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Presenilins and Alzheimer's disease
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Presenilins and Alzheimer's disease Tae-Wan Kim* and Rudolph E Tanzit Mutations in the genes encoding the presenilins cause the majority of early-onset cases of Alzheimer's disease (AD). The identification of the presenilin genes has provided new opportunities for elucidating the molecular mechanisms underlying the etiology and pathogenesis of AD. Recent progress has been made in attempts to understand the normal and pathological functions of the presenilins, emphasizing the effects of presenilin familial AD mutations on the amyloid 13-protein precursor, the presenilins themselves, and apoptotic cell death.
This review will describe recent advances that have been made in three major areas of AD research: biological functions, cellular metabolism, and pathological functions of presenilins in relation to ~-amyloid deposition and neuronal death (see also [6-8]). T h e major focus of this review will be on the effects of presenilin FAD mutations on cellular metabolism and processing of APP and the presenilins, as well as the relationship of these events to apoptotic cell death.
Cellular metabolism Addresses Genetics and Aging Unit, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA *e-mail: [email protected] re-mail: [email protected] Current Opinion in Neurobiology 1997, 7:683-688
http://biomednet.com/elecref/0959438800700683 © Current Biology Ltd ISSN 0959-4388 Abbreviations A~ amyloid I~-peptide AD Alzheimer'sdisease APP amyloid13-protein precursor ER endoplasmicreticuium FAD familial AD PS1 presenilin1 PS2 presenilin2
Introduction Alzheimer's disease (AD) is the most prevalent cause of dementia in the elderly. It is characterized pathologically by the presence of intracellular neurofibrillary tangles and extracellular neuritic plaques consisting of deposits of the amyloid [3-peptide (AI3), a 39~,3 amino acid peptide proteolytically derived from amyloid [3-protein precursor (APP). A significant portion of AD is attributed to heterogeneous genetic elements characterized as either 'causative gene defects' or 'genetic risk factors'. Approximately 5% of AD can be classified as early-onset (<60years) familial Alzheimer's disease (FAD), which is inherited in an autosomal dominant fashion. Relatively rare mutations causing early-onset FAD have been identified within the gene encoding APP located on chromosome 21 (for a review, see [1]). T h e discovery of additional genes accounting for up to 50% of early-onset FAD, termed presenilin 1 (PSI) on chromosome 14 [2] and presenilin 2 (PS2) on chromosome 1 [3,4], has provided new opportunities for understanding the molecular bases of the etiology and pathogenesis of AD. To date, 45 different PSI mutations have been described and two FAD mutations have been identified in its close homologue PS2 (for reviews, see [1,5]).
of presenilins
One of the critical starting points in attempts to understand presenilin biology has been the study of the cellular metabolism and structure of the presenilins (Figure 1). In mammalian cells transfected with full-length presenilin cDNAs, PS1 is observed as a 43-45kDa polypeptide [9°°,10,11], whereas PS2 is observed as a 53-55kDa protein [12"]. In native cell lines and brains, little or no full-length PS1 and PS2 are detected, whereas their proteolytic cleavage fragments can be readily observed, indicating that the presenilins constitutively undergo endoproteolytic processing. T h e fragments produced by this pathway are saturable and generated in a highly regulated manner [9"°,13",14"°]. The amino- and carboxy-terminal fragments generated by the proteolytic processing of PS1 have been shown to form oligomeric complexes in vitro and in vivo ([15°]; G Thinakaran et al., Soc Neurosd Abstr 1997, 22:728). These findings indicate that the presenilins undergo proteolytic processing and subsequent oligomerization as a part of their maturation process. Although the physiological significance of these events is not yet clear, PSI carboxy-terminal fragments have also been shown to be phosphorylated by protein kinase C (PKC) [15°,16°]. T h e phosphorylation of these PS1 carboxy-terminal fragments may regulate their turn-over rate or interaction with other cellular factors. As negligible levels of full-length presenilins are observed in native cell lines and brains, and as overexpression of the proteins leads to only a modest increase in the amount of cleavage products, a distinct proteolytic system most likely operates to prevent the accumulation of excess full-length presenilin molecules. Recent evidence suggests that the proteasome pathway plays a part in this process by deposing excess presenilin holoproteins in the endoplasmic reticulum (ER), independent of the regulated endoproteolytic processing pathway [12"] (Figure 1). This supposition is based on the finding that PS2 degradation is blocked by proteasome inhibitors [12°°]. It remains to determined why endogenous presenilins exist primarily as cleavage fragments and whether accumulation
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Figure 1 Degradation by a proteasome Presenilin cleavage sites Normal Alternative
Presenilin
'Normal'endoproteolyticprocessing
'Alternative'cleavage by a caspase
fC
I
I
I
Oligomerization
fC
I
I
I
Alternatively cleaved presenilin carboxy-terminal fragment
CurrentOpinionin Neurobiology Proposed metabolic pathways for the presenilins: 'normal' and 'alternative' cleavage and proteasomal degradation. The 'normal' endoproteolytic pathway yields saturable amounts of amino- and carboxy-terminal fragments [9"°], and the levels of the full-length presenilins seem to be tightly regulated by degradation via the ubiquitin-proteasome pathways [12°°]. The normal cleavage site is contained within a portion of PS1 encoded by exon 9 [58]. Once generated, the two normal cleavage products can undergo oligomerization. During apoptosis, or when the presenilins are overexpressed, the presenilins can be diverted into the 'alternative' cleavage pathway in which cleavage by a caspase-3 (CPP32) family protease occurs distal to the normal cleavage site and gives rise to a larger amino-terminal fragment and a smaller, detergent-insoluble carboxy-terminal fragment [14*'].
of excessive full-length presenilins confers potentially harmful effects to cells expressing these proteins.
