Lipid rafts have been identified as further actors of the autophagic process. Autophagy is a ubiquitous intracellular degradation pathway whose levels need to be tightly controlled to secure cell homeostasis. Alterations in lipid rafts components have been hypothesized to contribute to the loss of neural function and potentially to the cell death/cell survival or autophagy balance associated with neurodegeneration (ND). Lipid rafts have been also identified as subdomains on mitochondria-associated membranes (MAMs) proposed as tethering sites between the endoplasmic reticulum (ER) and mitochondrial membranes. The membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. In fact, this process acts at basal levels to maintain a continuous and effective intracellular clearance but it may also be induced under stress conditions. It has been hypothesized that alteration in MAM's function leads to several human disorders, and in particular can contribute to the loss of neural function and potentially to the cell death associated with ND.
Recently, we clarified the protective role of endogenous neuroglobin (NGB) against ND and its relocalization into the mitochondria lipid rafts. Thus, studying the autophagic machinery during stress condition, this research is aimed to evaluate if the protective role of NGB overexpression may depend on autophagy activation MAM raft-like microdomains mediated.
Thus, this research program is divided into 4 main objectives:
1. Proteomic analysis in neuroblastoma cells overexpressing NGB;
2. Analysis of NGB interaction with MAM raft-like microdomains in neuroblastoma cells after stress induction;
3. Evaluation of NGB MAM-association during autophagy activation;
4. Analysis of NGB neuroprotection using "in vitro" models.
These data could provide new insight on the role of NGB in neuroprotection and/or in the identification of novel pharmacological targets in the control of cell fate.
The fact that the MAMs embed subdomains enriched in cholesterol and glycosphingolipids, similar to the lipid rafts of the plasma membrane, emphasizes the direct involvement of these constituents in the regulated trafficking of molecules and ions between the two organelles, which can drive the cell fate towards survival/autophagy or death. Recently, we have identified and characterized the raft-like microdomains in mitochondrial membrane, demonstrating their key role in the structural and functional mitochondrial modifications associated with apoptosis execution. Lipid rafts may be considered as preferential sites on the cell membranes where several key reactions take place.
Thus, it is not surprising that changes in the MAM's lipid composition/ concentration in response to pathological conditions could alter the correct assembly of the vesicles and the scrambling among organelles hindering the autophagic process. Many fundamental questions on the biology of the MAMs remain unanswered.
This project through the study of the main objectives described above, will enlighten the molecular mechanisms controlling the upstream regulation of autophagy, a key process in cell metabolism and stress response and if Ngb may be related to the autophagic machinery involved in neurodegeneration.
It is still a matter of debate whether variations in the lipid and/or protein signature of the MAMs exist in different cell types or in response to physiologic/pathologic stimuli. What is clear is that specific lipid combinations within the MAMs dictate the recruitment and activity of distinct sets of proteins. In the last few years, increased emphasis has been devoted to understand the contribution of MAM to human pathology, in general, and to neurodegenerative diseases, in particular. A major reason for this is the central role that this subdomain of the ER plays in metabolic regulation and in mitochondrial biology. As such, aberrant MAM function may help explain the seemingly unrelated metabolic abnormalities often seen in neurodegeneration disease.
First, we will aim at identifying key molecules as components of a multimolecular complex in MAM, able to regulate autophagy in the initial organelle scrambling activity, leading to the formation of autophagosome. Techniques will include isolation of high-purity MAMs fraction, FRET, HPTLC and coimmunoprecipitation. This project could provide new findings not only in the biology of inter-organelles contacts (both in terms of protein components and lipid/lipid rafts metabolism), but also in the fields of molecular neuroscience disorders. In fact, second, in these research program we will dissect MAMs to verify the effect that these have on autophagy process, selective and in turn neuronal, thus in protecting mitochondrial network. In particular, using a model of cells stably transfected with a plasmid leading the expression of human NGB, we will study the association of NGB with lipid rafts at MAM level, in order to clarify whether its neuroprotective activity is active through MAM raft-like microdomains. These studies may also have a clinical relevance in terms of drug-delivery. In fact, statins, which are known to decrease the cellular levels of cholesterol, or cyclodextrins, that exert their effects via the formation of noncovalent inclusion complexes, are being used in an ever-increasing way to camouflage undesirable pharmaceutical characteristics or to improve therapeutic indices and site-targeted delivery of different drugs, including some nonsteroidal anti-inflammatory drugs. Hence, our research, pointing to the key role of lipid rafts in cell fate regulation mechanisms, could provide further insight in this field, suggesting new molecular targets for neurodegenerative therapy.