Alzheimer Disease: Berezovska O

Lleó A, Berezovska O, Growdon JH, Hyman BT. · Massachusetts General Hospital, Alzheimer Research Unit, Charleston, MA 02129, USA. · Am J Geriatr Psychiatry. · Pubmed #15010344 No free full text.

Abstract: Three genes have been implicated in the etiology of early-onset autosomal-dominant Alzheimer disease (AD): the amyloid precursor protein, the presenilin-1, and presenilin-2 genes. Approximately half of autosomal-dominant AD cases are associated with mutations in the presenilin-1 (PS-1) gene on the long arm of Chromosome 14. Marked allelic heterogeneity characterizes families with PS-1 gene mutations; more than 100 different mutations have been found in independent families thus far. With the exception of age at onset, the clinical phenotype is similar to late-onset AD, although some rare specific phenotypes have been described. These mutations lead to enhanced deposition of total Abeta and Abeta42 (but not Abeta40) in the brain, compared with sporadic AD. There is a considerable heterogeneity in the histological profiles among brains from patients with different mutations, and although some lead to predominantly parenchymal deposition of Abeta in the form of diffuse and cored plaques, others show predominantly vascular deposition, with severe amyloid angiopathy. Only some mutations are associated with enhanced neurofibrillary tangle formation and increased neuronal loss compared with sporadic AD. However, there is an important clinical and pathological variability even among family members with the same mutation, which suggests the involvement of other genetic or environmental factors that modulate the clinical expression of the disease. This represents a valuable model for identifying such factors and has potential implications for the development of new therapeutic strategies for delaying disease onset.

Serneels L, Van Biervliet J, Craessaerts K, Dejaegere T, Horré K, Van Houtvin T, Esselmann H, Paul S, Schäfer MK, Berezovska O, Hyman BT, Sprangers B, Sciot R, Moons L, Jucker M, Yang Z, May PC, Karran E, Wiltfang J, D'Hooge R, De Strooper B. · Department for Molecular and Developmental Genetics, VIB, KULeuven, Herestraat 49, 3000 Leuven, Belgium. · Science. · Pubmed #19299585 No free full text.

Abstract: The gamma-secretase complex plays a role in Alzheimer's disease and cancer progression. The development of clinically useful inhibitors, however, is complicated by the role of the gamma-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different gamma-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B gamma-secretase in a mouse Alzheimer's disease model led to improvements of Alzheimer's disease-relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total gamma-secretase activity in the human brain, and thus specific targeting of Aph1B-containing gamma-secretase complexes may help generate less toxic therapies for Alzheimer's disease.

Herl L, Thomas AV, Lill CM, Banks M, Deng A, Jones PB, Spoelgen R, Hyman BT, Berezovska O. · Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative, Diseases, Massachusetts General Hospital, 114 16th Street, Charlestown, MA 02129, USA. · Mol Cell Neurosci. · Pubmed #19281847 No free full text.

Abstract: Alzheimer's disease is characterized by accumulation of toxic beta-amyloid (Abeta) in the brain and neuronal death. Several mutations in presenilin (PS1) and beta-amyloid precursor protein (APP) associate with an increased Abeta(42/40) ratio. Abeta(42), a highly fibrillogenic species, is believed to drive Abeta aggregation. Factors shifting gamma-secretase cleavage of APP to produce Abeta(42) are unclear. We investigate the molecular mechanism underlying altered Abeta(42/40) ratios associated with APP mutations at codon 716 and 717. Using FRET-based fluorescence lifetime imaging to monitor APP-PS1 interactions, we show that I716F and V717I APP mutations increase the proportion of interacting molecules earlier in the secretory pathway, resulting in an increase in Abeta generation. A PS1 conformation assay reveals that, in the presence of mutant APP, PS1 adopts a conformation reminiscent of FAD-associated PS1 mutations, thus influencing APP binding to PS1/gamma-secretase. Mutant APP affects both intracellular location and efficiency of APP-PS1 interactions, thereby changing the Abeta(42/40) ratio.

Thomas AV, Berezovska O, Hyman BT, von Arnim CA. · Department of Neurology, University of Cologne, Germany. · Methods. · Pubmed #18374273 links to  free full text

Abstract: 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.

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.

Thomas AV, Herl L, Spoelgen R, Hiltunen M, Jones PB, Tanzi RE, Hyman BT, Berezovska O. · Alzheimer's Disease Research Laboratory, Harvard Medical School, Charlestown, Massachusetts 02129, USA. · J Biol Chem. · Pubmed #16815845 links to  free full text

Abstract: Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the gamma-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid beta (Abeta). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.

