Alzheimer Disease: Romano DM

Hiltunen M, Lu A, Thomas AV, Romano DM, Kim M, Jones PB, Xie Z, Kounnas MZ, Wagner SL, Berezovska O, Hyman BT, Tesco G, Bertram L, Tanzi RE. · Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Charlestown, MA 02129, USA. · J Biol Chem. · Pubmed #16945923 links to  free full text

Abstract: Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with alpha-, beta-, or gamma-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (alpha- and beta-forms). Moreover, UBQLN1 knockdown increased levels of secreted Abeta40 and Abeta42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic "gatekeeper" that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Abeta.

Romano DM, Dong Y, Hiltunen M, Tanzi RE, Xie Z. · Department of Neurology, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital and Harvard Medical School, MA 02129-2060, USA. · Amyloid. · Pubmed #16911962 No free full text.

Abstract: The study of amyloidogenic beta-amyloid precursor protein (AbetaPP) metabolism and amyloid beta protein (Abeta) production has been a major focus of Alzheimer's disease (AD) neuropathogenesis research. Cell transfection is a commonly employed method for assessing the effects of various genes on AbetaPP processing and Abeta production. Certain cell transfection reagents utilize lipid-based formulations that could potentially affect AbetaPP processing and Abeta production. Thus, we set out to assess the effects of cell transfection reagents with lipid formulations (TKO, FuGene6, RNAifect) on AbetaPP processing and Abeta level in H4 human neuroglioma cells overexpressing human AbetaPP. We found both TKO and RNAifect increase the protein levels of AbetaPP-C-terminal fragments (CTFs) and Abeta levels, while FuGene6 increases the protein levels of AbetaPP-CTFs without altering Abeta level. In contrast, electroporation-based cell transfection does not affect AbetaPP processing and Abeta production in our studies. These results suggest for the first time that lipid-based cell transfection reagents may artefactually affect AbetaPP processing and Abeta production, thereby confounding studies aimed at assessing the effects of transfected genes on AbetaPP metabolism.

Xie Z, Moir RD, Romano DM, Tesco G, Kovacs DM, Tanzi RE. · Genetics and Aging Research Unit, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Charlestown 02129-4404, USA. · Neurodegener Dis. · Pubmed #16908971 No free full text.

Abstract: BACKGROUND: At least half of all cases of early onset (<60) familial Alzheimer's disease (FAD) are caused by any of over 150 mutations in three genes: the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2). Mutant forms of PS1 have been shown to sensitize cells to apoptotic cell death. OBJECTIVE: We investigated the effects of hypocapnia, a risk factor for both cognitive and neurodevelopment deficits, on caspase-3 activation, apoptosis, and amyloid beta-protein (Abeta) production, and assessed the influence of the PS1Delta9 FAD mutation on these effects. METHOD: For this purpose, we exposed stably transfected H4 human neuroglioma cells to conditions consistent with hypocapnia (PCO2<40 mm Hg) and hypocapnia plus hypoxia (PO2<21%). RESULTS: Hypocapnia (20 mm Hg CO2 for 6 h) induced caspase-3 activation and apoptosis; the PS1Delta9 FAD mutation significantly potentiated these effects. Moreover, the combination of hypocapnia (20 mm Hg CO2) and hypoxia (5%O2) induced caspase-3 activation and apoptosis in a synergistic manner. Hypocapnia (5 and 20 mm Hg CO2 for 6 h) also led to an increased Abeta production. CONCLUSION: The findings suggest that hypocapnia (e.g. during general anesthesia) could exacerbate AD neuropathogenesis.

Xie Z, Romano DM, Tanzi RE. · MassGeneral Institute for Neurodegenerative Disease, Department of NeurologyMassachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129-2060, USA. · J Mol Neurosci. · Pubmed #15781968 No free full text.

