Final Report Summary - HSPB8 AND NEUROPATHY (Role of the HspB8/Bag3 chaperone complex in neurodegenerative disorders)
Summary: Accumulation of aggregated proteins is a hallmark of neurodegenerative and muscular disorders, including polyglutamine disorders and protein aggregate myopathies. Molecular chaperones and degradation systems (e. g. autophagy) allow cells to cope with misfolded mutated proteins. The HSP70/DNAJ and HSPB (HSPB1-10) families of molecular chaperones recognise and bind misfolded proteins, prevent their aggregation and facilitate their degradation. Molecular chaperones may exert neuroprotective functions and their modulation may delay the onset of protein conformation disorders.
Our project focuses on the molecular chaperone HSPB8. HSPB8 forms a stable complex with BAG3, a co-chaperone of HSC70. Interestingly, mutations in HSPB8 and BAG3 cause peripheral neuropathy and muscular dystrophy, thus suggesting that alteration of their function may have detrimental consequences for the viability of neuronal and muscular cells.
We previously demonstrated that overexpression of HSPB8-HSC70-BAG3 prevents the aggregation of mutated huntingtin and facilitates its degradation by stimulating autophagy, an essential process for aggregate-prone protein clearance and neuronal survival. On the basis of these findings, we hypothesize that HSPB8-HSC70-BAG3 may play a role in stress-mediated autophagy stimulation and may help cells to cope with misfolded-aggregating substrates.
Our research goals are:
1) Studying whether the modulation of HSPB8-HSC70-BAG3 function may protect in vivo against polyglutamine disorders.
2) Studying how the disease-related mutations in HSPB8 (K141E and K141N), associated with peripheral neuropathy, affect its function in protein quality control and autophagy (loss of function and/or toxic gain of function).
Overview of the main results and Conclusions:
1) Studying whether the modulation of HSPB8-HSC70-BAG3 function may protect in vivo against polyglutamine disorders.
We first dissected the mechanism of action of the HSPB8-HSC70-BAG3 complex. We identified the interconnection between HSPB8-HSC70-BAG3, autophagy and the eIF2 alpha pathway. We demonstrated that inhibition of eIF2 alpha phosphorylation completely abrogates HSPB8-BAG3 effect on autophagy and decreases their role in PQC. We recently focused on the molecular mechanisms regulating how the HSPB8-HSC70-BAG3 complex recognises, binds to and targets misfolded clients to the autophagosomes for degradation. We found that HSC70 plays an important role in the steps of recognition and binding to (polyubiquitinated) misfolded proteins.
Then we identified the Drosophila ortholog of human HSPB8 (Dm-HSP67Bc) and we analysed the ability of HSPB8 to protect against polyglutamine toxicity in vivo. Using a Drosophila model of Spinocerebellar Ataxia 3 (SCA3), we demonstrated that in vivo, overexpression of human HSPB8 and Dm-HSP67Bc protects against the degeneration induced by mutated SCA3. Also, downregulation of endogenous Dm-HSP67Bc significantly worsened SCA3-mediated eye degeneration.
To further investigate our hypothesis that HSPB8-BAG3 could play a protective role in protein aggregation diseases and might be specifically upregulated in response to aggregate-prone protein-mediated toxicity we finally investigated their expression levels in post mortem human brain tissue from patients suffering of the following protein conformation disorders: Alzheimer disease (AD), Parkinson disease (PD), Huntington disease (HD) and spinocerebellar ataxia type 3 (SCA3). In these diseases, we observed a strong upregulation of HSPB8 and a moderate upregulation of BAG3 specifically in astrocytes in the cerebral areas affected by neuronal damage and degeneration. Intriguingly, no significant change in the HSPB8-BAG3 expression levels was observed within neurons, irrespective of their localisation or of the presence of proteinaceous aggregates. These results strongly suggest that the upregulation of HSPB8 and BAG3 may enhance the ability of astrocytes to clear aggregated proteins released from neurons, cellular debris, maintain the local tissue homeostasis and/or participate in the cytoskeletal remodeling that astrocytes undergo during astrogliosis and further confirm their implication in protein aggregate diseases. In fact, these findings within the context of this grant have lead to the successful application for a grant at the Prinses Beatrix Foundation in which this hypothesis can be further tested.