Berezovska O, Lleo A, Herl LD, Frosch MP, Stern EA, Bacskai BJ, Hyman BT. · Alzheimer Research Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. · J Neurosci. · Pubmed #15772361 links to  free full text

Abstract: Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, an enzymatic activity that cleaves amyloid beta (Abeta) from the amyloid precursor protein (APP). More than 100 mutations spread throughout the PS1 molecule are linked to autosomal dominant familial Alzheimer's disease (FAD). All of these mutations lead to a similar phenotype: an increased ratio of Abeta42 to Abeta40, increased plaque deposition, and early age of onset. We use a recently developed microscopy approach, fluorescence lifetime imaging microscopy, to monitor the relative molecular distance between PS1 N and C termini in intact cells. We show that FAD-linked missense mutations located near the N and C termini, in the mid-region of PS1, and the exon 9 deletion mutation all change the spatial relationship between PS1 N and C termini in a similar way, increasing proximity of the two epitopes. This effect is opposite of that observed by treatment with Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) (Lleo et al., 2004b). Accordingly, treatment of M146L PS1-overexpressing neurons with high-dose NSAIDs somewhat offsets the conformational change associated with the mutation. Moreover, by monitoring the relative distance between a PS1 loop epitope and the APP C terminus, we demonstrate that the FAD PS1 mutations are also associated with a consistent change in the configuration of the PS1-APP complex. The nonpathogenic E318G PS1 polymorphism had no effect on PS1 N terminus-C terminus proximity or PS1-APP interactions. We propose that the conformational change we observed may therefore provide a shared molecular mechanism for FAD pathogenesis caused by a wide range of PS1 mutations.

Irizarry MC, Deng A, Lleo A, Berezovska O, Von Arnim CA, Martin-Rehrmann M, Manelli A, LaDu MJ, Hyman BT, Rebeck GW. · Alzheimer Disease Research Unit, Massachusetts General Hospital-East, Charlestown, Massachusetts, USA. · J Neurochem. · Pubmed #15312168 No free full text.

Abstract: Polymorphisms in the apolipoprotein E (APOE) gene affect the risk of Alzheimer disease and the amount of amyloid beta-protein (Abeta) deposited in the brain. The apoE protein reduces Abeta levels in conditioned media from cells in culture, possibly through Abeta clearance mechanisms. To explore this effect, we treated multiple neural and non-neural cell lines for 24 h with apoE at concentrations similar to those found in the cerebrospinal fluid (1-5 microg/mL). The apoE treatment reduced Abeta40 by 60-80% and Abeta42 to a lesser extent (20-30%) in the conditioned media. Surprisingly, apoE treatment resulted in an accumulation of amyloid precursor protein (APP)-C-terminal fragments in cell extracts and a marked reduction of APP intracellular domain-mediated signaling, consistent with diminished gamma-secretase processing of APP. All three isoforms of apoE, E2, E3 and E4, had similar effects on Abeta and APP-C-terminal fragments, and the effects were independent of the low-density lipoprotein receptor family. Apolipoprotein E had minimal effects on Notch cleavage and signaling in cell-based assays. These data suggest that apoE reduces gamma-secretase cleavage of APP, lowering secreted Abeta levels, with stronger effects on Abeta40. The apoE modulation of Abeta production and APP signaling is a potential mechanism affecting Alzheimer disease risk.

Micchelli CA, Esler WP, Kimberly WT, Jack C, Berezovska O, Kornilova A, Hyman BT, Perrimon N, Wolfe MS. · Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA. · FASEB J. · Pubmed #12424225 links to  free full text

Abstract: Signaling from the Notch (N) receptor is essential for proper cell-fate determinations and tissue patterning in all metazoans. N signaling requires a presenilin (PS)-dependent transmembrane-cleaving activity that is closely related or identical to the gamma-secretase proteolysis of the amyloid-beta precursor protein (APP) involved in Alzheimer's disease pathogenesis. Here, we show that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester, a potent gamma-secretase inhibitor reported to reduce amyloid-beta levels in transgenic mice, prevents N processing, translocation, and signaling in cell culture. This compound also induces developmental defects in Drosophila remarkably similar to those caused by genetic reduction of N. The appearance of this phenocopy depends on the timing and dose of compound exposure, and effects on N-dependent signaling molecules established its biochemical mechanism of action in vivo. Other gamma-secretase inhibitors caused similar effects. Thus, the three-dimensional structure of the drug-binding site(s) in Drosophila gamma-secretase is remarkably conserved vis-à-vis the same site(s) in the mammalian enzyme. These results show that genetics and developmental biology can help elucidate the in vivo site of action of pharmacological agents and suggest that organisms such as Drosophila may be used as simple models for in vivo prescreening of drug candidates.

