Periodic Reporting for period 1 - NeuroFreezing (Biophysical Properties of the Neuronal Cytosol and their Dynamics upon Nutrient Starvation, Aging, and in Neurodegenerative Diseases.)
Reporting period: 2019-06-01 to 2021-05-31
Neurodegenerative polyglutamine (polyQ) expansion disorders such as Huntington’s disease (HD) are devastating, progressive and ultimately fatal diseases with no effective treatment or cure. The molecular hallmark of the diseases is an age-dependent, neurotoxic aggregation of polyglutamine-proteins. The fundamental cellular processes triggering this neurotoxic protein aggregation are poorly understood, resulting in a scarcity of drug targets for developing therapeutic strategies. Interestingly, glucose uptake into the brain is reduced in both HD patients and animal models. Reduced brain glucose metabolism precedes both motor symptoms and tissue loss, and the degree of glucose-hypometabolism appears to affect age of symptom-onset, with larger decreases in glucose uptake correlating with earlier disease onset. Recent studies in yeast and bacteria in have shown that the material properties of the cytosol in these cells change upon reduced glucose-uptake, leading the liquid-like cytosol to appear more solid-like, which in turn results in increased protein aggregation. We hypothesized that a similar response to reduced glucose-metabolism may change the material properties of the neuronal cytosol and thereby contribute to triggering the neurotoxic aggregation of polyQ-proteins in HD and other neurodegenerative polyglutamine expansion disorders.
The overall objectives of the project were to determine whether:
1. mammalian neurons regulate cytoplasmic diffusion and display a stress response that changes the material properties of the cytosol,
and whether
2. this neuronal stress response upon nutrient-starvation or ageing is sufficient to trigger aggregation of polyglutamine-proteins.
The overall objectives of the project were to determine whether:
1. mammalian neurons regulate cytoplasmic diffusion and display a stress response that changes the material properties of the cytosol,
and whether
2. this neuronal stress response upon nutrient-starvation or ageing is sufficient to trigger aggregation of polyglutamine-proteins.
1. Biophysical properties of the neuronal cytosol
We expressed fluorescent nanoparticles within neurons and used single particle tracking to obtain information about the physical properties of the neuronal cytosol. Our analysis of the particle behaviour revealed that there are significant differences in the material properties of the cytosol within the neuronal cell body and the cytosol within the neuronal cell processes.
In order to analyse the effects of nutrient-stress on the biophysical properties of the neuronal cytosol, we starved neurons of glucose for 1 hour and repeated the particle tracking. Our analysis showed that glucose-starvation has a strong impact on particle movement: after 1 hour of glucose-starvation particle displacement is significantly reduced.
2. Impact of metabolic stress on aggregation of mutant huntingtin within neurons
A major unanswered question regarding polyglutamine expansion disorders such as Huntington’s Disease is which cellular events trigger aggregate formation in neurons around midlife, when they have for decades expressed the mutated polyQ proteins without accumulating aggregates. A central hypothesis of our project is that reduced glucose metabolism may be a trigger for aggregation of polyQ proteins such as mutant huntingtin (mHtt) within neurons. Preliminary data included in my project proposal showed that glucose starvation leads to enhanced aggregation of mutant huntingtin (mHtt) within cancer cells after 1 hour of glucose-starvation.
We expressed fluorescent mHtt within neurons and observed aggregation preceding and following 1 hour glucose-starvation. We observed that the number of mHtt aggregates increased by a third following glucose-starvation.
3. Impact of metabolic stress on neuronal cell volume and refractive index
Our analyses of particle displacement and aggregation of mHtt indicate that nutrient starvation induces changes in the biophysical properties of the neuronal cytosol. In order to further explore these changes we used digital holographic microscopy to measure the phase shift caused to light being transmitted through neurons pre- and post 1 hour glucose-starvation. By analysing the changes to the optical path length, we were able to determine that glucose-starvation significantly reduces neuronal cell volume.
These results have not yet been disseminated to the wider scientific community.
We expressed fluorescent nanoparticles within neurons and used single particle tracking to obtain information about the physical properties of the neuronal cytosol. Our analysis of the particle behaviour revealed that there are significant differences in the material properties of the cytosol within the neuronal cell body and the cytosol within the neuronal cell processes.
In order to analyse the effects of nutrient-stress on the biophysical properties of the neuronal cytosol, we starved neurons of glucose for 1 hour and repeated the particle tracking. Our analysis showed that glucose-starvation has a strong impact on particle movement: after 1 hour of glucose-starvation particle displacement is significantly reduced.
2. Impact of metabolic stress on aggregation of mutant huntingtin within neurons
A major unanswered question regarding polyglutamine expansion disorders such as Huntington’s Disease is which cellular events trigger aggregate formation in neurons around midlife, when they have for decades expressed the mutated polyQ proteins without accumulating aggregates. A central hypothesis of our project is that reduced glucose metabolism may be a trigger for aggregation of polyQ proteins such as mutant huntingtin (mHtt) within neurons. Preliminary data included in my project proposal showed that glucose starvation leads to enhanced aggregation of mutant huntingtin (mHtt) within cancer cells after 1 hour of glucose-starvation.
We expressed fluorescent mHtt within neurons and observed aggregation preceding and following 1 hour glucose-starvation. We observed that the number of mHtt aggregates increased by a third following glucose-starvation.
3. Impact of metabolic stress on neuronal cell volume and refractive index
Our analyses of particle displacement and aggregation of mHtt indicate that nutrient starvation induces changes in the biophysical properties of the neuronal cytosol. In order to further explore these changes we used digital holographic microscopy to measure the phase shift caused to light being transmitted through neurons pre- and post 1 hour glucose-starvation. By analysing the changes to the optical path length, we were able to determine that glucose-starvation significantly reduces neuronal cell volume.
These results have not yet been disseminated to the wider scientific community.
The overall objectives of the project were to test the two hypotheses that:
1. mammalian neurons regulate cytoplasmic diffusion and display a stress response that changes the material properties of the cytosol,
and that
2. this neuronal stress response upon nutrient-starvation or ageing is sufficient to trigger aggregation of polyQ-proteins.
Our results so far show for the first time that:
- material properties of the cytosol differ between the neuronal cell body and neuronal cell processes;
- reduced neuronal glucose-uptake changes the material properties of the neuronal cytosol;
- glucose-starvation triggers aggregation of the polyglutamine protein mutated in HD within neurons;
- neuronal cell volume is reduced upon glucose-starvation
Our data indicate the validity of our hypotheses, and may suggest glucose metbaolism as a potential future drug target for treatment of neurodegenerative polyglutamine disorders in general and HD in particular.
1. mammalian neurons regulate cytoplasmic diffusion and display a stress response that changes the material properties of the cytosol,
and that
2. this neuronal stress response upon nutrient-starvation or ageing is sufficient to trigger aggregation of polyQ-proteins.
Our results so far show for the first time that:
- material properties of the cytosol differ between the neuronal cell body and neuronal cell processes;
- reduced neuronal glucose-uptake changes the material properties of the neuronal cytosol;
- glucose-starvation triggers aggregation of the polyglutamine protein mutated in HD within neurons;
- neuronal cell volume is reduced upon glucose-starvation
Our data indicate the validity of our hypotheses, and may suggest glucose metbaolism as a potential future drug target for treatment of neurodegenerative polyglutamine disorders in general and HD in particular.