Biological f u n c t i o n s of p r e s e n i l i n s Valuable clues regarding the biological functions of the presenilins have been derived from studies of SEL-12, the presenilin homologue (50% identity) in Caenorhabditis elegans [17]. SEL-12 functions as a co-receptor for the nematode Notch receptor, LIN-12, and defects in the sel-12 gene in C. elegans causes constitutive activation of LIN-12, resulting in an egg-lying defect [17]. Recent studies have demonstrated that both human PS1 and PS2 can rescue the sel-12 mutant phenotype, suggesting that presenilins are functionally interchangeable with SEL-12 [18°,19°]. This raises the possibility that the presenilins play a role in Notch signaling during embryonic development and/or cellular differentiation. In support of this notion, null mice in which the PS1 gene has been inactivated (i.e. PS1 knock-out mice) die in utero or shortly after birth and exhibit defects in the axial skeleton and somite segmentation, as well as cerebral hemorrhage [20°°,21 °° ] and neuronal loss [21°°]. Similar defects in somite formation have also been demonstrated for the Notchl and Dill null mice [22,23]. Both the
presenilins [24,25] and Notchl [25] have been shown to be strongly expressed in the ventricular zone during embryonic development, and the expression of both Notch 1 and Dill are markedly decreased in the paraxial mesoderm of PS1 knock-out mice [20°°]. This suggests the possibility of a functional association between presenilins and other molecules involved in Notch signaling. How these functions contribute to the pathological properties of mutant presenilins in AD remains to be determined. To elucidate the normal biological role of the presenilins, efforts have also been made to identify molecules that interact with the presenilins. In a recent report [26"], PSI was shown to interact with two different members of the catenin family of proteins, which are mammalian homologues of the Drosophila Armadillo family that are predicted to participate in the Wingless signaling pathway. In the brain, PSI interacts with a newly identified catenin, termed 5-catenin; in non-neuronal cells, !3-catenin is the predominant interactor for PS1 {26°]. It is interesting to note that the Notch and Wingless signaling pathways have been shown to be mutually inhibitory and are functionally associated via convergence with the protein called Dishevelled [27]. Interestingly,
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the human homologue of Dishevelled has been mapped to a region of chromosome 3 that has recently been linked to late-onset AD (RE Tanzi, unpublished data). How the Notch, Wingless, and related signaling pathways control presenilin function is clearly an important topic for future investigations. It is highly possible that differential molecular interactions of the presenilins modulated in a tissue or developmental stage specific manner may control the specific roles of the normal and mutant forms of the presenilins during development and in adulthood.
hydrophilic loop, and the carboxyl terminus of PS1 are all oriented toward the cytoplasm [39°], interspersed by 6-8 transmembrane domains [39°,40]. As it has been shown that the cytoplasmic domain of APP is not required for the putative interaction between APP and the presenilins [38], the luminal domains of the presenilins is predicted to mediate their interaction with APP. In this regard, only the first luminal loop between transmembrane domains 1 and 2 is of adequate length (> 30 amino acids) to allow for such an interaction.
Presenilins and APP metabolism
Recent studies have demonstrated that A1342 is generated primarily in the ER [41°°-43°°], whereas A1340 has been shown to be generated through distinct secretory and endocytic pathways (for a review, see [44]), As PS1 and PS2 have previously been localized predominantly to the ER [45",46-48], it is conceivable that interaction between APP and the presenilins within the ER directly influences the generation of A1342 in this cellular compartment.
13-amyloid is present in the AD brain in the form of senile plaques and cerebral blood vessel deposits. A wealth of data suggests a central and essential role for the deposition of 13-amyloid along with the generation and aggregation of its major component, the 4 k D a peptide A!8, in the etiology and pathogenesis of AD (for a review, see [1]). T h e deposition of A[3 and, particularly, a 'long' form of the peptide, AI342, appears to be the common pathogenic event that ties together the pathogenic mechanisms of the various genetic defects leading to AD neuropathogenesis. Six different mutations have been identified in the APP gene, all lying near or within the AI3 domain [1,5]. T h e FAD mutations in APP all lead to increased production of AI3 or, specifically, to a relative increase in AI342 [1,5]. In plasma and fibroblasts from patients and at-risk carriers for the presenilin mutations, increases in the ratio of AIM2:AI340 have been observed [28"]. Similar increases in the AI342:AI340 ratio have also been observed in transfected cell lines and transgenic mice expressing FAD mutant forms of PSI or PS2 [13°,29°,30",31,32]. Neuropathological studies have also demonstrated the predominant deposition of AIM2/43 in the brains of FAD patients carrying mutations in PS1 or PS2 [33-36]. These data suggest that a common molecular consequence of all known presenilin FAD mutations is the increased production and deposition of AI342, the longer, more amyloidogenic form of AI3. Presenilin FAD mutations may directly influence the processing of APP by y-secretase(s), unidentified enzyme(s) that cleave at the carboxyl terminus of A]3 and determine the carboxy-terminal end of A~. However, it is still unclear whether the relative increase in the ratio of AI342 :AI340 is sufficient to cause disease or is closely correlated marker for the progression of the disease. A direct physical association of APP with PS1 or PS2 has been suggested by co-immunoprecipitation [37,38] and affinity cross-linking (RD Moir, RE Tanzi, W Wasco, unpublished data) experiments. T h e formation of the complex between APP and the presenilins appears to occur principally in the ER among the presenilins and immature forms of APP [37,38]. These studies support the idea that FAD mutant presenilins interact directly with APP and steer APP metabolism toward increased A1~42 production. Topological studies of the presenilins have shown that the amino terminus, the large
Presenilins and apoptosis Apoptotic cell death is a pathological feature in the AD brain [49], although the exact contribution and importance of apoptosis in the pathogcnesis of AD remains unclear (for reviews, see [50-52]). Expression of a partial cDNA encoding the carboxy-terminal portion of mouse PS2 has been reported to rescue a T cell hybridoma undergoing Fas-mediated apoptosis [53]. In more recent studies, overexpression of PS2 in transiently transfected, nerve growth factor (NGF)-differentiated PC12 cells enhanced apoptosis induced by trophic factor withdrawal, whereas transfection with an antisense PS2 construct rescued cells from apoptosis [54]. In this study and others, the Asnl41--+Ile FAD mutation was reported to confer enhanced basal activity for inducing apoptosis [54,55]. In addition, the PSI Leu286--+Val FAD mutation has been shown to sensitize neuronal cells to apoptosis [56]. These observations have led to the suggestion that mutant presenilins possess the 'constitutive' ability to render neurons more vulnerable to a variety of neuronal injuries, such as oxidative stress, which increase in frequency with age.
