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Isoaspartate Formation and Neurodegeneration in Alzheimer's Disease
Archives of Biochemistry and Biophysics Vol. 381, No. 2, September 15, pp. 225–234, 2000 doi:10.1006/abbi.2000.1955, available online at http://www.idealibrary.com on
Isoaspartate Formation and Neurodegeneration in Alzheimer’s Disease Takahiko Shimizu,* Atsushi Watanabe,† Midori Ogawara,* Hiroshi Mori,‡ and Takuji Shirasawa* ,1 *Department of Molecular Genetics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan; †Division of Biomolecular Characterization, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan; and ‡Department of Neuroscience, School of Medicine, Osaka Municipal University, Abeno-ku, Osaka 545-8585, Japan
Received April 27, 2000, and in revised form May 26, 2000
We reviewed here that protein isomerization is enhanced in amyloid- peptides (A) and paired helical filaments (PHFs) purified from Alzheimer’s disease (AD) brains. Biochemical analyses revealed that A purified from senile plaques and vascular amyloid are isomerized at Asp-1 and Asp-7. A specific antibody recognizing isoAsp-23 of A further suggested the isomerization of A at Asp-23 in vascular amyloid as well as in the core of senile plaques. Biochemical analyses of purified PHFs also revealed that heterogeneous molecular weight tau contains L-isoaspartate at Asp-193, Asn-381, and Asp-387, indicating a modification, other than phosphorylation, that differentiates between normal tau and PHF tau. Since protein isomerization as L-isoaspartate causes structural changes and functional inactivation, or enhances the aggregation process, this modification is proposed as one of the progression factors in AD. Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of isomerized proteins containing L-isoaspartate. We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in neurofibrillary tangles (NFTs) in AD. Taken together with the enhanced protein isomerization in AD brains, it is implicated that the upregulated PIMT may associate with increased protein isomerization in AD. We also reviewed studies on PIMT-deficient mice that confirmed that PIMT plays a physiological role in the repair of isomerized proteins containing L-isoaspartate. The knockout study also suggested that the brain of PIMT-deficient mice manifested neurodegenerative changes concomitant with accumulation 1 To whom correspondence should be addressed. Fax: ⫹813-35794776. E-mail: [email protected].
0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
of L-isoaspartate. We discuss the pathological implications of protein isomerization in the neurodegeneration found in model mice and AD. © 2000 Academic Press
Key Words: protein isomerization; Alzheimer’s disease; A; PHFs; tau; L-isoaspartate; PIMT.
In Alzheimer’s disease (AD), 2 characteristic changes occur in hippocampus and cerebral cortex, such as the accumulation of amyloid in senile plaques, abnormal neurite outgrowth, and the accumulation of neurofibrillary tangles (NFTs) (1, 2). Biochemical studies revealed that the main component of amyloid deposits in senile plaques is amyloid- peptide (A) of 39 – 42 amino acids, an internal proteolytic product of amyloid precursor protein (APP) in chromosome 21. In the accumulated amyloid deposits of senile plaques, A consists of peptides with different C-termini, A40 and A42 representing the major A species. Evidence obtained from the genetic study of familial AD cases, in which mutations existed in either APP or presenilin genes, suggested that the elevated secretion of A1-42 is likely an etiological event in AD pathogenesis. In vitro studies further suggested that A1-42 is more fibrillogenic than A1-40 (3). Neuropathological studies also suggested that A1-42 is more abundant than A1-40 in amyloid deposits of AD brains (4, 5). Thus, in addition to the proposed genetic mutations or a polymorphism of presenilins (6 –9), APP (10, 11), or apoli2 Abbreviations used: PHFs, paired helical filaments; AD, Alzheimer’s disease; PIMT, L-isoaspartyl methyltransferase; NFTs, neurofibrillary tangles; A, amyloid- peptides; APP, amyloid precurser protein; pGlu-3, pyroglutamate-3; isoAsp-1; isoaspartate-1; CD, circular dichroism.
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poprotein E (12, 13), the post-translational modifications of A by oxygen radicals, truncations, or isomerization/racemization are well speculated as enhancers for A aggregation and hence as progression factors for sporadic cases of AD. On the other hand, NFTs appear as abnormal aggregates of fibrous materials that are called paired helical filaments (PHFs) in the perikarya of affected neurons, predominantly in hippocampus, amygdala, cerebral cortex, and deep gray matter (14). PHFs are also present intracellularly in the swollen dystrophic neurites and neuropil threads that contribute to senile plaques (15–17). Extensive examinations performed to clarify the biochemical nature of PHFs revealed that PHFs are composed of tau (18 –23) and ubiquitin (24, 25). However, it is still poorly understood why PHFs accumulate in an insoluble form. The abnormal phosphorylation (19, 26) or the fragmentation of tau (27–29) could explain, at least partly, its biochemical alteration in PHFs (30). However, some other post-translational modifications such as oxidation, glycation, or isomerization of tau (31, 32) may contribute to the conformational abnormalities of tau/ubiquitin complex in addition to the hyperphosphorylation of tau. We review in this article that protein isomerization is enhanced in A as well as PHFs purified from AD brains, both of which are the characteristic pathological aggregates in AD. Since protein isomerization with L-isoaspartate residues causes structural changes and functional inactivation or enhances the aggregation process in the modified proteins, this post-translational modification is suggested to be one of the progression factors in AD. Protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT; E.C. 2.1.1.77) is a cytosolic enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues in isomerized proteins. It is then proposed that PIMT plays a physiological role in the repair of damaged proteins containing L-isoaspartyl residues that accumulate in aged proteins (33). We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in NFTs in AD brains. Taken together with the evidence that protein isomerization is enhanced in AD brains, it is implicated that the upregulated PIMT may be associated with the increased amount of protein isomerization in AD. We also review the studies on PIMT-deficient mice, which confirmed that PIMT plays a physiological role in the repair of isomerized proteins with L-isoaspartate. The knockout study also suggested that the brains of PIMT-deficient mice manifested neurodegenerative changes and concomitant accumulation of Lisoaspartate. We also discuss here the pathological implications of protein isomerization in the neurodegeneration of model mice and AD.
