ES cells were transfected with a targeting vector designed to delete a portion of exon 1 from the proSAAS gene locus containing the transcription and translational start sites (). Targeted ES cell clones were identified by Southern blotting using a 32
P radiolabeled 5′ external genomic DNA probe and were used for blastocyst microinjection. Injection of homozygous mutant ES cells produced a germline-transmitting proSAAS chimeric male that were used to establish the proSAAS colony. The X-linkage of proSAAS required that female mice carrying the mutant allele transmit this allele to male offspring. Interbreeding of wild type males with heterozygous proSAAS females produced the expected equal frequency of male mutants (N=38/83; 46%) and heterozygous females (N=29/68; 44%). Female proSAAS homozygous mice were also viable and fertile. The absence of proSAAS transcript was confirmed by Northern blot in whole brains from homozygous mutants (data not shown). To confirm that proSAAS mutant animals completely lacked proSAAS expression, adult whole brains were assayed for the proSAAS-derived peptide, PEN. PEN was not detected in the brains of either mutant male or female mice (). Additionally, PEN levels were reduced by ~25% in female heterozygote brains relative to amount detected in wild-type female brains (although this difference was just outside the criteria for statistical significance (p=0.056), and over 50% in pituitary from adult female mice (; P=0.022). Finally, a peptidomics approach was used to compare levels of brain neuropeptides in proSAAS knock out and wild-type mice. Seven proSAAS derived peptides were detected in wild type mouse hypothalami but none of these were detectable in the mutant mice (Table S1
ProSAAS KO mice don’t produce the proSAAS peptide PEN
Previous work suggested that proSAAS might play a role regulating PC1/3 processing in a manner similar to the role of 7B2 in the regulation of PC2 (Fricker et al. 2000
, Cameron et al. 2000
). Since 7B2 is a chaperone for PC2 as well as an inhibitor of this enzyme, the loss of 7B2 causes a sharp reduction in PC2 levels. Western blot analysis of whole brain was performed to determine whether proSAAS deletion could cause a similar reduction in either PC1/3 or PC2 levels. Western blots using antisera selective for both PC1/3 and PC2 show that proSAAS deletion did not change the levels of either PC1/3 or PC2 protein in either brain () or pituitary (data not shown). Western blot images of PC1/3, PC2, and tubulin were subjected to densitometry analysis. The amounts of inactive (87 kDa) and active PC1/3 (67kDa) protein brain were normalized to the amount of tubulin. No differences in relative PC1/3 and PC2 levels were detected in the pituitaries (data not shown) and brains of adult proSAAS mutant and wild-type mice ().
PC1/3 and PC2 protein levels are unaltered in the adult brain of proSAAS KO mice
Although we found no changes in PC1/3 protein levels, there could potentially still be functional alterations in PC1/3 activity. If PC1/3 activity were affected by proSAAS disruption, such an alteration would be expected to be reflected by altered levels of many hypothalamic peptides. To investigate this possibility in adult proSAAS KO mice, a quantitative peptidomics screen was used to compare levels of hypothalamic peptides in extracts of proSAAS mutant mice relative to wild-type mice. This analysis involved stable isotopic tags and mass spectrometry that allowed the precise molecular form of each peptide to be determined. Altogether, 40 peptides from secretory pathway proteins were identified in wild type hypothalamic extracts (Table S1
); in addition to these 40 peptides, another 30 peptides were identified which were derived from cytosolic, nuclear, or mitochondrial proteins, and more peptides were detected that could not be identified by MS/MS sequence analysis (data not shown). As expected, none of the cytosolic, nuclear, and mitochondrial protein fragments showed major changes between wild type and proSAAS mutant mice (data not shown). Seven of the secretory pathway peptides detected in the wild type mice were proSAAS-derived peptides and none of these were detected in the proSAAS mutant hypothalamic extracts. The other 33 secretory pathway peptides represent 16 additional secretory pathway precursor proteins: cerebellin 1 precursor protein, chromogranin A, chromogranin B, prodynorphin, proenkephalin, prohormone convertase 2, promelanin concentrating hormone, proneurotensin, proopiomelanocortin, propeptidyl-amidating-monooxygenase, protachykinin A, protachykinin B, prothyrotropin releasing hormone, provasopressin, secretogranin II, secretogranin III. The average ratio of these 33 peptides in the proSAAS mutant versus wild type mice was very close to 1.00, indicating that they are not increased or decreased in the proSAAS mutant mice. Taken together, our peptidomics analysis strongly suggests that PC1/3 activity is normal in the hypothalami of adult proSAAS mutant mice.
