A hallmark of Alzheimer's disease (AD) pathology is the appearance of senile plaques, which are composed of β-amyloid (Aβ) peptides. Aβ is produced by sequential cleavages of amyloid precursor protein (APP) by β- and γ-secretases. These cleavages take place in endosomes during intracellular trafficking of APP through the endocytic and recycling pathways. Genome-wide association studies have identified several risk factors for late-onset AD, one of which is CD2-associated protein (CD2AP), an adaptor molecule that regulates membrane trafficking. Although CD2AP's involvement in APP trafficking has recently been reported, how APP trafficking is regulated remains unclear. We sought to address this question by investigating the effect of CD2AP overexpression or knockdown on the intracellular APP distribution and degradation of APP in cultured COS-7 and HEK293 cells. We found that overexpression of CD2AP increases the localization of APP to Rab7-positive late endosomes, and decreases its localization to Rab5-positive early endosomes. CD2AP overexpression accelerated the onset of APP degradation without affecting its degradation rate. Furthermore, nutrient starvation increased the localization of APP to Rab7-positive late endosomes, and CD2AP overexpression stimulated starvation-induced lysosomal APP degradation. Moreover, the effect of CD2AP on the degradation of APP was confirmed by CD2AP overexpression and knockdown in primary cortical neurons from mice. We conclude that CD2AP accelerates the transfer of APP from early to late endosomes. This transfer in localization stimulates APP degradation by reducing the amount of time before degradation initiation. Taken together, these results may explain why impaired CD2AP function is a risk factor for AD.
Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death. Cyclin-dependent kinases (Cdks)2 are a family of Ser/Thr kinases that are activated by binding a regulatory subunit called cyclin. Most members of Cdks are expressed in proliferating cells to promote cell cycle progression (1). In contrast, Cdk5 is activated by p35 or p39 non-cyclin proteins, which are mainly expressed in post-mitotic neurons (2). Cdk5 is a versatile kinase that is involved in many neuronal activities, including neuronal cell layer formation, synaptic transmission, membrane trafficking, and neuron cell death (3). p35 and p39 appear to share common and/or distinct functions for Cdk5, with p35 being the predominant activator. This is shown by the phenotypes of knock-out (KO) mice; p35 KO mice display abnormal neural layers in the cerebral cortex (4), and p39 KO mice do not show apparent abnormalities, whereas p35 and p39 double KO mice are perinatal lethal with abnormal neural layers, as are the Cdk5 KO mice (5-7). To understand the precise function of Cdk5-p35 in various neuronal activities, it is important to reveal the regulation mechanism of Cdk5 activity.As well as being cell cycle Cdks, Cdk5 is a stable protein and is expressed more abundantly than p35 in neurons (8, 9). Therefore, Cdk5 activity is determined primarily by the available amount of activator protein p35, and the protein amounts of p35 are regulated by the balance between synthesis and degradation (2). Although the synthesis of p35 is stimulated by NGF or BDNF (10, 11), the...
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