Cysteine oxidation within N-terminal mutant huntingtin promotes oligomerization and delays clearance of soluble protein

Huntington disease (HD) is a progressive neurodegenerative disorder caused by expression of polyglutamine-expanded mutant huntingtin protein (mhtt). Most evidence indicates that soluble mhtt species, rather than insoluble aggregates, are the important mediators of HD pathogenesis. However, the differential roles of soluble monomeric and oligomeric mhtt species in HD and the mechanisms of oligomer formation are not yet understood. We have shown previously that copper interacts with and oxidizes the polyglutamine-containing N171 fragment of huntingtin. In this study we report that oxidation-dependent oligomers of huntingtin form spontaneously in cell and mouse HD models. Levels of these species are modulated by copper, hydrogen peroxide, and glutathione. Mutagenesis of all cysteine residues within N171 blocks the formation of these oligomers. In cells, levels of oligomerization-blocked mutant N171 were decreased compared with native N171. We further show that a subset of the oligomerization-blocked form of glutamine-expanded N171 huntingtin is rapidly depleted from the soluble pool compared with "native " mutant N171. Taken together, our data indicate that huntingtin is subject to specific oxidations that are involved in the formation of stable oligomers and that also delay removal from the soluble pool. These findings show that inhibiting formation of oxidation-dependent huntingtin oligomers, or promoting their dissolution, may have protective effects in HD by decreasing the burden of soluble mutant huntingtin.     

Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β42/40 Ratio

Background

Presenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.

Methodology/Principal Findings

To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ₄₂ production are associated with a consistent conformational change in PS1.

Conclusions/Significance

These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ_42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ₄₂ generation, which could be exploited therapeutically.     

Direct visualization of the gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus

Amyloid-beta, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase-mediated intramembranous cleavage. In addition to amyloid-beta, this cleavage produces a carboxyl-terminal intracellular fragment which has an unknown function. The carboxyl-terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl-terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP-CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP-CT-Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP-CT.     

Detection of presenilin-1 homodimer formation in intact cells using fluorescent lifetime imaging microscopy

Presenilin-1 (PS1) is a multipass transmembrane domain protein, which is believed to be the catalytic component of the gamma-secretase complex. The complex is comprised of four major components: PS1, nicastrin, Aph-1, and Pen-2. The exact stoichiometric relationship between the four components remains unclear. It has been shown that gamma-secretase exists as high molecular weight complexes, suggesting the possibility of dimer/multimer formation. We combined a biochemical approach with a novel morphological microscopy assay to analyze PS1 dimer formation and subcellular distribution in situ, in intact mammalian cells. Both coimmunoprecipitation and fluorescent lifetime imaging microscopy approaches showed that wildtype PS1 molecules form dimers. Moreover, PS1 holoproteins containing the D257A mutation also come into close enough proximity to form a dimer, suggesting that cleavage within the loop is not necessary for dimer formation. Taken together these data suggest that PS1 dimerization occurs during normal PS1 function.     

Genetic Structure of Populations of the Colorado Potato Beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The genetic structure of a Colorado potato beetle population from Kiev oblast was examined by cluster analysis of individual RAPD patterns. The obtained clustering indicates that the population is structured. This may be explained by adaptation to pyrethroid insecticides used for controlling the population size of this pest. Microevolutionary factors affecting the genetic structure of local populations of Colorado potato beetle are discussed.     

Signal peptide peptidase (SPP) dimer formation as assessed by fluorescence lifetime imaging microscopy (FLIM) in intact cells

Background

Alzheimer disease (AD) is clinically characterized by progressive memory loss, impairments in behavior, language and visual-spatial skills and ultimately, death. Epidemiological data reporting the predisposition of women to AD has led to a number of lines of evidence suggesting that age-related changes in hormones of the hypothalamic-pituitary-gonadal (HPG) axis following reproductive senescence, may contribute to the etiology of AD. Recent studies from our group and others have reported not only increases in circulating gonadotropins, namely luteinizing hormone (LH) in individuals with AD compared with control individuals, but also significant elevations of LH in vulnerable neuronal populations in individuals with AD compared to control cases as well as the highest density of gonadotropin receptors in the brain are found within the hippocampus, a region devastated in AD. However, while LH is higher in AD patients, the downstream consequences of this are incompletely understood. To begin to examine this issue, here, we examined the expression levels of steroidogenic acute regulatory (StAR) protein, which regulates the first key event in steroidogenesis, namely, the transport of cholesterol into the mitochondria, and is regulated by LH through the cyclic AMP second messenger pathway, in AD and control brain tissue.

Results

Our data revealed that StAR protein was markedly increased in both the cytoplasm of hippocampal pyramidal neurons as well as in the cytoplasm of other non-neuronal cell types from AD brains when compared with age-matched controls. Importantly, and suggestive of a direct mechanistic link, StAR protein expression in AD brains colocalized with LH receptor expression.

Conclusion

Therefore, our findings suggest that LH is not only able to bind to its receptor and induce potentially pathogenic signaling in AD, but also that steroidogenic pathways regulated by LH may play a role in AD.     

Visualizing interaction of proteins relevant to Alzheimer's disease in intact cells

To understand normal function of memory studying models of pathological memory decline is essential. The most common form of dementia leading to memory decline is Alzheimer's disease (AD), which is characterized by the presence of neurofibrillary tangles and amyloid plaques in the affected brain regions. Altered production of amyloid beta (Abeta) through sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases seems to be a central event in the molecular pathogenesis of the disease. Thus, the study of the complex interplay of proteins that are involved in or modify Abeta production is very important to gain insight into the pathogenesis of AD. Here, we describe the use of Fluorescence lifetime imaging microscopy (FLIM), a Fluorescence resonance energy transfer (FRET)-based method, to visualize protein-protein-interaction in intact cells, which has proven to be a valuable method in AD research.     

CHIP targets toxic alpha-Synuclein oligomers for degradation

alpha-Synuclein (alphaSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric alphaSyn intermediates may represent the most toxic alphaSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on alphaSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced alphaSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic alphaSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of alphaSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric alphaSyn may contribute to the successful development of drug therapies that target oligomeric alphaSyn by mimicking or enhancing the powerful effects of CHIP.     

Notch1 competes with the amyloid precursor protein for gamma-secretase and down-regulates presenilin-1 gene expression

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same gamma-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct gamma-secretase substrates, Notch 1 Delta extracellular domain (N1DeltaEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-beta protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for gamma-secretase activity; transfection with either of two direct substrates of gamma-secretase led to a reduction in the gamma-cleaved products, Notch intracellular domain or amyloid-beta protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1 Delta EC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages gamma-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation.