In the last decade, intramembrane proteases have gained increasing attention because of their many links to various diseases. Nevertheless, our understanding as to how they function or how they are regulated is still limited, especially when it comes to human homologues. In this regard, here we sought to unravel mechanisms of regulation of the protease rhomboid-like protein-4 (RHBDL4), one of five active human serine intramembrane proteases. In view of our recent finding that human RHBDL4 efficiently cleaves the amyloid precursor protein (APP), a key protein in the pathology of Alzheimer's disease, we used established reagents to modulate the cellular cholesterol content and analyzed the effects of this modulation on RHBDL4-mediated processing of endogenous APP. We discovered that lowering membrane cholesterol levels increased the levels of RHBDL4-specific endogenous APP fragments, whereas high cholesterol levels had the opposite effect. Direct binding of cholesterol to APP did not mediate these modulating effects of cholesterol. Instead, using homology modeling, we identified two potential cholesterol-binding motifs in the transmembrane helices 3 and 6 of RHBDL4. Substitution of the essential tyrosine residues of the potential cholesterol-binding motifs to alanine increased the levels of endogenous APP C-terminal fragments, reflecting enhanced RHBDL4 activity. In summary, we provide evidence that the activity of RHBDL4 is regulated by cholesterol likely through a direct binding of cholesterol to the enzyme.

Since the first genetic description of a rhomboid inDrosophila melanogaster, tremendous efforts have been geared towards elucidating the proteolytic mechanism of this particular class of intramembrane proteases. In particular, mammalian rhomboid proteases sparked our interest and we aimed to investigate the human homologue RHBDL4. In light of our recent finding of the amyloid precursor protein (APP) family as efficient substrates of RHBDL4, we were enticed to further study the specific proteolytic mechanism of this enzyme by comparing cleavage patterns of wild type APP and APP TMS chimeras. Here, we demonstrate that the introduction of positively charged amino acid residues in the TMS redirects the RHBDL4-mediated cleavage of APP from its ectodomain closer towards the TMS, possibly inducing an ER-associated degradation (ERAD) of the substrate. In addition, we concluded that the cytoplasmic tail and proposed palmitoylation sites in the ectodomain of APP are not essential for the RHBDL4-mediated APP processing. In summary, our previously identified APP ectodomain cleavages by RHBDL4 are a subsidiary mechanism to the proposed RHBDL4-mediated ERAD of substrates likely through a single cleavage near or within the TMS.

Dopaminergic neurons (DA) are the predominant cell type in the midbrain that synthesize dopamine, a neurotransmitter implicated in a number of behavioural processes, including motor function, the reward pathway, and satiety. In diseases affecting these neurons, such as in Parkinsons disease (PD), there is growing evidence that the gut-brain axis and selective vulnerability of DA neurons plays a crucial role in disease. Most investigations relating to DA neurons in the gut rely on immunoreactivity to tyrosine hydroxylase (TH) - a rate-limiting enzyme in the production of dopamine. However, the reliability of TH staining as a marker of DA neurons has been questioned in recent years. Our aim is to perform a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein under the dopamine transporter promoter (DAT). Our findings confirm a unique localisation of DA neurons in the gut, and also unveil that there are discrete subtypes of DA neurons in the gut, which we characterised using both immunofluorescence and single cell transcriptomics. We observed distinct subtypes of DAT neurons expressing co-transmitters and modulators; some of them likely co-releasing acetylcholine, and a smaller population likely releasing nitric oxide; while others were positive for a slew of canonical DA markers (Vmat2, Girk2, Foxa2). Given the clear heterogeneity of DA gut neurons, further investigation is warranted to define their functional signatures and discover their inherent biological differences that put these cells at risk for neurodegeneration.