Previous studies have also shown that 7B2 deletion causes hypersecretion of ACTH from the pituitary resulting in a sharp increase in the level of circulating corticosterone (Westphal et al. 1999
, Laurent et al. 2002
). This finding suggested that 7B2 could be a component of the secretory apparatus with the ability to tonically inhibit secretion. RIAs for ACTH and corticosterone were performed to investigate whether proSAAS deletion could alter the circulating levels of these peptides. The plasma levels of corticosterone and ACTH in proSAAS mutants were indistinguishable from the amount of these peptides detected in wild type littermates (Figure S1
). This finding suggests that unlike 7B2, proSAAS does not appear to be involved in modulating ACTH secretion from pituitary corticotrophs, nor is it involved in its processing.
In addition to its activity in the hypothalamus, PC1/3 is needed to produce several pituitary peptides including ACTH. Extracts were fractionated on gel filtration columns and analyzed by RIA to investigate if the amount and molecular weight of ACTH, a POMC derived peptide produced by PC1/3, was similar in mutant and wild type pituitaries. Since the ACTH antibody used for RIAs is directed to the C-terminus of ACTH, this antibody also recognizes CLIP, a C-terminal cleavage product of ACTH. In assays of whole pituitary, the peak detected in fractions 21-26 corresponds to a combination of ACTH and CLIP () and suggests that processing of POMC to ACTH and CLIP occurs normally in the mutant pituitary. Dissection of the pituitary made it possible to examine production of ACTH and CLIP in the anterior and neurointermediate lobes of the pituitary, respectively. Production of ACTH in anterior pituitary was normal in proSAAS mutants relative to wild type littermates (). Similarly, the amount of CLIP in the neurointermediate lobe of proSAAS mutants was similar to the level detected in wild type littermates (). The immunoreactive forms of ACTH and CLIP in the extracts of proSAAS mutant whole, anterior, and neurointermediate pituitaries had the same molecular weights as the forms of these peptides detected in pituitary tissues from wild type animals. This suggests that PC1/3 and PC2 processing of POMC into ACTH and CLIP is not affected by the absence of proSAAS which supports the findings of the peptidomic experiments showing normal levels of processed neuropeptides in mutant brains.
Processing of POMC into ACTH and CLIP is unaffected in AL and IL of proSAAS KO pituitary
We previously determined that the processing of prodynorphin by PC1/3 is altered in embryonic brain tissues where the C-terminal part of proSAAS (PEN-LEN) that inhibits PC1/3 is accumulated (Morgan et al. 2005
). In contrast to the developing brain, this inhibitory fragment of proSAAS is undetectable in adult mouse brain (Morgan et al. 2005
, Mzhavia et al. 2002
). These findings raised the possibility that proSAAS-mediated inhibition of PC1/3 might be temporally restricted to the developing tissues where accumulated PEN-LEN can be detected. This possibility was explored by examining prodynorphin processing in proSAAS mutant embryos. In e15.5 proSAAS mutant brain we detect accumulation of the high molecular weight prodynorphin intermediate (~8-10 kDa), corresponding to the 8 and/or 10 kDa intermediates produced by PC1/3 (). This finding is in contrast with e15.5 wild-type brains that express the inhibitory fragment of proSAAS. In the wild-type embryonic brain the high molecular weight prodynorphin intermediates are undetectable, likely due to the expression of the inhibitory forms of proSAAS ().
An adult rather than fetal processing pattern of prodynorphin is present in embryonic brain extracts lacking proSAAS
Finally, we have begun a global phenotypic analysis of proSAAS KO mice. Body weight measurements were taken weekly starting at four weeks of age and continued until mice were 20 weeks old (). Male proSAAS mutants have reduced body weights (~10-15%) relative to wild type littermates, which was first detected just after weaning with differences between genotypes being statistically significant at 7, 10-13, and 15-20 weeks of age (p<0.05). In contrast, no evidence for reduced body weight was observed in female proSAAS mutants. The body length of male proSAAS null mice is normal suggesting there is likely not a gross growth deficit (data not shown). Blood glucose homeostasis was examined by measuring fasting glucose levels as well as glucose levels in response to a 2 mg/g of body weight injection of D-glucose. Fasting blood glucose in male mutants was identical to the level measured in wild-type males (). Administration of glucose (2 mg/g) causes a rapid increase in blood glucose, peaking around 30 minutes post-injection, and declining to pre-injection levels by 120-180 minutes following glucose administration. We find that glucose clearance in proSAAS mutant males was normal with mutant males having wild type levels of glucose at 30, 60 and 120 minutes following glucose injection ().
ProSAAS males have reduced body weight
Open-field locomotor activity was measured for 30 minutes in experimentally naive proSAAS mutant and wild type male mice. ProSAAS KO males exhibit decreased horizontal activity (), number of total movements (), number of rearing movements (), and number of stereotypic movements (). Cumulatively, these measurements suggest that proSAAS mutants are slightly less active than wild type littermates. Decreased activity in the context of a novel open-field raises the possibility that proSAAS mice might possess a proclivity towards anxiety-like behaviors.
ProSAAS mutant mice are less active when placed in a novel open field