Enzymatic upcycling of wild-simulated ginseng leaves for enhancing biological activities and compound K.

Compound K (CK), a ginsenoside with high bioavailability, is present at low levels in wild-simulated ginseng leaves (WSGL). WSGL contains the CK precursors, Rd and F2, in amounts up to 26.4 ± 0.4 and 24.1 ± 1.9 mg/g extract, respectively. In this study, CK production in WGSL reached 25.9 ± 1.0 mg/g extract following treatment with Viscozyme, Celluclast 1.5 L, Pectinex Ultra SP-L, and their combination. The antioxidant activities indicated by oxygen radical absorbance capacity, ferric reducing antioxidant power, and ABTS- and DPPH radical scavenging activity of enzyme-treated WSGL were enhanced 1.69-, 2.51-, 2.88-, and 1.80-fold, respectively, compared to non-treated WSGL. Furthermore, the CK-enriched WSGL demonstrated a 1.94-fold decrease in SA-ß-galactosidase expression in human dermal fibroblasts and a 3.8-fold enhancement of inhibition of nitric oxide release in lipopolysaccharide-induced RAW 264.7 cells relative to non-treated WSGL. Consequently, WSGL subjected to enzymatic upcycling has potential as a functional material in the food and pharmaceutical industries.

In Vivo Dopamine Neuron Imaging-Based Small Molecule Screen Identifies Novel Neuroprotective Compounds and Targets.

Parkinson's disease (PD) is the second most common neurodegenerative disorder with prominent dopamine (DA) neuron degeneration. PD affects millions of people worldwide, but currently available therapies are limited to temporary relief of symptoms. As an effort to discover disease-modifying therapeutics, we have conducted a screen of 1,403 bioactive small molecule compounds using an in vivo whole organism screening assay in transgenic larval zebrafish. The transgenic model expresses the bacterial enzyme nitroreductase (NTR) driven by the tyrosine hydroxylase (th) promotor. NTR converts the commonly used antibiotic pro-drug metronidazole (MTZ) to the toxic nitroso radical form to induce DA neuronal loss. 57 compounds were identified with a brain health score (BHS) that was significantly improved compared to the MTZ treatment alone after FDR adjustment (padj<0.05). Independently, we curated the high throughput screening (HTS) data by annotating each compound with pharmaceutical classification, known mechanism of action, indication, IC50, and target. Using the Reactome database, we performed pathway analysis, which uncovered previously unknown pathways in addition to validating previously known pathways associated with PD. Non-topology-based pathway analysis of the screening data further identified apoptosis, estrogen hormone, dipeptidyl-peptidase 4, and opioid receptor Mu1 to be potentially significant pathways and targets involved in neuroprotection. A total of 12 compounds were examined with a secondary assay that imaged DA neurons before and after compound treatment. The z'-factor of this secondary assay was determined to be 0.58, suggesting it is an excellent assay for screening. Etodolac, nepafenac, aloperine, protionamide, and olmesartan showed significant neuroprotection and was also validated by blinded manual DA neuronal counting. To determine whether these compounds are broadly relevant for neuroprotection, we tested them on a conduritol-b-epoxide (CBE)-induced Gaucher disease (GD) model, in which the activity of glucocerebrosidase (GBA), a commonly known genetic risk factor for PD, was inhibited. Aloperine, olmesartan, and nepafenac showed significant protection of DA neurons in this assay. Together, this work, which combines high content whole organism in vivo imaging-based screen and bioinformatic pathway analysis of the screening dataset, delineates a previously uncharted approach for identifying hit-to-lead candidates and for implicating previously unknown pathways and targets involved in DA neuron protection.

The effect of renin-angiotensin-aldosterone system inhibitors on organ-specific ace2 expression in zebrafish and its implications for COVID-19.

Among cases of SARS-CoV-2 infections that result in serious conditions or death, many have pre-existing conditions such as hypertension and are on renin-angiotensin-aldosterone system (RAAS) inhibitors. The angiotensin-converting-enzyme-2 (ACE2), a key protein of the RAAS pathway, also mediates cellular entry of SARS-CoV-2. RAAS inhibitors might affect the expression levels of ace2, which could impact patient susceptibility to SARS-CoV-2. However, multi-organ-specific information is currently lacking and no species other than rodents have been examined. To address this knowledge gap, we treated adult zebrafish with the RAAS inhibitors aliskiren, olmesartan, and captopril for 7 consecutive days and performed qRT-PCR analysis of major RAAS pathway genes in the brain, gill, heart, intestine, kidney, and liver. Both olmesartan and captopril significantly increased ace2 expression in the heart, gill, and kidney. Olmesartan also increased ace2 expression in the intestine. Conversely, aliskiren significantly decreased ace2 expression in the heart. Discontinuation of compound treatments for 7 days did not return ace2 expression to baseline levels. While potential risks or benefits of antihypertensive RAAS inhibitors to SARS-CoV-2 infections in humans remain uncertain, this study provides new insights regarding the impact of RAAS inhibitors on organ-specific ace2 expression in another vertebrate model, thereby providing comparative data and laying scientific groundwork for future clinical decisions of RAAS inhibitor use in the context of COVID-19.

A zebrafish screen reveals Renin-angiotensin system inhibitors as neuroprotective via mitochondrial restoration in dopamine neurons.

Parkinson's disease (PD) is a common neurodegenerative disorder without effective disease-modifying therapeutics. Here, we establish a chemogenetic dopamine (DA) neuron ablation model in larval zebrafish with mitochondrial dysfunction and robustness suitable for high-content screening. We use this system to conduct an in vivo DA neuron imaging-based chemical screen and identify the Renin-Angiotensin-Aldosterone System (RAAS) inhibitors as significantly neuroprotective. Knockdown of the angiotensin receptor 1 (agtr1) in DA neurons reveals a cell-autonomous mechanism of neuroprotection. DA neuron-specific RNA-seq identifies mitochondrial pathway gene expression that is significantly restored by RAAS inhibitor treatment. The neuroprotective effect of RAAS inhibitors is further observed in a zebrafish Gaucher disease model and Drosophila pink1-deficient PD model. Finally, examination of clinical data reveals a significant effect of RAAS inhibitors in delaying PD progression. Our findings reveal the therapeutic potential and mechanisms of targeting the RAAS pathway for neuroprotection and demonstrate a salient approach that bridges basic science to translational medicine.

Parkinson's disease is caused by the slow death and deterioration of brain cells, in particular of the neurons that produce a chemical messenger known as dopamine. Certain drugs can mitigate the resulting drop in dopamine levels and help to manage symptoms, but they cause dangerous side-effects. There is no treatment that can slow down or halt the progress of the condition, which affects 0.3% of the population globally. Many factors, both genetic and environmental, contribute to the emergence of Parkinson's disease. For example, dysfunction of the mitochondria, the internal structures that power up cells, is a known mechanism associated with the death of dopamine-producing neurons. Zebrafish are tiny fish which can be used to study Parkinson's disease, as they are easy to manipulate in the lab and share many characteristics with humans. In particular, they can be helpful to test the effects of various potential drugs on the condition. Here, Kim et al. established a new zebrafish model in which dopamine-producing brain cells die due to their mitochondria not working properly; they then used this assay to assess the impact of 1,403 different chemicals on the integrity of these cells. A group of molecules called renin-angiotensin-aldosterone (RAAS) inhibitors was shown to protect dopamine-producing neurons and stopped them from dying as often. These are already used to treat high blood pressure as they help to dilate blood vessels. In the brain, however, RAAS worked by restoring certain mitochondrial processes. Kim et al. then investigated whether these results are relevant in other, broader contexts. They were able to show that RAAS inhibitors have the same effect in other animals, and that Parkinson's disease often progresses more slowly in patients that already take these drugs for high blood pressure. Taken together, these findings therefore suggest that RAAS inhibitors may be useful to treat Parkinson's disease, as well as other brain illnesses that emerge because of mitochondria not working properly. Clinical studies and new ways to improve these drugs are needed to further investigate and capitalize on these potential benefits.