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Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

The prevalence of age-related neurodegenerative diseases, such as Parkinson's disease (PD) and related disorders continues to grow worldwide. Increasing evidence links intracellular inclusions of misfolded alpha-synuclein (α-syn) aggregates, so-called Lewy bodies (LB) and Lewy neuritis, to the progressive pathology of PD and other synucleinopathies. Our previous findings established that α-syn oligomers induce S-nitrosylation and deregulation of the E3-ubiquitin ligase Parkin, leading to mitochondrial disturbances in neuronal cells. The accumulation of damaged mitochondria as a consequence, together with the release of mitochondrial-derived damage-associated molecular patterns (mtDAMPs) could activate the innate immune response and induce neuroinflammation ("mito-inflammation"), eventually accelerating neurodegeneration. However, the molecular pathways that transmit pro-inflammatory signals from damaged mitochondria are not well understood. One of the proposed pathways could be the cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING) (cGAS–STING) pathway, which plays a pivotal role in modulating the innate immune response. It has recently been suggested that cGAS–STING deregulation may contribute to the development of various pathological conditions. Especially, its excessive engagement may lead to neuroinflammation and appear to be essential for the development of neurodegenerative brain diseases, including PD. However, the precise molecular mechanisms underlying cGAS–STING pathway activation in PD and other synucleinopathies are not fully understood. This review focuses on linking mitochondrial dysfunction to neuroinflammation in these disorders, particularly emphasizing the role of the cGAS–STING signaling. We propose the cGAS–STING pathway as a critical driver of inflammation in α-syn-dependent neurodegeneration and hypothesize that cGAS–STING–driven "mito-inflammation" may be one of the key mechanisms promoting the neurodegeneration in PD. Understanding the molecular mechanisms of α-syn–induced cGAS–STING–associated "mito-inflammation" in PD and related synucleinopathies may contribute to the identification of new targets for the treatment of these disorders. [ABSTRACT FROM AUTHOR]

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.

Cinnamic acid, ferulic acid, and the flavonoids quercetin and taxifolin (dihydroquercetin) are naturally occurring compounds found in plants. They are often referred to as polyphenols and are known, among others, for their pharmacological effects supporting health through the inhibition of aging processes and oxidative stress. To improve their bioavailability, pharmacological activities, and safety, the creation of novel flavonoid–phenolic acid hybrids is an area of active research. Previous work showed that such hybridization products of phenolic acids and flavonoids enhanced the resilience of neuronal cells against oxidative stress in vitro, and attenuated cognitive impairment in a mouse model of Alzheimer's disease (AD) in vivo. Notably, the therapeutic effects of the hybrid compounds we obtained were more pronounced than the protective activities of the respective individual components. The underlying mechanisms mediated by the flavonoid–phenolic acid hybrids, however, remained unclear and may differ from the signaling pathways activated by the originating structures of the respective individual phenolic acids or flavonoids. In this study, we characterized the effects of four previously described potent flavonoid–phenolic acid hybrids in models of oxidative cell death through ferroptosis. Ferroptosis is a type of iron-dependent regulated cell death characterized by lipid peroxidation and mitochondrial ROS generation and has been linked to neurodegenerative conditions. In models of ferroptosis induced by erastin or RSL3, we analyzed mitochondrial (lipid) peroxidation, mitochondrial membrane integrity, and Ca2+ regulation. Our results demonstrate the strong protective effects of the hybrid compounds against ROS formation in the cytosol and mitochondria. Importantly, these protective effects against ferroptosis were not mediated by radical scavenging activities of the phenolic hybrid compounds but through inhibition of mitochondrial complex I activity and reduced mitochondrial respiration. Our data highlight the effects of flavonoid–phenolic acid hybrids on mitochondrial metabolism and further important mitochondrial parameters that collectively determine the health and functionality of mitochondria with a high impact on the integrity and survival of the neuronal cells. [ABSTRACT FROM AUTHOR]