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Proc Natl Acad Sci U S A. Dec 10, 2002; 99(Suppl 4): 16407–16411.
PMCID: PMC139901
Colloquium Paper

Chaperoning brain degeneration

Abstract

Drosophila has emerged as a première model system for the study of human neurodegenerative disease. Genes associated with neurodegeneration can be expressed in flies, causing phenotypes remarkably similar to those of the counterpart human diseases. Because human neurodegenerative diseases, including Huntington's and Parkinson's diseases, are disorders for which few cures or treatments are available, Drosophila brings to bear powerful genetics to the problem of these diseases. The molecular chaperones were the first modifiers defined that interfere in the progression of such disease phenotypes in Drosophila. Hsp70 is a potent suppressor of both polyglutamine disease and Parkinson's disease in Drosophila. These studies provide the promise of treatments for human neurodegeneration through the up-regulation of stress and chaperone pathways.

Huntington's and Parkinson's diseases are late-onset, progressive human neurodegenerative diseases associated with selective neuronal loss and abnormal protein accumulations. Huntington's disease is one of a class of human diseases known as the polyglutamine repeat diseases (see ref. 1 for review). This class also includes dentatorubropallidoluysian atrophy (DRPLA), spinobulbar muscular atrophy (SBMA) and spinocerebellar ataxias type 1, 2, 3 (also known as Machado–Joseph disease, MJD), 6, 7, and 17. The polyglutamine diseases are characterized by the expansion of a run of the amino acid glutamine within the ORF of the respective proteins. The expanded polyglutamine domain confers dominant toxicity on the respective disease proteins, leading to neuronal dysfunction and degeneration. These diseases are also associated with abnormal protein accumulations containing the disease protein, typically in the form of nuclear inclusions. These inclusions immunostain for ubiquitin, suggesting that they contain misfolded or abnormally folded protein, potentially targeted for proteasomal degradation.

Dominant Parkinson's disease is characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta. Abnormal protein accumulations, known as Lewy bodies, typify the disease. Lewy bodies are cytoplasmic aggregates composed primarily of the protein α-synuclein (2); they contain ubiquitinated protein, suggesting that the accumulating protein has been targeted for degradation. Causal association of abnormal α-synuclein function with Parkinson's disease was found when two mutations in α-synuclein, A30P and A53T, were described in rare familial forms (3, 4).

Drosophila is a powerful genetic model system, which has been well studied as a developmental system. Many genes are conserved between humans and flies, including entire gene pathways (5). Drosophila has a complex nervous system and displays complex behaviors, including learning and memory. Many genes known to be involved in pathways of behavior, including learning and memory, circadian behavior, and phototaxis, were first described in Drosophila mutants (68). Given these striking homologies between Drosophila and humans, we reasoned that the power of Drosophila genetics could be brought to bear on the problem of human neurodegenerative disease.

Whereas mutations have been known for many years that lead to loss of integrity of the fly brain (8, 9), we reasoned that another way to generate such models of specific interest for their application to human neurodegeneration would be to express in the fly the pathogenic human disease gene. With the phenotype in the fly resembling that of the human disease in fundamental properties, this would indicate at least some aspects of the disease process are also conserved between flies and humans. This conservation therefore would allow fly genetics to be applied to define mechanisms of disease progression and modifiers that interfere with the disease process, thus opening up the realm of Drosophila neurogenetics toward the cure and treatment of these devastating human disorders. Here I present a review of previous findings on Drosophila models of neurodegeneration, with some additional new findings.

Materials and Methods

Details of the methods used in the studies summarized here are described in previously published research reports (see refs. 1013). The Hsp70 dominant-negative transgene encoding Hsp70.K71E was generated by mutagenesis of the Hsp70 transgene described (12).

