The well-documented commonality between protein misfolding or aggregation and a wide range of neurological diseases has resulted in significant efforts directed toward gaining an understanding of molecular mechanisms responsible for this process. In 1994, Max Perutz postulated that the formation of "polar zippers" consisting of antiparallel β-sheets of expanded polyglutamine protein tracts may be responsible for self-aggregation underlying the toxicity associated with Huntington’s disease (HD) and other disorders (Perutz et al., 1994). More recently, elegant studies by the lab of Ron Wetzel at the University of Tennessee (Thakur & Wetzel, 2002), demonstrated requirements for optimum length and conformation of synthetic polyglutamine-containing peptides to nucleate and aggregate. The search for gene products and compounds designed to ameliorate the cellular consequences of protein aggregation has already yielded several important leads, none...  Read more

The well-documented commonality between protein misfolding or aggregation and a wide range of neurological diseases has resulted in significant efforts directed toward gaining an understanding of molecular mechanisms responsible for this process. In 1994, Max Perutz postulated that the formation of "polar zippers" consisting of antiparallel β-sheets of expanded polyglutamine protein tracts may be responsible for self-aggregation underlying the toxicity associated with Huntington’s disease (HD) and other disorders (Perutz et al., 1994). More recently, elegant studies by the lab of Ron Wetzel at the University of Tennessee (Thakur & Wetzel, 2002), demonstrated requirements for optimum length and conformation of synthetic polyglutamine-containing peptides to nucleate and aggregate. The search for gene products and compounds designed to ameliorate the cellular consequences of protein aggregation has already yielded several important leads, none more promising than that described in the January 23 Nature from the lab of Junying Yuan. These authors report a comprehensive analysis of the numerous cytoprotective effects associated with the azo-dye, Congo red, in the inhibition of polyglutamine oligomerization.

Previous studies (Klunk et al. 1989 and Carter & Chao, 1998) illustrated that Congo red had the capacity to specifically associate with β-amyloid fibrils; it was subsequently shown to inhibit huntingtin fibrillogenesis in a dose-dependent manner (Heiser et al., 2000). In this latest paper, Sanchez et al. utilize a battery of experimental approaches to systematically address the intracellular mechanism by which Congo red reduces polyglutamine aggregation and cytotoxicity. These authors demonstrate that Congo red acts independent of molecular chaperones and the protein synthesis and degradation machinery by directly inhibiting nucleation of expanded polyglutamine aggregates. Moreover, Congo red dissolved preformed aggregates of a Q79 peptide from cell lysates in the absence of other proteins. In contrast, chrysamine G, a structurally similar compound that binds expanded polyglutamine, was incapable of disrupting aggregates or inhibiting polyglutamine-induced cell death.

Through the use of fluorescence resonance energy transfer (FRET), these investigators further showed that Congo red inhibited oligomerization of dual-labeled Q79 fluorescent protein variants and that this inhibition correlated with an increased solubility of Q79 protein in cell lysates. Bence et al., 2001 previously showed that an in vivo consequence of polyglutamine oligomerization was inhibition of the ubiquitin-proteosome system (UPS). The increased solubility of Congo red-treated polyglutamine protein resulted in a selective increase in Q79 degradation, indicating that the dye rendered misfolded proteins more accessible to the UPS-mediated degradation and simultaneously alleviated the cellular stress induced by the presence of polyglutamine aggregates. Importantly, Congo red was also demonstrated to be effective at significantly abrogating multiple features of the disease state when intraperitoneally infused into an HD mouse model at micromolar concentrations. In the absence of any overt toxic effects in these animals, Congo red-treated mice showed a reduction in severe weight loss, hindlimb dyskinesia, and blood glucose levels. Brain slices also showed reductions of polyglutamine immunostaining in the basal ganglia and hippocampus of Congo red-treated mice vs. control animals.

It will be interesting to see what native proteins may coordinately act to enhance cytoplasmic clearance of expanded polyglutamine-repeat containing proteins in the presence of Congo red. Further work aimed at defining gene products that may function to protect cells may expand our understanding of existing cellular mechanisms that might be induced to facilitate or mimic Congo red activity. The exceptional work of Sanchez et al. have set the stage for similar studies and mutational analyses on full-length polyglutamine target proteins. This will likely reveal the specificity of Congo red for these proteins and lead to the rational design of related compounds with potentially therapeutic value.

