Protein Aggregates and Polyglutamine Tracts In Neurodegenerative Disease

The incidence of neurodegenerative diseases such as Alzheimer\u27s Disease, Parkinson\u27s Disease, Huntington\u27s Disease and other Polyglutamine Diseases is projected to dramatically increase throughout the developed world, and yet the pathology of these diseases remains poorly understood. One pathway that these neurodegenerative diseases share is the accumulation of pathologic proteins which are not only harmful in their soluble form but may go on to form toxic aggregates. In many cases, a consensus has yet to be reached concerning the mechanism for protein aggregation. Therefore, the exploration of the roles of these proteins and their possible mechanisms, along with potential techniques for treatment, are more important than ever

Suppression of polyglutamine toxicity by a Drosophila homolog of myeloid leukemia factor 1

The toxicity of an abnormally long polyglutamine [poly(Q)] tract within specific proteins is the molecular lesion shared by Huntington's disease (HD) and several other hereditary neurodegenerative disorders. By a genetic screen in Drosophila, devised to uncover genes that suppress poly(Q) toxicity, we discovered a Drosophila homolog of human myeloid leukemia factor 1 (MLF1). Expression of the Drosophila homolog (dMLF) ameliorates the toxicity of poly(Q) expressed in the eye and central nervous system. In the retina, whether endogenously or ectopically expressed, dMLF co-localized with aggregates, suggesting that dMLF alone, or through an intermediary molecular partner, may suppress toxicity by sequestering poly(Q) and/or its aggregates

Multiple discrete soluble aggregates influence polyglutamine toxicity in a Huntington\u27s disease model system

Huntington’s disease (HD) results from expansions of polyglutamine stretches (polyQ) in the huntingtin protein (Htt) that promote protein aggregation, neurodegeneration, and death. Since the diversity and sizes of the soluble Htt-polyQ aggregates that have been linked to cytotoxicity are unknown, we investigated soluble Htt-polyQ aggregates using analytical ultracentrifugation. Soon after induction in a yeast HD model system, non-toxic Htt-25Q and cytotoxic Htt-103Q both formed soluble aggregates 29S to 200S in size. Because current models indicate that Htt-25Q does not form soluble aggregates, reevaluation of previous studies may be necessary. Only Htt-103Q aggregation behavior changed, however, with time. At 6 hr mid-sized aggregates (33S to 84S) and large aggregates (greater than 100S) became present while at 24 hr primarily only mid-sized aggregates (20S to 80S) existed. Multiple factors that decreased cytotoxicity of Htt-103Q (changing the length of or sequences adjacent to the polyQ, altering ploidy or chaperone dosage, or deleting anti-aging factors) altered the Htt-103Q aggregation pattern in which the suite of mid-sized aggregates at 6 hr were most correlative with cytotoxicity. Hence, the amelioration of HD and other neurodegenerative diseases may require increased attention to and discrimination of the dynamic alterations in soluble aggregation processes

From gene to therapy in spinal and bulbar muscular atrophy: Are we there yet?

Abnormal polyglutamine expansions in the androgen receptor (AR) cause a muscular condition, known as Kennedy's disease or spinal and bulbar muscular atrophy (SBMA). The disease is transmitted in an X-linked fashion and is clinically characterized by weakness, atrophy and fasciculations of the limb and bulbar muscles as a result of a toxic gain-of-function of the mutant protein. Notably, affected males also show signs of androgen insensitivity, such as gynaecomastia and reduced fertility. The characterization of the natural history of the disease, the increasing understanding of the mechanism of pathogenesis and the elucidation of the functions of normal and mutant AR have offered a momentum for developing a rational therapeutic strategy for this disease. In this special issue on androgens and AR functions, we will review the molecular, biochemical, and cellular mechanisms underlying the pathogenesis of SBMA. We will discuss recent advances on therapeutic approaches and opportunities for this yet incurable disease, ranging from androgen deprivation, to gene silencing, to an expanding repertoire of peripheral targets, including muscle. With the advancement of these strategies into the clinic, it can be reasonably anticipated that the landscape of treatment options for SBMA and other neuromuscular conditions will change rapidly in the near future

Unexpected cell type-dependent effects of autophagy on polyglutamine aggregation revealed by natural genetic variation in C. elegans.

BACKGROUND: Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown. RESULTS: We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells. CONCLUSIONS: Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types

Intrabodies Binding the Proline-Rich Domains of Mutant Huntingtin Increase Its Turnover and Reduce Neurotoxicity

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as VL12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells

Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT

Huntington’s disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ

Impaired Nuclear Export of Polyglutamine-Expanded Androgen Receptor in Spinal and Bulbar Muscular Atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR). Prior studies have highlighted the importance of AR nuclear localization in SBMA pathogenesis; therefore, in this study, we sought to determine the role of AR nuclear export in the pathological manifestations of SBMA. We demonstrate here that the nuclear export of polyQ-expanded AR is impaired, even prior to the formation of intranuclear inclusions of aggregated AR. Additionally, we find that promoting AR export with an exogenous nuclear export signal substantially reduces its aggregation and blocks hormone-induced toxicity. Moreover, we show that these protective effects are conferred by destabilization of the mutant protein due to an increase in proteasomal degradation of the cytoplasmic AR. Despite a growing body of evidence that global disruption of nucleo/cytoplasmic transport occurs in ALS and HD, our data suggest that no such global disruption occurs in models of SBMA; rather, AR-specific mechanisms, including reduced phosphorylation at Serine 650, are likely responsible for the impaired nuclear export of polyQ-expanded AR

Identification and Characterization of Phenotypically Distinct Aggregates within Huntingtin-inducible PC12 Cell Models

The role of various polyglutamine (polyGln) aggregated states in the disease mechanism of expanded CAG repeat disorders continues to be a perplexing subject. We are interested in learning more about these species and the relationship between the aggregation pathways. We have developed an innovative staining technique that allows, for the first time, the visualization of small polyGln aggregates, which we refer to as aggregation foci (AF), that are functionally distinguished by their ability to serve as seeds for amyloid growth. The aggregation foci stain should prove useful in the Huntington’s disease research as a tool for the identification of a functional class of aggregates that was previously undescribed. The ability to account for different aggregates may provide a better correlation with neurodegeneration. Using this method we were able to detect at least four phenotypic classes of aggregates in stable PC12 cells engineered for inducible expression of different polyGln repeat length forms of a green fluorescent protein-fused huntingtin (htt) exon-1 fragment: 1) small, recruitment-competent, cytoplasmic AF, 2) large, cytoplasmic, perinuclear, green-fluorescent, recruitment-inert htt aggregates, 3) cytoplasmic, perinuclear, green-fluorescent, recruitment-competent aggregates, and 4) small, nuclear, recruitment-inert, green-fluorescent aggregates. Each species can be found either in isolation or co-existing within the same microscope field depending on the time post-induction and Gln repeat-length. In our studies, AF tend to be the earliest species that appear in the cells. The PC12 cells generally did not exhibit indications of toxicity due to the polyGln species, and thus provided a good model to study the aggregation pathway without the bias of cell death. Biochemical data were gathered to understand the properties of each distinct aggregate specie. We also examined the ability of two separate compounds (curcumin and demecolcine) to alter the aggregation pathway. Each experiment was designed to obtain knowledge about possible precursor/product relationships that exist among the species, with the ultimate goal of gaining insight into the aggregation pathways responsible for the formation of the amyloid-like AF and larger inert aggregates that arise in these the cell lines expressing various polyGln repeat-lengths. We interpret this data as indicating that the two species are on competing assembly pathways