ISOMERIZATION AND RACEMIZATION OF ASPARTYL AND ASPARAGINYL RESIDUES
In many organisms, peptides and proteins are susceptible to spontaneous deamidation and isomerization that could alter their structural integrity as proteins, resulting in the loss of their biological activity (34 –36). These nonenzymatic covalent modifications occur primarily at aspargine and aspartate residues (Fig. 1; Refs. 36 – 43). The reaction proceeds spontaneously under physiological conditions through a cyclic succinimidyl intermediate formed by the nucleophilic attack of the peptide bond nitrogen of the following residue on the side chain carbonyl group. The succinimidyl intermediate then undergoes a relatively rapid hydrolysis at either the ␣- or -carbonyl group to generate isoaspartate and normal aspartate residues in a ratio of approximately 3:1 in a variety of substrates (38). The reactions can also be accompanied by an enhanced racemization at the ␣-carbon to generate a mixture of L- and D-amino acid derivatives (37). For example, free aspartic acid in a neutral solution (pH 7.5, 37°C) racemized with a half-time of 460 years (44) while the half-time of racemization in a succinimidyl residue of a synthetic hexapeptide was 19.5 h (pH 7.4, 37°C) (38). A similar series of reactions would occur in glutamine and glutamate residues, generating D- and L-isoglutamate and glutamate residues, but at a markedly slower rate (36). D-aspartyl, L-isoaspartyl, and Disoaspartyl residues have been detected in a variety of cellular proteins, including eye lens crystallin (45, 46), myelin basic protein (47, 48), serine hydroxymethyltransferase (49), calmodulin (50), and A (4). Evidence has been accumulated that calmodulin (51), epidermal growth factor (52), calbindin (53), and HPr (54) are less active when altered aspartyl residues are incorporated. ISOASPARTATE FORMATION OF A IN SENILE PLAQUES AND BLOOD VESSELS
AD is a neurodegenerative disease characterized by the extracellular accumulation of amyloid in the cortical regions of the brain and in the walls of the cerebral and leptomeningeal blood vessels. The deposited amyloid is primarily composed of insoluble fibrillar A of 40 or 42 amino acids derived by the proteolysis of APP (55). A42 is the major form of A present in the cortical neuritic plaques, which has been chemically shown to be less soluble than A40 (3). On the other hand, A40, which is more soluble than A42, is the major species of A deposited in the walls of the cerebral vasculature (4, 56). After the cleavage of APP, A undergoes various post-translational modifications such as truncation (57– 62), pyroglutamylation (58, 59,
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FIG. 1. Pathways for spontaneous deamidation, isomerization, and racemization for aspartyl and asparaginyl residues. L-Aspartyl and L-asparginyl residues (boxed) can degrade spontaneously via succinimidyl intermediate to form L-isoaspartyl and racemized residues. The rates of the nonenzymatic reactions denoted by the thick arrows are faster than those denoted by the thin arrows. (Modified from Ref. 99.)
61– 66), isomerization (4, 56, 61, 62, 64 – 66), and racemization (4, 56, 61, 62, 64 – 67). Recently, Kuo et al. (61) characterized the N-terminal structure of A, purified from senile plaques as well as from vascular deposits by amino acid analysis and mass spectrometric analysis of trypsin-digested A. As shown in Fig. 2, pyroglutamate-3 (pGlu-3), the cyclization of N-terminal glutamyl residue, (51%) was the major structure of A42 N-terminus in senile plaques, followed by isoaspartate-1 (isoAsp-1) (20%) and Asp-1 (10%) (Fig. 2). In vascular amyloid, however, Asp-1 (63%), Ala-2 (20%), and pGlu-3 (11%) were the major structures of A N-termini (Fig. 2). In addition to truncation and Nterminal cyclization, isomerization and racemization of Asp-7 has also been detected by amino acid analysis of extracted senile plaques from AD brains. Roher et al. (4) also showed the presence of L-isoAsp-7 (55.7%) and D-isoAsp-7 (19.2%) in A isolated from AD brains (Fig. 2). In addition, both D-Asp-1 and D-Asp-7 were found in the isolated A (4). Four aspartate isomers, L-Asp, D-Asp, L-isoAsp, and D-isoAsp, were all present at the Asp-7 site of A42. Examination of A from leptomeningeal vessels further revealed the presence of all four
aspartate isomers except for they were found in A40 molecules (61). As to Asp-23, however, no stereo- or isomerized isomer has yet been identified in the biochemical analysis of AD brains.
FIG. 2. Post-translational modifications of A purified from senile plaque or vascular amyloid of AD brains. Amino acid residues and their frequencies found in the N-termini of A samples are shown. IsoAsp and pGlu indicate isoaspartic acid and pyroglutamic acid, respectively. Data are modified from papers (4, 61).
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FIG. 3. Immunohistochemical analysis of AD brains using anti-isoAsp antibodies. (A–C) Temporal cortex sections from aged individual and (D–F) Temporal cortex sections from another aged individual were stained with anti-A1-42 (A, D), anti-isoAsp7 (B, E), and anti-isoAsp23 antibodies (C, F). Arrows indicate the core of mature plaques. Original magnification, ⫻100 in A–F.
To characterize the pathological significance of the isomerization at positions 7 and 23 of A, we developed specific antibodies (anti-isoAsp7 or anti-isoAsp23) raised against A carrying the isoAsp residue at position 7 or 23. Immunohistochemical studies in AD brains revealed that almost all plaques and amyloidbearing vessels were positively stained with antiisoAsp7 antibody, in a manner similar to that of antiA42 antiserum (Fig. 3). On the other hand, isoAsp-23 was characteristically localized in amyloid-bearing vessels as well as in the core of mature plaques (Fig. 3). In Western blot analysis, A(isoAsp23) and A(isoAsp7) peptides were also immunochemically detected in formic acid extracts of AD brains (data not shown). Although previous work on AD brains failed to identify the modification of A at Asp-23, our finding clearly demonstrated that Asp-23 of A was evidently isomerized in plaques and amyloid-bearing vessels of
AD brains. Furthermore, to clarify the biochemical properties of isomerized A, we synthesized A1-42 peptides and their isomers with an isoaspartyl residue at position 7 or 23 [A1-42(L-isoAsp7) and A1-42(LisoAsp23)]. The aggregation ability of the synthetic peptides was measured by thioflavin T (68). A1-42(LisoAsp23) aggregated more extensively than native A1-42 and A1-42(L-isoAsp7) (69). To assess further the potential -sheet structure of each A, we performed circular dichroism (CD) spectral analysis. All A initially exhibited a random structure in solution when the peptides were freshly dissolved. During incubation at 37°C, all A1-42 peptides transformed to typical -sheet structure, irrespective of isomerization or L/D configuration of the Asp residue at position 23 (data not shown). In fourier transform infrared spectroscopy and CD studies, Fabian et al. (70) suggested that A1-42 with isomerizations at positions 1 and 7
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FIG. 4. Isoaspartate formation sites of tau in PHFs. The bar represents a 441-residue human tau isoform (81). The amino-terminal inserts are in brackets. Microtubule-binding domains (repeats) are shown by boxes. Three-repeat isoform lacks the second repeat. Phosphorylation sites in PHF tau are marked by P. Outline-face letters, D and N, denote isoaspartate formation sites. The proportion of each isoasparatyl residue was roughly estimated as follows. They were calculated from the HPLC profiles and/or the yields in Edman degradation before the blocked cycle. The proportions of isoAsp for these peptides from the smeared tau were about ⬃24, ⬃30, and ⬃50% for Asp-193, Asn-381, and Asp-387, respectively (80).
