Study of the effect of mitochondrial deficiency on the neuronal morphology of the nematode <em>Caenorhabditis elegans</em>

Neurons are highly polarized cells that constantly undergo remodeling of their branches during and after their development. This energy demanding process depends on energy provision from mitochondria via the oxidative phosphorylation (OXPHOS). Although the molecular mechanisms connecting the two are important for understanding pathologies linked to defective neuronal branching due to mitochondrial deficiency, they still remain elusive. In this study, we approached this problem by using as a model a highly branched sensory neuron in Caenorhabditis elegans which demonstrates altered branching pattern in conditions of mitochondrial deficiency. In particular, we show that animals carrying mutations in genes encoding mitochondrial complex I subunits have more dendrites on their sensory neurons compared to wild type animals. The increased outgrowth is not a cell type-specific phenomenon since other types of sensory neurons demonstrate ectopic structures due to complex I deficiency, as well. Importantly, the increased branching is not a result of oxidative stress, but it is regulated by a distinct signaling cascade. Specifically, activation of AMP-activated protein kinase AMPK due to mitochondrial deficiency is one of the regulatory events and we show that its downstream target phosphoinositide 3-kinase PI3K participates in the same pathway. Finally, we demonstrated that the increased neuronal arborizations displayed by OXPHOS mutants are independent of their longevity. Overall, our findings reveal a signaling pathway which regulates tightly dendritic branching in conditions of mitochondrial deficiency and contribute to the understanding of more complicated phenomena occurring in relevant neurodegenerative diseases

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

Neuron-specific proteasome activation exerts cell non-autonomous protection against amyloid-beta (Aβ) proteotoxicity in Caenorhabditis elegans

Proteostasis reinforcement is a promising approach in the design of therapeutic interventions against proteinopathies, including Alzheimer's disease. Understanding how and which parts of the proteostasis network should be enhanced is crucial in developing efficient therapeutic strategies. The ability of specific tissues to induce proteostatic responses in distal ones (cell non-autonomous regulation of proteostasis) is attracting interest. Although the proteasome is a major protein degradation node, nothing is known on its cell non-autonomous regulation. We show that proteasome activation in the nervous system can enhance the proteasome activity in the muscle of Caenorhabditis elegans. Mechanistically, this communication depends on Small Clear Vesicles, with glutamate as one of the neurotransmitters required for the distal regulation. More importantly, we demonstrate that this cell non-autonomous proteasome activation is translated into efficient prevention of amyloid-beta (Αβ)-mediated proteotoxic effects in the muscle of C. elegans but notably not to resistance against oxidative stress. Our in vivo data establish a mechanistic link between neuronal proteasome reinforcement and decreased Aβ proteotoxicity in the muscle. The identified distal communication may have serious implications in the design of therapeutic strategies based on tissue-specific proteasome manipulation

A disease-associated Aifm1 variant induces severe myopathy in knockin mice

Objective: Mutations in the AIFM1 gene have been identified in recessive X-linked mitochondrial diseases. Functional and molecular consequences of these pathogenic AIFM1 mutations have been poorly studied in vivo. Methods/results: Here we provide evidence that the disease-associated apoptosis-inducing factor (AIF) deletion arginine 201 (R200 in rodents) causes pathology in knockin mice. Within a few months, posttranslational loss of the mutant AIF protein induces severe myopathy associated with a lower number of cytochrome c oxidase-positive muscle fibers. At a later stage, Aifm1 (R200 del) knockin mice manifest peripheral neuropathy, but they do not show neurodegenerative processes in the cerebellum, as observed in age-matched hypomorphic Harlequin (Hq) mutant mice. Quantitative proteomic and biochemical data highlight common molecular signatures of mitochondrial diseases, including aberrant folate-driven one-carbon metabolism and sustained Akt/mTOR signaling. Conclusion: Our findings indicate metabolic defects and distinct tissue-specific vulnerability due to a disease-causing AIFM1 mutation, with many pathological hallmarks that resemble those seen in patients. Keywords: Akt/mTOR signaling, Apoptosis-inducing factor (AIF), 1C metabolism, Mitochondria, Mitochondrial diseases, Oxidative phosphorylatio

Systems biology analysis identifies impairment of mitochondrial and glycolytic metabolism in a genetic model of Alzheimer\u2019s disease.

Mitochondrial dysfunction is implicated in most neurodegenerative diseases, including Alzheimer\u2019s disease (AD). We here combined experimental and computational approaches to investigate mitochondrial health and bioenergetic function in neurons from a double transgenic animal model of AD (PS2APP/B6.152H). Experiments in primary cortical neurons demonstrated that AD neurons had reduced mitochondrial respiratory capacity. Interestingly, the computational model predicted that this mitochondrial bioenergetic phenotype could not be explained by any defect in the mitochondrial respiratory chain (RC), but could be closely resembled by a simulated impairment in the mitochondrial NADH flux. Further computational analysis predicted that such an impairment would reduce levels of mitochondrial NADH, both in the resting state and following pharmacological manipulation of the RC. To validate these predictions, we utilised fluorescence lifetime imaging microscopy (FLIM) and autofluorescence imaging and confirmed that transgenic AD neurons had reduced mitochondrial NAD(P)H levels at rest, and impaired power of mitochondrial NAD(P)H production. Of note, FLIM measurements also highlighted reduced cytosolic NAD(P)H in these cells, and extracellular acidification experiments showed an impaired glycolytic flux. The impaired glycolytic flux was identified to be responsible for the observed mitochondrial hypometabolism, since bypassing glycolysis with pyruvate restored mitochondrial health. This study highlights the benefits of a systems biology approach when investigating complex, non-intuitive molecular processes such as mitochondrial bioenergetics, and indicates that primary cortical neurons from a transgenic AD model have reduced glycolytic flux, leading to reduced cytosolic and mitochondrial NAD(P)H and reduced mitochondrial respiratory capacity

Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer's disease neurons

Mitochondrial dysfunction is implicated in most neurodegenerative diseases, including Alzheimer's disease (AD). We here combined experimental and computational approaches to investigate mitochondrial health and bioenergetic function in neurons from a double transgenic animal model of AD (PS2APP/B6.152H). Experiments in primary cortical neurons demonstrated that AD neurons had reduced mitochondrial respiratory capacity. Interestingly, the computational model predicted that this mitochondrial bioenergetic phenotype could not be explained by any defect in the mitochondrial respiratory chain (RC), but could be closely resembled by a simulated impairment in the mitochondrial NADH flux. Further computational analysis predicted that such an impairment would reduce levels of mitochondrial NADH, both in the resting state and following pharmacological manipulation of the RC. To validate these predictions, we utilized fluorescence lifetime imaging microscopy (FLIM) and autofluorescence imaging and confirmed that transgenic AD neurons had reduced mitochondrial NAD(P)H levels at rest, and impaired power of mitochondrial NAD(P)H production. Of note, FLIM measurements also highlighted reduced cytosolic NAD(P)H in these cells, and extracellular acidification experiments showed an impaired glycolytic flux. The impaired glycolytic flux was identified to be responsible for the observed mitochondrial hypometabolism, since bypassing glycolysis with pyruvate restored mitochondrial health. This study highlights the benefits of a systems biology approach when investigating complex, nonintuitive molecular processes such as mitochondrial bioenergetics, and indicates that primary cortical neurons from a transgenic AD model have reduced glycolytic flux, leading to reduced cytosolic and mitochondrial NAD(P)H and reduced mitochondrial respiratory capacity.</p