Biology subjects

Cirilli, I.

Publications and source records attributed to Cirilli, I..

3 recordsLinked to original sources

bioRxiv2024

SARM1 activation induces reversible mitochondrial dysfunction and can be prevented in human neurons by antisense oligonucleotides

SARM1 is a key regulator of a conserved program of axon degeneration increasingly linked to human neurodegenerative diseases. Pathological SARM1 activation causes rapid NAD consumption, disrupting cellular homeostasis and leading to axon degeneration. In this study, we develop antisense oligonucleotides targeting human SARM1, demonstrating robust neuroprotection against morphological, metabolic, and mitochondrial impairment in human iPSC-derived dopamine neurons induced by the lethal neurotoxin vacor, a potent SARM1 activator. Furthermore, our findings reveal that axon fragmentation can be prevented, and mitochondrial dysfunction reversed using the NAD precursor nicotinamide, a form of vitamin B3, even after SARM1 activation has occurred, when neurons are already unhealthy. This research identifies ASOs as a promising therapeutic strategy to block SARM1, and provides an extensive characterisation and further mechanistic insights that demonstrate the reversibility of SARM1 toxicity in human neurons. It also identifies the SARM1 activator vacor as a specific and reversible neuroablative agent in human neurons.

neuroscience↗

bioRxiv2023

Adaptation of a commercial NAD quantification kit to assay the base exchange activity of SARM1

Here we report an adapted protocol using the Promega NAD/NADH-Glo Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3- acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate. In fact, AcPyrAD generates amplification signals of larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where AcPyrAD is the product. We also propose an application of this method based on competition between AcPyr and other free bases to rank their preference for SARM1. This has significant advantages over traditional methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base exchange activity.

biochemistry↗

bioRxiv2019

Palmitate-induced toxicity is associated with impaired mitochondrial respiration and accelerated oxidative stress in cultured cardiomyocytes: the critical role of Coenzyme Q9/10

Impaired mitochondrial function concomitant to enhanced oxidative stress-induced damage are well established mechanisms involved in hyperlipidemia-induced cardiotoxicity. Coenzyme Q9/10 (CoQ) is known to be a critical component of the mitochondrial electron transport chain that efficiently supports the process of bioenergetics in addition to its antioxidant activities. However, there is very limited information on the direct effect of myocardial lipid overload on endogenous CoQ levels in association with mitochondrial respiration and oxidative stress status. Here, such effects were explored by exposing H9c2 cardiomyocytes to various doses (0.15 to 1 mM) of palmitate for 24 hours. The results demonstrated that palmitate doses [≥] 0.25 mM are enough to impair mitochondrial respiration and cause oxidative stress. Although endogenous CoQ levels are enhanced by palmitate doses [≤] 5 mM, this is not enough to counteract oxidative stress, but is sufficient to maintain cell viability of cardiomyocytes, suggesting a compensation mechanism. Palmitate doses > 5 mM caused severe mitochondrial toxicity, including reduction of cell viability. Interestingly, enhancement of CoQ levels with the lowest dose of palmitate (0.15 mM) was accompanied by a significantly reduction of CoQ oxidation status, as well as low cytosolic production of reactive oxygen species. From the overall findings, it appears that CoQ response may be crucial to improve mitochondrial function and thus protect against hyperlipidemia-induced insult. These results further suggest that therapeutic agents that can stimulate endogenous levels of CoQ may be beneficial in protecting the myocardium against diabetes associated complications.

biochemistry↗