Alternative endoproteolysis of presenilins Valuable new clues to the etiology and neuropathogenesis of AD have recently been derived from the observation that the presenilins can also undergo 'alternative' endoproteolytic cleavage at a site distal to the normal cleavage site [14 °°] (Figure 1). Alternative endoproteolysis of presenilins is mediated by a member of the apoptotic cysteine protease family known as the caspases, which are activated during apoptosis and play a critical role in the apoptotic cell death process. Alternative cleavage of the presenilins can be inhibited either by treatment with caspase inhibitors or by substitution of asparagine residue(s) at the consensus caspase cleavage sites (Asp326 and Asp329 in PS2) located distal to the 'normal' cleavage sites [14°°]. These findings suggest that presenilins probably
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serve as cell death substrates and raise the possibility that the apoptosis-associated alternative presenilin fragments harbor pro-apoptotic properties. A potential role for caspase-mediated, alternative cleavage of the presenilins in the pathogenesis of AD has also been suggested. This is based on the observation that the ratio of the alternative to normal carboxy-terminal fragments is significantly elevated (approximately threefold) in stably transfected inducible H4 human neuroglioma cells expressing PS2 with the Asnl41--+Ile FAD mutation as compared to those expressing wild-type PS2 [14°°]. Interestingly, huntingtin, a protein that is defective in another neurodegenerative disorder, Huntington's disease, has also been shown to be cleaved by a caspase [57]. The polyglutamine tract of huntingtin, which is expanded in Huntington's disease patients, has been reported to modulate the extent of caspase-mediated cleavage [57]. It is thus conceivable that a conformational change in PS2 caused by the Asnl41--+Ile FAD mutation contributes to the apparent enhanced susceptibility of PS2 to alternative cleavage mediated by a caspasc-3 family protease [14"]. It is interesting to note that one of the PS1 FAD mutations characterized by the deletion of exon 9 in PSI [58], which removes the 'normal' cleavage site [9•',11], is still susceptible to alternative endoproteolysis because the more distal alternative cleavage site is preserved (Figure 1). These findings raise the intriguing possibility that, similar to the case of altered proteolytic processing of APP associated with FAD in that protein, altered endoproteolysis of presenilins could also represent an etiopathogenic event in FAD. Alternative proteolytic processing of the presenilins has also been described recently for primary neurons undergoing differentiation [59,60]. However, it is currently unclear whether caspase activity is also involved in this pathway. Alternative presenilin cleavage fragments have also been found specifically in aged brains [59]. Thus, it is possible that a caspase becomes activated and regulates presenilin processing in the late stages of neuronal differentiation and in aged brains. On the other hand, alternative presenilin cleavage fragments generated at late stages in the neuronal differentiation of primary cultures could simply originate from a subset of neurons undergoing apoptosis after 12-14 days in culture.
alternative presenilin fragments, respectively. Exactly how changes in cellular metabolism of the presenilins and APP, induction of apoptosis, and the generation of A1342 inter-play to contribute to AD neuropathogenesis will be an important topic for future studies. Novel therapeutic strategies aimed at the above targets may carry the greatest potential for developing effective treatments for AD in the future.
Acknowledgements Funding for these studies is provided by the National Institute of Aging (NIA), National Institute of Neurological Disorders and Stroke (NINDS), and the Metropolitan Life Foundation.
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Presenilins and Alzheimer's disease Kim and Tanzi
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26. •
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Guo Q, Furukawa K, Sopher BL, Pham DG, Xie J, Robinson N, Martin GM, Mattson MP: Alzheimer's PS-1 mutation perturbs calcium homeostasis and sensitizes PC12 cells to death induced by amyloid 13-peptide. Neuroreport 1996, 8:379-383.
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Goldberg YP, Nicholson DW, Rasper DM, Kalchman MA, Koide HB, Graham RK, Bromm M, Kazerni-Esfarjani P, Thornberry NA, Vailancourt JP, Hyden MR: Cleavage of huntingtin by apopain, proapoptotic cysteine protease, is modulated by the polyglutamine tract. Nat Genet 1996, 13:442-449.