A Drosophila Model for Human Neurodegenerative Disease

To establish the fly as a model system for human neurodegeneration, we decided to express in the fly the normal form and a mutant disease form of the gene encoding spinocerebellar ataxia type 3, or MJD. We used a truncated form of the disease protein in these studies, as this protein had been shown to have effects when expressed in transgenic mice (14). To do this, we subcloned cDNAs encoding a protein with a polyglutamine repeat within the normal range, MJDtrQ27, and a protein with a polyglutamine repeat within the pathogenic range, MJDtrQ78, into fly transformation vectors. The two-component GAL4-UAS system was used for transgene expression (15). Transgenic flies were obtained, and expression was directed to neural tissues. Typically, expression is directed to the eye with gmr-GAL4 or to the entire nervous system with elav-GAL4.

Expression of the control protein MJDtrQ27 has no discernable phenotype—flies are born with eyes indistinguishable from normal. Expression of the disease form of the protein, MJDtrQ78, however, has profound effects. Flies are born with eyes mildly to strongly degenerate when the gmr-GAL4 eye driver is used, with loss of red pigmentation, loss of internal eye integrity, and severe degeneration of the photoreceptor neurons (Fig. (Fig.11 A–D) (13). The strength of the phenotype depends on expression level of the transgene encoding the pathogenic protein—weak expression induces mild degeneration, whereas strong expression is associated with severe degeneration.

Figure 1
Polyglutamine degeneration and suppression by the molecular chaperone Hsp70. (Upper) External eyes. (Lower) Horizontal sections through the eye to reveal the internal eye structure. (A and B) Normal fly with just the driver gmr-GAL4. (C and D) Fly expressing ...

The phenotype was also progressive over time. Although the flies are born with various degrees of degeneration depending upon the specific transgenic insertion, degeneration becomes progressively more severe over the lifetime of the adult fly. This degeneration is seen as progressive loss of pigmentation, enhanced deterioration of internal eye integrity, and early death of the animal, associated with tremors and shaking movement.

Examination of the tissue for protein expression revealed that the pathogenic polyglutamine protein forms abnormal inclusions within the cell nuclei. Such abnormal inclusions are characteristic of the human polyglutamine diseases, where they are described as nuclear inclusions or cytoplasmic inclusions, depending upon their particular subcellular localization (1618). In our MJD model, the inclusions are nuclear. Whereas these inclusions form early in the fly cells, degeneration of the cells does not occur for many days. The inclusions form in all cells in which the pathogenic protein is expressed, even in cells that do not degenerate and are insensitive to the pathogenic actions of the disease protein. This observation suggests that whereas the inclusions may be part of the disease process or indicative of the abnormal folding of the pathogenic protein, the mere presence of an inclusion is not sufficient for cellular degeneration. Nevertheless, such abnormal protein accumulations are characteristic of the human diseases, providing another point of similarity between the fly model and the human disorders.

These features of the fly model—late onset and progressive neurodegeneration accompanied by the formation of abnormal protein accumulations—are fundamental features of human polyglutamine disease. This fact indicates that Drosophila can display mechanisms of human polyglutamine degeneration. These findings indicate that Drosophila genetics can be applied toward defining mechanisms, cures, and treatments for such human neurodegeneration.

The Molecular Chaperone Hsp70 Is a Potent Suppressor of Polyglutamine Pathogenicity

Given that polyglutamine disease is associated with an abnormal protein conformation, the molecular chaperones may play a role in disease progression. In flies, the major stress-induced chaperone is Hsp70. Therefore, we asked whether Hsp70 might be involved in the disease process. We found that the nuclear inclusions immunostained, from initial stages of their formation, with antibodies that detect the Hsp70 proteins (12). This finding suggested the possibility that the cells were mounting a stress response against the pathogenic protein. Therefore, we asked whether it would make a difference to supply the cells with additional Hsp70 activity. To do this, we made transgenic flies that overexpress human Hsp70, which is highly conserved with fly Hsp70, but can be detected with species-specific antibodies.

