PREPL (lane 1) is used as a positive control to test the polyclonal antibody used to in this blot. Analysis of spinal cord samples from PREPL^−/− (lanes 2–4) and PREPL^+/+ (lanes 5–7) demonstrate the PREPL^−/− mice lack full-length PREPL protein. Removal of exon 11 could result in a truncated PREPL protein, but no evidence of a shorter protein is evident in the western blot of PREPL^−/− samples (lanes 2–4). B) Analysis of Prepl mRNA by qPCR in mouse liver reveals that there is substantially less Prepl transcript in the liver of PREPL^−/− mice when compared to PREPL^+/+, indicating that removal of exon 11 also impacts mRNA levels. (Error bars depict SEM. Statistical significance calculated by a Student's t-test, p-value<0.05, *, N = 4).

Figure 3. PREPL^−/− mice have diminished growth rates and are smaller than PREPL^+/+ animals.

A) Body lengths of one-month old PREPL^−/− and PREPL^+/+ mice were compared. Both female and male PREPL^−/− mice are visibly smaller than PREPL^+/+ mice. B) Female and male mice of both genotypes were weighed every day for the first forty days after birth to obtain growth curves. Female PREPL^−/− mice begin to gain less weight than PREPL^+/+ mice at about three weeks old, with the data being statistically significant at day 26, while male PREPL^−/− mice show significantly less weight gain than PREPL^+/+ mice from postnatal day 21. (Error bars show SEM and significance calculated using the Holm-Sidak method to correct for multiple comparisons, alpha = 5%, each row was analyzed individually, without assuming a consistent SD).

Figure 4. Grip strength and righting assay to assess hypotonia in PREPL^−/− mice.

A) Six month old female mice had no difference in grip strength between genotypes when normalized to their respective body weights. B) A comparison of pups of both genders and genotypes in a righting assay. In this experiment, mice are placed on their backs and the time it takes for them to right themselves is measured. At day 5, PREPL^−/− pups are much slower at righting themselves than PREPL^+/+ mice. (Error bars show SEM and Statistical significance calculated by a Student's t-test, p-value<0.05, *, N = 4–5 for grip strength and N = 13–14 for the righting assay).