References:
Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science. 2001 May 25;292(5521):1552-5. Abstract

Carter DB, Chou KC. A model for structure-dependent binding of Congo red to Alzheimer beta-amyloid fibrils. Neurobiol Aging. 1998 Jan-Feb ;19(1):37-40. Abstract

Heiser V, Scherzinger E, Boeddrich A, Nordhoff E, Lurz R, Schugardt N, Lehrach H, Wanker EE. Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6739-44. Abstract

Klunk WE, Pettegrew JW, Abraham DJ. Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem. 1989 Aug;37(8):1273-81. Abstract

Perutz MF, Johnson T, Suzuki M, Finch JT. Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5355-8. Abstract

Thakur AK, Wetzel R. Mutational analysis of the structural organization of polyglutamine aggregates. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):17014-9. Abstract

View all comments by Guy Caldwell

The accumulation of SCA7 in the knockin model could mean that the expanded polyQ forms oligomers that are invisible under the microscope. Depending on the endogenous level of SCA7, which apparently is very low, a critical threshold to form visible inclusions may not have been reached until later. It is quite possible that several thousand molecules have to pile up in order to be seen under the microscope. It needs to be tested whether increased polyQ-polyQ interaction is required for the accumulation of SCA7 and if disruption of such interactions might ameliorate neurodegeneration in this model.

View all comments by Junying Yuan

Cellular homeostasis is an exceedingly complex process. Conceptually, one may consider two regimes within which the phenomenon operates, extra- and intracellular. The extracellular regime requires dynamic responses of the cell to external stimuli. The intracellular regime involves metabolic processes that neurologists might refer to as “activities of daily living,” those processes that the cell must execute continuously to function normally. One of these activities is the synthesis and folding of proteins. This activity is highly efficient overall, but imperfect. A significant percentage of nascent proteins fold improperly, even with the help of folding chaperones, and thus must be “recycled” through proteolysis in the proteasome system. What happens if the capacity of the protein folding and degradation machinery is exceeded?

In a paper published on 9 February in Sciencexpress, Morimoto and colleagues at Northwestern University address the general question raised above from the perspective of diseases of protein aggregation that cause neurodegenerative disorders. These...  Read more

Cellular homeostasis is an exceedingly complex process. Conceptually, one may consider two regimes within which the phenomenon operates, extra- and intracellular. The extracellular regime requires dynamic responses of the cell to external stimuli. The intracellular regime involves metabolic processes that neurologists might refer to as “activities of daily living,” those processes that the cell must execute continuously to function normally. One of these activities is the synthesis and folding of proteins. This activity is highly efficient overall, but imperfect. A significant percentage of nascent proteins fold improperly, even with the help of folding chaperones, and thus must be “recycled” through proteolysis in the proteasome system. What happens if the capacity of the protein folding and degradation machinery is exceeded?

In a paper published on 9 February in Sciencexpress, Morimoto and colleagues at Northwestern University address the general question raised above from the perspective of diseases of protein aggregation that cause neurodegenerative disorders. These diseases include Alzheimer’s, Parkinson’s, prion, and Huntington’s. The last disease is an archetypal member of a family of diseases caused by an increase in the number of contiguous glutamine residues within normal proteins, the “polyglutamine diseases.” A number of pathogenetic mechanisms have been postulated to explain the cellular and organismal effects of polyglutamine expansion. Morimoto et al. sought mechanistic insights through an examination of the effects of simultaneous expression in the worm C. elegans of temperature-sensitive (ts) protein mutants and fluorescent proteins bearing varying numbers of polyglutamine tracts. The key strategic kernel was the study of phenotype at permissive temperatures. In a visually and intellectually beautiful set of experiments, effects were observed in sarcomere morphology, movement, body shape, egg-laying, and early development. Affected animals displayed large numbers of protein aggregates, and evidence was presented that proteins expressed in the presence of a polyglutamine-containing protein acquired increased protease resistance (one characteristic of amyloids and other protein accretions).

All proteins constantly sample different areas of conformational space. In the ts mutants studied, amino acid substitutions alter the energy-dependence of this sampling process, allowing the protein to fold into pathologic conformations if sufficient activation energy can be obtained (at the non-permissive temperature). One intriguing part of the work of Morimoto et al. is the finding that polyglutamine tracts can decrease this activation energy, allowing the ts proteins to fold pathologically at relatively low temperatures. Importantly, this effect is bidirectional. The ts proteins themselves contribute to the self-assembly of the polyglutamine proteins, as evidenced by a synergistic increase in aggregate number when both proteins are expressed.

