Cellular mechanisms of protection against protein misfolding in neurodegenerative disease. Role of oxidative stress.

Projektförslag för kandidatarbete inom inst. Kemi och kemiteknik och Biologi och bioteknik
​Projektexamenskod: BBTX01-21-03 

Avdelningen för Systembiologi
Institutionen för Biologi och Bioteknik, Chalmers tekniska högskola 

Protein biosynthesis is an energetically demanding process and it is regulated in response to environment (nutrient availability, stress …). Proteins must be folded in order to perform their functions. To adapt to this, cells have exquisite molecular machineries, for example molecular chaperones that aid in protein folding and in maintaining proteins folded.
Under stress conditions, proteins may unfold or fail to fold correctly. This misfolding protein can lead to aggregate formation and that are problems in the brain, being the underlying cause of several severe and incurable age-related disorders including Alzheimer’s and Parkinson’s diseases as the motor neuron disorder Amyotrophic Lateral Sclerosis (ALS). The pathophysiology of ALS is also characterized by increased oxidative stress. Different types of protein aggregates occur in most neurodegenerative disease including ALS.
Interestingly, a particular type of anti-oxidant protein, peroxiredoxin, was found to be absolutely required for molecular chaperones to engage age-related aggregates. Peroxiredoxins function as H₂O₂-controlled chaperones that slow down aging and decrease the incidence of age-related disease in organisms ranging from yeast to mice.
TDP-43 is an RNA binding protein that has been identified as a major component of the protein aggregates characterizing ALS. By expressing TDP-43 in baker’s yeast, several novel regulators of ALS have been identified in genome-wide screens, notably genes encoding ribosomal proteins and regulators of so-called stress granules. These are a type of beneficial inclusions where mRNA and translational components are stored transiently under unfavorable conditions until translation can resume.
The exact relationship between beneficial aggregates and pathophysiological TDP-43 aggregates is still mostly unclear. Our previous research in yeast show that the absence of the peroxiredoxin Tsa1 results in increased TDP-43 toxicity.

On going studies in our lab suggest that Tsa1 regulates protein biosynthesis and may be involved in transfer RNA (tRNA) modification. This since Tsa1 shares negative genetic interactions with several tRNA modification enzymes, e.g. Deg1 (depressed growth rate, tRNA pseudouridine synthase) suggesting a functional interaction. In fact, cells lacking Deg1 display a strong growth defect upon H₂O₂ which cannot be overcome by overexpression of the TSA1 gene, suggesting that Tsa1 may elicit H₂O₂ resistance through modifying tRNAs.

With a global increase in life expectancy, the prevalence of neurodegenerative disease, for instance ALS, increases. These diseases are characterized by, among other things, progressive loss of neurons. As of today, these diseases cannot be cured. Neurodegenerative diseases can be linked both oxidative stress and accumulation of toxic proteins aggregates in neurons. In ALS, these aggregates consist mainly of the protein TDP-43. Previous studies performed in yeast suggest that the absence of the peroxiredoxin Tsa1 result in increased TDP-43 toxicity.

We will study:
1.    Role of tRNA modification enzymes in TDP-43 aggregation: Using TDP-43 plasmids and fluorescence microscopy we will study if Deg1 and other tRNA modification enzymes have a role in TDP-43 aggregation.
2.    Role of Ataxin-2 in TDP-43 aggregation under oxidative stress: The yeast gene PBP1 (Poly (A)- binding protein (Pab1p)-binding protein) is an enhancer of TDP-43 toxicity. Interesting, Pbp1 is an ortholog of the human Ataxin-2-gene, mutations in which cause the neurodegenerative disease spinocerebellar ataxia type 2 (SCA2). In SCA2, as in ALS, motor neurons degenerate. Interestingly, Pbp1 interacts with Pab1 to regulate stress granule assembly. Recent studies suggest that Pbp1 regulates the nutrient sensing hub TORC1 through methionine residues, sensitive to oxidation by H₂O₂. We will study the role of Pbp1 aggregation in TDP-43 toxicity and its connection with oxidative stress and Tsa1.
Genomförande /Viktiga moment/teknikinnehåll 
1. PCR amplification of both plasmids. DNA electrophoresis to check if the amplify is well done.
2. Plasmid extraction from bacteria through growing bacteria in LB medium + ampicillin and extraction using a midiprep kit.
3. Yeast transformation with both plasmids and growth in selective medium.
4. Growth curves and characterization of TDP-43 expressing humanized yeast using spot tests with estradiol (to induce TDP-43 expression) and different stress conditions.
5. Protein analysis including Western Blot and biochemical techniques to extract insoluble protein aggregates.
6. Fluorescence microscopy and live cell confocal microscopy imaging. Image analysis including cell tracking.

Speciella förkunskapskrav: Basic knowledge of microbiology techniques and image analysis including cell tracking is meritorious.

Möjlig målgrupp Bt

Gruppstorlek: 4–6 studenter 

Förslagsställare/kontaktperson/huvudhandledare: ​​Mikael Molin and Cecilia Picazo, SysBio, BIO, Chalmers.
Övriga handledare:

Sidansvarig Publicerad: fr 30 okt 2020.