HSF1-controlled and age-associated chaperone capacity in neurons and muscle cells of C. elegans

Abstract

Protein stability under changing conditions is of vital importance for the cell and under the control of a fine-tuned network of molecular chaperones. Aging and age-related neurodegenerative diseases are directly associated with enhanced protein instability. Employing C. elegans expressing GFP-tagged luciferase as a reporter for evaluation of protein stability we show that the chaperoning strategy of body wall muscle cells and neurons is significantly different and that both are differently affected by aging. Muscle cells of young worms are largely resistant to heat stress, which is directly mediated by the stress response controlled through Heat Shock Transcription Factor 1. During recovery following heat stress the ability to refold misfolded proteins is missing. Young neurons are highly susceptible to chronic heat stress, but show a high potency to refold or disaggregate proteins during subsequent recovery. The particular proteome instability in neurons results from a delayed induction of the heat shock response. In aged neurons protein stability is increased during heat stress, whereas muscle cells show enhanced protein instability due to a deteriorated heat shock response. An efficient refolding activity is absent in both aged tissues. These results provide molecular insights into the differential protein stabilization capacity in different tissues and during aging.

Figure 1. Tissue-specific analysis of Luc::GFP denaturation/aggregation.

(A) Fluorescence micrographs of nematodes expressing Luc::GFP in neurons or muscle cells. (B) Analysis of luciferase activity during heat stress at 35°C. Luciferase activity from total lysates of 1 day adult Luc::GFP expressing worms was determined at indicated times of heat stress and compared to unstressed worms. Asterisks represent the statistical significance between muscular and neuronal luciferase activity at a given time point. **P<0.01, Student's t-test, n = 5. (C) Fluorescence micrographs of Luc::GFP in neuronal and muscle cells at indicated times of heat stress. Arrows indicate aggregates. (D) Immunoblotting of the soluble (S) and aggregated (P) fraction of Luc::GFP after 3.5 h heat stress. Luc::GFP was detected by an antibody directed against luciferase. Tubulin served for control of the preparation. Graphical representations of the ratio P/S were calculated using optical band densities. *P<0.05, Student's t-test, n = 3.

Figure 2. Tissue-specific analysis of Luc::GFP recovery.

(A) Analysis of luciferase activity during recovery at 20°C after 3.5 h heat stress. Luciferase activity from total lysates of 1 day adult Luc::GFP expressing worms was determined at indicated times and was compared to unstressed controls. *P<0.05, **P<0.01, Student's t-test, n = 5. (B) Fluorescence micrographs of Luc::GFP in neurons at indicated times of recovery at 20°C after 3.5 h heat stress. Arrows indicate aggregates. (C) Immunoblotting of the soluble (S) and aggregated (P) fraction of neuronal Luc::GFP at 3.5 h heat stress and 1.5 h recovery. Luc::GFP was detected by an antibody directed against luciferase. Tubulin served for control of the preparation. Graphical representations of the ratio P/S were calculated using optical band densities. *P<0.05, Student's t-test, n = 3. (D) Refolding of Luc::GFP after inhibition of protein translation. Neuronal Luc::GFP expressing worms were heat stressed for 3.5 h at 35°C. During the following recovery period worms were incubated with 0.6 mg/ml cycloheximide (CHX) or vehicle, respectively. Luciferase activity was determined at 3.5 h heat stress and 1.5 h recovery and was compared to unstressed controls. *P<0.05, Student's t-test, n = 4.

Figure 3. Neurons are susceptible to heat stress due to a delayed heat shock response.

(A) Analysis of HSF1 activity, using the expression of P_hsp-16.2::GFP. 1 day adult CL2070 worms were heat stressed for indicated times and the appearance of a GFP fluorescence in different tissues was analyzed in a total of at least 80 worms (n = 4). The temperature for heat stress was reduced to 32°C due to an increased susceptibility of the strain. (B) Analysis of luciferase activity during heat stress at 35°C after hsf-1 RNAi. Luciferase activity from total lysates of muscle Luc::GFP expressing worms was determined at indicated times and was normalized to unstressed worms. Control worms were treated with empty vector (eV). Asterisks represent the statistical significance between the two conditions at a given time point. *P<0.05, **P<0.01, Student's t-test, n = 4. (C) Fluorescence micrographs of Luc::GFP in muscles at indicated times of heat stress after hsf-1 RNAi. Control worms were treated with eV. Arrows indicate aggregates. (D) Analysis of neuronal luciferase stability in HSP1::DsRed co-expressing worms. Luciferase activity from total lysates of Luc::GFP expressing worms was determined after 3.5 h heat stress and was normalized to unstressed worms. **P<0.01, Student's t-test, n = 4. (E) Total luminescence of unstressed neuronal Luc::GFP expressing and HSP1::DsRed co-expressing worms. n = 3. (F) Fluorescence micrographs of Luc::GFP and HSP1::DsRed in neurons at indicated times of heat stress. Arrows assign aggregates. HSP1::DsRed accumulated in inclusions already at unstressed conditions, which did not interfere with its ability to rescue Luc::GFP from denaturation.

Figure 4. Tissue-specific alterations in chaperone capacity during aging.

(A,B) Neuronal luciferase activity in young and aged worms during heat stress and subsequent recovery. Luciferase activity from total lysates of 1 and 14 day adult worms was determined at indicated times of heat stress and recovery and was compared to appropriate unstressed worms. Asterisks in (A) represent the statistical significance between young and aged worms at a given time point. *P<0.05, **P<0.01, Student's t-test, n = 3. (C,D) Muscular luciferase activity in young and aged worms during heat stress and subsequent recovery. Luciferase activity from total lysates of 1 and 14 day adult worms was determined at indicated times of heat stress and recovery and was compared to appropriate unstressed worms. Asterisks in (C) represent the statistical significance between young and aged worms at a given time point. *P<0.05, **P<0.01, Student's t-test, n = 3. (E) Analysis of HSF1 activity in aged worms, using the expression of P_hsp-16.2::GFP. 10 day adult CL2070 worms were heat stressed for indicated times and the appearance of a GFP fluorescence in different tissues was analyzed in a total of at least 50 worms (n = 3). Due to an enhanced sensibility of the strain to heat stress and a reduced life span, experiments were carried out at 32°C and after 10 days of adulthood.