Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that have been suggested to interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Moreover, lipid microdomains, i.e. 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. In fact, this process acts at basal levels to maintain a continuous and effective intracellular clearance but it may also be induced in a number of stress conditions. It has been hypothesized that alteration in MAM's function leads to majority of human disorders, and in particular can contribute to the loss of neural function and potentially to the cell death associated with neurodegeneration.
Thus, this research program is divided into 4 main objectives:
1. Evaluation of raft-like microdomains in the regulation of autophagy.
2. Analysis of a possible implication of MAM raft-like microdomains during early steps of autophagosome generation.
3. Proteomic analysis of MAMs in the absence or following autophagic stimulus.
4. Analysis of a possible role of autophagy on neuroprotection through MAM raft-like microdomains in neuroblastoma cells.
The fact that the MAMs embed subdomains enriched in cholesterol and glycosphincolipids, similar to the lipid rafts of the PM, 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 combined data from the multidisciplinary approaches described above, will enlighten the molecular mechanisms controlling the upstream regulation of autophagy, a key process in cell metabolism and stress response.
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 understanding the contribution of MAMs to human pathology in general, and 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.
First we will aim at identifying key molecules as components of a multimolecular complex in MAMs, able to regulate autophagy in the initial organelle scrambling activity leading to the formation of autophagosome. Techniques will include isolation of high-purity MAM's fraction, FRET, HPTLC and coimmunoprecipitation. This project, could provide 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. Infact, second in our research program we will dissect MAMs to verify the effect that these have on global autophagy, selective mitophagy 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.