Age- and region-dependent alterations in Aβ-degrading enzymes: implications for Aβ-induced disorders

https://doi.org/10.1016/j.neurobiolaging.2004.06.013Get rights and content

Abstract

Accumulation of amyloid β-protein (Aβ) is a fundamental feature of certain human brain disorders such as Alzheimer's disease (AD) and Down syndrome and also of the skeletal muscle disorder inclusion body myositis (IBM). Emerging evidence suggests that the steady-state levels of Aβ are determined by the balance between production and degradation. Although the proteolytic processes leading to Aβ formation have been extensively studied, less is known about the proteases that degrade Aβ, which include insulin-degrading enzyme (IDE) and neprilysin (NEP). Here we measured the steady-state levels of these proteases as a function of age and brain/muscle region in mice and humans. In the hippocampus, which is vulnerable to AD pathology, IDE and NEP steady-state levels diminish as function of age. By contrast, in the cerebellum, a brain region not marked by significant Aβ accumulation, NEP and IDE levels either increase or remain unaltered during aging. Moreover, the steady-state levels of IDE and NEP are significantly higher in the cerebellum compared to the cortex and hippocampus. We further show that IDE is more oxidized in the hippocampus compared to the cerebellum of AD patients. In muscle, we find differential levels of IDE and NEP in fast versus slow twitch muscle fibers that varies with aging. These findings suggest that age- and region-specific changes in the proteolytic clearance of Aβ represent a critical pathogenic mechanism that may account for the susceptibility of particular brain or muscle regions in AD and IBM.

Introduction

Brain and skeletal muscle are the only two human tissues where accumulation of the amyloid β-protein (Aβ) protein is known to have clinicopathological consequences. Accrual of Aβ occurs in selective brain regions in Alzheimer's disease (AD) and in related disorders such as Down syndrome or hereditary cerebral hemorrhage with amyloidosis [30]. Accumulation of the Aβ peptide also occurs in the age-related muscle disorder inclusion body myositis (IBM), a paralytic condition for which there is no known cure [1], [19]. In certain cohorts of patients, Aβ accumulation is known to occur either as a result of increased expression of the amyloid precursor protein (APP) such as in Down syndrome and IBM [28], or from APP misprocessing induced by mutations in genes associated with familial AD [32]. For the remaining cases, however, age- and region-related changes in Aβ clearance may also be part of the underlying pathogenic mechanism. Even in transgenic mice overexpressing high APP levels during early stages of life (see, for example, [23]), Aβ deposits typically develop in a progressive and age-related fashion, supporting the hypothesis that accumulation is dependent on the balance between production and clearance-based mechanisms.

Several proteases have recently been shown to play a role in regulating steady-state levels of cerebral Aβ, and among these, insulin-degrading enzyme (IDE) and neprilysin (NEP) have shown the largest effect and have received the most experimental confirmation [31]. IDE, also called insulysin, is a ∼110 kDa thiol metallo-endopeptidase that degrades small peptides such as insulin and Aβ [31]. An unbiased screen of the conditioned medium of neuronal and non-neuronal cell lines for the ability to degrade naturally secreted Aβ led to the identification of IDE as a major protease that degrades extracellular Aβ [25], [26]. Emerging genetic evidence suggests that IDE is linked to some late-onset familial AD cases [3], [5], [8], [21], [24], although pathogenic mutations have yet to be identified. Neuronal overexpression of IDE suffices to reduce Aβ levels and greatly retard cerebral plaque formation in transgenic mice expressing high levels of APP with the Swedish and Indiana mutations, and the reduction in cerebral Aβ levels is even able to rescue the premature lethality present in this model [17]. By contrast, mice with a homozygous deletion of the IDE gene show a 50% decrease in Aβ degradation in both brain membrane fractions and cultured primary neurons, along with an increase in endogenous Aβ in the brain [9], [20]. Moreover, besides degrading Aβ, IDE also degrades the APP intracellular domain (AICD), the other γ-secretase-generated product of APP [7], [9].

NEP, a membrane-anchored zinc endopeptidase, is another Aβ-degrading protease that has been extensively studied. The injection of radiolabeled Aβ1–42 into the hippocampi of two-month-old rats in the presence or absence of various protease inhibitors indicated that NEP is a major Aβ42-degrading enzyme [14]. Whereas IDE exclusively degrades soluble monomeric but not oligomeric Aβ species [25], NEP is capable of degrading monomeric and oligomeric Aβ [15]. NEP-deficient mice show a significant dose-dependent increase in cerebral Aβ levels, suggesting that even a partial reduction of NEP levels can lead to a buildup of Aβ [13]. This hypothesis is further strengthened by data indicating that NEP levels are reduced in the peripheral organs compared to the brain and within the AD brain NEP levels are lower in high plaques areas [36], [37]. Conversely, overexpression of NEP in vitro and in vivo leads to a reduction in cerebral Aβ levels in a dose-dependent manner [10], [11], [17], [18].

To assess the potential role that IDE and NEP may play in the pathogenesis of Aβ-induced disorders, we analyzed their steady-state levels during aging in different brain and muscle regions. Here we report that the levels of these catabolic enzymes change differentially in brain regions more susceptible to Aβ accumulation as compared to the cerebellum, which is usually devoid of extensive Aβ deposits. We further show that IDE and NEP levels are higher in the cerebellum of humans and mice relative to the hippocampus and cortex. IDE function in the hippocampus may be further limited in the aging brain, as we detect more oxidized IDE in this brain region compared to the cerebellum of AD patients. Given the critical role that IDE and NEP play in regulating Aβ degradation, and by extension, disease progression, our findings suggest that region- and age-specific changes in their levels may account for the sensitivity and resistance of certain brain and muscle regions to Aβ accumulation.

Section snippets

Protein extractions

C57BL6/129 and 3xTg-AD mice [23] were sacrificed by CO2 asphyxiation. Brain (hippocampus, cortex, and cerebellum) and muscle (WMG, plantaris, and soleus) were isolated under a dissecting microscope and snap frozen on dry ice. Human AD and control brains were obtained from the Brain Tissue Repository of the Institute for Aging and Dementia (University of California, Irvine). Tissue was homogenized in 50 mM Tris, pH 8.0 containing 0.7 mg/ml Pepstatin A supplemented with a complete mini protease

Temporal and spatial modulation of IDE and NEP in mouse and human brain

It is well-established that the hippocampus and the cortex are two brain areas highly susceptible to Aβ accumulation in AD, whereas the cerebellum is usually spared. To determine if regional differences in the levels of the proteases that degrade Aβ might contribute to this differential accumulation, we analyzed IDE and NEP levels in the cerebellum, cortex, and hippocampus from wild-type and transgenic mice and from human AD patients and controls.

In the hippocampus, we found that IDE and NEP

Discussion

In this study, we show age- and region-related alterations in the levels of two major Aβ-degrading enzymes in both brain and skeletal muscle. We specifically report the following key observations: (i) IDE and NEP levels decrease in the hippocampus with aging and are elevated in the cerebellum of older mice, (ii) IDE and NEP levels are significantly higher in the cerebellum compared to AD vulnerable regions such as the hippocampus and cortex in both mice and humans, (iii) IDE is more oxidized in

Acknowledgments

We thank Dr. Dennis Selkoe for the anti-IDE antibodies and the Institute for Brain Aging and Dementia (University of California, Irvine) for the postmortem AD and control brain specimens. This work was supported by grants to FML from the Alzheimer's Association and by the National Institutes of Health (AG26175 and AG0212982).

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