showed an enhanced fibrillogenesis by facilitating formation of -sheet structure. Kuo et al. also suggested that the isomerization at positions 1 and 7 of A1-42 may contribute to the enhanced insolubility of A and its resistance to enzymatic degradation (71). These results further indicate that the isomerization of Asp residues greatly alters the aggregation properties of A1-42. In conclusion, it is suggested that isomerization of the aspartyl residues in A plays an important role in fibril formation and progression of amyloid deposits in AD brains. Alternatively, it is also suggested that the chemical modifications accumulated in the progression phase of amyloid deposition further accelerate the pathological process by enhancing fibrillogenesis in the neurodegeneration of AD. ISOASPARTATE FORMATION OF TAU IN PAIRED HELICAL FILAMENTS (PHFs)
One of the hallmarks of AD is the formation of innumerable NFTs throughout the cortex. The extensive loss of selected populations of neurons in AD is closely related to the formation of NFTs (72). The unit fibrils of NFTs are called PHFs. The major components of PHFs are tau and ubiquitin (24, 28, 29), the former of which constitutes the framework of PHFs (73, 74). The striking characteristics of tau in PHFs are insolubility and hyperphosphorylation. Thus far, the abnormal phosphorylation of PHF tau (hyperphosphorylated fulllength tau) has been intensively investigated, and the phosphorylation sites have been determined by immunochemical and protein chemical procedures (75–77). However, it is now known that the microtubule-binding domain makes up the framework of PHFs (73) and that phosphorylation in the flanking regions is not required for the assembly of tau into PHFs in vitro (74).
To characterize the biochemical nature of PHFs, the smearing on the electrophoretic blot of the PHF-enriched fraction was analyzed (78). The smear on the blot ranged from the gel top to ⬃10 kDa, with high molecular mass regions showing the most intense immunoreactivity with tau antibodies. The smear may be composed of vast numbers of oligomers and polymers of various sizes (79). Thus, the analysis of the smear should provide us with important insight into the assembly of tau into PHFs in vivo. We purified the smeared tau and compared its high-performance liquid chromatography (HPLC) peptide map profiles with that of soluble (normal) tau obtained from control aged brains or AD brains (80). In the peptide map of smeared tau-derived fragments, there were distinct peaks that eluted at unusual locations, when compared with the map of normal tau-derived fragments. Amino acid sequence and mass spectrometric analyses revealed that these distinct peptides bear isoaspartate at Asp-193, Asn-381, and Asp-387 numbered according to the longest human tau isoform (Fig. 4; Refs. 80 and 81). Thus, in the smear specific Asn and Asp residues are selectively deamidated and/or isomerized. Previously it had been reported that proteins in the PHF-enriched fraction from AD brain were isomerized and/or racemized at their Asp residues (82, 83) and that the preparations of PHF tau contained D-Asp (84). However, in these studies neither the sites of D-Asp nor the presence of isoAsp was confirmed. We showed that one of the differences between normal tau and tau in PHFs, besides phosphorylation, was the isoaspartate formation in PHF tau (80). It is of particular interest that isoaspartate residues are located just before the hyperphosphorylation sites of tau in PHFs. It is possible that the isoaspartate formation in tau induces a profound conformational change by which the tau can
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self-assemble into PHFs and be unusually stable in vivo. PIMT, A REPAIR ENZYME FOR ISOASPARTATE, IS LOCALIZED TO NFTs IN AD BRAIN
PIMT, which specifically transfers the methyl residues to L-isoaspartyl residues, but not to L-aspartyl residues, has been widely detected in various organisms (33, 85– 87). Experiments performed in vitro have demonstrated that incubation of synthetic L-isoaspartyl-containing peptides with PIMT results in the conversion of at least 50% of the peptide to the L-aspartylcontaining form (51, 85). This finding led to the hypothesis that the function of PIMT in vivo is to minimize the accumulation of L-isoaspartyl residues in long-lived proteins. We therefore investigated the expression of PIMT by immunohistochemistry in AD brains. As shown in Fig. 5, anti-PIMT antibody stained NFTs (Figs. 5C–5H) but failed to stain any senile plaque in AD brains. We then compared the localization of PIMT with that of other components of NFTs; MAP-5 (Figs. 5C–5E) and ubiquitin (Figs. 5F–5H). In a confocal image of temporal lobe sections from an AD individual, PIMT accumulated at the distinct localization of a NFT from that of MAP-5 (Figs. 5C–5E). PIMT was microscopically localized to the outer structure of NFTs (Fig. 5C), whereas MAP-5 was localized in both the inner and outer structure of the NFTs (Fig. 5E), suggesting that PIMT may be involved in the later stage of NFT progression. The immunoreactivity with anti-PIMT antibody was then studied for neuropil threads (curly fibers), another PHF-containing structure in AD brains. In affected regions of the cortex, we detected the immunoreactivity for PIMT in a small number of neuropil threads although the majority of threads were positive for MAP-5 and tau-2 in the serial sections. We also investigated colocalization of PIMT with ubiquitin in confocal images, showing that some neuropil threads were single-positive for ubiquitin (Figs. 5F–5H, arrowheads) while other neuropil threads were single-positive for PIMT (Figs. 5F–5H, arrows) and some were doublepositive for PIMT and ubiquitin. The results suggested that two distinct repair/degradation systems, ubiquitin-dependent degradation pathway and PIMT-dependent repair/degradation pathway, may independently function in AD brains. It is also noteworthy that the neurodegenerative neurons from AD brains showed a stronger immunostaining for PIMT in the cytoplasm (Fig. 5A). This homogeneous immunostaining in cytoplasm was highly characteristic of PIMT while neither tau-2 nor MAP-5 was diffusely localized in cytoplasm. These strongly stained neurons characteristically appeared in affected regions of AD brains
FIG. 5. Confocal images of an AD brain stained with anti-PIMT, anti-MAP-5, and anti-ubiquitin antibodies. (A, B) Temporal cortex sections of an AD brain (B) and a control brain (A) were stained with anti-PIMT antibody. PIMT (green) is upregulated in neurons and neuropil threads of an AD brain (B). Yellow pigments in these images are lipofuscin. (C–E) A NFTs double-stained for PIMT/MAP-5; antiPIMT (red in C, but yellow when overlapped with green in D) and anti-MAP-5 (green in E). PIMT is localized to the outer structure of a NFT, whereas MAP-5 is localized to both the internal and outer structure of the NFTs. (F–H) A NFT and neuropil threads doublestained for PIMT/ubiquitin; anti-PIMT (red) and anti-ubiquitin (green). The NFT is double positive for PIMT and ubiquitin. Arrows indicate PIMT-positive but ubiquitin-negative neuropil thread. Arrowheads indicate ubiquitin-positive but PIMT-negative neuropil thread. Original magnification, ⫻300 in A–B and ⫻800 in C–H.
