Formation Of Nuclear Aggregates Research Articles

Bound to Aggregate

A common feature of some neurodegenerative diseases is the abnormal accumulation of proteins in neurons. In spinocerebellar ataxia type 1 (SCA1), an expanded polyglutamine tract in the protein ataxin-1 causes it to aggregate and become toxic in neurons, a process that appears dependent on the phosphorylation of a particular serine residue. Chen et al. have now discovered that phosphorylation of this serine in a toxic form of ataxin-1 [ataxin-1(82Q)] facilitates interaction with the regulatory signaling molecule 14-3-3. Overexpression of both 14-3-3 and ataxin-1(82Q) in transfected cells increased the stability of ataxin-1 and enhanced the formation of nuclear aggregates, where both proteins also localized. Expression of 14-3-3 in transgenic flies enhanced the toxic effects of ataxin-1(82Q) expression alone. The authors further determined that the serine-threonine kinase Akt phosphorylates ataxin-1 on the critical serine residue in vitro and in transfected cells. Although the function of 14-3-3 binding is not clear, it may increase ataxin-1 toxicity in cells by stabilizing a conformation that resists degradation. H.-H. Chen, P. Fernandez-Funez, S. F. Acevedo, Y. C. Lam, M. D. Kaytor, M. H. Fernandez, A. Aiken, E. M. C. Skoulakis, H. T. Orr, J. Botas, H. Y. Zoghbi, Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1. Cell 113 , 457-468 (2003). [Online Journal]

SUMO-1 co-localized with mutant atrophin-1 with expanded polyglutamines accelerates intranuclear aggregation and cell death.

To investigate the implication of small ubiquitin-related modifier-1 (SUMO-1) in the formation of neuronal intranuclear inclusions in polyglutamine diseases, we examined the localization of SUMO-1 in dentatorubral-pallidoluysian atrophy (DRPLA) brain tissues and PC12 cells expressing truncated atrophin-1 with expanded poly-glutamine stretches. SUMO-1 was co-localized with neuronal intranuclear inclusions in DRPLA brain and the DRPLA model cells, which showed that the aggregates formed by expanded polyglutamine stretches were highly SUMOlylated. In addition, to examine the role of SUMO-1 in nuclear aggregate formation and cell death, either SUMO-1 or DeltaSUMO-1, which is a SUMOlylation defective mutant lacking the C-terminal motif, was co-transfected with atrophin-1 with expanded polyglutamine stretches. Co-transfection of DeltaSUMO-1 decreased number of the cells with nuclear aggregates and consequent apoptosis of PC12 cells, both of which were markedly enhanced by co-transfection of SUMO-1 with atrophin-1 with expanded polyglutamine stretches. These results suggest that SUMO-1 is implicated in the pathogenesis of DRPLA and accelerates aggregate formation and cell death.

Cellular localization and development of neuronal intranuclear inclusions in striatal and cortical neurons in R6/2 transgenic mice.

The cellular localization and development of neuronal intranuclear inclusions (NIIs) in cortex and striatum of R6/2 HD transgenic mice were studied to ascertain the relationship of NIIs to symptom formation in these mice and gain clues regarding the possible relationship of NII formation to neuropathology in Huntington's disease (HD). All NIIs observed in R6/2 mice were ubiquitinated, and no evidence was observed for a contribution to them from wild-type huntingtin; they were first observed in cortex and striatum at 3.5 weeks of age. In cortex, NIIs increased rapidly in size and prevalence after their appearance. Generally, cortical projection neurons developed NIIs more rapidly than cortical interneurons containing calbindin or parvalbumin. Few cortical somatostatinergic interneurons, however, formed NIIs. In striatum, calbindinergic projection neurons and parvalbuminergic interneurons rapidly formed NIIs, but they formed more gradually in cholinergic interneurons, and few somatostatinergic interneurons developed NIIs. Striatal NIIs tended to be smaller than those in cortex. The early accumulation of NIIs in cortex and striatum in R6/2 mice is consistent with the early appearance of motor and learning abnormalities in these mice, and the eventual pervasiveness of NIIs at ages at which severe abnormalities are evident is consistent with their contribution to a neuronal dysfunction underlying the abnormalities. That cortex develops larger NIIs than striatum, however, is inconsistent with the preferential loss of striatal neurons in HD but is consistent with recent evidence of early morphological abnormalities in cortical neurons in HD. That calbindinergic and parvalbuminergic striatal neurons develop large NIIs is consistent with a contribution of nuclear aggregate formation to their high degree of vulnerability in HD.

Glucocorticoid modulation of androgen receptor nuclear aggregation and cellular toxicity is associated with distinct forms of soluble expanded polyglutamine protein.