Kinoshita A, Whelan CM, Smith CJ, Berezovska O, Hyman BT. · Alzheimer Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA. · J Neurochem. · Pubmed #12358789 No free full text.

Abstract: 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.

Zaidi NF, Berezovska O, Choi EK, Miller JS, Chan H, Lilliehook C, Hyman BT, Buxbaum JD, Wasco W. · Genetics and Aging Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USA. · Neuroscience. · Pubmed #12207970 No free full text.

Abstract: Mutations in the presenilin 1 and 2 genes cause the majority of early onset familial forms of Alzheimer's disease. Here we describe the biochemical and immunohistochemical characterization of calsenilin, a novel calcium binding protein that we have previously shown to interact with presenilins 1 and 2, in mouse brain. The co-immunoprecipitation of endogenous calsenilin and presenilin 1 demonstrates that these proteins are physiologic binding partners. Although calsenilin has been predicted to be a soluble protein, we have found that the majority of it is tightly associated with the cytoplasmic face of intracellular membranes and that it can only be dissociated using harsh treatments such as urea. In addition, we have demonstrated that calsenilin is a developmentally regulated protein that is mainly present in the brain, where it localizes to both the hippocampus and cerebellum. Calsenilin staining co-localized with the somatodendritic marker microtubule-associated protein-2 primarily in the granular cell layer of the cerebellum, indicating that calsenilin expression is primarily neuronal. In primary cultured neurons, calsenilin immunoreactivity was observed in cell bodies as well as in some neuronal processes. Co-localization experiments using specific axonal and dendritic markers indicate that these processes were mainly axonal in nature, although a smaller subset of dendrites also appears to contain calsenilin. In summary, we have established that calsenilin and presenilin 1 can interact at physiologic levels, and that calsenilin is a developmentally regulated protein that is expressed primarily in the cerebellum and hippocampus. Although calsenilin is a soluble protein, it is tightly associated with the membrane. Finally, the expression pattern of calsenilin, which is similar to that of the presenilin(s), suggests that the common locations of these two proteins provide an opportunity for physical interaction in vivo.

Kinoshita A, Whelan CM, Berezovska O, Hyman BT. · Alzheimer's Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA. · J Biol Chem. · Pubmed #12032152 links to  free full text

Abstract: The amyloid precursor protein (APP), a large glycoprotein highly expressed in neurons, is cleaved in its intramembranous domain by gamma secretase to generate amyloid-beta and a free carboxyl-terminal intracellular fragment (APP-CT), which has previously been suggested to interact with the adapter protein Fe65 and the histone acetyltransferase Tip60. An identical gamma secretase activity mediates cleavage of Notch, releasing an intracellular signaling domain that translocates to the nucleus. We examined the effect of an ectopically expressed 58-amino acid APP-CT fragment (APP-C58) on human H4 neuroglioma cells. We demonstrate by confocal microscopy and fluorescence resonance energy transfer analysis that APP-C58 translocates to the nucleus and forms a complex in the nucleus with the Tip60, independent of interactions with Fe65. APP-C58 transfected H4 cells undergo apoptosis within 48-72 h, marked by nuclear blebbing, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, and blockade by a caspase inhibitor. When nuclear access of APP-C58 is prevented by fusing with a strong membrane-targeting farnesylation domain, apoptosis is blocked. APP-C58-induced apoptosis was markedly enhanced by co-transfection with wild type Tip60 and decreased by mutant Tip60 lacking histone acetyltransferase activity, suggesting that Tip60 mediates APP-CT-induced cell death. Thus, gamma secretase cleavage of APP may contribute to Alzheimer's disease-related neurodegeneration in two ways: release of amyloid-beta and liberation of a bioactive carboxyl-terminal domain from membrane-bound APP.

Jack C, Berezovska O, Wolfe MS, Hyman BT. · Alzheimer's Disease Research Laboratory, Department of Neurology, Harvard Medical School, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA. · Brain Res Mol Brain Res. · Pubmed #11245918 No free full text.