Co-expression of Hsp70 dramatically suppresses the degeneration normally associated with the pathogenic polyglutamine protein MJDtr-Q78 (Fig. (Fig.1).1). The external eye structure is fully restored to normal, and internal eye structure is strongly restored. Moreover, not only is initial degeneration arrested but also progressive degeneration is prevented. We verified that there were no differences in transgene expression and that, rather, the added Hsp70 is protecting or compensating for the toxicity of the pathogenic disease protein.

To address whether the enzymatic ATPase activity of Hsp70 is important for the suppression, we examined transgenic flies that express a form of the constitutively expressed Hsp70, Hsc4, with a point mutation in the ATPase domain that acts in vivo in a dominant-negative manner (19). Co-expression of this protein with the disease protein not only fails to suppress, but actually enhances, degeneration. This finding suggests that toxicity to the polyglutamine protein is sensitive to the levels of the Hsp70 family of molecular chaperones, with added Hsp70 preventing degeneration, whereas interference with endogenous chaperone activity promotes degeneration.

We also investigated the role of the Hsp70 co-chaperone Hsp40 in protein pathogenicity, by creating transgenic flies that overexpress the fly counterpart of the human Hdj1 class of molecular chaperone, dHdj1. These flies, like those expressing Hsp70, also show strong suppression of polyglutamine toxicity (ref. 11, also ref. 20). The Hsp40 proteins show specificity in that dHdj1 is effective, whereas dHdj2 is poor at protecting against polyglutamine toxicity (11). This difference is consistent with idea that different Hsp40 class chaperones have distinct substrate specificities [see review by Hartl and colleagues in this issue (21) for more extensive discussion of Hsp70/Hsp40 functional interactions]. There appears to be selectivity for the polyglutamine protein, such that dHdj1 is more effective.

We also examined potential interactions between Hsp70 and dHdj-1. As dHdj-1 is presumably a co-chaperone for Hsp70, we anticipated that we might detect a synergy between the two proteins in suppression of pathogenicity. Indeed, although either Hsp70 or dHdj1 on its own is a strong suppressor, when they are co-expressed suppression of polyglutamine degeneration is even stronger (Fig. (Fig.2).2). These findings emphasize the importance of providing a sufficient complement of chaperones for suppression. In our disease model, it appears that there are sufficient levels of Hsp70 and Hsp40 alone to allow initial suppression. The late-onset nature of the degeneration may signify that the chaperone system eventually becomes overwhelmed. However, cells with a generally poor basal stress or chaperone system may require more than just Hsp70 or Hsp40 alone for significant suppression, instead requiring a complement of chaperones to effect protection.

Figure 2
Hsp70 and Hsp40 synergize in suppression of polyglutamine toxicity. Two copies of the polyglutamine protein are expressed in A–D, which makes the phenotype severe enough that synergy may readily be seen. (A) The polyglutamine protein MJDtr-Q78 ...

It is of interest that the potent suppression of the adult eye degenerative phenotype by Hsp70 and dHdj1 occurs in the absence of an effect on the morphology of the aggregates formed by the pathogenic protein, as visualized by immunocytochemistry. Nuclear inclusions are formed in the fly upon chaperone suppression, and they are present in the same number and the same size as in the absence of additional chaperones. The nuclear inclusions appear the same, except that the exogenous Hsp70 and dHdj1 are now also found in the inclusions, suggesting an interaction with the pathogenic protein.

To address this question in another manner, we performed Western immunoblot analysis on the flies and examined the solubility properties of the pathogenic protein. By this assay, the pathogenic protein remains largely insoluble in SDS-resistant complexes that fail to enter the protein gel, remaining within the stacking gel, and transferring poorly in immunoblot analysis. However, in flies that are co-expressing the chaperones, a large amount of the pathogenic protein is now SDS-soluble and detected as a monomeric protein by Western immunoblot (Fig. (Fig.22E) (11).