while such neurons were hardly detectable in non-AD control brains, arguing that higher concentrations of PIMT may represent neurodegeneration without the presence of NFTs. The average PIMT concentration in confocal images of neural cells showed a significant upregulation in AD brain as compared to non-AD control brain (AD: 115.2 ⫾ 12.6 fluorescence intensity (mean ⫾ 1 SD, n ⫽ 10); non-AD: 63.9 ⫾ 5.1 fluorescence intensity (mean ⫾ 1 SD, n ⫽ 10); P ⬍ 0.001,
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Student’s t test; Figs. 5A and 5B). Taken together, the localization of PIMT suggests that a distinct repair/ degradation system other than ubiquitin may be involved in neurodegeneration and the formation of NFTs and neuropil threads in AD. THE IMPLICATION OF ISOASPARTATE FORMATION IN AD
The presence of isomerization in senile plaques and NFTs has several implications in AD neuropathogenesis. It is generally accepted that many proteins are post-translationally modified during aging and that isomerization/racemization is one of the characteristic modifications in aged proteins (88, 89). Since isomerization/racemization was detected in senile plaques and NFTs, these post-translational modifications in aspartyl or asparaginyl residue may change the properties of proteins: the solubility, the conformation, and the function of proteins, accelerating the progression of abnormal deposition in AD brains. Thus, in addition to the proposed aggregation mechanisms for A, such as longer form of A (3), presenilin mutations (6 –9), mutations in APP gene (10, 11), or metal-catalyzed oxidation system (90), it is tempting to speculate that the isomerization/racemization of A may also play a pathological role in the aggregation process of A (69, 91, 92). Since an aggregated insoluble form of crystallin has more racemized aspartate (D-Asp) than its soluble form in eye lens, the racemization may likely precede the aggregation process (45, 93). By analogy with this, the isomerized form of A or isomerized tau may make the proteins insoluble and trigger the abnormal aggregations in senile plaques or NFTs. It is shown here that two abnormally accumulated proteins, A and tau, are more isomerized in AD brains than in normal brains, arguing that the isomerizing mechanisms may be enhanced in AD, or alternatively an altered cellular or extracellular physiological condition may favor protein isomerization in AD, although the molecular basis involved in this post-translational modification has yet to be clarified. As evidently shown in Fig. 5, cytoplasmic enzyme PIMT is associated with NFTs but not detected in senile plaques because of the simple reason that NFTs localize intracellularly while amyloid depositions accumulate in extracellular space where PIMT failed to exist. In both abnormal accumulations, the mechanism by which isomerizations of NFTs or A are involved in or linked to other modifications such as abnormal phosphorylation on tau (26, 94 –96) or pathological aggregation of A should be further addressed to understand the complicated mechanisms of AD progression.
FIG. 6. Accumulation of isoaspartate in the brain of PIMT-deficient mice. The brain extracts were prepared from PIMT-deficient mice of indicated ages from Embryonic Day 18 (E18) to 9 weeks (9W) or from littermate control mice. The isoaspartate contained in the brain extracts was determined by ISOCOUNT protein deamidation kit (Promega) and plotted in the graph against the age. Data are presented as an average ⫾ SD of three individual mice.
ISOMERIZED PROTEINS WERE ACCUMULATED IN BRAIN OF YOUNG ADULT PIMT-DEFICIENT MICE
PIMT has been suggested to play a role in the repair of aged proteins spontaneously incorporated with isoaspartyl residues. Thus the lack of this repair system was expected to show an abnormal accumulation of isoaspartate in the brain of knockout animals with reference to the pathological implications of protein isomerization during aging. Recently, another group and we reported knockout mice lacking the enzymatic activity for PIMT (97, 98). The PIMT-deficient mice unexpectedly showed a fatal epileptic seizure at a rather young age and failed to survive beyond 12 weeks, making it impossible to investigate the pathological implications of isomerized proteins in aged brains. Therefore, we next investigated the dynamics of accumulated isoaspartate in the brain during the development of PIMT-deficient mice. Biochemical analysis revealed that isomerized proteins have already accumulated in neonatal brains of PIMT-deficient mice compared to control mice (Fig. 6). The accumulation of isomerized proteins then markedly progressed after 2 weeks of age and continued to increase as long as we could observe it until 9 weeks of age (Fig. 6). These data indicated that protein isomerization is not restricted to long-lived proteins of aged brains, but is observed as early as the embryonic stage in PIMTdeficient mice. The result also suggested that the amount of isoaspartate was kept low with PIMT in the wild type brain, with an equilibrium between the repairing activity of PIMT and the spontaneous generation of protein isomerization. On the other hand, the isomerized proteins are hardly subjected for the repair process without PIMT and are instead vulnerable for
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the abnormal accumulation in PIMT-deficient mice. Interestingly, PIMT-deficient mice start to show refractory seizures at 4 weeks, which are initially controlled by an antiepileptic agent, but become resistant to medical treatment at 9 weeks (98). The amount of isomerized proteins prominently accumulated in the brains of PIMT-deficient mice at 4 weeks of age when mice show the clinical symptom of seizures. The development of resistance to antiepileptic agents at 7–9 weeks would be well explained by the fact that medical treatment cannot repair or degrade the isomerized proteins accumulated in the brain of PIMT-deficient mice (Fig. 6). Morphological analysis of PIMT-deficient mice showed that the dentate granule cells in hippocampi of PIMT-deficient mice manifested neurodegenerative changes (Ikegaya et al., submitted for publication). Physiological study also suggested that neuronal plasticity at dentate gyrus–CA3 synapses was impaired with long-term potentiation being depressed in PIMTdeficient mice (Ikegaya et al., submitted for publication). These results implied that protein isomerization may play a pathological role in neurodegeneration in an animal model as well as in AD brains. Although seizure is not a common symptom of AD patients, rapid accumulation of isomerized proteins may cause an epileptic seizure while chronic accumulation may cause neurodegeneration. Alternatively, it is also speculated that some proteins specifically isomerized during the early phase of the development are responsible for the symptom of epileptic seizure while other proteins isomerized in matured or aged brains are associated with the progression of neurodegeneration. The identification of substrate proteins for PIMT should clarify this issue in the future study of PIMT-deficient mice.