Abstract: Presenilin1 (PS1) has been implicated in normal Notch1 processing and signaling in several experimental systems. In the present study, the relationship between PS1 and Notch1 in mammalian neurons is studied by analyzing Notch1 cleavage and C-terminal nuclear translocation as well as Notch1 signaling via the transactivation of a CBF1-luciferase reporter construct. We show that full-length Notch1 [N1(FL)] transfected into wild type (WT) primary neurons is cleaved in the presence of its biological ligand Delta (Dl) and translocated to the nucleus within 1--3 min of ligand addition. PS1 deficient neurons show normal Notch1 insertion into the cellular membrane, yet lack Notch1 activation resulting in markedly inhibited nuclear translocation of the C-terminal Notch fragment (NICD). PS1 deficient neurons also have impaired Notch1 signaling which can be restored fully or partially to levels seen in WT littermates by transfection with WT or familial Alzheimer's disease-associated M146L mutant PS1, respectively. We also show that pharmacological inhibition of PS1-associated gamma-secretase activity parallels the effects of genetic PS1 deficiency in these assays. These results support the hypothesis that PS1 deficiency blocks neuronal Notch1 processing and signaling.

Berezovska O, Jack C, McLean P, Aster JC, Hicks C, Xia W, Wolfe MS, Weinmaster G, Selkoe DJ, Hyman BT. · Alzheimer's Disease Research Laboratory, Massachusetts General Hospital, 149-13th Street, Charlestown, MA 02129, USA. · Ann N Y Acad Sci. · Pubmed #11193154 No free full text.

Abstract: Recent data suggest an intimate relationship between the familial Alzheimer disease gene presenilin 1 (PS1) and proteolytic processing of both the amyloid precursor protein (APP) and the important cell signaling molecule, Notch1. We now show, using mammalian cells transfected with full-length Notch1, that the C terminal domain of Notch1 rapidly translocates to the nucleus upon stimulation with the physiologic ligand Delta and initiates a CBF1-dependent signal transduction cascade. Using this assay, we demonstrate that the same aspartate mutations in PS1 that block APP processing also prevent Notch1 cleavage and translocation to the nucleus. Moreover, we show that two APP gamma-secretase inhibitors also diminish Notch1 nuclear translocation in a dose-dependent fashion. However, Notch1 signaling, assessed by measuring the activity of CBF1, a downstream gene, was reduced but not completely abolished in the presence of either aspartate mutations or gamma-secretase inhibitors. Our results support the hypothesis that similar PS1-related enzymatic activity is necessary for both APP and Notch1 processing, yet suggest that Notch signaling may remain relatively preserved with moderate levels of gamma-secretase inhibition.

Berezovska O, Frosch M, McLean P, Knowles R, Koo E, Kang D, Shen J, Lu FM, Lux SE, Tonegawa S, Hyman BT. · Alzheimer Research Unit, Massachusetts General Hospital, Neurology Service, Rm. 6405, 149 13th Street, Charlestown, MA 02129, USA. · Brain Res Mol Brain Res. · Pubmed #10366748 No free full text.

Abstract: The normal functional neurobiology of the Alzheimer's disease (AD) related gene presenilin 1 (PS1) is unknown. One clue comes from a genetic screen of Caenorhabditis elegans, which reveals that the presenilin homologue sel-12 facilitates lin-12 function [D. Levitan, I. Greenwald, Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene, Nature 377 (1995) 351-355]. The mammalian homologue of lin-12, Notch1, is a transmembrane receptor that plays an important role in cell fate decisions during development, including neurogenesis, but does not have a known function in fully differentiated cells. To better understand the potential role of Notch1 in mammalian postmitotic neurons and to test the hypothesis that Notch and PS 1 interact, we studied the effect of Notch1 transfection on neurite outgrowth in primary cultures of hippocampal/cortical neurons. We demonstrate that Notch1 inhibits neurite extension, and thus has a function in postmitotic mature neurons in the mammalian CNS. Furthermore, we present evidence demonstrating that there is a functional interaction between PS1 and Notch1 in mammalian neurons, analogous to the sel-12/lin-12 interaction in vulval development in C. elegans [D. Levitan, T. Doyle, D. Brousseau, M. Lee, G. Thinakaran, H. Slunt, S. Sisodia, I. Greenwald, Assessment of normal and mutant human presenilin function in Caenorhabditis elegans, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 14940-14944; D. Levitan, I. Greenwald, Effect of Sel-12 presenilin on Lin-12 localization and function in Caenorhabditis elegans, Development, 125 (1998) 3599-3606]. The inhibitory effect of Notch1 on neurite outgrowth is markedly attenuated in neurons from PS1 knockout mice, and enhanced in neurons from transgenic mice overexpressing wild type PS1, but not mutant PS1. These data suggest that PS1 facilitates Notch1 function in mammalian neurons, and support the hypothesis that a functional interaction exists between PS1 and Notch1 in postmitotic mammalian neurons.