The degree of SDS-solubility strikingly correlates with pathogenicity of the protein—dHdj2, which suppresses poorly, shows little or no change in monomer, despite high levels of coexpressed chaperone. These data suggest that the properties of the pathogenic protein have changed in the presence of the chaperones. Potentially, the protein is being maintained in a more native or normal conformation, with toxic interactions being abated, seen as a change in SDS-solubility.

Chaperone Suppression of α-Synuclein Toxicity in a Drosophila Model for Parkinson's Disease

The demonstration that Drosophila can be used to model a human neurodegenerative disease by directed expression of the respective human disease protein opened the possibility of modeling human neurodegenerative diseases other than polyglutamine diseases in the fly. Indeed, directed expression of α-synuclein, a component of Lewy bodies and mutated in familial forms of Parkinson's disease, causes adult-onset degeneration of dopaminergic neurons in Drosophila, thereby providing a model for Parkinson's disease (Fig. (Fig.3)3) (10, 22). We have also determined whether we could apply the principles learned from chaperone suppression of polyglutamine pathogenicity to the problem of protein toxicity of α-synuclein in Drosophila.

Figure 3
α-Synuclein Lewy-body-like aggregates in flies and in Parkinson's disease patient tissue. (A and B) Brain sections through a 20-day-old fly expressing wild-type α-synuclein show Lewy-body-like aggregates in the cortex (arrow) and neuropil ...

When α-synuclein expression is directed to dopaminergic neurons in the Drosophila brain, select clusters of neurons show adult-onset progressive loss of cells. To define this cell loss, we prepared serial brain sections and immunostained for tyrosine hydroxylase expression, which selectively detects the dopaminergic neurons. We then counted the number of dopaminergic neurons present in the dorsomedial (DM) and dorsolateral (DL-1) clusters in the adult fly over time. We found a consistent 50% loss of dopaminergic neurons in the DM cluster, and a variable 0–50% loss of cells within the DL-1 cluster (10). Normal cell numbers were present at eclosion of the adult fly from the pupal case, with the cells degenerating over 20 days of adult life. We did not see further loss of cells, indicating that by 20 days all of the cells sensitive to α-synuclein toxicity had degenerated. Moreover, we did not detect a difference in the toxicity of normal α-synuclein, or the two mutant forms A30P and A53T.

In flies, normal α-synuclein and the mutant forms form abnormal Lewy-body-like and Lewy-neurite-like accumulations in the brain over time (Fig. (Fig.3).3). The Lewy-body-like aggregates appear as smaller, more loosely formed accumulations at 1 day, and become progressively larger by 20 days. As with human Lewy bodies, the fly Lewy-body-like aggregates immunolabel with antibodies to ubiquitin, indicating they may reflect accumulation of misfolded protein targeted for degradation by the proteasome. Most Parkinson's disease is sporadic and associated with accumulation of normal α-synuclein in Lewy bodies (2), consistent with the toxicity and Lewy-body-like formation by normal α-synuclein, in a manner similar to the mutant forms, in Drosophila.

We then asked whether co-expression of Hsp70 had an effect on α-synuclein toxicity. We co-expressed human Hsp70 with α-synuclein and counted the number of dopaminergic neurons in the DM clusters, where we detect a consistent loss of neurons upon α-synuclein expression alone.

Hsp70 had a dramatic effect to maintain dopaminergic neural numbers and prevent the degeneration of dopaminergic neurons (10). Whereas normally upon α-synuclein expression, 50% of neurons in the DM cluster were lost over 20 days in the adult, now all neurons were maintained over the 20-day period. This was the case upon expression of the wild-type α-synuclein, as well as the mutant forms A30P and A53T. We examined whether this protection was accompanied by a change in formation of the Lewy-body-like aggregates; however, as with polyglutamine degeneration, we detected no change. This result indicates that Hsp70 fully protects against α-synuclein toxicity, despite the continued presence of aggregates. The aggregates immunolabel for the exogenous Hsp70, however, indicating a potential direct interaction of the chaperone with α-synuclein. We then asked whether there was a change in distribution of endogenous chaperones, and indeed we found that the aggregates immunolabel for the stress-induced form of fly Hsp70 (Fig. (Fig.3).3). This finding indicates that there might be an involvement of endogenous chaperones, and potentially a stress response, in α-synuclein toxicity.

