Alzheimer’s disease and the reversal of pathology and behavior in human APP transgenic mice by mutation of Asp664

Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA, Bredesen DE. Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664. Proc Natl Acad Sci USA 2007 Apr 17; 104(16): 6876.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous system (CNS) and is associated with memory loss, behavior and personality changes, and cognitive decline. The neuropathophysiology of AD is accompanied by three major structural changes: extracellular deposition of β-amyloid (Aβ), intracellular deposits called neurofibrillary tangles (NFT) consisting of hyperphosphorylated tau proteins, and selective neuronal loss, cholinergic deficits and astrogliosis (astrocyte proliferation) in the cerebral cortex, hippocampus, and other brain regions essential for memory and cognitive ability (1).

There are two hypotheses that postulate the molecular mechanisms of this disease. The cholinergic hypothesis suggests a dysfunctional cholinergic system is sufficient to cause memory loss as demonstrated animal models analogous to AD-related dementia. The brains of AD patients show selective degeneration of cholinergic neurons of the basal forebrain (nucleus basalis of Meynert) resulting in a substantial decline in choline acetyltransferase (ChAT) and acetyl cholinesterase (AChase). These enzymes are responsible for the production and regulation of acetylcholine (ACh) (a neurotransmitter) and its loss is associated with cognitive decline (2).

The amyloid cascade hypothesis states that the pathophysiology of AD is the result of abnormal processing of the amyloid precursor protein (APP), which is a membrane protein expressed during cell stress (3). Genetic studies have implicated four genes in the APP proteolytic cascade: APP gene (gene product is substrate) (21q21.2) (4), presenilin 1 (PSEN1) (14q24.3) and presenilin 2 (PSEN2) (1q31-q42) (5, 6), and apolipoprotein E (ApoE) (19q13.2) (7). Mutations in these genes are linked to the autosomal dominant or familial early onset AD (FAD) (8). The production of Aβ is dependant on the activities of two enzymes: β-secretase and γ-secretase. Depending on the type of APP mutation, either β- or γ-secretase activity is altered. PSEN proteins (PS) have been proposed to function in the APP processing pathway. PS1 is hypothesized to be either an essential cofactor or is itself γ-secretase (9). PS have also been proposed to function in apoptosis. Mutated PS expression or PS overexpression leads to increased sensitivity to apoptosis in transfected cells (10). PS mutations destabilize Ca2+ homeostasis (11) and may play a direct role in Fas-mediated apoptosis (12).

ApoE is a 34kDa lipid transport protein that has also been implicated in the pathogenesis of AD (13). It has also been shown to repair nerve cells in response to oxidative damage by reducing secondary glutamate (primary CNS neurotransmitter) excitotoxicity (14). ApoE exists in three allelic variants: E2, E3, and E 4. Biochemical tests have shown that ApoE can directly interact with Aβ. ApoE3 has been reported to protect neurons against Aβ neurotoxicity, through association and internalization of Aβ (15). However, the ApoE4 allele is associated with anticipation of AD as it binds more rapidly to Aβ and leads to its increased deposition.

Tau (MAPT gene), a microtubule associated protein, has multiple phosphorylation sites and binds tubulin molecules and facilitates microtubule assembly and stability (16). In AD, tau is hyperphosphorylated and this impairs its ability to perform its function and undermines microtubule stability (17).

The CNS is especially vulnerable to oxidative stress due to the brain's high oxygen consumption, abundance of lipid content, and relative absence of antioxidants compared to other tissues (18). This oxidation may come from the reaction of peroxynitrite, a powerful oxidant from the reaction of O2- and NO. This occurs from the overstimulation of neurons during cell stress when they are unable to uptake glutamate (CNS neurotransmitter) and allows the accumulation of intracellular calcium. Excess calcium activates neuronal NO synthases, enzymes that form NO from L-arginine, which leads to excitotoxicity.

Astrocytes and microglia are also involved in causing damage to the AD brain. In AD, the number of astrocytes is increased (astrogliosis) and the expression of phospholipase A2 is upregulated leading to arachidonic acid/prostaglandins inflammatory products. Activated microglial cells are also abundant in the AD brain and produce a number of neurotoxic compounds: superoxide anions, glutamate, and NO (19). They upregulate MHC expression and secrete cytokines and chemokines, including interleukin-1 (which increases APP expression), TNF-α, and interferon-γ (both involved in the cellular immune attack).