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Isoaspartate Formation and Neurodegeneration in Alzheimer’s Disease Takahiko Shimizu,* Atsushi Watanabe,† Midori Ogawara,* Hiroshi Mori,‡ and Takuji Shirasawa* ,1 *Department of Molecular Genetics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan; †Division of Biomolecular Characterization, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan; and ‡Department of Neuroscience, School of Medicine, Osaka Municipal University, Abeno-ku, Osaka 545-8585, Japan
Received April 27, 2000, and in revised form May 26, 2000
We reviewed here that protein isomerization is enhanced in amyloid- peptides (A) and paired helical filaments (PHFs) purified from Alzheimer’s disease (AD) brains. Biochemical analyses revealed that A purified from senile plaques and vascular amyloid are isomerized at Asp-1 and Asp-7. A specific antibody recognizing isoAsp-23 of A further suggested the isomerization of A at Asp-23 in vascular amyloid as well as in the core of senile plaques. Biochemical analyses of purified PHFs also revealed that heterogeneous molecular weight tau contains L-isoaspartate at Asp-193, Asn-381, and Asp-387, indicating a modification, other than phosphorylation, that differentiates between normal tau and PHF tau. Since protein isomerization as L-isoaspartate causes structural changes and functional inactivation, or enhances the aggregation process, this modification is proposed as one of the progression factors in AD. Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of isomerized proteins containing L-isoaspartate. We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in neurofibrillary tangles (NFTs) in AD. Taken together with the enhanced protein isomerization in AD brains, it is implicated that the upregulated PIMT may associate with increased protein isomerization in AD. We also reviewed studies on PIMT-deficient mice that confirmed that PIMT plays a physiological role in the repair of isomerized proteins containing L-isoaspartate. The knockout study also suggested that the brain of PIMT-deficient mice manifested neurodegenerative changes concomitant with accumulation 1 To whom correspondence should be addressed. Fax: ⫹813-35794776. E-mail: [email protected].
0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
of L-isoaspartate. We discuss the pathological implications of protein isomerization in the neurodegeneration found in model mice and AD. © 2000 Academic Press
Key Words: protein isomerization; Alzheimer’s disease; A; PHFs; tau; L-isoaspartate; PIMT.
In Alzheimer’s disease (AD), 2 characteristic changes occur in hippocampus and cerebral cortex, such as the accumulation of amyloid in senile plaques, abnormal neurite outgrowth, and the accumulation of neurofibrillary tangles (NFTs) (1, 2). Biochemical studies revealed that the main component of amyloid deposits in senile plaques is amyloid- peptide (A) of 39 – 42 amino acids, an internal proteolytic product of amyloid precursor protein (APP) in chromosome 21. In the accumulated amyloid deposits of senile plaques, A consists of peptides with different C-termini, A40 and A42 representing the major A species. Evidence obtained from the genetic study of familial AD cases, in which mutations existed in either APP or presenilin genes, suggested that the elevated secretion of A1-42 is likely an etiological event in AD pathogenesis. In vitro studies further suggested that A1-42 is more fibrillogenic than A1-40 (3). Neuropathological studies also suggested that A1-42 is more abundant than A1-40 in amyloid deposits of AD brains (4, 5). Thus, in addition to the proposed genetic mutations or a polymorphism of presenilins (6 –9), APP (10, 11), or apoli2 Abbreviations used: PHFs, paired helical filaments; AD, Alzheimer’s disease; PIMT, L-isoaspartyl methyltransferase; NFTs, neurofibrillary tangles; A, amyloid- peptides; APP, amyloid precurser protein; pGlu-3, pyroglutamate-3; isoAsp-1; isoaspartate-1; CD, circular dichroism.
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poprotein E (12, 13), the post-translational modifications of A by oxygen radicals, truncations, or isomerization/racemization are well speculated as enhancers for A aggregation and hence as progression factors for sporadic cases of AD. On the other hand, NFTs appear as abnormal aggregates of fibrous materials that are called paired helical filaments (PHFs) in the perikarya of affected neurons, predominantly in hippocampus, amygdala, cerebral cortex, and deep gray matter (14). PHFs are also present intracellularly in the swollen dystrophic neurites and neuropil threads that contribute to senile plaques (15–17). Extensive examinations performed to clarify the biochemical nature of PHFs revealed that PHFs are composed of tau (18 –23) and ubiquitin (24, 25). However, it is still poorly understood why PHFs accumulate in an insoluble form. The abnormal phosphorylation (19, 26) or the fragmentation of tau (27–29) could explain, at least partly, its biochemical alteration in PHFs (30). However, some other post-translational modifications such as oxidation, glycation, or isomerization of tau (31, 32) may contribute to the conformational abnormalities of tau/ubiquitin complex in addition to the hyperphosphorylation of tau. We review in this article that protein isomerization is enhanced in A as well as PHFs purified from AD brains, both of which are the characteristic pathological aggregates in AD. Since protein isomerization with L-isoaspartate residues causes structural changes and functional inactivation or enhances the aggregation process in the modified proteins, this post-translational modification is suggested to be one of the progression factors in AD. Protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT; E.C. 2.1.1.77) is a cytosolic enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues in isomerized proteins. It is then proposed that PIMT plays a physiological role in the repair of damaged proteins containing L-isoaspartyl residues that accumulate in aged proteins (33). We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in NFTs in AD brains. Taken together with the evidence that protein isomerization is enhanced in AD brains, it is implicated that the upregulated PIMT may be associated with the increased amount of protein isomerization in AD. We also review the studies on PIMT-deficient mice, which confirmed that PIMT plays a physiological role in the repair of isomerized proteins with L-isoaspartate. The knockout study also suggested that the brains of PIMT-deficient mice manifested neurodegenerative changes and concomitant accumulation of Lisoaspartate. We also discuss here the pathological implications of protein isomerization in the neurodegeneration of model mice and AD.