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Perez-TurJ, Froelich S, Prihar G, Crook R, Baker M, Duff K, Wragg M, Busfield F, Lendon C, Clark RF et aL: A mutation in Alzheimer's disease destroying a splice acceptor site in the presenilin-1 gene. Neuroreport 1996, 7:297-301.
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HartmannH, Busciglio J, Baumann K-H, Staufenbiel M, Yankner BA: Developmental regulation of presenilin-1 processing in the brain suggests a role in neuronal differentiation. J Biol Chem 199?, 272:14505-14508.
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Capell A, Saffrich R, Olivo J-C, Meyn L, Walter J, GrLinberg J, Dotti C, Haass C: Cellular expression and proteolytic processing of presenilin proteins is developmentally regulated during neuronal differentiation. J Neurochem 1997, in press.
39. •
LehmannS, Chiesa R, Harris DA: Evidence for a sixtransmembrane domain structure of presenilin 1. J Biol Chem 199?, 272:12047-12051.
41. •.
Wild-Bode C, Yamazaki T, Capell A, Leimer U, Steiner H, Ihara Y, Haass C: Intracellular generation and accumulation of amyloid ~-peptide terminating at amino acid 42. J Bio/Chem 1997, 272:16085-16068. This report demonstrates that A~42 is generated primarily in the endoplasmic reticulum. The generation of intracellular AI3 could be an initial step in AD neuropathogenesis. 42. ••
Cook DG, Forman MS, Sung JC, Leight S, Kolson DL, Iwatsubo T, Lee VM-Y, Doms RW: Alzheimer's A~(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat Med 1997, 3:1021-1023. This paper definitely shows that AI342, primary component of senile plaques in AD, can be generated in the endoplasmic reticulum, suggesting that this may be the initial site of AD pathogenesis. See also annotation [43"]. 43. •.
HartmannT, Bieger SC, BrGhl B, Tienari PJ, Ida N, AIIsop D, Roberts GW, Masters CL, Dotti CG, Unsicker K, Beyreuther K: Distinct sites of intracellular production of Alzheimer's disease A~10/42 amyloid peptides. Nat Med 199"7, 3:1016-1020. This is the first report to show that while A~42 is mainly localized in the endoplasmic reticulum, All40 is present in the Golgi and cell surface. Along with other reports [41 "°,42"°], these data point to the endoplasmic reticulum as an initial cellular compartment of AD neuropathogenesis. 44.
Selkoe DJ: Amyloid 13-protein and the genetics of Alzheimer's disease. J Bio/Chem 1996, 271:18295-18298.
45. •
Kovacs DM, Fausett HJ, Page KJ, Kim T-W, Moir RD, Merriam DE, Hollister RD, Hallmark OG, Mancini R, Felsenstein KM et al.: Alzheimer associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells. Nat Med 1996, 2:224-229. The first report to demonstrate the predominant neuronal expression of the presenilins in brain and their subcellular localization in the endoplasmic reticulum and Golgi of mammalian cells. 46.
Walter J, Capell A, GrLinberg J, Pesold B, Schindzielorz A, Prior R, Podlisny MB, Fraser P, St George Hyslop P, Selkoe DJ, Haass C: The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum. Mol Med 199"7, 2:673-691.
4?.
Cook DG, Sung JC, Golde TE, Felsenstein KM, Wojczk BS, Tanzi RE, Trojanowski JQ, Lee VM-Y, Doms RW: Expression and analysis of presenilin 1 in a human neuronal system:
Presenilins and Alzheimer's disease Tae-Wan Kim* and Rudolph E Tanzit Mutations in the genes encoding the presenilins cause the majority of early-onset cases of Alzheimer's disease (AD). The identification of the presenilin genes has provided new opportunities for elucidating the molecular mechanisms underlying the etiology and pathogenesis of AD. Recent progress has been made in attempts to understand the normal and pathological functions of the presenilins, emphasizing the effects of presenilin familial AD mutations on the amyloid 13-protein precursor, the presenilins themselves, and apoptotic cell death.
This review will describe recent advances that have been made in three major areas of AD research: biological functions, cellular metabolism, and pathological functions of presenilins in relation to ~-amyloid deposition and neuronal death (see also [6-8]). T h e major focus of this review will be on the effects of presenilin FAD mutations on cellular metabolism and processing of APP and the presenilins, as well as the relationship of these events to apoptotic cell death.
Cellular metabolism Addresses Genetics and Aging Unit, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA *e-mail: [email protected] re-mail: [email protected] Current Opinion in Neurobiology 1997, 7:683-688
http://biomednet.com/elecref/0959438800700683 © Current Biology Ltd ISSN 0959-4388 Abbreviations A~ amyloid I~-peptide AD Alzheimer'sdisease APP amyloid13-protein precursor ER endoplasmicreticuium FAD familial AD PS1 presenilin1 PS2 presenilin2
Introduction Alzheimer's disease (AD) is the most prevalent cause of dementia in the elderly. It is characterized pathologically by the presence of intracellular neurofibrillary tangles and extracellular neuritic plaques consisting of deposits of the amyloid [3-peptide (AI3), a 39~,3 amino acid peptide proteolytically derived from amyloid [3-protein precursor (APP). A significant portion of AD is attributed to heterogeneous genetic elements characterized as either 'causative gene defects' or 'genetic risk factors'. Approximately 5% of AD can be classified as early-onset (<60years) familial Alzheimer's disease (FAD), which is inherited in an autosomal dominant fashion. Relatively rare mutations causing early-onset FAD have been identified within the gene encoding APP located on chromosome 21 (for a review, see [1]). T h e discovery of additional genes accounting for up to 50% of early-onset FAD, termed presenilin 1 (PSI) on chromosome 14 [2] and presenilin 2 (PS2) on chromosome 1 [3,4], has provided new opportunities for understanding the molecular bases of the etiology and pathogenesis of AD. To date, 45 different PSI mutations have been described and two FAD mutations have been identified in its close homologue PS2 (for reviews, see [1,5]).
