Cellular and animal models for high-throughput screening of therapeutic agents for the treatment of the diseases of the elderly in general and Alzheimer's disease in particular(†)

Abstract

It is currently thought that the dementia of Alzheimer's disease is due to the neurotoxicity of the deposits or aggregates of amyloid-β (Aβ) in the extracellular space of the cerebral cortex. This model has been widely criticized because there is a poor correlation between deposits and dementia. Others have questioned whether Aβ is truly neurotoxic. Yet, in spite of these concerns, the search for therapeutic agents has been based on the development of mouse models transfected with mutant genes associated in humans with early onset Alzheimer's disease. A major limitation of these models is that although they exhibit many of the pathological and clinical manifestation of the human disease, the bulk of individuals who develop the dementia of Alzheimer's disease have none of these mutant genes. Furthermore, nine clinical trials of drugs that were effective in transgenic mice failed to show any benefit in patients. Finally, a major unresolved issue with the Aβ model is that since Aβ is produced in everyone, why are deposits only seen in the elderly? This issue must be resolved if we are to understand the etiology of the disease and develop test systems for both diagnosis and drug discovery. Published studies from my laboratory demonstrate that in human cerebrospinal fluid immunoreactive Aβ is only present as a complex with two chaperones, ERp57 and calreticulin and is N-glycosylated. This complex formation is catalyzed by the posttranslational protein processing system of the endoplasmic reticulum (ER). Others have reported that in plaque Aβ is present only as the naked peptide. Together these results suggest that both plaque and dementia are secondary to an age related decline in the capacity of the ER to catalyze protein, posttranslational processing. Since the synaptic membrane proteins necessary for a functioning memory are also processed in the ER, these findings would suggest that the loss of cognition is due to a decline in the capacity of the neuron to produce and maintain functioning synapses. Work from my laboratory and from others further indicate that the components of the ER, posttranslational, protein processing pathway do dramatically decline with age. These data suggest that this decline may be found in all cells and could account not only for the dementia of Alzheimer's disease, but also for many of the other manifestations of the aging process. These observations also suggest that declining ER function has a role in two well-recognized phenomena associated with aging: a loss of mitochondrial function and a decrease in myelin. Finally, based on this paradigm I propose new cellular and animals models for high-throughput screening for drug discovery.

Keywords: N-glycosylation; amyloid-β; chaperones; dementia; protein processing.

FIGURE 1

Amino acid sequence of APP.

FIGURE 2

The effect of Ginkgo biloba on Cognitive Function in the GEM Trial (taken from DeKosky et al., 2008).

FIGURE 3

The effect of estrogen alone on Cognitive Function in the Women’s Health Initiative (taken from Espeland et al., 2004).

FIGURE 4

Western blots of human, CSF with antibodies to ERp57 and Aβ.

FIGURE 5

Immunoopurification of the Aβ-ERp57 Complex from Human CSF. Aβ and ERp57 were isolated by immunoprecipitation. The samples were then purified by western blotting on sodium docecyl sulfate-polylacrylamide gel electrophoresis. The bands were identified by antibodies to ERp57 (A) and Aβ (B). (Taken from Erickson et al., 2005)

FIGURE 6

Western blot of the Aβ–ERp57–calreticulin complex from human CSF after separation on a native gel (taken from Erickson et al., 2005).

FIGURE 7

Immunoblot of Aβ in CSF after treatments to dissociate the Aβ–ERp57 complex. Channel 1 – molecular weight markers; channel 2 – untreated CSF; channel 3 – CSF treated with 70% formic acid; channel 4 – CSF treated with 80% trifluoroacetic acid; channel 5 – CSF treated with 6 M guanidine isothiocyante; channel 6 – CSF treated with 6 M urea; channel 7 – CSF proteins which did not bind to a boronate column; channel 8 – CSF proteins which bound to a boronate column; channel 9 – treatment of the complex with glycine buffer, pH 9.0 (taken from Erickson et al., 2005).

FIGURE 8

The effect of age on the ERp57 content of rat, hepatic microsomes (taken from Erickson et al., 2006).

FIGURE 9

The N-glycosylation pathway (Helenius and Aebi, 2002).

FIGURE 10

The tethering of the ER to the mitochondria (taken from Merkwirth and Langer, 2008).

FIGURE 11

Schwann cell.

FIGURE 12

The effect of feeding of methoxychlor for 3 weeks on the hepatic level of ERp57 (data taken from Morrell et al., 2000).