ISOMERIZATION AND RACEMIZATION OF ASPARTYL AND ASPARAGINYL RESIDUES
In many organisms, peptides and proteins are susceptible to spontaneous deamidation and isomerization that could alter their structural integrity as proteins, resulting in the loss of their biological activity (34 –36). These nonenzymatic covalent modifications occur primarily at aspargine and aspartate residues (Fig. 1; Refs. 36 – 43). The reaction proceeds spontaneously under physiological conditions through a cyclic succinimidyl intermediate formed by the nucleophilic attack of the peptide bond nitrogen of the following residue on the side chain carbonyl group. The succinimidyl intermediate then undergoes a relatively rapid hydrolysis at either the ␣- or -carbonyl group to generate isoaspartate and normal aspartate residues in a ratio of approximately 3:1 in a variety of substrates (38). The reactions can also be accompanied by an enhanced racemization at the ␣-carbon to generate a mixture of L- and D-amino acid derivatives (37). For example, free aspartic acid in a neutral solution (pH 7.5, 37°C) racemized with a half-time of 460 years (44) while the half-time of racemization in a succinimidyl residue of a synthetic hexapeptide was 19.5 h (pH 7.4, 37°C) (38). A similar series of reactions would occur in glutamine and glutamate residues, generating D- and L-isoglutamate and glutamate residues, but at a markedly slower rate (36). D-aspartyl, L-isoaspartyl, and Disoaspartyl residues have been detected in a variety of cellular proteins, including eye lens crystallin (45, 46), myelin basic protein (47, 48), serine hydroxymethyltransferase (49), calmodulin (50), and A (4). Evidence has been accumulated that calmodulin (51), epidermal growth factor (52), calbindin (53), and HPr (54) are less active when altered aspartyl residues are incorporated. ISOASPARTATE FORMATION OF A IN SENILE PLAQUES AND BLOOD VESSELS
AD is a neurodegenerative disease characterized by the extracellular accumulation of amyloid in the cortical regions of the brain and in the walls of the cerebral and leptomeningeal blood vessels. The deposited amyloid is primarily composed of insoluble fibrillar A of 40 or 42 amino acids derived by the proteolysis of APP (55). A42 is the major form of A present in the cortical neuritic plaques, which has been chemically shown to be less soluble than A40 (3). On the other hand, A40, which is more soluble than A42, is the major species of A deposited in the walls of the cerebral vasculature (4, 56). After the cleavage of APP, A undergoes various post-translational modifications such as truncation (57– 62), pyroglutamylation (58, 59,
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FIG. 1. Pathways for spontaneous deamidation, isomerization, and racemization for aspartyl and asparaginyl residues. L-Aspartyl and L-asparginyl residues (boxed) can degrade spontaneously via succinimidyl intermediate to form L-isoaspartyl and racemized residues. The rates of the nonenzymatic reactions denoted by the thick arrows are faster than those denoted by the thin arrows. (Modified from Ref. 99.)
61– 66), isomerization (4, 56, 61, 62, 64 – 66), and racemization (4, 56, 61, 62, 64 – 67). Recently, Kuo et al. (61) characterized the N-terminal structure of A, purified from senile plaques as well as from vascular deposits by amino acid analysis and mass spectrometric analysis of trypsin-digested A. As shown in Fig. 2, pyroglutamate-3 (pGlu-3), the cyclization of N-terminal glutamyl residue, (51%) was the major structure of A42 N-terminus in senile plaques, followed by isoaspartate-1 (isoAsp-1) (20%) and Asp-1 (10%) (Fig. 2). In vascular amyloid, however, Asp-1 (63%), Ala-2 (20%), and pGlu-3 (11%) were the major structures of A N-termini (Fig. 2). In addition to truncation and Nterminal cyclization, isomerization and racemization of Asp-7 has also been detected by amino acid analysis of extracted senile plaques from AD brains. Roher et al. (4) also showed the presence of L-isoAsp-7 (55.7%) and D-isoAsp-7 (19.2%) in A isolated from AD brains (Fig. 2). In addition, both D-Asp-1 and D-Asp-7 were found in the isolated A (4). Four aspartate isomers, L-Asp, D-Asp, L-isoAsp, and D-isoAsp, were all present at the Asp-7 site of A42. Examination of A from leptomeningeal vessels further revealed the presence of all four
aspartate isomers except for they were found in A40 molecules (61). As to Asp-23, however, no stereo- or isomerized isomer has yet been identified in the biochemical analysis of AD brains.
FIG. 2. Post-translational modifications of A purified from senile plaque or vascular amyloid of AD brains. Amino acid residues and their frequencies found in the N-termini of A samples are shown. IsoAsp and pGlu indicate isoaspartic acid and pyroglutamic acid, respectively. Data are modified from papers (4, 61).
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FIG. 3. Immunohistochemical analysis of AD brains using anti-isoAsp antibodies. (A–C) Temporal cortex sections from aged individual and (D–F) Temporal cortex sections from another aged individual were stained with anti-A1-42 (A, D), anti-isoAsp7 (B, E), and anti-isoAsp23 antibodies (C, F). Arrows indicate the core of mature plaques. Original magnification, ⫻100 in A–F.
To characterize the pathological significance of the isomerization at positions 7 and 23 of A, we developed specific antibodies (anti-isoAsp7 or anti-isoAsp23) raised against A carrying the isoAsp residue at position 7 or 23. Immunohistochemical studies in AD brains revealed that almost all plaques and amyloidbearing vessels were positively stained with antiisoAsp7 antibody, in a manner similar to that of antiA42 antiserum (Fig. 3). On the other hand, isoAsp-23 was characteristically localized in amyloid-bearing vessels as well as in the core of mature plaques (Fig. 3). In Western blot analysis, A(isoAsp23) and A(isoAsp7) peptides were also immunochemically detected in formic acid extracts of AD brains (data not shown). Although previous work on AD brains failed to identify the modification of A at Asp-23, our finding clearly demonstrated that Asp-23 of A was evidently isomerized in plaques and amyloid-bearing vessels of
AD brains. Furthermore, to clarify the biochemical properties of isomerized A, we synthesized A1-42 peptides and their isomers with an isoaspartyl residue at position 7 or 23 [A1-42(L-isoAsp7) and A1-42(LisoAsp23)]. The aggregation ability of the synthetic peptides was measured by thioflavin T (68). A1-42(LisoAsp23) aggregated more extensively than native A1-42 and A1-42(L-isoAsp7) (69). To assess further the potential -sheet structure of each A, we performed circular dichroism (CD) spectral analysis. All A initially exhibited a random structure in solution when the peptides were freshly dissolved. During incubation at 37°C, all A1-42 peptides transformed to typical -sheet structure, irrespective of isomerization or L/D configuration of the Asp residue at position 23 (data not shown). In fourier transform infrared spectroscopy and CD studies, Fabian et al. (70) suggested that A1-42 with isomerizations at positions 1 and 7
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FIG. 4. Isoaspartate formation sites of tau in PHFs. The bar represents a 441-residue human tau isoform (81). The amino-terminal inserts are in brackets. Microtubule-binding domains (repeats) are shown by boxes. Three-repeat isoform lacks the second repeat. Phosphorylation sites in PHF tau are marked by P. Outline-face letters, D and N, denote isoaspartate formation sites. The proportion of each isoasparatyl residue was roughly estimated as follows. They were calculated from the HPLC profiles and/or the yields in Edman degradation before the blocked cycle. The proportions of isoAsp for these peptides from the smeared tau were about ⬃24, ⬃30, and ⬃50% for Asp-193, Asn-381, and Asp-387, respectively (80).