Endogenous Chaperone Activity Plays a Role in α-Synuclein Toxicity in Drosophila and Potentially also in Parkinson's Disease

To ask whether endogenous chaperone levels may normally help protect against α-synuclein toxicity, the dominant-negative form of Hsc4 was co-expressed with the disease protein. This form of Hsc4 will interfere with endogenous activity of the Hsp70 family of chaperones, in effect lowering endogenous chaperone activity (19). In this situation, we noted an acceleration of α-synuclein toxicity. Whereas normally upon expression of α-synuclein, flies are born with the full complement of dopaminergic neurons in the DM clusters, in the presence of Hsc4.K71S, flies are born with a 50% loss of dopaminergic neurons. This cell loss did not progress further over time in the adult; rather, Hsc4.K71S accelerated the toxicity of α-synuclein to those cells sensitive to α-synuclein. However, we also noted that Hsc4.K71S has some toxicity to dopaminergic neurons when expressed in the absence of α-synuclein. The fact that α-synuclein and Hsc4.K71S are acting similarly with regard to dopaminergic neural loss indicates that the toxicity in both cases may share common mechanisms.

To extend our findings back to the human disease condition, we asked whether Parkinson's disease was associated with a change in chaperone function. To do this, we immunostained patient tissue with antibodies to Hsp70 and its co-chaperone Hsp40. Indeed, Lewy bodies and Lewy neurites in disease brain immunolabel for the chaperones (Fig. (Fig.3).3). The significance of this finding is best evaluated in the context of the fly study: in Drosophila the abnormal inclusions of α-synuclein immunolabel for Hsp70 and up-regulation of Hsp70 activity by directed transgenic expression mitigates the toxicity. This finding suggests that it may be of value in Parkinson's disease to up-regulate chaperone function.

Discussion

Our initial and other subsequent studies have established Drosophila as a model genetic system to bring to bear in the arsenal of approaches toward the combat of human neurodegenerative disease (10, 12, 13, 20, 2226). The striking homology of a large number of fly genes with human genes—indeed of entire gene pathways—indicates that fundamental properties of degeneration modeled in flies may be conserved in humans. Whereas the demonstration of the fly as an outstanding in vivo model for human neurodegeneration is significant, of great importance is the use of those models to reveal disease mechanisms and pioneer ways to interfere in the disease process.

The implication that chaperones may be of interest in such neurodegenerative diseases finds its roots in an even simpler model system than Drosophila—the yeast Saccharomyces cerevisiae. Indeed, protein conformational changes relevant to human prion disease are found in yeast in the study of endogenous yeast prions such as Sup35 (27), where Hsp104 was described as a regulator of the prion state (28). The Drosophila studies establish that chaperone modulation can be applied to the nervous system in vivo in the context of neurodegeneration.

In Drosophila, the chaperones are found to be potent modulators both of polyglutamine toxicity and of α-synuclein toxicity in models for human polyglutamine disease and Parkinson's disease. Not only does added chaperone activity prevent disease, but interfering with endogenous chaperone activity accelerates pathogenesis. This finding indicates that chaperone activity is central to the disease process, being modulated upon both up-regulation and interference. Moreover, these studies in Drosophila for polyglutamine toxicity have been found to translate to mammalian models for polyglutamine toxicity. Up-regulation of Hsp70 in transgenic mice expressing the Ataxin-1 pathogenic polyglutamine disease protein leads to protection against behavioral and cellular pathology (29).

