The propeptide precursor proSAAS is involved in fetal neuropeptide processing and body weight regulation

Abstract

Mice with a targeted mutation in proSAAS have been generated to investigate whether peptides derived from this precursor could function as an inhibitor of prohormone convertase 1/3 (PC1/3) in vivo as well as to determine any alternate roles for proSAAS in nervous and endocrine tissues. Fetal mice lacking proSAAS exhibit complete, adult-like processing of prodynorphin in the prenatal brain instead of the incomplete processing seen in the brains of wild-type fetal mice where inhibitory proSAAS intermediates are transiently accumulated. This study provides evidence that proSAAS is directly involved in the prenatal regulation of neuropeptide processing in vivo. However, adult mice lacking proSAAS have normal levels of all peptides detected using a peptidomics approach, suggesting that PC1/3 activity is not affected by the absence of proSAAS in adult mice. ProSAAS knockout mice exhibit decreased locomotion and a male-specific 10-15% decrease in body weight, but maintain normal fasting blood glucose levels and are able to efficiently clear glucose from the blood in response to a glucose challenge. This work suggests that proSAAS-derived peptides can inhibit PC1/3 in embryonic brain, but in the adult brain proSAAS peptides may function as neuropeptides that regulate body weight and potentially other behaviors.

Figure 1. Production of proSAAS KO mice

A. A proSAAS genomic fragment was isolated from a murine Sw129/ReJ genomic library and genomic fragments upstream and downstream and of exon 1 were used to make the targeting vector. The targeting vector was made by subcloning these genomic DNA fragments, a neomycin (NEO) cassettee, and the HSV-tk gene into a pKO Scrambler V919 targeting vector. The NEO and HSV-TK cassettes were subcloned into Asc I (NEO) and Rsr II (HSV-TK) restriction sites. The 2.5 kb Xba I/Sac II genomic fragment, 5′ of exon 1 (A-arm) was inserted into Xba I/Sac II restriction sites within the targeting vector. A 5.5 kb Xba I genomic fragment located 3′ of exon 1 (B-arm) was sub-cloned into pBluescript, excised with Not I and Kpn I, and inserted into Bgl II (blunt) and Kpn I sites in the targeting vector. A 500 bp genomic fragment (Xba I/Spe I), just 5′ of the Xba I/Sac II A-arm fragment was used as a Southern blot screening probe. B. Male proSAAS chimeras were used to establish a colony of proSAAS knock-out (−/− or −/Y), female heterozygote (+/−), and wild type (+/+, +/Y) mice. Southern blotting of BamHI digested genomic DNA yields a 9 kB band from the wild type allele and a 6 kB band from the mutant allele.

Figure 2. ProSAAS KO mice don’t produce the proSAAS peptide PEN

A. Peptide extracts from the whole brain of adult wild type and proSAAS KO male mice as well as adult female wild type (+/+), heterozygote (+/−), and mutants (−/−) mice were assayed using RIA for the proSAAS derived peptide PEN. PEN was not detectable using our assay in brain of proSAAS male hemizygous and female homozygous KO mice, while female heterozygous mice possess a reduced amount of PEN relative to wild type controls. B. Peptide analysis of PEN in male and female proSAAS pituitaries. Error bars indicate the SEM and p-values were calculated using unpaired Student t-tests (* p< 0.05).

Figure 3. PC1/3 and PC2 protein levels are unaltered in the adult brain of proSAAS KO mice

The levels of PC1/3 (A) and PC2 (B) protein forms from whole brains of wild-type (WT) and proSAAS mutant (KO) animals were analyzed by Western blot. The blot for PC1/3 shows bands corresponding to the mature fully active 66 kDa form of PC1/3 as well as the inactive 87 kDa pro-PC1/3 form. The fully mature 68 kDa form of PC2 as well as an intermediate 71 kDa form of PC2 are shown. To ensure that the qualitative appearance of the PC1 blot accurately reflected the lack of effect of proSAAS mutation on either the inactive (68 kDA) or active PC1/3 isoforms (87 kDa), the ratio of these bands was determined in brains from wild-type (white bar) and proSAAS k/o (black bar) mice. Peptide sizes were estimated using Bio-Rad Kaliedoscope pre-stained standards (216, 132, 78, 45.7, 32.5, 18.4, and 7.6 kDa).

Figure 4. Processing of POMC into ACTH and CLIP is unaffected in AL and IL of proSAAS KO pituitary

The processing of POMC into ACTH was analyzed in pooled extracts of whole (A), anterior (B), or neurointermediate (C) pituitaries. Extracts from three proSAAS mutant (circles and dashed lines) or wild type (squares and solid lines) pituitaries were pooled and subjected to gel filtration chromatography. RIAs were performed using anti-sera that recognize ACTH (1-39). The following molecular weight calibration standards were used: cytochrome c, 12.4 kDa; ACTH, 4.6 kDa; β-endorphin, 3.5 kDa; α-MSH, 1.7 kDa, and Dyn A-8, 1.0 kDa. The void volume of the column occurs in fraction 12 and the salt fraction elutes in fraction 52.

Figure 5. An adult rather than fetal processing pattern of prodynorphin is present in embryonic brain extracts lacking proSAAS

A. Extracts from brain of e15.5 mouse embryos (n=25-40) were pooled and separated by gel-filtration chromatography, treated with trypsin/carboxypeptidase and ir-Leucine enkephalin (ir-Leu-enk) was measured by RIA. High molecular weight prodynorphin intermediates were absent in wild type e15.5 whole brains (squares and solid line). A substantial amount of higher molecular weight prodynorphin characteristic of PC1/3 processing (seen in peak I) was detected in e15.5 brains lacking proSAAS (circles, dotted line) but not in e15.5 WT brains (square, bold line). Other lower molecular weight prodynorphin intermediates (Dynorphin A-17, B-13, and others in fractions 25-28; Peak II) were generated in mutant embryonic brains (circles, dotted line). The following molecular weight calibration standards were used: cytochrome c, 12.4 kDa; ACTH, 4.6 kDa; β-endorphin, 3.5 kDa; α-MSH, 1.7 kDa, and Dyn A-8, 1.0 kDa. The void volume of the column occurs in fraction 12 and the salt fraction elutes in fraction 52. B. Schematic diagram showing the processing of prodynorphin by PC1/3 into 8 and 10 kDa intermediates that are subsequently processed by PC2 to generate terminally processed dynorphin peptides (Dynorphin B-17, B-29, A-8, A-17, alpha-neo-endorphin (αNE), and leucine-enkephalin).

Figure 6. ProSAAS males have reduced body weight

A. The body weight of proSAAS male mutants (circles and dashed line) is plotted weekly and is shown relative to wild type littermates (squares and solid line). Mice were fed a standard rodent chow diet. Error bars correspond to the SEM and the differences at each time point were analyzed using Student’s t-test (* P<0.05). B. Fasting blood glucose (t=0) and blood glucose levels at 30, 60, and 120 minutes following intraperitoneal administration of 2mg/g glucose were plotted for proSAAS mutants (circles and dashed line) and wild type littermates (squares and solid line).

Figure 7. ProSAAS mutant mice are less active when placed in a novel open field

Locomotor activity was monitored for proSAAS male mutants (SAAS KO) and wild type littermates (WT) placed in a novel open-field for 30 minutes. Horizontal activity (A), number of movements (B), number of rearing movements (C), and the number of stereotypic movements (D) were monitored during the open field test. Error bars indicate the SEM and p-values calculated using unpaired Student t-tests (* p< 0.05).