2) Studying how the disease-related mutations in HSPB8 (K141E and K141N), associated with peripheral neuropathy, affect its function in protein quality control and autophagy (loss of function and/or toxic gain of function). The impact of the K141E and K141N mutations on HSPB8 role in PQC was investigated using cell models expressing either mutated SCA3 (64) Q or the P182L mutant of HSPB1, which is also associated with peripheral neuropathy and aggregates in cells. Overexpression of HSPB8 or Dm-HSP67Bc significantly decreased the aggregation of SCA3 (64) Q and P182L-HSPB1. In contrast, the K141E and K141N mutated forms of human HSPB8 were significantly less efficient than wild-type HSPB8, suggesting that the mutated forms of HSPB8 are characterised by a loss of function in PQC. Furthermore, in collaboration with Prof. Angelo Poletti, we demonstrated that in cellular models of Amyotrophic Lateral Sclerosis (ALS) upregulation of HSPB8, which naturally occurs in motor neurons in patients affected by ALS, protects against protein aggregation and motor neuron degeneration. These results, combined with our data suggestive of mutant HSPB8 loss of function in peripheral motor neuropathy, further point to upregulation and potentiation of HSPB8 function as potential therapeutic approach to protect motor neurons from stress and death.
Socio-economic impacts of the project: Deregulated autophagy is associated with neurodegenerative and neuromuscular disorders, whereas stimulation of autophagy is beneficial in diseases characterised by aggregating proteins. These items underscore the importance of understanding how autophagy is normally regulated and how it can be stimulated under specific diseases. Our project allowed a better understanding of how HSPB8-HSC70-BAG3 modulates PQC and autophagy, thus allowing the clearance of misfolded aggregate-prone proteins and provided insights in its role as modulator of polyglutamine diseases, as well as the motor neuron disease ALS. Also, our in vitro and in vivo results provided sufficient evidence to support that upregulation of HSPB8-HSC70-BAG3 might indeed represent a valid therapeutic tool to slow-down protein aggregate disease progression. Finally, they also strongly suggest that potentiation of HSPB8 function might be relevant especially to cure motor neuron disease, pointing to the importance of finding in the future drugs able to specifically target HSPB8 expression as therapeutic tool. Protein aggregate diseases like AD, PD, polyglutamine diseases and ALS, in which we demonstrated an implication of the HSPB8-HSC70-BAG3 complex have an increasing frequency (they mainly affect older people and the age worldwide population is increasing) and a very high socio-economic impact. Our project may allow in the future to develop pharmaceutical drugs able to slow-down (e. g. via HSPB8 upregulation) the progression of age-related aggregate protein disorders, therefore decreasing their socio-economic costs.
Our project focuses on the molecular chaperone HSPB8. HSPB8 forms a stable complex with BAG3, a co-chaperone of HSC70. Interestingly, mutations in HSPB8 and BAG3 cause peripheral neuropathy and muscular dystrophy, thus suggesting that alteration of their function may have detrimental consequences for the viability of neuronal and muscular cells.
We previously demonstrated that overexpression of HSPB8-HSC70-BAG3 prevents the aggregation of mutated huntingtin and facilitates its degradation by stimulating autophagy, an essential process for aggregate-prone protein clearance and neuronal survival. On the basis of these findings, we hypothesize that HSPB8-HSC70-BAG3 may play a role in stress-mediated autophagy stimulation and may help cells to cope with misfolded-aggregating substrates.
Our research goals are:
1) Studying whether the modulation of HSPB8-HSC70-BAG3 function may protect in vivo against polyglutamine disorders.
2) Studying how the disease-related mutations in HSPB8 (K141E and K141N), associated with peripheral neuropathy, affect its function in protein quality control and autophagy (loss of function and/or toxic gain of function).
Overview of the main results and Conclusions:
1) Studying whether the modulation of HSPB8-HSC70-BAG3 function may protect in vivo against polyglutamine disorders.
We first dissected the mechanism of action of the HSPB8-HSC70-BAG3 complex. We identified the interconnection between HSPB8-HSC70-BAG3, autophagy and the eIF2 alpha pathway. We demonstrated that inhibition of eIF2 alpha phosphorylation completely abrogates HSPB8-BAG3 effect on autophagy and decreases their role in PQC. We recently focused on the molecular mechanisms regulating how the HSPB8-HSC70-BAG3 complex recognises, binds to and targets misfolded clients to the autophagosomes for degradation. We found that HSC70 plays an important role in the steps of recognition and binding to (polyubiquitinated) misfolded proteins.