Spinobulbar muscular atrophy is a progressive motor neuron disease caused by abnormal polyglutamine tract expansion in the androgen receptor (AR) gene, and is part of a family of central nervous system (CNS) neurodegenerative diseases, including Huntington's disease (HD). Each pathologic protein is widely expressed, but the cause of neuronal degeneration within the CNS remains unknown. Many reports now link abnormal polyglutamine protein aggregation to pathogenesis. A previous study reported that activation of the wild-type glucocorticoid receptor (wtGR) suppressed the aggregation of expanded polyglutamine proteins derived from AR and huntingtin, whereas a mutant receptor containing an internal deletion, GRDelta108-317, increased polyglutamine protein aggregation, in this case primarily within the nucleus. In this study, we use these two forms of GR to study expanded polyglutamine AR protein in different cell contexts. Using cell biology and biochemical approaches, we find that wtGR promotes soluble forms of the protein and prevents nuclear aggregation in NIH3T3 cells and cultured neurons. In contrast, GRDelta108-317 decreases polyglutamine protein solubility, and causes formation of nuclear aggregates in non-neuronal cells. Nuclear aggregates recruit hsp72 more rapidly than cytoplasmic aggregates, and are associated with decreased cell viability. Limited proteolysis and chemical cross-linking suggest unique soluble forms of the expanded AR protein underlie these distinct biological activities. These observations provide an experimental framework to understand why expanded polyglutamine proteins may be toxic only to certain populations of cells, and suggest that unique protein associations or conformations of expanded polyglutamine proteins may determine subsequent cellular effects such as nuclear localization and cellular toxicity.

Lysine-Rich Histone (H1) Is a Lysyl Substrate of Tissue Transglutaminase: Possible Involvement of Transglutaminase in the Formation of Nuclear Aggregates in (CAG)n/Qn Expansion Diseases

Histone H1, which contains about 27% lysine, is an excellent lysyl donor substrate of Ca²⁺-activated guinea pig liver tissue transglutaminase as judged by rapid fluorescence enhancement in the presence of the glutaminyl-donor substrate 1-N-(carbobenzoxy-L-glutaminylglycyl)-5-N-(5′N′N′-dimethylaminonaphthalenesulfonyl) diamidopentane. Sodium dodecyl sulfate gel electrophoresis of a 30-min reaction mixture revealed the presence of fluorescent high-M_r aggregates, which are also formed when histone H1 is incubated solely with activated tissue transglutaminase. Aggregate formation is even more pronounced when histone H1 is incubated with activated tissue transglutaminase and dimethylcasein (glutaminyl donor only). The findings suggest not only that histone H1 is an especially good lysyl substrate of tissue transglutaminase, but that it is also a glutaminyl substrate. Histone H1 is a good lysyl substrate of transglutaminase purified from Streptoverticillium mobaraense, suggesting that the ability of histone H1 to act as a transglutaminase lysyl substrate is widespread. In agreement with previous studies, it was found that human β-endorphin is a moderately good substrate of tissue transglutaminase. At least 8 neurodegenerative diseases, including Huntington’s disease, are caused by (CAG)_n expansions in the genome and by an expansion of the corresponding polyglutamine domain within the expressed, mutated protein. Polyglutamine domains are excellent substrates of liver and brain transglutaminases. A hallmark of many of the (CAG)_n/polyglutamine expansion diseases is the presence of polyglutamine-containing aggregates within the cytosol and nuclei of affected neurons. Transglutaminase activity occurs in both of these compartments in human brain. In future studies, it will be important to determine whether transglutaminases play a role in (1) cross-linking of histone H1 to glutaminyl donors (including polyglutamine domains) in nuclear chromatin, (2) the formation of nuclear aggregates in (CAG)_n/polyglutamine expansion diseases, (3) DNA laddering and cell death in neurodegenerative diseases and (4) depletion of neuropeptides in vulnerable regions of Huntington’s disease brain.

Polyglutamine-Mediated Aggregation and Cell Death

The expansion of CAG repeats is the genetic defect underlying eight neurodegenerative diseases. A common feature of these disorders is the presence of intracellular aggregates in neuronal cells. It is still unclear the significance of these cellular inclusions in the neurodegenerative process, since cell death without aggregate formation has been reported. We have constructed a synthetic fusion protein containing 17 or 43 CAG repeats and the green fluorescent protein that recapitulates the features of CAG-expanded alleles. Expression of 43, but not 17 CAG repeats results in formation of nuclear aggregates in human neuroblastoma cells. Moreover, the normal allele (17 CAG) is sequestered in the inclusion bodies. The presence of nuclear inclusions tightly correlates with apoptosis in cells expressing the protein encoding 43 CAG repeats. Cells harboring nuclear aggregates stop proliferation and undergo apoptosis. Moreover, the inhibition of protein degradation pathway increases intracellular aggregates and cell death. These data indicate that intranuclear aggregates induce apoptosis and suggest that the degradation of unfolded proteins improves cell survival.

Polyglutamine expansions: proteolysis, chaperones, and the dangers of promiscuity.

Polyglutamine expansions: proteolysis, chaperones, and the dangers of promiscuity.

Pathogenesis of Polyglutamine-Induced Disease: A Model for SCA1

During the past 7 years several inheritable neurological disorders have been found to be due to the expansion of an unstable CAG trinucleotide repeat that leads to an increase in the length of a polyglutamine tract within a disease-specific protein. Based on pathological evidence obtained from the brains of affected individuals and transgenic mice expressing a mutant human gene, it was proposed that the formation of nuclear aggregates of the polyglutamine protein plays a critical role in pathogenesis. However, recent evidence indicates that this may not be the case. This review focuses on our results for one of these disorders, spinocerebellar ataxia type 1 (SCA1), and presents a model for SCA1 pathogenesis.