Mechanisms of Molecular Chaperones in Neurodegenerative Diseases
Despre această carte electronică
Molecular chaperones make up the cellular machinery that handle unfolded, misfolded, and aggregated proteins. Throughout evolution chaperones have been a prerequisite for the existence of complex proteins. This has made chaperones essential for biological life as we know it.
Utilizing chaperones as a pharmaceutical drug is a novel treatment approach for neurodegenerative disease. By adding molecular chaperones to the system, one could potentially improve the cells’ ability to handle the cellular stress that comes because of the imbalance of the proteostasis. Today there are several chaperone candidates that are being explored for this purpose.
HSP10 is an essential co-chaperone to HSP60. However, HSP10 has also been found to act alone. HSP10 has been shown to inhibit autoimmune activity. The co-chaperone is systemically overexpressed in the early stages of pregnancy. The overabundance of HSP10 in the brain compared to HSP60 and association with neurodegenerative disease suggests that the co-chaperone could have more functions than previously documented.
This thesis investigates how HSP10 interacts with several different amyloid proteins during amyloid fibrillation both in an in vitro and in an in vivo setting. For in vitro studies, interactions between recombinant proteins were scrutinized using a range of biophysical measurement techniques, e.g. aggregation kinetics, transmission electron microscopy, small angle X-ray scattering among others. The in vivo model Drosophila melanogaster was utilized to monitor how the combination of HSP10 and aggregation prone protein expression affects a complex organism.
Aggregation kinetics of the neurodegenerative peptide Aβ1-42, associated with Alzheimer´s disease, was performed in the presence of different concentrations of HSP10. Aggregation inhibition of Aβ1-42 was evident when HSP10 was present at high concentrations. However, low concentration of HSP10 in contrast accelerated the aggregation of Aβ1-42. This was followed by aggregation kinetics with α-synuclein, a protein associated with Parkinson´s disease. The presence of HSP10 at high concentration yielded a complete inhibition of α- synuclein aggregation. However, no acceleration of α-synuclein fibrillation was seen when the concentration was lowered. Further investigation of aggregation kinetics with the familial disease associated α-synuclein mutant A30P revealed the dual property of inhibition at high concentration of HSP10 and acceleration at lower concentration of HSP10.
To investigate how HSP10 would act in vivo, we used Drosophila melanogaster (Drosophila) as a model system. HSP10 was overexpressed in the neurons of Drosophila with and without the presence Aβ1-40 and Aβ1-42 and the lifespan and activity of the flies were monitored and were compared with controls. Overexpression of HSP10 in wild type flies without Aβ1-42 significantly prolonged the lifespan of Drosophila compared to control flies demonstrating that HSP10 rendered a protective effect against aging. Overexpression of Aβ1-42 decreased the lifespan of Drosophila. Surprisingly, the overexpression of both HSP10 and Aβ1-42 lead to an even shorter lifespan than expression of Aβ1-42 alone, suggesting an accelerated fibril formation process with enhanced neurotoxicity.
To investigate if the dual property of concentration dependent inhibition and acceleration of amyloid formation was unique to HSP10, fibril formation studies of Aβ1-40 and Aβ1-42 was conducted in the presence of the molecular chaperones αB-crystallin (from the small HSP chaperone family) and DNAJB1 (from the HSP40 chaperone family). DNAJB1 and αB-crystallin are classified as canonical ATP-independent holdases. Both DNAJB1 and αB-crystallin chaperones showed a clear inhibition of amyloid fibril formation when they were present at high concentration while they accelerated the fibril formation when the chaperone concentrations were lowered. Hence, we concluded that chaperone acceleration of amyloid formation appeared to be a general chaperone mechanism of holdases and not a unique feature for HSP10.
Herein, we have investigated fundamental mechanisms of molecular chaperones in the context of modulation of amyloid fibril formation. Our results uncovered a new chaperone property: acceleration of amyloid fibril formation and provided valuable insights into the molecular mechanisms of chaperones. This thesis contributed with crucial knowledge needed for development of molecular chaperones as pharmaceutical agents targeting neurodegenerative disease.