of presenilins
One of the critical starting points in attempts to understand presenilin biology has been the study of the cellular metabolism and structure of the presenilins (Figure 1). In mammalian cells transfected with full-length presenilin cDNAs, PS1 is observed as a 43-45kDa polypeptide [9°°,10,11], whereas PS2 is observed as a 53-55kDa protein [12"]. In native cell lines and brains, little or no full-length PS1 and PS2 are detected, whereas their proteolytic cleavage fragments can be readily observed, indicating that the presenilins constitutively undergo endoproteolytic processing. T h e fragments produced by this pathway are saturable and generated in a highly regulated manner [9"°,13",14"°]. The amino- and carboxy-terminal fragments generated by the proteolytic processing of PS1 have been shown to form oligomeric complexes in vitro and in vivo ([15°]; G Thinakaran et al., Soc Neurosd Abstr 1997, 22:728). These findings indicate that the presenilins undergo proteolytic processing and subsequent oligomerization as a part of their maturation process. Although the physiological significance of these events is not yet clear, PSI carboxy-terminal fragments have also been shown to be phosphorylated by protein kinase C (PKC) [15°,16°]. T h e phosphorylation of these PS1 carboxy-terminal fragments may regulate their turn-over rate or interaction with other cellular factors. As negligible levels of full-length presenilins are observed in native cell lines and brains, and as overexpression of the proteins leads to only a modest increase in the amount of cleavage products, a distinct proteolytic system most likely operates to prevent the accumulation of excess full-length presenilin molecules. Recent evidence suggests that the proteasome pathway plays a part in this process by deposing excess presenilin holoproteins in the endoplasmic reticulum (ER), independent of the regulated endoproteolytic processing pathway [12"] (Figure 1). This supposition is based on the finding that PS2 degradation is blocked by proteasome inhibitors [12°°]. It remains to determined why endogenous presenilins exist primarily as cleavage fragments and whether accumulation
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Figure 1 Degradation by a proteasome Presenilin cleavage sites Normal Alternative
Presenilin
'Normal'endoproteolyticprocessing
'Alternative'cleavage by a caspase
fC
I
I
I
Oligomerization
fC
I
I
I
Alternatively cleaved presenilin carboxy-terminal fragment
CurrentOpinionin Neurobiology Proposed metabolic pathways for the presenilins: 'normal' and 'alternative' cleavage and proteasomal degradation. The 'normal' endoproteolytic pathway yields saturable amounts of amino- and carboxy-terminal fragments [9"°], and the levels of the full-length presenilins seem to be tightly regulated by degradation via the ubiquitin-proteasome pathways [12°°]. The normal cleavage site is contained within a portion of PS1 encoded by exon 9 [58]. Once generated, the two normal cleavage products can undergo oligomerization. During apoptosis, or when the presenilins are overexpressed, the presenilins can be diverted into the 'alternative' cleavage pathway in which cleavage by a caspase-3 (CPP32) family protease occurs distal to the normal cleavage site and gives rise to a larger amino-terminal fragment and a smaller, detergent-insoluble carboxy-terminal fragment [14*'].
of excessive full-length presenilins confers potentially harmful effects to cells expressing these proteins.
Biological f u n c t i o n s of p r e s e n i l i n s Valuable clues regarding the biological functions of the presenilins have been derived from studies of SEL-12, the presenilin homologue (50% identity) in Caenorhabditis elegans [17]. SEL-12 functions as a co-receptor for the nematode Notch receptor, LIN-12, and defects in the sel-12 gene in C. elegans causes constitutive activation of LIN-12, resulting in an egg-lying defect [17]. Recent studies have demonstrated that both human PS1 and PS2 can rescue the sel-12 mutant phenotype, suggesting that presenilins are functionally interchangeable with SEL-12 [18°,19°]. This raises the possibility that the presenilins play a role in Notch signaling during embryonic development and/or cellular differentiation. In support of this notion, null mice in which the PS1 gene has been inactivated (i.e. PS1 knock-out mice) die in utero or shortly after birth and exhibit defects in the axial skeleton and somite segmentation, as well as cerebral hemorrhage [20°°,21 °° ] and neuronal loss [21°°]. Similar defects in somite formation have also been demonstrated for the Notchl and Dill null mice [22,23]. Both the
presenilins [24,25] and Notchl [25] have been shown to be strongly expressed in the ventricular zone during embryonic development, and the expression of both Notch 1 and Dill are markedly decreased in the paraxial mesoderm of PS1 knock-out mice [20°°]. This suggests the possibility of a functional association between presenilins and other molecules involved in Notch signaling. How these functions contribute to the pathological properties of mutant presenilins in AD remains to be determined. To elucidate the normal biological role of the presenilins, efforts have also been made to identify molecules that interact with the presenilins. In a recent report [26"], PSI was shown to interact with two different members of the catenin family of proteins, which are mammalian homologues of the Drosophila Armadillo family that are predicted to participate in the Wingless signaling pathway. In the brain, PSI interacts with a newly identified catenin, termed 5-catenin; in non-neuronal cells, !3-catenin is the predominant interactor for PS1 {26°]. It is interesting to note that the Notch and Wingless signaling pathways have been shown to be mutually inhibitory and are functionally associated via convergence with the protein called Dishevelled [27]. Interestingly,
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the human homologue of Dishevelled has been mapped to a region of chromosome 3 that has recently been linked to late-onset AD (RE Tanzi, unpublished data). How the Notch, Wingless, and related signaling pathways control presenilin function is clearly an important topic for future investigations. It is highly possible that differential molecular interactions of the presenilins modulated in a tissue or developmental stage specific manner may control the specific roles of the normal and mutant forms of the presenilins during development and in adulthood.
hydrophilic loop, and the carboxyl terminus of PS1 are all oriented toward the cytoplasm [39°], interspersed by 6-8 transmembrane domains [39°,40]. As it has been shown that the cytoplasmic domain of APP is not required for the putative interaction between APP and the presenilins [38], the luminal domains of the presenilins is predicted to mediate their interaction with APP. In this regard, only the first luminal loop between transmembrane domains 1 and 2 is of adequate length (> 30 amino acids) to allow for such an interaction.
Presenilins and APP metabolism
Recent studies have demonstrated that A1342 is generated primarily in the ER [41°°-43°°], whereas A1340 has been shown to be generated through distinct secretory and endocytic pathways (for a review, see [44]), As PS1 and PS2 have previously been localized predominantly to the ER [45",46-48], it is conceivable that interaction between APP and the presenilins within the ER directly influences the generation of A1342 in this cellular compartment.
13-amyloid is present in the AD brain in the form of senile plaques and cerebral blood vessel deposits. A wealth of data suggests a central and essential role for the deposition of 13-amyloid along with the generation and aggregation of its major component, the 4 k D a peptide A!8, in the etiology and pathogenesis of AD (for a review, see [1]). T h e deposition of A[3 and, particularly, a 'long' form of the peptide, AI342, appears to be the common pathogenic event that ties together the pathogenic mechanisms of the various genetic defects leading to AD neuropathogenesis. Six different mutations have been identified in the APP gene, all lying near or within the AI3 domain [1,5]. T h e FAD mutations in APP all lead to increased production of AI3 or, specifically, to a relative increase in AI342 [1,5]. In plasma and fibroblasts from patients and at-risk carriers for the presenilin mutations, increases in the ratio of AIM2:AI340 have been observed [28"]. Similar increases in the AI342:AI340 ratio have also been observed in transfected cell lines and transgenic mice expressing FAD mutant forms of PSI or PS2 [13°,29°,30",31,32]. Neuropathological studies have also demonstrated the predominant deposition of AIM2/43 in the brains of FAD patients carrying mutations in PS1 or PS2 [33-36]. These data suggest that a common molecular consequence of all known presenilin FAD mutations is the increased production and deposition of AI342, the longer, more amyloidogenic form of AI3. Presenilin FAD mutations may directly influence the processing of APP by y-secretase(s), unidentified enzyme(s) that cleave at the carboxyl terminus of A]3 and determine the carboxy-terminal end of A~. However, it is still unclear whether the relative increase in the ratio of AI342 :AI340 is sufficient to cause disease or is closely correlated marker for the progression of the disease. A direct physical association of APP with PS1 or PS2 has been suggested by co-immunoprecipitation [37,38] and affinity cross-linking (RD Moir, RE Tanzi, W Wasco, unpublished data) experiments. T h e formation of the complex between APP and the presenilins appears to occur principally in the ER among the presenilins and immature forms of APP [37,38]. These studies support the idea that FAD mutant presenilins interact directly with APP and steer APP metabolism toward increased A1~42 production. Topological studies of the presenilins have shown that the amino terminus, the large
Presenilins and apoptosis Apoptotic cell death is a pathological feature in the AD brain [49], although the exact contribution and importance of apoptosis in the pathogcnesis of AD remains unclear (for reviews, see [50-52]). Expression of a partial cDNA encoding the carboxy-terminal portion of mouse PS2 has been reported to rescue a T cell hybridoma undergoing Fas-mediated apoptosis [53]. In more recent studies, overexpression of PS2 in transiently transfected, nerve growth factor (NGF)-differentiated PC12 cells enhanced apoptosis induced by trophic factor withdrawal, whereas transfection with an antisense PS2 construct rescued cells from apoptosis [54]. In this study and others, the Asnl41--+Ile FAD mutation was reported to confer enhanced basal activity for inducing apoptosis [54,55]. In addition, the PSI Leu286--+Val FAD mutation has been shown to sensitize neuronal cells to apoptosis [56]. These observations have led to the suggestion that mutant presenilins possess the 'constitutive' ability to render neurons more vulnerable to a variety of neuronal injuries, such as oxidative stress, which increase in frequency with age.