These results suggest two answers to the question posed above, one obvious from first principles and one new and unexpected. The obvious answer is that the quality and quantity of pathologically folded proteins can overwhelm a cell’s homeostatic mechanisms and cause disease. The novel insight is that pathologically folded proteins may act relatively nonspecifically to alter folding pathways of many other nascent proteins—an effect opposite to that of folding chaperones—and thus affect many different physiologic processes. How does the work further our understanding of pathogenetic mechanisms of polyglutamine diseases in particular, vis-à-vis the beneficial or detrimental effect of aggregation (a question posed by Dr. Eddie Koo at UC, San Diego). In my opinion, the work increases the complexity of the problem and emphasizes the difficulty of ascribing a single pathologic process to a protein-linked disease. Specifically, macroscopic aggregates of polyglutamine-containing proteins may be “protective” through their ability to sequester toxic protein polymers. However, monomers, low-order oligomers, or protofibrils, prior to accretion on deposits, all may contribute to the effects observed by Morimoto et al. If so, strategies to eliminate deposits may fail because they do not target smaller, intracellular, toxic assemblies.

View all comments by David Teplow

The authors have elegantly demonstrated the importance of the presence of intracellular misfolded proteins in mediating cellular dysfunction in neurodegenerative disease. Coexpressing the temperature-sensitive (ts) mutants with polyQ in C. elegans at permissive conditions resulted in phenotypes similar to those exhibited by ts mutants under restrictive conditions. This conversion of relatively harmless ts mutants into those which exhibit mutant phenotypes under permissive conditions is a fascinating and enlightening observation. The experiments with various other strains of ts mutants make the case that the expression of aggregation-prone polyQ protein meddles with the structure and function of unrelated proteins. Specifically, the authors suggest that the levels of polyQ influence the folding of ts protein and that perhaps the opposite is also true, as though a positive feedback mechanism exists to augment the imbalance in cellular folding.

In interpreting the results, the authors propose that marginally stable proteins do not in and of themselves cause disease;...  Read more

The authors have elegantly demonstrated the importance of the presence of intracellular misfolded proteins in mediating cellular dysfunction in neurodegenerative disease. Coexpressing the temperature-sensitive (ts) mutants with polyQ in C. elegans at permissive conditions resulted in phenotypes similar to those exhibited by ts mutants under restrictive conditions. This conversion of relatively harmless ts mutants into those which exhibit mutant phenotypes under permissive conditions is a fascinating and enlightening observation. The experiments with various other strains of ts mutants make the case that the expression of aggregation-prone polyQ protein meddles with the structure and function of unrelated proteins. Specifically, the authors suggest that the levels of polyQ influence the folding of ts protein and that perhaps the opposite is also true, as though a positive feedback mechanism exists to augment the imbalance in cellular folding.

In interpreting the results, the authors propose that marginally stable proteins do not in and of themselves cause disease; rather, they misfold and subsequently modify the aggregation and toxicity of aggregation-prone proteins associated with conformational disease, and this overwhelms the balance of cellular folding and clearing processes. However, if in the presence of aggregation-prone proteins these metastable proteins misfold, this hints at a possible interaction between the two that perpetuates their mutual misfolding and accumulation. To expand on the authors’ interpretation, perhaps the expression of the aggregation-prone protein somehow induces the misfolding of the metastable mutant proteins, contributing to the progressive disruption of cellular processes that maintain the folding environment.

This imbalance of clearance and folding may be initially caused by the early accumulation of such aggregation-prone proteins as polyQ, which not only stress the overall folding capacity of the cell, but also play a role in stimulating the misfolding of metastable proteins. In fact, in our hands, many aggregation-prone proteins in vitro are capable of interacting with other marginally stable proteins and inducing subsequent conformational changes and aggregation.

Alternatively, the expression of aggregation-prone proteins might in some way stimulate the synthesis of more of these metastable proteins, thus increasing the chances for even more misfolding and increasing their accumulation. If that is the case, again, the cellular clearance processes may become overwhelmed and tilt the delicate balance of protein homeostasis.

Whatever the case may be, clearly these data suggest that disruption of cellular processes does not result from a single defect. Rather, in combination, aggregation-prone and metastable proteins can affect protein homeostasis and help to explain the gradual accumulation of damaged proteins observed in misfolding diseases. It would be interesting to try these same experiments with other aggregation-prone proteins.

View all comments by Rakez Kayed