Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells

Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III–IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.

ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes

Glomerular podocytes are instrumental in ensuring glomerular permselectivity and regulating the integrity of glomerular biology. However, podocytes are vulnerable to various noxious stimuli such as hypoxia, and podocyte injury presented with glomerulosclerosis and impaired kidney function. The mechanism of hypoxia-induced podocyte injury vis-a-vis glomerulosclerosis has remained enigmatic. Hypoxia inducible factor 1α (HIF1α) that transduces hypoxic adaptations, induces Transglutaminase 2 (TG2), a calcium dependent enzyme that catalyzes intramolecular ε-(γ-glutamyl) lysine cross-links of extracellular matrix (ECM) proteins. In this study, we investigated the mechanism of regulation of TG2 by HIF1α. Stabilization of HIF1⍺ by FG4592 (Roxadustat) and physiological hypoxia, resulted in elevated expression of ZEB2 (zinc-finger E-box-homeobox 2) and its downstream target TRPC6 (transient receptor potential channel 6). ZEB2 transcriptionally activates TG2 expression, whereas, via TRPC6, it induces calcium influx, inturn it increases the TG2 activity. Blocking the TRPC6 action or suppressing its expression only partially attenuated FG4592 induced TG2 activity, whereas suppression of ZEB2 expression significantly abolished TG2 activity. This study demonstrates that stabilization of HIF1α stimulates both TG2 expression and activity, whereas abrogation of HIF1⍺ by metformin prevented HIF1⍺ regulated TG2 and consequent glomerular injury.

Identification of novel modulators of a schistosome transient receptor potential channel targeted by praziquantel

Given the worldwide burden of neglected tropical diseases, there is ongoing need to develop novel anthelmintic agents to strengthen the pipeline of drugs to combat these burdensome infections. Many diseases caused by parasitic flatworms are treated using the anthelmintic drug praziquantel (PZQ), employed for decades as the key clinical agent to treat schistosomiasis. PZQ activates a flatworm transient receptor potential (TRP) channel within the melastatin family (TRPMPZQ) to mediate sustained Ca2+ influx and worm paralysis. As a druggable target present in many parasitic flatworms, TRPMPZQ is a promising target for a target-based screening campaign with the goal of discovering novel regulators of this channel complex. Here, we have optimized methods to miniaturize a Ca2+-based reporter assay for Schistosoma mansoni TRPMPZQ (Sm.TRPMPZQ) activity enabling a high throughput screening (HTS) approach. This methodology will enable further HTS efforts against Sm.TRPMPZQ as well as other flatworm ion channels. A pilot screen of ~16,000 compounds yielded a novel activator of Sm.TRPMPZQ, and numerous potential blockers. The new activator of Sm.TRPMPZQ represented a distinct chemotype to PZQ, but is a known chemical entity previously identified by phenotypic screening. The fact that a compound prioritized from a phenotypic screening campaign is revealed to act, like PZQ, as an Sm.TRPMPZQ agonist underscores the validity of TRPMPZQ as a druggable target for antischistosomal ligands.

Transient Receptor Potential Channel 4 Small-Molecule Inhibition Alleviates Migraine-Like Behavior in Mice

Migraine is a common neurological disorder with few available treatment options. Recently, we have demonstrated the role of transient receptor potential cation channel subfamily C member 4 (TRPC4) in itch and the modulation of the calcitonin gene-related peptide (CGRP), a biomarker and emerging therapeutic target for migraine. In this study, we characterized the role of TRPC4 in pain and evaluated its inhibition as anti-migraine pain therapy in preclinical mouse models. First, we found that TRPC4 is highly expressed in trigeminal ganglia and its activation not only mediates itch but also pain. Second, we demonstrated that the small-molecule inhibitor ML204, a specific TRPC4 antagonist, significantly reduced episodic and chronic migraine-like behaviors in male and female mice after injection of nitroglycerin (NTG), a well-known migraine inducer in rodents and humans. Third, we found a significant decrease in CGRP protein levels in the plasma of both male and female mice treated with ML-204, which largely prevented the development of chronic migraine-like behavior. Using sensory neuron cultures, we confirmed that activation of TRPC4 elicited release of CGRP, which was significantly diminished by ML-204. Collectively, our findings identify TRPC4 in peripheral sensory neurons as a mediator of CGRP release and NTG-evoked migraine. Since a TRPC4 antagonist is already in clinical trials, we expect that this study will rapidly lead to novel and effective clinical treatments for migraineurs.

Plasma Membrane and Organellar Targets of STIM1 for Intracellular Calcium Handling in Health and Neurodegenerative Diseases

Located at the level of the endoplasmic reticulum (ER) membrane, stromal interacting molecule 1 (STIM1) undergoes a complex conformational rearrangement after depletion of ER luminal Ca2+. Then, STIM1 translocates into discrete ER-plasma membrane (PM) junctions where it directly interacts with and activates plasma membrane Orai1 channels to refill ER with Ca2+. Furthermore, Ca2+ entry due to Orai1/STIM1 interaction may induce canonical transient receptor potential channel 1 (TRPC1) translocation to the plasma membrane, where it is activated by STIM1. All these events give rise to store-operated calcium entry (SOCE). Besides the main pathway underlying SOCE, which mainly involves Orai1 and TRPC1 activation, STIM1 modulates many other plasma membrane proteins in order to potentiate the influxof Ca2+. Furthermore, it is now clear that STIM1 may inhibit Ca2+ currents mediated by L-type Ca2+ channels. Interestingly, STIM1 also interacts with some intracellular channels and transporters, including nuclear and lysosomal ionic proteins, thus orchestrating organellar Ca2+ homeostasis. STIM1 and its partners/effectors are significantly modulated in diverse acute and chronic neurodegenerative conditions. This highlights the importance of further disclosing their cellular functions as they might represent promising molecular targets for neuroprotection.