ProSAAS-Derived Peptides are Colocalized with Neuropeptide Y and Function as Neuropeptides in the Regulation of Food Intake

ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY), but not α-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1–2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpefat/fat mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpefat/fat mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake

ProSAAS-derived peptides are differentially processed and sorted in mouse brain and AtT-20 cells.

ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes) and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides

Comparison of the distribution of proSAAS-derived peptides and NPY-expressing cells in the arcuate nucleus of the hypothalamus.

<p>Immunofluorescence was performed in mice expressing GFP under the NPY promoter, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a>. Left panels show the distribution of GFP, which is localized to the cytosol and reveals the cell bodies of NPY-positive neurons. Middle panels show immunofluorescence of the indicated proSAAS-derived peptide. Right panels show the merged images from the left and right panels. Cell nuclei were visualized with DAPI (blue color, right panels). Scale bar = 10 µm.</p

Effect of mutation of the two major furin cleavage sites on the localization of SAAS and PEN in AtT-20 cells.

<p>Wild type rat proSAAS was transiently expressed in AtT20 cells and peptides examined by immunofluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a> using antiserum to PEN (Cy3) and SAAS (Alexa 488 directly labeled). A: Wild type rat proSAAS. B: Rat proSAAS with the two major furin sites (RxRR) mutated to KxRR, which eliminates the furin consensus site. C. Quantification of colocalization as measured by Pearson's Coefficient. At least 30 cells were averaged per condition. Error bars represent standard error of the mean. *, p<0.05 using Student's t-test.</p

Schematic diagram of proSAAS and proSAAS-derived peptides.

<p>The major peptides derived from proSAAS are indicated, along with the enzymes involved in the cleavages. The amino acids within the cleavage site are shown (top row); residues in black are removed by the carboxypeptidase (CPD or CPE), and residues in purple remain on the C-terminus of the upstream peptide. The sites that are efficiently cleaved by furin/CPD are indicated by solid arrows; these cleavages generate big SAAS, an intermediate peptide representing GAV and the mid-portion of proSAAS, and PEN-LEN. Furin can also cleave PEN-LEN into PEN and big LEN (dashed arrow) but this cleavage does not go to completion in mouse brain. Within secretory vesicles, big SAAS is cleaved into KEP and little SAAS, the middle portion of proSAAS is cleaved into GAV and an un-named peptide, PEN-LEN is cleaved into PEN and big LEN, and big LEN is converted into little LEN and a 4-residue peptide LLPP. The conversion of big LEN into little LEN and LLPP is primarily catalyzed by PC1/3 and CPE. In addition to these major forms of the proSAAS-derived peptides, smaller forms of little SAAS, GAV, and PEN are present in mouse brain although the levels of these truncated peptides are generally lower than the levels of the peptides indicated in this figure.</p

Localization of proSAAS-derived peptides in AtT-20 cells.

<p>Immunofluorescence was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a>. A: Big LEN (Cy2) and PEN-LEN (Cy3). B: Big LEN (Alexa 488 directly labeled) and PEN (cy3). C: Big LEN (Alexa 488 directly labeled) and little LEN (Cy3). D: SAAS (Alexa 488 directly labeled) and big LEN (Cy3). E: SAAS (Alexa 488 directly labeled, left panel) and PEN (Cy3, middle panel). The right panel is a merge of the two images. F. SAAS (Alexa 488 directly labeled, left panel) and little LEN (Cy3, middle panel). Right panel is a merge. Scale bars in panels A, B, C, and D indicate 10 µm. Scale bars in panels E and F represent 1 µm.</p

Comparison of proSAAS-derived peptide immunofluorescence in the arcuate nucleus of the hypothalamus in wild type and proSAAS knock-out mice.

<p>Immunofluorescence was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a>, using a Cy3-labeled secondary antiserum (red). Cell nuclei were visualized with DAPI (blue). Antisera to SAAS, PEN-LEN, PEN, big LEN, and little LEN show a strong punctate staining pattern in wild type mouse hypothalamus (left panels) which is absent in the knock-out mouse brain (right panels).</p

Localization of proSAAS-derived peptides in the arcuate nucleus.

<p>Immunofluorescence was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a> using antibodies directly labeled with Alexa 488 (big LEN and SAAS), or with unlabeled primary antisera followed by Cy2- or Cy3-labeled anti-rabbit or anti-chicken secondary antibodies. A: PEN-LEN (in red, Cy3) and big LEN (in green, Cy2). B: PEN-LEN (Cy3) and SAAS (Cy2). C: PEN (Cy3) and big LEN (Alexa 488 directly labeled). D: PEN (Cy3) and SAAS (Alexa 488 directly labeled). E: Little LEN (Cy3) and big LEN (Alexa 488 directly labeled). F: Little LEN (Cy3) and SAAS (Alexa 488 directly labeled). G: SAAS (Cy3) and big LEN (Alexa 488 directly labeled). Scale bar = 10 µm.</p

Transcriptional profiling of transport mechanisms and regulatory pathways in rat choroid plexus

BACKGROUND: Dysregulation of brain fluid homeostasis associates with brain pathologies in which fluid accumulation leads to elevated intracranial pressure. Surgical intervention remains standard care, since specific and efficient pharmacological treatment options are limited for pathologies with disturbed brain fluid homeostasis. Such lack of therapeutic targets originates, in part, from the incomplete map of the molecular mechanisms underlying cerebrospinal fluid (CSF) secretion by the choroid plexus. METHODS: The transcriptomic profile of rat choroid plexus was generated by RNA Sequencing (RNAseq) of whole tissue and epithelial cells captured by fluorescence-activated cell sorting (FACS), and compared to proximal tubules. The bioinformatic analysis comprised mapping to reference genome followed by filtering for type, location, and association with alias and protein function. The transporters and associated regulatory modules were arranged in discovery tables according to their transcriptional abundance and tied together in association network analysis. RESULTS: The transcriptomic profile of choroid plexus displays high similarity between sex and species (human, rat, and mouse) and lesser similarity to another high-capacity fluid-transporting epithelium, the proximal tubules. The discovery tables provide lists of transport mechanisms that could participate in CSF secretion and suggest regulatory candidates. CONCLUSIONS: With quantification of the transport protein transcript abundance in choroid plexus and their potentially linked regulatory modules, we envision a molecular tool to devise rational hypotheses regarding future delineation of choroidal transport proteins involved in CSF secretion and their regulation. Our vision is to obtain future pharmaceutical targets towards modulation of CSF production in pathologies involving disturbed brain water dynamics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-022-00335-x