Alternative endoproteolysis of presenilins Valuable new clues to the etiology and neuropathogenesis of AD have recently been derived from the observation that the presenilins can also undergo 'alternative' endoproteolytic cleavage at a site distal to the normal cleavage site [14 °°] (Figure 1). Alternative endoproteolysis of presenilins is mediated by a member of the apoptotic cysteine protease family known as the caspases, which are activated during apoptosis and play a critical role in the apoptotic cell death process. Alternative cleavage of the presenilins can be inhibited either by treatment with caspase inhibitors or by substitution of asparagine residue(s) at the consensus caspase cleavage sites (Asp326 and Asp329 in PS2) located distal to the 'normal' cleavage sites [14°°]. These findings suggest that presenilins probably
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serve as cell death substrates and raise the possibility that the apoptosis-associated alternative presenilin fragments harbor pro-apoptotic properties. A potential role for caspase-mediated, alternative cleavage of the presenilins in the pathogenesis of AD has also been suggested. This is based on the observation that the ratio of the alternative to normal carboxy-terminal fragments is significantly elevated (approximately threefold) in stably transfected inducible H4 human neuroglioma cells expressing PS2 with the Asnl41--+Ile FAD mutation as compared to those expressing wild-type PS2 [14°°]. Interestingly, huntingtin, a protein that is defective in another neurodegenerative disorder, Huntington's disease, has also been shown to be cleaved by a caspase [57]. The polyglutamine tract of huntingtin, which is expanded in Huntington's disease patients, has been reported to modulate the extent of caspase-mediated cleavage [57]. It is thus conceivable that a conformational change in PS2 caused by the Asnl41--+Ile FAD mutation contributes to the apparent enhanced susceptibility of PS2 to alternative cleavage mediated by a caspasc-3 family protease [14"]. It is interesting to note that one of the PS1 FAD mutations characterized by the deletion of exon 9 in PSI [58], which removes the 'normal' cleavage site [9•',11], is still susceptible to alternative endoproteolysis because the more distal alternative cleavage site is preserved (Figure 1). These findings raise the intriguing possibility that, similar to the case of altered proteolytic processing of APP associated with FAD in that protein, altered endoproteolysis of presenilins could also represent an etiopathogenic event in FAD. Alternative proteolytic processing of the presenilins has also been described recently for primary neurons undergoing differentiation [59,60]. However, it is currently unclear whether caspase activity is also involved in this pathway. Alternative presenilin cleavage fragments have also been found specifically in aged brains [59]. Thus, it is possible that a caspase becomes activated and regulates presenilin processing in the late stages of neuronal differentiation and in aged brains. On the other hand, alternative presenilin cleavage fragments generated at late stages in the neuronal differentiation of primary cultures could simply originate from a subset of neurons undergoing apoptosis after 12-14 days in culture.
alternative presenilin fragments, respectively. Exactly how changes in cellular metabolism of the presenilins and APP, induction of apoptosis, and the generation of A1342 inter-play to contribute to AD neuropathogenesis will be an important topic for future studies. Novel therapeutic strategies aimed at the above targets may carry the greatest potential for developing effective treatments for AD in the future.
Acknowledgements Funding for these studies is provided by the National Institute of Aging (NIA), National Institute of Neurological Disorders and Stroke (NINDS), and the Metropolitan Life Foundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • = of outstanding interest 1.
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ThinakaranG, Borchelt DR, Lee MK, Slunt HH, Spitzer L, Kim G, Ratovitsky T, Davenport F, Nordstedt C, Seeger Met aL: Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo. Neuron 1996, 17:181-190. The first paper to describe the endoproteolysis and regulated accumulation of the proteolytic fragments of PS1. The authors reported the important findings that presenilin cleavage fragments may be the functional component of these proteins. 10.
Mercken M, Takahashi H, Honda T, Sate K, Murayama M, Nakazato Y, Noguchi K, Imahori K, Takashima A: Characterization of human presenilin 1 using N-terminal specific monoclonal antibodies: evidence that Alzheimer mutations affect proteolytic processing. FEBS Lett 1996, 389:297-303.
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Conclusions While the precise mechanism by which known presenilin gene defects lead to the onset of AD remains unclear, new data regarding the cellular metabolism and potential biological functions of the presenilins have begun to shed new light on these mysterious proteins. Moreover, specific molecular phenotypic features of the FAD mutant forms of the presenilins include aberrant proteolytic processing of APP and the presenilins themselves, leading to the increased production and deposition of amyloidogenic AIM2 and the enhanced generation of apoptosis-associated,
12. •.
Kim T-W, Pettingell WH, Hallmark OG, Moir RD, Wasco W, Tanzi RE: Endoproteolytic processing and proteasomal degradation of presenilin 2 in transfected cells. J Bio/Chem 1997, 272:11006-11010. The first paper to report that PS2 undergoes endoproteolytic processing and is degraded via the ubiquitin-proteasome pathway. The authors raise
Presenilins and Alzheimer's disease Kim and Tanzi
the possibility that proteasomal degradation may be used to regulate the levels of full-length presenilins, thereby permitting regulated endoproteolytic cleavage. 13. •
Borchelt DR, Thinakaran G, Eckman CB, Lee MK, Davenport F, Ratovitsky T, Prada C-M, Kim G, Seekins S, Yager D et aL: Familial Alzheimer's disease-linked presenilin 1 variants elevate A~1-42/1-40 ratio in vitro and in vivo. Neuron 1996, 17:1005-1013. One of three simultaneous reports (see [29",30"]) demonstrating increases in the A~42 :A~40 ratio in transfected cells and transgenio mice expressing FAD mutant forms of the presenilins. The results in this paper are extremely similar and confirmatory findings to those described in [29",30"]. Kim T-W, Pettingell WH, Jung YK, Kovacs DM, Tanzi RE: Alternative cleavage of Alzheimer-associated presenilins during apoptosis by a caspase-3 family protease. Science 1997, 277:373-376. The first report showing that the presenilins undergo alternative endoproteolysis, which is mediated by an apoptosis-associated caspase-3 family protease and which is enhanced by the Asn141--~lle FAD mutation. These findings suggest that the presenilins may be cell death substrates and that FAD mutations may lead to the enhanced activation of caspase-3 family proteases.
25.
