Mervyn J. Monteiro

Effects of overexpression of Huntingtin proteins on mitochondrial integrity

Huntingtons disease (HD) is caused by an expansion of a CAG trinucleotide sequence that encodes a polyglutamine tract in the huntingtin (Htt) protein. Expansion of the polyglutamine tract above 35 repeats causes disease, with the age of onset inversely related to the degree of expansion above this number. Growing evidence suggests that mitochondrial function is compromised during HD pathogenesis, but how this occurs is not understood. We examined mitochondrial properties of HeLa cells that expressed green fluorescent protein (GFP)- or FLAG-tagged N-terminal portions of the Htt protein containing either, 17, 28, 74 or 138 polyglutamine repeats. Immunofluorescence staining of cells using antibodies against Tom20, a mitochondrion localized protein, revealed that cells expressing Htt proteins with 74 or 138 polyglutamine repeats were more sensitized to oxidative stress-induced mitochondria fragmentation and had reduced ATP levels compared with cells expressing Htt proteins with 17 or 28 polyglutamine repeats. By measuring changes in fluorescence of a photoactivated GFP protein targeted to mitochondria, we found that cells expressing red fluorescent protein (RFP)-tagged Htt protein containing 74 polyglutamine repeats had mitochondria that displayed reduced movement and fusion than cells expressing RFP-Htt protein with 28 polyglutamine repeats. Overexpression of Drp-1(K38A), a dominant-negative mitochondria-fission mutant, or Mfn2, a protein that promotes mitochondria fusion, suppressed polyglutamine-induced mitochondria fragmentation, the reduction of ATP levels and cell death. In a Caenorhabditis elegans model of HD, we found that reduction of Drp-1 expression by RNA interference rescued the motility defect associated with the expression of Htt proteins with polyglutamine repeats. These results suggest that the increase in cytotoxicity induced by Htt proteins containing expanded polyglutamine tracts is likely mediated, at least in part, by an alteration in normal mitochondrial dynamics, which results in increased mitochondrial fragmentation. Furthermore, our results suggest that it might be possible to reverse polyglutamine-induced cytotoxicity by preventing mitochondrial fragmentation.

Cyclin' toward dementia: cell cycle abnormalities and abortive oncogenesis in Alzheimer disease.

Recent evidence has associated the aberrant, proximal re‐expression of various cell cycle control elements with neuronal vulnerability in Alzheimer disease, a chronic neurodegeneration. Such ectopic localization of various cyclins, cyclin‐dependent kinases, and cyclin inhibitors in neurons can be seen as an attempt to re‐enter the cell cycle. Given that primary neurons are terminally differentiated, any attempted re‐entry into the cell division cycle in this postmitotic environment will be dysregulated. Since successful dysregulation of the cell cycle is also the hallmark of a neoplasm, early cell‐cycle pathophysiology in Alzheimer disease may recruit oncogenic signal transduction mechanisms and, hence, can be viewed as an abortive neoplastic transformation. J. Neurosci. Res. 61:128–133, 2000.

Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy

Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimers disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.

Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD

Loss of ubiquilin or erasin activates ER stress, increases accumulation of polyubiquitinated proteins, and shortens lifespan in worms.

Overexpression of ubiquilin decreases ubiquitination and degradation of presenilin proteins

