Investigating the role of DnaJ proteins in the suppression of tau aggregation

Abstract (Swedish)

Tauopathies belong to a group of neurodegenerative diseases that are characterized by the presence of neurofibrillary tangles in neurons and includes diseases such as Alzheimer´s disease and frontotemporal dementia. Neurofibrillary tangles are insoluble deposits that are composed mainly of tau, which normally acts to stabilize microtubules. In disease, tau forms fibrillar and oligomeric species, which can be toxic to cells. DnaJ co-chaperone proteins are involved in preventing or inhibiting the aggregation of other aggregation-prone proteins, such as huntingtin, amyloid-β, and α-synuclein However, the role of DnaJ co-chaperone proteins, specifically DNAJB1 and DNAJB6, has not been explored in the aggregation of tau. Therefore, the goal of this project was to determine whether the lack of DNAJB1 or DNAJB6 would lead to an increase in tau aggregates. This was accomplished by generating DNAJB1 or DNAJB6 knock-outs (KOs) in a HEK293 system either overexpressing human tau attached to the fluorophore mCherry (mCherry-tau) or overexpressing human tau with a with a proline to leucine mutation at tau site 301 (mCherry-P301L ta), which is located in exon 10. Generation of KOs was successfully accomplished by using a CRISPR/Cas9 system. However, none of the mCherry-tau DNAJB1 or DNAJB6 KOs differed significantly in their aggregation potential when compared to the parental control. Interestingly, mCherry-P301L tau DNAJB1 and DNAJB6 KOs exhibited lower percentages of cells with aggregates when compared to parental control. All parental and KO cell lines were then characterized using the alamarBlue assay. The alamarBlue assay revealed no significant difference among the mCherry-tau and mCherry-P301L tau lines.

Popular Abstract

Helpers of helpers – co-chaperone proteins and protein aggregation

Proteins are one of the essential molecules that make up our bodies and the bodies of all living beings. Chaperone proteins exist as a protein ¨helper,¨ folding, maintaining, and regulating other proteins. To help these helpers, co-chaperone proteins, ¨helpers of helpers,¨ regulate chaperone proteins and help them by carrying out specialized functions. Nevertheless, some proteins can get into trouble by aggregating together. Protein aggregates can cause problems in cells, such as leading to cell death. The problems of protein aggregates are one of the contributing factors to the debilitating effects of neurodegenerative diseases. The goal of this project was to determine whether certain co-chaperone proteins play a role in the aggregation of one protein that is implicated in certain neurodegenerative diseases.

Proteins play a vital role in the function of all living things (including us!). Therefore, the dysfunction of certain proteins can have debilitating consequences on a being as a whole. In the case of neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease, proteins form aggregates that may be toxic to the cells in the brain. Here, we observed aggregates of a particular protein, microtubule associated protein tau. Tau protein normally helps cells form their ¨skeleton,¨ but, in AD, tau protein aggregates are implicated in causing brain cell degeneration.

How does the cell fight protein aggregation? One line of defense includes a class of proteins called chaperone proteins and their helpers, co-chaperone proteins. In particular, we were interested in these ¨helpers of helpers¨ because not much was known about their role in tau protein aggregation. For this project, we picked two co-chaperone proteins based on previous protein aggregation research.

Here, the loss of certain co-chaperone proteins did not play a significant role in tau protein aggregation. However, the aggregation of tau protein with a mutation was affected by the removal of co-chaperone proteins. In this case, the loss of the co-chaperone proteins led to more mutant tau protein aggregation, suggesting co-chaperone proteins can be good or bad for protein aggregation. Because we did not see much tau protein aggregation in general, we tried to encourage aggregation by adding either purified partially aggregated tau protein or brain homogenate from AD patients, which presumably contained tau protein aggregates. Both experiments were only carried out once, so the results were statistically inconclusive, but they may provide inspiration for future experiments.

By understanding the role these ¨helpers of helpers¨ play, we can piece together the factors that contribute to protein aggregation in neurodegenerative diseases and, one day, develop treatments that can target these factors. This thesis work is one stepping stone in that process and may help guide future experiments that clarify the role of co-chaperone proteins in tau protein aggregation.

Master’s Degree Project in Molecular Biology, Medical Biology 60 credits 2017-2018
Department of Biology, Lund University

Advisor: Christian Hansen
Molecular Neurobiology, Department of Experimental Medical Science, Lund University

• author: Garcia, Megg
• supervisor: Christian Hansen
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20172
• year: 2018
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8952794
• date added to LUP: 2018-06-25 15:19:02
• date last changed: 2018-06-25 15:19:02