How are the chaperones modulating protein toxicity? In vivo in flies, chaperones modulate the solubility properties of the polyglutamine protein concomitant with a modulation in the toxicity. However, no morphological change in the aggregates is detected. This is the case for both polyglutamine and α-synuclein toxicity. Studies of polyglutamine aggregation and protein solubility in yeast remarkably parallel the fly findings, where chaperones modulate solubility but aggregates are still present (30). One possibility is that the chaperones are modulating the structure of the protein, and this is not visible in the large aggregates. Prefibrils or protofibrils may be the toxic entity (see refs. 31 and 32)—smaller clumps and misconformations of the disease protein that precede or are independent of large, visible aggregates. Chaperones may be modulating these abnormal and toxic conformations, thereby preventing neurodegeneration. This conceivably can happen in the absence of an effect on large, visible aggregates, which are a different form of the disease protein, perhaps even inert or protective.

Another possibility is that chaperones, by interacting with the disease protein, prevent abnormal interactions with other proteins in the cell that are causal in toxicity. However, clearly, just anything that physically interacts with the disease protein appears not to have an effect to suppress, as demonstrated by the dHdj2 studies for polyglutamine disease. Whereas dHdj-2 is in association with the polyglutamine protein in the aggregates, as it co-localizes by immunocytochemistry, it fails to suppress toxicity (11).

Another possibility is that of chaperone depletion. Because of an abnormal or misfolded conformation, pathogenic disease proteins may cause cellular depletion of chaperone activity. Chaperones are required for the proper folding and function of many cellular proteins with diverse roles. Therefore, slow depletion of chaperones from the cellular milieu could lead to the failure of many cellular processes.

The role of chaperone depletion is intriguing because of the ability of chaperones to phenocopy aspects of disease protein expression. In the α-synuclein studies, expression of Hsc4.K71S on its own caused some loss of dopaminergic neurons, indicating that dopaminergic neurons are sensitive to compromised chaperone activity [also supported by other studies of α-synuclein and Parkinson's disease-associated proteins (33, 34)]. In the studies of the Hsc4.K71S transgene in flies, Elefant and Palter (19) noted that in some situations the protein was toxic, inducing a misfolded protein response accompanied by a degeneration reminiscent of neurodegenerative disease. We have also found that expressing Hsp70.K71E in the eye causes an external eye phenotype strikingly similar to the polyglutamine disease phenotype (Fig. (Fig.4).4). These studies suggest that compromising chaperone levels alone is phenotypically similar to the pathogenic actions of polyglutamine and α-synuclein proteins, indicating that chaperone interference is a major contributing factor to neurodegeneration. Cell specificity differs between polyglutamine and α-synuclein toxicity, with pathogenic polyglutamine protein appearing to be much more generally toxic in Drosophila than α-synuclein. However, this observation could be explained by cellular differences in the chaperone response to the specific protein in different tissues.

Figure 4
Interfering with endogenous chaperone activity causes a severely degenerate eye phenotype. (A) Expression of a dominant-negative form of Hsp70 causes loss of pigmentation and deterioration of eye structure, similar to the expression of the polyglutamine ...

With the ever-accelerating development of fly models for various human neurodegenerative diseases, and tremendous interest in such models for both standard genetic and pharmacological approaches, Drosophila may reveal new cures and treatments of relevance to human neurodegeneration, including polyglutamine and Parkinson's diseases.

Acknowledgments

I thank Mark Fortini and Anthony Cashmore for comments. I receive funding support from the David and Lucile Packard Foundation and the National Institutes of Health, and I am an Assistant Investigator of the Howard Hughes Medical Institute.

Abbreviations

MJD
Machado–Joseph disease
DM
dorsomedial

Footnotes

This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “Self-Perpetuating Structural States in Biology, Disease, and Genetics,” held March 22–24, 2002, at the National Academy of Sciences in Washington, DC.

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