Mutations in presenilin proteins (PS1 and PS2) are associated with most cases of early-onset Alzheimers disease. Several proteins appear to regulate accumulation of PS proteins in cells. One such protein is ubiquilin-1, which increases levels of coexpressed PS2 protein in a dose-dependent manner. We now report that overexpression of ubiquilin-2, which is 80% identical to ubiquilin-1, also increases the levels of coexpressed PS1 and PS2 proteins in cells. To investigate the mechanism by which ubiquilin proteins increase levels of PS proteins, we examined how overexpression of ubiquilin-1, which possesses all of the key signature motifs present in ubiquilin proteins, affects PS2 gene transcription and PS2 protein turnover and ubiquitination. HeLa cells overexpressing both PS2 and ubiquilin-1 had PS2 mRNA levels lower than HeLa cells overexpressing PS2 alone, indicating that ubiquilin-1 overexpression, in fact, decreases PS2 transcription. Cells overexpressing ubiquilin-1 and PS2 displayed decreased turnover of high molecular weight (HMwt) forms of PS2 but not of full-length PS2 proteins. The reduced turnover of HMwt PS2 proteins appears to be mediated by the binding of the ubiquitin-associated domain (UBA) of ubiquilin to ubiquitin chains conjugated onto PS2 proteins. Immunoprecipitation studies indicated that ubiquilin-1 overexpression decreases ubiquitination of coexpressed PS2 proteins, suggesting that binding of ubiquilin might block ubiquitin chain elongation. Consistent with this model, we found that the UBA domain of ubiquilin-1 binds poly-ubiquitin chains in vitro. In addition, we show that ubiquilin proteins colocalize with ubiquitin-immunoreactive structures in cells and that ubiquilin proteins are present within the inner core of aggresomes, which are structures associated with accumulation of misfolded proteins in cells. Our results suggest that ubiquilin proteins play an important role in regulating PS protein levels in cells.

Presenilin Overexpression Arrests Cells in the G1 Phase of the Cell Cycle : Arrest Potentiated by the Alzheimer’s Disease PS2(N141I) Mutant

To investigate the mechanism by which presenilin (PS) overexpression induces apoptosis, we studied the effects of these proteins on cell cycle progression. Transiently transfected HeLa cells were bromodeoxyuridine (BrdU) labeled to visualize DNA synthesis by immunofluorescence and stained with propidium iodide to measure DNA content by fluorescence-activated cell sorting (FACS). BrdU labeling was decreased in cells expressing presenilin-1 (PS1), presenilin-2 (PS2), an Alzheimers disease-associated missense mutation PS2(N141I), and the carboxyl-terminally deleted PS2 construct PS2(166aa), compared with mock and neurofilament-light (NF-L) transfected cells. Analysis of BrdU incorporation in mitotically synchronized HeLa cells suggested that cells were arresting in the G1 phase of the cell cycle, and this was confirmed by FACS analysis. Interestingly, cell cycle progression was more inhibited by the expression of PS2(N141I) compared with wild-type PS2. In addition, ATM, the gene product mutated in ataxia-telangiectasia, does not appear to be a downstream effector of PS-induced cell cycle arrest as transfection of PS constructs into an ataxia-telangiectasia cell line also resulted in cell cycle inhibition. Quantitative immunoblotting of whole-cell lysates from PS-transfected cells did not reveal increases or decreases in the steady-state levels of p21, p27, p53, pRb, or c-myc, suggesting that the presenilins mediate cell cycle arrest by mechanisms other than simple changes in the steady-state levels of these cell-cycle-related proteins.

Characterization of erasin (UBXD2): a new ER protein that promotes ER-associated protein degradation

Ubiquitin regulator-X (UBX) is a discrete protein domain that binds p97/valosin-containing protein (VCP), a molecular chaperone involved in diverse cell processes, including endoplasmic-reticulum-associated protein degradation (ERAD). Here we characterize a human UBX-containing protein, UBXD2, that is highly conserved in mammals, which we have renamed erasin. Biochemical fractionation, immunofluorescence and electron microscopy, and protease protection experiments suggest that erasin is an integral membrane protein of the endoplasmic reticulum and nuclear envelope with both its N- and C-termini facing the cytoplasm or nucleoplasm. Localization of GFP-tagged deletion derivatives of erasin in HeLa cells revealed that a single 21-amino-acid sequence located near the C-terminus is necessary and sufficient for localization of erasin to the endoplasmic reticulum. Immunoprecipitation and GST-pulldown experiments confirmed that erasin binds p97/VCP via its UBX domain. Additional immunoprecipitation assays indicated that erasin exists in a complex with other p97/VCP-associated factors involved in ERAD. Overexpression of erasin enhanced the degradation of the ERAD substrate CD3δ, whereas siRNA-mediated reduction of erasin expression almost completely blocked ERAD. Erasin protein levels were increased by endoplasmic reticulum stress. Immunohistochemical staining of brain tissue from patients with Alzheimers disease and control subjects revealed that erasin accumulates preferentially in neurons undergoing neurofibrillary degeneration in Alzheimers disease. These results suggest that erasin may be involved in ERAD and in Alzheimers disease.