Recent evidence suggests that APP may be cleaved intracytoplasmically at Asp664 by caspases (activated during apoptosis) (20, 21) to release a cytotoxic carboxy-terminal peptide: APP-C31 (21, 22). This finding suggests a model where Aβ oligomerizes to APP leading to cytosolic cleavage and initiation of synaptic and neuronal damage (23). However, it is unknown whether APP intracytoplasmic cleavage in vivo results in AD pathogenesis. Galvan et al. (2005) generated transgenic mice expressing human APP transgene with the familial AD-associated Swedish and Indiana mutations (24, 25) except that the cytosolic Asp was mutated to Ala. These mice were designated as PDAPP(D664A).

The authors incubated 3 month-old (mo) brain tissue samples with an antibody that binds the C-terminal APP cleavage peptide but not full length APP (20, 22). Immunoreactivity in PDAPP(D664A) mice was substantially lower than PDAPP mice suggesting that Asp664 mutation prevented hAPP C-terminal cleavage in vivo. The authors also examined Aβ production and deposition, hippocampal presynaptic density and dentate gyrus (DG) volume, astrogliosis, and cognitive and behavioral abnormalities.

Immunoprecipitation and western blot analysis for Aβ revealed that Asp664 mutation had no demonstrable effect on Aβ production in vivo. ELISA quantification revealed that mouse lines with APP overexpression and D664A mutation still had high levels of Aβ. Plaque formation was also unaffected by Asp664 mutation as shown by Aβ antibody (4G8) immunoreactivity and positive thioflavin-S results.

Synaptophysin is a synaptic vesicle glycoprotein involved in synaptic transmission. Its reduction in the hippocampus and prefrontal cortex is correlated with cognitive decline (26). PDAPP mice show decreased numbers of hippocampal-synaptophysin-immunoreactive presynaptic densities (HSPDs). However, the PDAPP(D664A) mice have HSPD levels indistinguishable from nontransgenic (wildtype) mice suggesting that Asp664 mutation blocks hippocampal synaptic loss.

DG volume in 3 mo transgenic mice were determined using digital 3D reconstruction of Nissl-stained sections (IMARIS 3D (Bitplane)) and by manual Calvieri analysis. DG volume reduction, as observed in PDAPP mice, was not seen in PDAPP(D664A) mice indicating that Asp664 mutation can rescue DG neuronal loss.

Neuronal loss allows astrocyte activation, infiltration, and proliferation (27). Hippocampal samples of 12 mo transgenic mice were incubated with antibodies specific for an astrocyte marker: glial fibrillary acidic protein (GFAP). A significant increase in GFAP immunoreactivity was observed in PDAPP mice but not in PDAPP(D664A) or nontransgenic mice. Thus, Asp664 mutation prevents astrogliosis.

AD patients, and animal models, exhibit many cognitive deficits and behavioral abnormalities (1). Learning and spatial memory, which are hippocampus-dependant, were evaluated using the Morris water maze (MWM) test (28). Performance of PDAPP mice was considerably impaired compared to PDAPP(D664A) and nontransgenic mice. Behavioral abnormalities, such as neophobia (29) where the exploration time of novel object or region is reduced, have also been described in AD transgenic models. Neophobia is associated with decreased glucose utilization in the entorhinal cortex. This behavior pattern was detected in some PDAPP mice but nonexistent in PDAPP(D664A) mice. These results demonstrate that learning, spatial memory, and behavior problems associated with AD can be rescued by ASP664 mutation.

Although Aβ production and deposition remain unchanged, all consequential pathophysiological features of AD are improved in PDAPP(D664A) mice. This shows that Asp664 is critical for the downstream pathological consequences of Aβ and it is likely to be either cleavage or protein interaction site. The results obtained by Galvan et al. (2005) lends support to the new proposed model of AD, where Aβ binds and oligomerizes APP leading to cleavage at Asp664 and neurotoxicity (23).


References:

(1)Lesli RA. Imaging Alzheimer’s disease in vivo: not so implaque-able anymore. Trends Neurosci. 2002; 25(5):232-233.
(2)Bowen DM et al. Classical neurotransmitters in Alzheimer’s disease. In: Terry RD (ed) Aging and the Brain. Raven Press, New York 1988; 115-128.
(3)Checler F et al. Alzheimer’s and Prion diseases: distinct pathologies, common proteolytic denominators. Trends Neurosci. 2002; 25(12):616-620.
(4)Citron M et al. Mutations of the β-amyloid precrusor protein in familial Alzheimer’s disease increase β-protein production. Nature 1992; 360: 672-674.
(5)Levy-Lahad E et al. Candidate gene for the chromosome I familial Alzheimer’s disease locus. Science 1995; 269:973-977
(6)Sherrington R et al. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer’s disease. Nature 1995; 375: 754-760.
(7)Poirier J et al. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 1993; 342: 697-699.
(8)Selkoe DJ et al. Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 1999; 399: A23-31.
(9)Kimberly WT et al. The transmembrane asparatases in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid-beta protein generation. J. Biol. Chem. 2000; 275: 3173-3178.
(10)Wolozin B et al. Regulation of apoptosis by presenilin 1. Neurobiol. Aging 1998; 19: 523-527.
(11)Buxbaum JD et al. Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the level of a presenilin fragment. Nature Med. 1998; 4: 1177-1181.
(12)Drouet B et al. Molecular basis of Alzheimer’s disease. Cell Mol. Life Sci. 2000; 57: 705-715.
(13)Marques MA et al. Apolipoprotein E-mediated neurotoxicity as a therapeutic target for Alzheimer’s disease. J. Mol. Neurosci. 2003; 20: 327-337.
(14)Lee Y et al. Apolipoprotein E protects against oxidative stress in mixed neuronal-glial cell cultures by reducing glutamate toxicity. Neurochem. Int. 2004; 44: 107-18.
(15)Jordan J et al. Isoform specific effect of apolipoprotein E on cell survival and β-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures. J. Neurosci. 1998; 18: 195-204.
(16)Guela C et al. Aging renders the brain vulnerable to amyloid β-protein neurotoxicity. Nature Med. 1998; 4: 827-831.
(17)Frank RA et al. Biological markers for therapeutic trails in Alzheimer’s disease proceedings of the biological markers working group; NIA on neuroimaging in Alzheimer’s disease. Neurobiol. Aging 2003; 24: 521=536.
(18)Reiter RJ. Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J. 1995; 9: 526-533.
(19)Brown GC, Bal-Price A. Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol. Neurobiol. 2003; 27: 325-355.
(20)Gervais, F. G., Xu, D., Robertson, G. S., Vaillancourt, J. P., Zhu, Y., Huang, J., LeBlanc, A., Smith, D., Rigby, M., Shearman, M. S., et al. Involvement of Caspases in Proteolytic Cleavage of Alzheimer’s Amyloid-β Precursor Protein and Amyloidogenic Aβ Peptide Formation Cell 1999; 97: 395–406.
(21)Lu, D. C., Rabizadeh, S., Chandra, S., Shayya, R. F., Ellerby, L. M., Ye, X., Salvesen, G. S., Koo, E. H. & Bredesen, D. E. A second cytotoxic proteolytic peptide derived from amyloid β-protein precursor. Nat. Med.200; 6: 397–404.
(22)Galvan, V., Chen, S., Lu, D., Logvinova, A., Goldsmith, P., Koo, E. H. & Bredesen, D. E. Caspase cleavage of members of the amyloid precursor family of proteins. J. Neurochem.2002; 82: 283–294.
(23)Lu, D. C., Shaked, G. M., Masliah, E., Bredesen, D. E. & Koo, E. H. Amyloid βprotein toxicity mediated by the formation of amyloid-β protein precursor complexes. Ann. Neurol. 2003; 54: 781–789.
(24)Hsia, A. Y., Masliah, E., McConlogue, L., Yu, G.-Q., Tatsuno, G., Hu, K., Kholodenko, D., Malenka, R. C., Nicoll, R. A. & Mucke, L. Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc. Natl. Acad. Sci. USA 1999; 96: 3228–3233.
(25)Mucke, L., Masliah, E., Yu, G. Q., Mallory, M., Rockenstein, E. M., Tatsuno, G., Hu, K., Kholodenko, D., Johnson-Wood, K. & McConlogue, L. High-Level Neuronal Expression of Aβ1-42 in Wild-Type Human Amyloid Protein Precursor Transgenic Mice: Synaptotoxicity without Plaque Formation. J. Neurosci. 2000; 20: 4050–4058.
(26)Terry, R. D., Masliah, E., Salmon, D. P., Butters, N., DeTeresa, R., Hill, R., Hansen, L. A. & Katzman, R. Physical basis of cognitive alterations in alzheimer's disease: Synapse loss is the major correlate of cognitive impairment. Ann. Neurol.1991; 30, 572–580.
(27)Streit, W. J. Microglia and Alzheimer's disease pathogenesis. J. Neurosci. Res. 2004; 77: 1–8.
(28)Morris, R. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 1984; 11: 47–60.
(29)Hsiao, K. K., Borchelt, D. R., Olson, K., Johannsdottir, R., Kitt, C., Yunis, W., Xu, S., Eckman, C., Younkin, S., Price, D., et al. Age-related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins. Neuron 1995; 15: 1203–1218.