Abstract: The gamma-secretase complex consists of PS1/PS2, nicastrin, APH-1a, and PEN-2. PS1 undergoes endoproteolytic processing to yield two fragments: PS1-NTF and PS1-CTF. Changes in PEN-2 levels have been shown previously to affect the endoproteolytic processing of wild-type (wt)-PS1. However, the effects of PEN-2 on the proteolytic processing of familial Alzheimer's disease (FAD) mutant forms of PS1 have not yet been reported. To determine whether PEN-2 affects the proteolytic processing of mutant PS1 in the same manner as that of wt-PS1, we established RNA interference (RNAi) for PEN-2 in H4 human neuroglioma cells stably transfected to express wt or FAD mutant forms of PS1 including L286V, A246E, and that lacking exon 9 (Delta9). As expected, in H4 cells expressing wt-PS1, RNAi for PEN-2 increased levels of PS1-FL and attenuated PS1 endoproteolysis. Likewise, in cells expressing PS1 with the FAD missense mutations, L286V and A246E, RNAi for PEN-2 increased PS1-FL and reduced PS1 endoproteolysis. However, in H4 cells stably transfected to express the FAD-linked Delta9 mutation (PS1 lacking exon 9), RNAi for PEN-2 did not increase but, instead, decreased PS1-FL. In contrast, RNAi for nicastrin and APH-1a decreased PS1-FL in H4 cells expressing either wt-PS1 or Delta9-PS1. In summary, the metabolism of wt-PS1 and FAD-linked Delta9-PS1 is specifically and differentially affected by loss of function of PEN-2.

Xie Z, Romano DM, Tanzi RE. · Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129-4404, USA. · J Biol Chem. · Pubmed #15699037 links to  free full text

Abstract: Processing of the beta-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by beta- and alpha-secretase to produce APP-C99 and APP-C83, which are further cleaved by gamma-secretase to produce amyloid beta-protein (Abeta) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11alpha (MINT1, encoded by gene APBA1) and X11beta (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11alpha and X11beta alters APP processing and Abeta production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11alpha and X11beta expression on APP processing and Abeta production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Abeta levels in both cell lines by inhibiting gamma-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Abeta levels, but apparently not via attenuation of gamma-secretase cleavage of APP. The notion of attenuating gamma-secretase cleavage of APP via the APP adaptor protein X11alpha is particularly attractive with regard to therapeutic potential given that side effects of gamma-secretase inhibition due to impaired proteolysis of other gamma-secretase substrates, e.g. Notch, might be avoided.

Moir RD, Atwood CS, Romano DM, Laurans MH, Huang X, Bush AI, Smith JD, Tanzi RE. · Genetics and Aging Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts 02129-2060, USA. · Biochemistry. · Pubmed #10194381 No free full text.

Abstract: The epsilon 4 allele of apolipoprotein E (APOE) has been found to be a risk factor for late-onset Alzheimer's disease (AD). While the pathogenic mechanism of APOE in AD is not yet clear, APOE isoforms appear to differentially influence the aggregation of A beta, the principal component of Alzheimer-associated beta-amyloid deposits. To date, no data are available for the propensity of A beta to aggregate in the presence of APOE under conditions where these components are at physiological concentrations (in cerebrospinal fluid, APOE and A beta are approximately 100 nM and approximately 5 nM, respectively). We employed a novel in vitro filtration assay for detecting zinc(II)- and copper(II)-induced aggregation of A beta in solutions containing concentrations of the peptide that are similar to those reported for human cerebrospinal fluid. The potential for resolubilization with EDTA and the relative densities of zinc- and copper-induced A beta aggregates were also compared. Zinc-induced A beta aggregates were found to be denser and less easily resolubilized than copper-induced precipitates. Metal-induced aggregation of A beta was studied in the presence of purified apolipoprotein E2, apolipoprotein E3, and apolipoprotein E4 under conditions that approximate the physiological concentrations and ratios of these proteins. In the presence of all three APOE isoforms, zinc-induced aggregation of A beta was attenuated, while precipitation with copper was enhanced. Consistent with the increased risk for AD associated with the epsilon 4 allele of APOE, metal-induced aggregation of A beta was highest for both zinc and copper in the presence of apolipoprotein E4. Our data are consistent with a role for APOE as an in vivo molecular chaperone for A beta.