showed an enhanced fibrillogenesis by facilitating formation of -sheet structure. Kuo et al. also suggested that the isomerization at positions 1 and 7 of A1-42 may contribute to the enhanced insolubility of A and its resistance to enzymatic degradation (71). These results further indicate that the isomerization of Asp residues greatly alters the aggregation properties of A1-42. In conclusion, it is suggested that isomerization of the aspartyl residues in A plays an important role in fibril formation and progression of amyloid deposits in AD brains. Alternatively, it is also suggested that the chemical modifications accumulated in the progression phase of amyloid deposition further accelerate the pathological process by enhancing fibrillogenesis in the neurodegeneration of AD. ISOASPARTATE FORMATION OF TAU IN PAIRED HELICAL FILAMENTS (PHFs)
One of the hallmarks of AD is the formation of innumerable NFTs throughout the cortex. The extensive loss of selected populations of neurons in AD is closely related to the formation of NFTs (72). The unit fibrils of NFTs are called PHFs. The major components of PHFs are tau and ubiquitin (24, 28, 29), the former of which constitutes the framework of PHFs (73, 74). The striking characteristics of tau in PHFs are insolubility and hyperphosphorylation. Thus far, the abnormal phosphorylation of PHF tau (hyperphosphorylated fulllength tau) has been intensively investigated, and the phosphorylation sites have been determined by immunochemical and protein chemical procedures (75–77). However, it is now known that the microtubule-binding domain makes up the framework of PHFs (73) and that phosphorylation in the flanking regions is not required for the assembly of tau into PHFs in vitro (74).
To characterize the biochemical nature of PHFs, the smearing on the electrophoretic blot of the PHF-enriched fraction was analyzed (78). The smear on the blot ranged from the gel top to ⬃10 kDa, with high molecular mass regions showing the most intense immunoreactivity with tau antibodies. The smear may be composed of vast numbers of oligomers and polymers of various sizes (79). Thus, the analysis of the smear should provide us with important insight into the assembly of tau into PHFs in vivo. We purified the smeared tau and compared its high-performance liquid chromatography (HPLC) peptide map profiles with that of soluble (normal) tau obtained from control aged brains or AD brains (80). In the peptide map of smeared tau-derived fragments, there were distinct peaks that eluted at unusual locations, when compared with the map of normal tau-derived fragments. Amino acid sequence and mass spectrometric analyses revealed that these distinct peptides bear isoaspartate at Asp-193, Asn-381, and Asp-387 numbered according to the longest human tau isoform (Fig. 4; Refs. 80 and 81). Thus, in the smear specific Asn and Asp residues are selectively deamidated and/or isomerized. Previously it had been reported that proteins in the PHF-enriched fraction from AD brain were isomerized and/or racemized at their Asp residues (82, 83) and that the preparations of PHF tau contained D-Asp (84). However, in these studies neither the sites of D-Asp nor the presence of isoAsp was confirmed. We showed that one of the differences between normal tau and tau in PHFs, besides phosphorylation, was the isoaspartate formation in PHF tau (80). It is of particular interest that isoaspartate residues are located just before the hyperphosphorylation sites of tau in PHFs. It is possible that the isoaspartate formation in tau induces a profound conformational change by which the tau can
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self-assemble into PHFs and be unusually stable in vivo. PIMT, A REPAIR ENZYME FOR ISOASPARTATE, IS LOCALIZED TO NFTs IN AD BRAIN
PIMT, which specifically transfers the methyl residues to L-isoaspartyl residues, but not to L-aspartyl residues, has been widely detected in various organisms (33, 85– 87). Experiments performed in vitro have demonstrated that incubation of synthetic L-isoaspartyl-containing peptides with PIMT results in the conversion of at least 50% of the peptide to the L-aspartylcontaining form (51, 85). This finding led to the hypothesis that the function of PIMT in vivo is to minimize the accumulation of L-isoaspartyl residues in long-lived proteins. We therefore investigated the expression of PIMT by immunohistochemistry in AD brains. As shown in Fig. 5, anti-PIMT antibody stained NFTs (Figs. 5C–5H) but failed to stain any senile plaque in AD brains. We then compared the localization of PIMT with that of other components of NFTs; MAP-5 (Figs. 5C–5E) and ubiquitin (Figs. 5F–5H). In a confocal image of temporal lobe sections from an AD individual, PIMT accumulated at the distinct localization of a NFT from that of MAP-5 (Figs. 5C–5E). PIMT was microscopically localized to the outer structure of NFTs (Fig. 5C), whereas MAP-5 was localized in both the inner and outer structure of the NFTs (Fig. 5E), suggesting that PIMT may be involved in the later stage of NFT progression. The immunoreactivity with anti-PIMT antibody was then studied for neuropil threads (curly fibers), another PHF-containing structure in AD brains. In affected regions of the cortex, we detected the immunoreactivity for PIMT in a small number of neuropil threads although the majority of threads were positive for MAP-5 and tau-2 in the serial sections. We also investigated colocalization of PIMT with ubiquitin in confocal images, showing that some neuropil threads were single-positive for ubiquitin (Figs. 5F–5H, arrowheads) while other neuropil threads were single-positive for PIMT (Figs. 5F–5H, arrows) and some were doublepositive for PIMT and ubiquitin. The results suggested that two distinct repair/degradation systems, ubiquitin-dependent degradation pathway and PIMT-dependent repair/degradation pathway, may independently function in AD brains. It is also noteworthy that the neurodegenerative neurons from AD brains showed a stronger immunostaining for PIMT in the cytoplasm (Fig. 5A). This homogeneous immunostaining in cytoplasm was highly characteristic of PIMT while neither tau-2 nor MAP-5 was diffusely localized in cytoplasm. These strongly stained neurons characteristically appeared in affected regions of AD brains
FIG. 5. Confocal images of an AD brain stained with anti-PIMT, anti-MAP-5, and anti-ubiquitin antibodies. (A, B) Temporal cortex sections of an AD brain (B) and a control brain (A) were stained with anti-PIMT antibody. PIMT (green) is upregulated in neurons and neuropil threads of an AD brain (B). Yellow pigments in these images are lipofuscin. (C–E) A NFTs double-stained for PIMT/MAP-5; antiPIMT (red in C, but yellow when overlapped with green in D) and anti-MAP-5 (green in E). PIMT is localized to the outer structure of a NFT, whereas MAP-5 is localized to both the internal and outer structure of the NFTs. (F–H) A NFT and neuropil threads doublestained for PIMT/ubiquitin; anti-PIMT (red) and anti-ubiquitin (green). The NFT is double positive for PIMT and ubiquitin. Arrows indicate PIMT-positive but ubiquitin-negative neuropil thread. Arrowheads indicate ubiquitin-positive but PIMT-negative neuropil thread. Original magnification, ⫻300 in A–B and ⫻800 in C–H.