Then we identified the Drosophila ortholog of human HSPB8 (Dm-HSP67Bc) and we analysed the ability of HSPB8 to protect against polyglutamine toxicity in vivo. Using a Drosophila model of Spinocerebellar Ataxia 3 (SCA3), we demonstrated that in vivo, overexpression of human HSPB8 and Dm-HSP67Bc protects against the degeneration induced by mutated SCA3. Also, downregulation of endogenous Dm-HSP67Bc significantly worsened SCA3-mediated eye degeneration.
To further investigate our hypothesis that HSPB8-BAG3 could play a protective role in protein aggregation diseases and might be specifically upregulated in response to aggregate-prone protein-mediated toxicity we finally investigated their expression levels in post mortem human brain tissue from patients suffering of the following protein conformation disorders: Alzheimer disease (AD), Parkinson disease (PD), Huntington disease (HD) and spinocerebellar ataxia type 3 (SCA3). In these diseases, we observed a strong upregulation of HSPB8 and a moderate upregulation of BAG3 specifically in astrocytes in the cerebral areas affected by neuronal damage and degeneration. Intriguingly, no significant change in the HSPB8-BAG3 expression levels was observed within neurons, irrespective of their localisation or of the presence of proteinaceous aggregates. These results strongly suggest that the upregulation of HSPB8 and BAG3 may enhance the ability of astrocytes to clear aggregated proteins released from neurons, cellular debris, maintain the local tissue homeostasis and/or participate in the cytoskeletal remodeling that astrocytes undergo during astrogliosis and further confirm their implication in protein aggregate diseases. In fact, these findings within the context of this grant have lead to the successful application for a grant at the Prinses Beatrix Foundation in which this hypothesis can be further tested.
2) Studying how the disease-related mutations in HSPB8 (K141E and K141N), associated with peripheral neuropathy, affect its function in protein quality control and autophagy (loss of function and/or toxic gain of function). The impact of the K141E and K141N mutations on HSPB8 role in PQC was investigated using cell models expressing either mutated SCA3 (64) Q or the P182L mutant of HSPB1, which is also associated with peripheral neuropathy and aggregates in cells. Overexpression of HSPB8 or Dm-HSP67Bc significantly decreased the aggregation of SCA3 (64) Q and P182L-HSPB1. In contrast, the K141E and K141N mutated forms of human HSPB8 were significantly less efficient than wild-type HSPB8, suggesting that the mutated forms of HSPB8 are characterised by a loss of function in PQC. Furthermore, in collaboration with Prof. Angelo Poletti, we demonstrated that in cellular models of Amyotrophic Lateral Sclerosis (ALS) upregulation of HSPB8, which naturally occurs in motor neurons in patients affected by ALS, protects against protein aggregation and motor neuron degeneration. These results, combined with our data suggestive of mutant HSPB8 loss of function in peripheral motor neuropathy, further point to upregulation and potentiation of HSPB8 function as potential therapeutic approach to protect motor neurons from stress and death.
Socio-economic impacts of the project: Deregulated autophagy is associated with neurodegenerative and neuromuscular disorders, whereas stimulation of autophagy is beneficial in diseases characterised by aggregating proteins. These items underscore the importance of understanding how autophagy is normally regulated and how it can be stimulated under specific diseases. Our project allowed a better understanding of how HSPB8-HSC70-BAG3 modulates PQC and autophagy, thus allowing the clearance of misfolded aggregate-prone proteins and provided insights in its role as modulator of polyglutamine diseases, as well as the motor neuron disease ALS. Also, our in vitro and in vivo results provided sufficient evidence to support that upregulation of HSPB8-HSC70-BAG3 might indeed represent a valid therapeutic tool to slow-down protein aggregate disease progression. Finally, they also strongly suggest that potentiation of HSPB8 function might be relevant especially to cure motor neuron disease, pointing to the importance of finding in the future drugs able to specifically target HSPB8 expression as therapeutic tool. Protein aggregate diseases like AD, PD, polyglutamine diseases and ALS, in which we demonstrated an implication of the HSPB8-HSC70-BAG3 complex have an increasing frequency (they mainly affect older people and the age worldwide population is increasing) and a very high socio-economic impact. Our project may allow in the future to develop pharmaceutical drugs able to slow-down (e. g. via HSPB8 upregulation) the progression of age-related aggregate protein disorders, therefore decreasing their socio-economic costs.