The role of cell cycle‐mediated events in Alzheimer's disease

The mechanism(s) underlying selective neuronal death in Alzheimers disease remain unresolved. However, recently, we and others showed that susceptible hippocampal neurones in Alzheimers disease express markers common to cells in various phases of the cell cycle. Since neuronal maturation is associated with effective escape from the cell division cycle, emergence out of quiescence may be deleterious. Here, we review a number of current findings indicating that disregulated ectopic re‐activation of cell cycle‐mediated events, akin to neoplasia, represent an important early pathway associated with neuronal death and, more importantly, one that involves virtually the entire spectrum of the pathological events described in Alzheimers disease.

Ubiquilin regulates presenilin endoproteolysis and modulates γ-secretase components, Pen-2 and nicastrin

Mutations in presenilin proteins (PS1 and PS2) lead to early-onset Alzheimers disease. PS proteins are endoproteolytically cleaved into two main fragments: the NTF (PS N-terminal fragment) and the CTF (PS C-terminal fragment). The two fragments are believed to constitute the core catalytic enzyme activity called gamma-secretase, which is responsible for cleaving beta-amyloid precursor protein to release Abeta. Thus, studying factors that modulate PS fragment levels could provide important information about gamma-secretase. Previously, we demonstrated that the protein, ubiquilin-1, interacts both in vivo and in vitro with PS and that overexpression of ubiquilin-1 or -2 leads to increased accumulation of full-length PS proteins. Using wild-type HEK-293 cells (human embryonic kidney 293 cells) and PS-inducible cells, we now show that overexpression of either ubiquilin-1 or -2 decreases the PS NTF and CTF levels. Conversely, siRNA (small interfering RNA)-mediated knockdown of ubiquilin-1 and -2 proteins increased the PS NTF and CTF levels. We considered that ubiquilin might alter PS fragment accumulation by acting as a shuttle factor escorting PS fragments to the proteasome for degradation. However, through proteasome inhibition studies, we show that this does not occur. Instead, our results suggest that ubiquilin regulates PS fragment production. We also examined whether other components of the gamma-secretase complex are affected by ubiquilin expression. Interestingly, overexpression of ubiquilin resulted in a decrease in Pen-2 and nicastrin levels, two essential components of the gamma-secretase complex. In contrast, knockdown of ubiquilin-1 and -2 protein expression by RNAi (RNA interference) increased Pen-2 and nicastrin levels. Finally, we show that inhibition of the proteasome results in decreased PS fragment production and that reversal of proteasome inhibition restores PS fragment production, suggesting that the proteasome may be involved in PS endoproteolysis. These studies implicate ubiquilin as an important factor in regulating PS biogenesis and metabolism.

Huntingtin Interacts with the Cue Domain of gp78 and Inhibits gp78 Binding to Ubiquitin and p97/VCP

Huntingtons disease (HD) is caused by polyglutamine expansion in huntingtin (htt) protein, but the exact mechanism of HD pathogenesis remains uncertain. Recent evidence suggests that htt proteins with expanded polyglutamine tracts induce endoplasmic reticulum (ER) stress, probably by interfering with ER-associated degradation (ERAD). Here we report that mutant htt interacts and interferes with the function of gp78, an ER membrane-anchored ubiquitin ligase (E3) involved in ERAD. Mapping studies showed that the HEAT repeats 2&3 of htt interact with the cue domain of gp78. The interaction competitively reduces polyubiquitinated protein binding to gp78 and also sterically blocks gp78 interaction of p97/VCP, a molecular chaperone that is essential for ERAD. These effects of htt negatively regulate the function of gp78 in ERAD and are aggravated by polyglutamine expansion. Paradoxically, gp78 is still able to ubiquitinate and facilitate degradation of htt proteins with expanded polyglutamine. The impairment of ERAD by mutant htt proteins is associated with induction of ER stress. Our studies provide a novel molecular mechanism that supports the involvement of ER stress in HD pathogenesis.