Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases

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

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

Healthspan improvement and anti-aggregation effects induced by a marine-derived structural proteasome activator

Proteasome activation has been shown to promote cellular and organismal healthspan and to protect against aggregation-related conditions, such as Alzheimer's disease (AD). Various natural compounds have been described for their proteasome activating properties but scarce data exist on marine metabolites that often possess unique chemical structures, exhibiting pronounced bioactivities with novel mechanisms of action. In this study, we have identified for the first time a marine structural proteasome activator, namely (1R,3E,6R,7Z,11S,12S)-dolabella-3,7,18-trien-6,17-olide (DBTO). DBTO activates the 20S proteasome complex in cell-free assays but also in cellulo. Continuous supplementation of human primary fibroblasts with DBTO throughout their cellular lifespan confers an improved healthspan while ameliorated health status is also observed in wild type (wt) Caenorhabditis elegans (C. elegans) nematodes supplemented with DBTO. Furthermore, treatment of various AD nematode models, as well as of human cells of neuronal origin challenged with exogenously added Aβ peptide, with DBTO results in enhanced protection against Aβ-induced proteotoxicity. In total, our results reveal the first structural proteasome activator derived from the marine ecosystem and highlight its potential as a compound that might be used for healthspan maintenance and preventive strategies against proteinopathies, such as AD. © 2022 The Author

The SWI/SNF subunit Bcl7a contributes to motor coordination and Purkinje cell function.

Chromatin remodelers have emerged as prominent regulators of epigenetic processes and potential drivers of various human pathologies. The multi-subunit chromatin-remodeling SWI/SNF complex determines gene expression programs and, consequently, contributes to the differentiation, maturation and plasticity of neurons. Here, we investigate the elusive biological function of Bcl7a and Bcl7b, two newly identified subunits of the SWI/SNF complex that are highly expressed throughout the brain. We generated ubiquitous and neuron-specific Bcl7a and Bcl7b single and double knockout mice. We provide evidence that Bcl7b is dispensable for animal survival as well as behavioral plasticity. Conversely, ubiquitous Bcl7a knockout results in perinatal lethality, while genetic deletion of Bcl7a in postmitotic neurons elicits motor abnormalities and affects dendritic branching of Purkinje cells, with no obvious synergistic relationship with Bcl7b. Collectively, our findings reveal novel insights into the cellular processes linked to BCL7-containing SWI/SNF complexes and their unrecognized roles in the brain

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases