Projects / Programmes
Fusion Pore Regulation and Lysosomal Storage Disorders
| Code |
Science |
Field |
Subfield |
| 3.03.00 |
Medical sciences |
Neurobiology |
|
| Code |
Science |
Field |
| B470 |
Biomedical sciences |
Physiology |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
exocytosis, fusion pore, cholesterol, lysosome, lysosomal storage diseases
Researchers (7)
Organisations (2)
Abstract
The proposed project aims to understand one of the most important mechanisms by which cells communicate with each other; regulated exocytosis. This process engages cytoplasmic, membrane bound vesicles, carrying cargo or simply vesicle membrane resident molecules, to be delivered to the plasmalemma. Vesicles are of different kinds and this project proposal will study lysosomes, that are typically larger than synaptic-like and dense-core vesicles.
Lysosomes were discovered in 1955 as one of the first cellular organelle, which opened the field of intracellular vesicle traffic studies in eukaryotic cells. It is well accepted that lysosomes play a pivotal role in the cell homeostasis, however not only in degradation, autophagy, storage and recycling processes, as originally proposed, but also in signalling. The function of lysosomes is mediated by their lumenal enzymes and the membrane with the ability to efficiently fuse with several target membranes including the plasma membrane.
Lysosomal dysfunction, as evidenced in over 50 types lysosomal storage disorders (LSDs), results mainly from mutations in enzymes that are active in the lysosome, although a handful are caused by defects in lysosomal transport or vesicular trafficking. The phenotypic consequences in patients are extremely varied, ranging from asymptomatic or sub-clinical manifestations; to chronic visceral, musculoskeletal or immunological disease, to lethal acute neuropathic disease, with early and late symptoms. Roughly two thirds of LSDs are neuropathic.
Although particular forms of LSDs are quite rare, however when taken together all of the LDSs, they usually express multi-organ defects, and because of a continuing lack of truly effective treatments for many LSDs, these diseases are often characterized by high mortality and morbidity. Current therapies are extremely costly and are often lifelong. LSDs therefore constitute a significant burden on affected individuals and their families and on healthcare systems. Moreover, a growing appreciation of the role of lysosomal dysfunction in neurodegenerative disorders has led to a recent surge of interest in LSDs. Understanding of the common denominator in LSDs and neurodegeneration, at the level of a cellular mechanism, represents an important problem and an urgent challenge to be addressed.
Since the common feature of LSDs is accumulation of lipids and cholesterol in lysosomes, this project proposal will study the mechanism of how cholesterol affects fusion pore gating. As the fusion pore is mediating the exit of lumenal vesicle cargo and is thus key in the regulation of transfer of molecules between cell interior and cell exterior, this knowledge is of wide scientific significance. We hypothesize that cholesterol regulates fusion pore gating and that this is impaired under conditions when cholesterol levels are in excess as is the case in LSDs. For this we will study the nature of vesicle-fusion with the plasma membrane at single vesicle level in real time, by the most sensitive electrophysiological monitoring of membrane capacitance, a parameter linearly related to plasma membrane area. Moreover, we will use novel fluorescent markers to label cholesterol-enriched membrane domains and super-resolution fluorescence microscopy to learn whether cholesterol-rich domains are associated with membranes where fusion of vesicles occurs. Furthermore, we will study cells isolated form animal models of LSDs, such as the Niemann Pick type C disease.
As such this project proposal, interdisciplinary in nature, will unravel one of the outstanding unsolved problems in eukaryotic cell biology. Moreover, this knowledge is required to rationalize the strategy of developing therapies for treating, for example LSDs, for which the cellular mechanism that is impaired is unknown, and needs to be revealed for the therapy to be developed. Thus the results of this study are essential and are bearing a significant transla
Significance for science
The proposed project aims to understand one of the most important mechanisms by which cells communicate with each other; regulated exocytosis. The relevance of this field of neurobiology is, among others, empahsized by two Nobel prizes that have been awarded in the last five decades (in 1970 to B. Katz, U. Euler, J. Axelrod, and in 2013 to J.E. Rothman, R.W. Schekman, T. Sudhof). Moreover, the impairment of exocytosis can lead to the development of several pathological conditions, including to lysosomal storage diseases. However, the key impairments are still unknown. One of the unresolved questions is linked to the role of lipids, such as cholesterol, in one of the last steps of exocytosis, i.e. in the fusion of vesicles with the plasma membrane. In lysosomal storage disease, where lipids accumulate in vesicles, this likely affects the nature of vesicle fusion with the plasma membrane. In part the knowledge of fusion pore regulation is still fragmental due to experimental inaccessibility. Therefore, we will use methods, which we introduced and developed in our laboratory to monitor physiological parameters at single cell, single organelle, and at single molecule level. These methods include STED microscopy (we have developed a two color STED system in colaboration with the research group of prof. dr. Stefan W. Hell) and electrophysiological monitoring of the membrane capacitance (we have developed our own lock-in patch-clamp amplifier, low-pass filter unit and software for acquisition and analysis).
In summary, the proposed project will:
a) generate new insights into the field of neuroscience,
b) be useful for discovering new therapeutic processes and targets,
c) help to address question on mechanisms in lysosomal storage diseases and will advance some of the already cutting edge technologies.
Significance for the country
The proposed project aims to understand one of the most important mechanisms by which cells communicate with each other; regulated exocytosis. The relevance of this field of neurobiology is, among others, empahsized by two Nobel prizes that have been awarded in the last five decades (in 1970 to B. Katz, U. Euler, J. Axelrod, and in 2013 to J.E. Rothman, R.W. Schekman, T. Sudhof). Moreover, the impairment of exocytosis can lead to the development of several pathological conditions, including to lysosomal storage diseases. However, the key impairments are still unknown. One of the unresolved questions is linked to the role of lipids, such as cholesterol, in one of the last steps of exocytosis, i.e. in the fusion of vesicles with the plasma membrane. In lysosomal storage disease, where lipids accumulate in vesicles, this likely affects the nature of vesicle fusion with the plasma membrane. In part the knowledge of fusion pore regulation is still fragmental due to experimental inaccessibility. Therefore, we will use methods, which we introduced and developed in our laboratory to monitor physiological parameters at single cell, single organelle, and at single molecule level. These methods include STED microscopy (we have developed a two color STED system in colaboration with the research group of prof. dr. Stefan W. Hell) and electrophysiological monitoring of the membrane capacitance (we have developed our own lock-in patch-clamp amplifier, low-pass filter unit and software for acquisition and analysis).
In summary, the proposed project will:
a) generate new insights into the field of neuroscience,
b) be useful for discovering new therapeutic processes and targets,
c) help to address question on mechanisms in lysosomal storage diseases and will advance some of the already cutting edge technologies.
Most important scientific results
Interim report
Most important socioeconomically and culturally relevant results
Interim report