Seeger M, Nordstedt C, Petanoeska S, Kovacs DM, Gouras GK, Hahne S, Fraser P, Levesque L, Czernik AJ, St George-Hyslop P et aL: Evidence for phosphorylation and oligomeric assembly of presenilin 1. Proc Nat/Acad Sci USA 1997, 94:5090-5094. This report demonstrates the oligomeric association between amino- and carboxy-terminal PS1 endoproteolytic fragments and shows that the carboxyl terminus of PS1 can be phosphorylated (see also [16"]). 16. •
Walter J, Grfinberg J, Capell A, Pesold B, Schindzielorz A, Citron M, Mendla K, St George-Hyslop P, Multhaup G, Selkoe DJ, Haass C: Proteolytic processing of the Aizheimer diseaseassociated presenilin-1 gene generates an in vivo substrate for protein kinase C. Proc Nat/Acad Sci USA 1997, 94:5349-5354. This report shows that the carboxy-terminal cleavage fragments of PS1 can be phosphorylated. 17.
Levitan D, Greenwald h Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans $182 Alzheimer's disease gene. Nature 1995, 377:351-354.
18. •
Levitan D, Doyle TG, Brousseau D, Lee MK, Thinakaran G, Slunt HH, Sisodia SS, Greenwald I: Assessment of normal and mutant human presenilin function in Caenorhabditis elegans. Proc Nat/Acad Sci USA 1996, 93:14940-14944. One of two reports (see [19°]) showing that human presenilins can rescue the sel-12 mutant phenotype. 19. •
Baumeister R, Leimer U, Zweckbronner J, Jakubek C, Gruenberg J, Haass C: The Se1-12 mutant phenotype of C. elegans is rescued independent of proteolytic processing by Wt but not mutant presenilin. Genes Function 1997, 1:149-159. One of two reports (see also [18"]) showing that human preseni]ins can rescue the se/-12 mutant phenotype. 20. •,
Wong P, Zheng H, Chen H, Becher MW, Sidnathsinghji DJS, TrumbauerME, Proce DL, Van der Ploeg LHT, Sisodia SS: Presenilin 1 is required for Notch1 and DIll expression in the paraxial mesoderm. Nature 1997, 387:288-292. One of two reports (see [21 °°]) that were published simultaneously describing the phenotypes of the PS1 null mice. These data further suggest a role for PS1 in the Notch developmental pathway. PS1 expression may be required during development for proper temporal expression of Notch and Delta. 21. ••
Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S: Skeletal and CNS defects in presenilin-l-deficient mice. Cell 1997, 89:629-539. One of two reports (see [20"]) that were published simultaneously describing the phenotypes of the PSI null mice. In this paper, additional knock-out phenotype involving neuronal loss was observed. 22.
Conlon RA, Reaume AG, Rossant J: Notch1 is required for the coordinate segmentation of somites. Development 1995, 121:1533-1545.
23.
Hrabe de Angelis M, Mcylntyre J II, Gossler A: Maintenance of somite borders in mice requires the Delta homologue DIll. Nature 199?, 386:717-721.
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Lee MK, Slunt HH, Martin I.J, Thinakaran G, Kim G, Gandy SE, Seeger M, Koo E, Price DL, Sisodia SS: Expression of presenilin 1 and 2 (PS1 and PS2) in human and mouse tissues. J Neurosci 1996, 16:7513-7525.
Berezovska O, Xia M, Page K, Wasco W, Tanzi R, Hyman B: Developmental regulation of presenilin mRNA expression parallels notch expression. J Neuropath Exp Neuro/1997, 56:4044.
26. •
Zhou J, Liyanage U, Medina M, Ho C, Simmons AD, Lovett M, Kosik KS: Presenilin 1 interaction in the brain with a novel member of the Armadillo family. Neuroreport 1997, 8:14891494. This report shows that in the brain, PS1 interacts with &catenin, a putative novel mammalian homologue of the Drosophila Armadillo family, suggesting a role for PS1 in the Wingless signaling pathway. 2?.
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28. •.
Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N, Bird TD, Hardy J, Hutton M, Kukull W e t al.: AI342(43) is increased in vivo by the PS1/2 and APP mutations linked to familial Alzheimer's disease. Nat Med 1996, 2:864-870. The first report to demonstrate increased A~42 in plasma and fibroblasts derived from FAD patients carrying PS1, PS2, or APP mutations. This seminar paper was the first to demonstrate a common phenotype for FAD mutations in APP and presenilins, causing a relative increase in the highly amyloidogenic peptide A~42. 29. •
Citron M, Westaway D, Xia W, Carlson G, Diehl T, Levesque G, Johnson-Wood K, Lee M, Seubert P, Davis A, Kholodenko D eta/.: Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. Nat Med 1996, 3:67-72. One of three simultaneous reports (see [13°,30°]) demonstrating increases in the A~42 : A~40 ratio in transfected cells and transgenic mice expressing FAD mutant forms of the presenilins. The results in this paper are extremely similar and confirmatory findings to those described in [13",30"]. 30. •
Duff K, Eckman C, Zehr C, Yu X, Prada C-M, Perez-Tur J, Hutton M, Buee L, Harigaya Y, Yager D et aL: Increased amyloid1342(43) in brains of mice expressing mutant presenilin 1. Nature 1996, 383:710-713. One of three simultaneous reports (see [13",29"]) demonstrating increases in the A~42 : A~40 ratio in transfected cells and transgenic mice expressing FAD mutant forms of the presenilins. The results in this paper are extremely similar and confirmatory findings to those described in [13",29"]. Uniquely, in this paper, increase of endogenous mouse A~42/A~40 is observed due to the expression of FAD mutant PS1 transgene. 31.
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