Neuroglobin high levels: an inducible pathway to prevent the mitochondrial dysfunctionality accompanying neurodegenerative diseases
Descrizione
The increased life expectancy further increases the impact of neurodegenerative diseases (NDs) on society. NDs are characterized by loss of specific neurons, which progressively lead to motor and cognitive deficits. NDs occur in acute pathological conditions and in chronic disorders such as Alzheimer’s disease (AD), Parkinson’s Disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). Although the differences in ND symptomatology, these diseases share similar biochemical hallmarks that include, among other, Reactive Oxygen Species (ROS) accumulation, reduced respiratory activity linked to respiratory complex deficiencies, neuron apoptosis, and mitochondrial dysfunction. Curative treatments for these disorders are lacking or inefficient; thus, the looming predicament for the economics of health care systems worldwide mandates the formulation of a strategic goal to discover and develop wide-ranging interventions to delay and/or prevent the onset of chronic disabling conditions. The rationale for adopting such a tactical objective relies on the premise that half costs and prevalence of chronic disabling conditions will be obtained even if a modest delay of 5 years in the onset of disability is obtained. However, the ‘ideal intervention’ for complex diseases as NDs requires re-evaluating current drug development paradigms, which rely on a set or hierarchy of assumptions derived from reductionist approach to explain the underlying biological of neurodegenerative process. The discovery of neuroglobin (NGB) and characterization of its preferential expression in brain tissue presented a new opportunity to understand the mechanisms underlying neural pathologies, as well as to discover new therapeutic approaches. Nowadays, NGB is regarded as an endogenous neuroprotective heme-protein being active against several brain injuries, including hypoxia, ischemia, toxicity, and O2/nutrient deprivation. The correlation between NGB expression and protection against nervous system pathologies has been demonstrated in cell systems, in vivo models of ND, and in human beings. Aside from the nature of the insult considered, NGB protective effects seem to depend on its overexpression and mitochondrial localization. Here, we propose a novel approach to develop interventions to delay and/or to prevent the onset of ND by increasing the levels of NGB that could open a new scenario in the possibility to discover new therapeutic approaches.
Finalità
This proposal aims to find if high levels of intracellular (i.e., induced) and extracellular (e.g., released from exosomes) NGB could preserve mitochondrial morphology and functionality in neurons maintaining its protective role in alleviating the symptoms and slowing down the progress of neurodegeneration. To reach this main goal, this joint and multidisciplinary proposal will reach the following scientific sub-objectives:
- Impact of high level of NGB induced by cell autonomous pathways on mitochondrial functions;
- Impact of exosomes containing high level of NGB on mitochondrial functions in cellular models of ND.
Risultati attesi
Work Package 1. Impact of high level of NGB induced by cell autonomous pathways on mitochondrial functions in neuronal cells
Action 1.1 Impact of high levels of NGB on mitochondrial functions.
1.1.1. Mitochondrial dynamics (completed)
1.1.2. Mitochondrial membrane potential (ΔΨ) (completed)
1.1.3. Total/mitochondrial ROS (completed)
1.1.4. Lipid peroxidation (completed)
1.1.5. Apoptosis (completed)
Action 1.2. Impact of high levels of NGB on mitophagy
1.2.1. Association NGB/gangliosides and NGB/cardiolipin (CL) during autophagy (ongoing)
1.2.2. Lipidic profile of NGB overexpressing cells (ongoing)
1.2.3. Association of NGB with CL with specific markers of mitophagy (ongoing)
1.2.4. Impact of high levels of NGB in raft like microdomains associated to MAM (completed)
1.2.5. Role of rafts in autophagy/mitophagy processes (completed)
1.2.6. Role of high levels of NGB in MAM-regulated autophagy process (completed)
Action 1.3. Impact of high levels of NGB on mitochondrial bioenergetic
1.3.1 Activity of respiratory chain complexes (Complex I, II/III, IV) (completed).
The result obtained in this WP allowed us to define (i) which level of NGB is necessary to maintain the mitochondrial functionality injured by oxidative stress (Milestone 1) and (ii) the mitochondrial function(s) that are more susceptible to the increase of globin levels (Milestone 2).
Work Package 2. Impact of exosomes containing high level of NGB on mitochondrial functions in in vitro models of HD and ASL.