while such neurons were hardly detectable in non-AD control brains, arguing that higher concentrations of PIMT may represent neurodegeneration without the presence of NFTs. The average PIMT concentration in confocal images of neural cells showed a significant upregulation in AD brain as compared to non-AD control brain (AD: 115.2 ⫾ 12.6 fluorescence intensity (mean ⫾ 1 SD, n ⫽ 10); non-AD: 63.9 ⫾ 5.1 fluorescence intensity (mean ⫾ 1 SD, n ⫽ 10); P ⬍ 0.001,
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Student’s t test; Figs. 5A and 5B). Taken together, the localization of PIMT suggests that a distinct repair/ degradation system other than ubiquitin may be involved in neurodegeneration and the formation of NFTs and neuropil threads in AD. THE IMPLICATION OF ISOASPARTATE FORMATION IN AD
The presence of isomerization in senile plaques and NFTs has several implications in AD neuropathogenesis. It is generally accepted that many proteins are post-translationally modified during aging and that isomerization/racemization is one of the characteristic modifications in aged proteins (88, 89). Since isomerization/racemization was detected in senile plaques and NFTs, these post-translational modifications in aspartyl or asparaginyl residue may change the properties of proteins: the solubility, the conformation, and the function of proteins, accelerating the progression of abnormal deposition in AD brains. Thus, in addition to the proposed aggregation mechanisms for A, such as longer form of A (3), presenilin mutations (6 –9), mutations in APP gene (10, 11), or metal-catalyzed oxidation system (90), it is tempting to speculate that the isomerization/racemization of A may also play a pathological role in the aggregation process of A (69, 91, 92). Since an aggregated insoluble form of crystallin has more racemized aspartate (D-Asp) than its soluble form in eye lens, the racemization may likely precede the aggregation process (45, 93). By analogy with this, the isomerized form of A or isomerized tau may make the proteins insoluble and trigger the abnormal aggregations in senile plaques or NFTs. It is shown here that two abnormally accumulated proteins, A and tau, are more isomerized in AD brains than in normal brains, arguing that the isomerizing mechanisms may be enhanced in AD, or alternatively an altered cellular or extracellular physiological condition may favor protein isomerization in AD, although the molecular basis involved in this post-translational modification has yet to be clarified. As evidently shown in Fig. 5, cytoplasmic enzyme PIMT is associated with NFTs but not detected in senile plaques because of the simple reason that NFTs localize intracellularly while amyloid depositions accumulate in extracellular space where PIMT failed to exist. In both abnormal accumulations, the mechanism by which isomerizations of NFTs or A are involved in or linked to other modifications such as abnormal phosphorylation on tau (26, 94 –96) or pathological aggregation of A should be further addressed to understand the complicated mechanisms of AD progression.
FIG. 6. Accumulation of isoaspartate in the brain of PIMT-deficient mice. The brain extracts were prepared from PIMT-deficient mice of indicated ages from Embryonic Day 18 (E18) to 9 weeks (9W) or from littermate control mice. The isoaspartate contained in the brain extracts was determined by ISOCOUNT protein deamidation kit (Promega) and plotted in the graph against the age. Data are presented as an average ⫾ SD of three individual mice.
ISOMERIZED PROTEINS WERE ACCUMULATED IN BRAIN OF YOUNG ADULT PIMT-DEFICIENT MICE
PIMT has been suggested to play a role in the repair of aged proteins spontaneously incorporated with isoaspartyl residues. Thus the lack of this repair system was expected to show an abnormal accumulation of isoaspartate in the brain of knockout animals with reference to the pathological implications of protein isomerization during aging. Recently, another group and we reported knockout mice lacking the enzymatic activity for PIMT (97, 98). The PIMT-deficient mice unexpectedly showed a fatal epileptic seizure at a rather young age and failed to survive beyond 12 weeks, making it impossible to investigate the pathological implications of isomerized proteins in aged brains. Therefore, we next investigated the dynamics of accumulated isoaspartate in the brain during the development of PIMT-deficient mice. Biochemical analysis revealed that isomerized proteins have already accumulated in neonatal brains of PIMT-deficient mice compared to control mice (Fig. 6). The accumulation of isomerized proteins then markedly progressed after 2 weeks of age and continued to increase as long as we could observe it until 9 weeks of age (Fig. 6). These data indicated that protein isomerization is not restricted to long-lived proteins of aged brains, but is observed as early as the embryonic stage in PIMTdeficient mice. The result also suggested that the amount of isoaspartate was kept low with PIMT in the wild type brain, with an equilibrium between the repairing activity of PIMT and the spontaneous generation of protein isomerization. On the other hand, the isomerized proteins are hardly subjected for the repair process without PIMT and are instead vulnerable for
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the abnormal accumulation in PIMT-deficient mice. Interestingly, PIMT-deficient mice start to show refractory seizures at 4 weeks, which are initially controlled by an antiepileptic agent, but become resistant to medical treatment at 9 weeks (98). The amount of isomerized proteins prominently accumulated in the brains of PIMT-deficient mice at 4 weeks of age when mice show the clinical symptom of seizures. The development of resistance to antiepileptic agents at 7–9 weeks would be well explained by the fact that medical treatment cannot repair or degrade the isomerized proteins accumulated in the brain of PIMT-deficient mice (Fig. 6). Morphological analysis of PIMT-deficient mice showed that the dentate granule cells in hippocampi of PIMT-deficient mice manifested neurodegenerative changes (Ikegaya et al., submitted for publication). Physiological study also suggested that neuronal plasticity at dentate gyrus–CA3 synapses was impaired with long-term potentiation being depressed in PIMTdeficient mice (Ikegaya et al., submitted for publication). These results implied that protein isomerization may play a pathological role in neurodegeneration in an animal model as well as in AD brains. Although seizure is not a common symptom of AD patients, rapid accumulation of isomerized proteins may cause an epileptic seizure while chronic accumulation may cause neurodegeneration. Alternatively, it is also speculated that some proteins specifically isomerized during the early phase of the development are responsible for the symptom of epileptic seizure while other proteins isomerized in matured or aged brains are associated with the progression of neurodegeneration. The identification of substrate proteins for PIMT should clarify this issue in the future study of PIMT-deficient mice.
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