The recently discovered adipokinetic hormone/corazonin-related peptide (ACP) is an insect neuropeptide structurally intermediate between corazonin (CRZ) and adipokinetic (AKH) hormones, which all demonstrate homology to the vertebrate gonadotropin-releasing hormone (GnRH). To date, the function of the ACP signaling system remains unclear. In the present study, we molecularly identified the complete open reading frame encoding the Aedes aegypti ACP receptor (ACPR), which spans nine exons and undergoes alternative splicing giving rise to three transcript variants. Only a single variant, AedaeACPR-I, yielding a deduced 577 residue protein, contains all seven transmembrane domains characteristic of rhodopsin-like G protein-coupled receptors. Functional deorphanization of AedaeACPR-I using a heterologous cell culture-based system revealed highly-selective and dose-dependent receptor activation by AedaeACP (EC50 = 10.25 nM). Analysis of the AedaeACPR-I and AedaeACP transcript levels in all post-embryonic developmental stages using quantitative RT-PCR identified enrichment of both transcripts after adult eclosion. Tissue-specific expression profiling in adult mosquitoes reveals expression of the AedaeACPR-I receptor transcript in the central nervous system, including significant enrichment within the abdominal ganglia. Further, the AedaeACP transcript is prominently detected within the brain and thoracic ganglia. Collectively, these results indicate a neuromodulator or neurotransmitter role for ACP and suggest this neuropeptide may function in regulation of post-ecdysis activities.

Insect CAPA neuropeptides are homologs of mammalian neuromedin U and are known to influence ion and water balance by regulating the activity of the Malpighian ‘renal’ tubules (MTs). Several diuretic hormones are known to increase primary fluid and ion secretion by insect MTs and, in adult female mosquitoes, a calcitonin-related peptide (DH31) called mosquito natriuretic peptide, increases sodium secretion to compensate for the excess salt load acquired during blood-feeding. An endogenous mosquito anti-diuretic hormone was recently described, having potent inhibitory activity against select diuretic hormones, including DH31. Herein, we functionally deorphanized, both in vitro and in vivo, a mosquito anti-diuretic hormone receptor (AedaeADHr) with expression analysis indicating highest enrichment in the MTs where it is localized within principal cells. Characterization using a heterologous in vitro system demonstrated the receptor was highly sensitive to mosquito CAPA neuropeptides while in vivo, AedaeADHr knockdown abolished CAPA-induced anti-diuretic control of DH31-stimulated MTs. CAPA neuropeptides are produced within a pair of neurosecretory cells in each of the abdominal ganglia, whose axonal projections innervate the abdominal neurohaemal organs, where these neurohormones are released into circulation. Lastly, pharmacological inhibition of nitric oxide synthase (NOS) and protein kinase G (PKG) signaling eliminated anti-diuretic activity of CAPA, highlighting the role of the second messenger cGMP and NOS/PKG in this anti-diuretic signaling pathway.

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Adipokinetic hormone (AKH), corazonin (CRZ), and the AKH/CRZ-related peptide (ACP) are neuropeptides considered homologous to the vertebrate gonadotropin-releasing hormone (GnRH). All three Aedes aegypti GnRH-related neuropeptide receptors have been characterized and functionally deorphanized. Individually they exhibit high specificity for their native ligands, prompting us to investigate the contribution of ligand structures in conferring receptor specificity for two of these receptors. Here, we designed a series of analogs based on the native ACP sequence and screened them using a heterologous system to identify critical residues required for ACP receptor (ACPR) activation. Analogs lacking the carboxy-terminal amidation, replacing aromatics, as well as truncated analogs were either completely inactive or had very low activities on ACPR. The polar threonine (position 3) and the blocked amino-terminal pyroglutamate are also critical, whereas ACP analogs with alanine substitutions at position 2 (valine), 5 (serine), 6 (arginine), and 7 (aspartate) were less detrimental including the substitution of charged residues. Replacing asparagine (position 9) with an alanine resulted in a 5-fold more active analog. A naturally-occurring ACP analog, with a conserved substitution in position two, was well tolerated yet displayed significantly reduced activity compared to the native mosquito ACP peptide. Chain length contributes to ligand selectivity in this system, since the endogenous octapeptide Aedae-AKH does not activate the ACPR whereas AKH decapeptides show low albeit significant activity. Similarly, we utilized this in vitro heterologous assay approach against an A. aegypti AKH receptor (AKHR-IA) testing carefully selected naturally-occurring AKH analogs from other insects to determine how substitutions of specific residues in the AKH ligand influence AKHR-IA activation. AKH analogs having single substitutions compared to Aedae-AKH revealed position 7 (either serine or asparagine) was well tolerated or had slightly improved activation whereas changes to position 6 (proline) compromised receptor activation by nearly 10-fold. Substitution of position 3 (threonine) or analogs with combinations of substitutions were quite detrimental with a significant decrease in AKHR-IA activation. Collectively, these results advance our understanding of how two GnRH-related systems in A. aegypti sharing the most recent evolutionary origin sustain independence of function and signaling despite their relatively high degree of ligand and receptor homology.

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.