Action 2.1. The impact of exosomes or extracellular vesicles or Conditioned Media enriched with NGB on mitochondrial functionality
2.1.1 Quantification of intracellular NGB levels after cell treatment with enriched extracellular media (completed)
2.1.2. Impact of NGB enriched media on mitochondrial functions selected in WP1 (ongoing)
2.1.3. Impact of NGB enriched media on ATP levels (ongoing)
2.1.4. Impact of NGB enriched media on cell viability after treatment with the oxidative stressors (ongoing)
Stato dell’arte
Neuroglobin (NGB) is an inducible globin, whose high intracellular levels exert neuroprotective effects against several types of insults and neurological disorders. Indeed, NGB over-expression protects cortical and hippocampal neurons against hypoxia-induced neuron death. A transgenic mouse engineered to overexpress NGB preserves mitochondrial respiration, attenuates formation of oxidative DNA damage and the persistent inflammatory changes induced by smoke exposure. In human SH-SY5Y neuroblastoma cells, NGB over-expression protects against anoxia and glucose deprivation. In vivo experiments using double transgenic mice overexpressing both NGB and amyloid precursor protein (APP) suggested that NGB overexpression plays a protective role against β-amyloid-induced neurotoxicity, as well as AD pathogenesis. NGB overexpression, protected against α-synuclein inclusion formation, hallmark of PD, in human neuroglioma cells. In animal model of amyotrophic lateral sclerosis (ALS) pathology progression, characterized by a hypermetabolism and a negative energy balance, is paralleled to a progressive loss of weight. Though no significant difference in weight was detected between double transgenic mice model Ngb-/-SODG93A and NgbWt/WtSOD1G93A mice, NGB expression seems to be necessary for the preservation of a normal mitochondrial phenotype and permeability transition pore opening. Nowadays, there is an ample consensus that the neuroprotective effects depend on NGB high levels and its mitochondrial localization. Although, NGB lacks a mitochondrial target sequence, it has been reported that NGB within the mitochondrial matrix interacts with electron-transferring flavoprotein α-subunit (Etfα) probably taking part of the respiratory chain. Other reports indicate NGB interactions with cytochrome c and voltage-dependent anion channel 1 (VDAC). Our findings indicate that in mitochondria of human neuroblastoma cells and of other cells, cytochrome c-NGB association occurs just in presence of high levels of NGB. Therefore, NGB-cytochrome c association into mitochondria may both avoid the release of cytochrome c in cytosol, its association with APAF-1, and the triggering of apoptotic cascade. The interaction between NGB and VDAC opens the possibility for NGB to modulate directly the permeability of the outer mitochondrial membrane, which represents a key event leading to apoptotic cell death. VDAC is a structural component of multimolecular signaling complexes associated with mitochondrial raft-like microdomains. Components of these domains, under pro-apoptotic stimulation, can be recruited from plasma membrane and Golgi apparatus to the mitochondria and can participate in the apoptotic cascade.
Since an NGB-VDAC physical interaction may regulate the intrinsic apoptotic pathway, the possible role of mitochondrial raft-like microdomains in the NGB anti-apoptotic function were evaluated. The overexpression of NGB abrogated the inhibition of complex IV place if the mitochondrial lipid rafts-like microdomains were intact. Indeed, NGB failed to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-β-cyclodextrin. Although the mechanism regulating NGB mitochondrial localization remains unclear, an association of NGB with huntingtin (HTT) has been reported. This association allows NGB to shuttle from cytosol to the mitochondria and protects SK-N-BE neuroblastoma cells and murine striatal cells against H2O2-induced apoptosis. Intriguingly, in striatal neurons expressing the mutated form of HTT (i.e., HTTQ111), a model of HD, HTT loses its function as scaffold protein, impairing the NGB localization into mitochondria.
The concept that high levels of NGB exert neuroprotective effects against several types of insults and neurological disorders is now widely accepted. Over the years, it has been proved that NGB is a mitochondrial protein which direct interaction with mitochondrial proteins is not limited to a structural connection, but it also implies a functional one. Nowadays, the finding that NGB impacts on mitochondrial dynamics/trafficking, preserves mitochondrial functionality in terms of ATP and ROS production and regulation of cell death and that, in turn, mitochondria are needed for the NGB-dependent cytoprotection opens new avenues in the field of NGB research.
The identification of molecular mechanisms and intracellular pathway regulating the role of NGB in mitochondrial homeostasis, could define new therapeutic targets for developing strategies and drugs to counteract the progression of mitochondrial-associated NDs.
Riferimento: PRIN 2022 – Codice progetto: 20223RXEEC – CUP F53D23005240006
Investimento totale del progetto: 246.606 €
Partner/proponente: Università Roma Tre (proponente), Università Sapienza (partner), CNRR (partner)
Coordinatore dell’UdR Università degli Studi Roma Tre: Maria Marino
