Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibers containing misfolded protein with a beta-sheet conformation, termed amyloid. There is partial but not perfect overlap among the cells in which abnormal proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves. For several diseases, genetic variants assist in explaining the pathogenesis of the more common sporadic forms and developing mouse and other models. There is now increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity. These are leading to approaches toward rational therapeutics.

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Protein aggregation and neurodegenerative disease.

The causes of amyotrophic lateral sclerosis (ALS), a devastating human neurodegenerative disease, are poorly understood, although the protein TDP-43 has been suggested to have a critical role in disease pathogenesis. Here we show that ataxin 2 (ATXN2), a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2, is a potent modifier of TDP-43 toxicity in animal and cellular models. ATXN2 and TDP-43 associate in a complex that depends on RNA. In spinal cord neurons of ALS patients, ATXN2 is abnormally localized; likewise, TDP-43 shows mislocalization in spinocerebellar ataxia type 2. To assess the involvement of ATXN2 in ALS, we analysed the length of the polyQ repeat in the ATXN2 gene in 915 ALS patients. We found that intermediate-length polyQ expansions (27-33 glutamines) in ATXN2 were significantly associated with ALS. These data establish ATXN2 as a relatively common ALS susceptibility gene. Furthermore, these findings indicate that the TDP-43-ATXN2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.

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Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS

Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases.

Many neurodegenerative disorders are characterized by conformational changes in proteins that result in misfolding, aggregation and intra- or extra-neuronal accumulation of amyloid fibrils. Molecular chaperones provide a first line of defence against misfolded, aggregation-prone proteins and are among the most potent suppressors of neurodegeneration known for animal models of human disease. Recent studies have investigated the role of molecular chaperones in amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and polyglutamine diseases. We propose that molecular chaperones are neuroprotective because of their ability to modulate the earliest aberrant protein interactions that trigger pathogenic cascades. A detailed understanding of the molecular basis of chaperone-mediated protection against neurodegeneration might lead to the development of therapies for neurodegenerative disorders that are associated with protein misfolding and aggregation.

Modulation of neurodegeneration by molecular chaperones.

The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and prion disease, is thought to occur after failure of the cellular protein quality control mechanisms. Here we examine the formation of misfolded protein inclusions in the eukaryotic cytosol of yeast and mammalian cell culture models. We identify two intracellular compartments for the sequestration of misfolded cytosolic proteins. Partition of quality control substrates to either compartment seems to depend on their ubiquitination status and aggregation state. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Notably, disease-associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Enhancing ubiquitination of a prion protein suffices to promote its delivery to the juxtanuclear inclusion. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.

/pdf/misfolded-proteins-partition-between-two-distinct-quality-43pvgmfqk0.pdf

Misfolded proteins partition between two distinct quality control compartments

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.

https://www.pnas.org/content/pnas/97/4/1589.full.pdf

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Our work supports the hypothesis that a protein can serve as an element of genetic inheritance. This protein–only mechanism of inheritance is propagated in much the same way as hypothesized for the transmission of the protein–only infectious agent in the spongiform encephalopathies; hence these protein factors have been called yeast prions. Our work has focused on [ PSI + ], a dominant cytoplasmically inherited factor that alters translational fidelity.This change in translation is produced by a self–perpetuating change in the conformation of the translation–termination factor, Sup35. Most recently, we have determined that new elements of genetic inheritance can be created by deliberate genetic engineering, opening prospects for new methods of manipulating heredity. We have also uncovered evidence that other previously unknown elements of protein–based inheritance are encoded in the yeast genome. Finally, we have begun to use yeast as a model system for studying human protein folding diseases, such as Huntington9s disease. Proteins responsible for some of these diseases have properties uncannily similar to those that produce protein–based mechanisms of inheritance.

Investigating protein conformation-based inheritance and disease in yeast.

Screening methods for identifying substances that provide therapeutic value for various diseases associated with protein misfolding are provided. Genetic and chemical screening methods are provided using a yeast system. The methods of the invention provide a rapid and cost-effective method to screen for compounds that prevent protein misfolding and/or protein fibril formation and/or protein aggregation which includes numerous neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease as well as non-neuronal diseases such as type 2 diabetes.

Sylvia Krobitsch

Papers

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Investigating protein conformation-based inheritance and disease in yeast.

Yeast screens for agents affecting protein folding