CD2AP in Alzheimer's disease: Key mechanisms and therapeutic potential

CD2AP Single-Nucleotide Polymorphisms Are Associated with AD

In 2011, Naj et al. and Hollingworth et al. independently conducted genome-wide association studies (GWAS) using large samples and consistently identified that the single-nucleotide polymorphism (SNP) rs9349407 in CD2AP intron 1 significantly associated with LOAD (8, 9). Subsequent replicative studies reported controversial results on the association between rs9349407 and AD, with both positive (40–42) and negative (43–46) correlations suggested. However, most negative studies used relatively small sample sizes. Another work studying deceased individuals who underwent complete neuropathological evaluations found that the rs9349407 locus was related to neuritic plaque pathology (47).

The work by Hollingworth et al. further identified that the SNP rs9296559 in CD2AP intron 1 was also associated with LOAD (9), and this association was confirmed in the southern Han Chinese population (42). Other studies identified additional SNPs in the CD2AP gene to associate with AD, such as rs10948363 in CD2AP intron 2 (48, 49) and rs116754410 in CD2AP exon 18 (50), of which the latter results in a missense mutation (K633R) in CD2AP. The amino acid K633 is located in the coiled-coil domain of CD2AP, and one recent study found that CD2AP K633R overexpression in neurons could increase spine density and volume. In contrast, its overexpression failed to rescue impaired spine density in CD2AP-deficient neurons (37). Herein, we also evaluated the conservation of K633 and found it conserved from humans to zebrafish (Figure 1B), further implicating the importance of K633 for CD2AP function; this requires further investigation. Moreover, The SNP rs9473117 located upstream of CD2AP was reported to be associated with EOAD (51).

CD2AP Expression Alteration in AD

It was reported that CD2AP expression decreased in peripheral blood lymphocytes in patients with LOAD (42). However, we recently noticed that in one proteomic study with large datasets (52), CD2AP protein levels were significantly increased in the brain of patients with AD compared to control subjects and asymptomatic patients with AD (39). We also found that CD2AP levels significantly increased in the hippocampus of the 5xFAD AD model mice at pathological stages but not at pre-pathological stages, and such an increase probably occurred specifically in microglia (39). Our findings implicate that CD2AP alterations in AD occur after pathological changes. Consistent with our results, an earlier study also reported that CD2AP levels were increased in the brain of the APP/PS1 AD model mice (53).

Although several SNPs around CD2AP have been reported to be highly associated with AD, only a few studies have explored the effects of some of these SNPs on CD2AP expression so far. In a study analyzing human brain gene expression data, researchers found that the minor allele of rs9349407 was only associated with decreased CD2AP expression in the cerebellar cortex (54). While the study showed that rs9473117 associates with EOAD, the minor allele of rs9473117 was found to associate with increased CD2AP expression in the thalamus and cerebellar cortex (51). In addition, Pavešković et al. leveraged new paired single-nucleus RNA-sequence and whole genome sequencing data and found that the risk variant of rs9473117, together with those of rs7767350 and rs9369716 that are in strong linkage disequilibrium with rs9473117, increases human CD2AP mRNA expression in both excitatory and inhibitory neurons, as well as in microglia (36).

CD2AP Regulates APP Transport and Aβ Production

One major pathological feature of AD is the formation of amyloid plaques composed of Aβ, which is derived from APP through sequential cleavages. First, APP is cleaved by the β-secretase (BACE1) to generate soluble extracellular APP (sAPPβ) and a C-terminal fragment (CTFβ). APP CTFβ is then cleaved by the γ-secretase to produce Aβ fragments of 40 or 42 amino acids in length, namely Aβ40 and Aβ42 (55, 56). The cleavage of APP is determined by the subcellular localization of APP and its interactions with BACE1 and γ-secretase during membrane, endosome, and lysosome trafficking (57, 58).

CD2AP is an actin-associated adaptor protein that, along with several members of the Rab family, participates in the docking process of vesicles to target membranes and regulates endosome morphology (19). Several studies have suggested that CD2AP is involved in the intracellular transport and cleavage of APP for Aβ production. Furusawa et al. found that overexpression of CD2AP at the cellular level accelerated the transport of APP from early endosomes to late endosomes, thus initiating the degradation process of APP without affecting its degradation rate. In contrast, knocking down CD2AP in cells significantly increased intracellular APP levels (59). Ubelmann et al. also showed that CD2AP could keep APP and BACE1 apart in early endosomes in neurons: CD2AP deficiency increased the trapping of APP at the limiting membrane of early endosomes and thus reduced its sorting for degradation in dendrites so that APP and BACE1 had elevated convergence in early endosomes for increased Aβ generation (34). However, Liao et al. reported that in the N2a-APP695 cell line, overexpressing APP, knocking down CD2AP reduced cell membrane APP levels and Aβ release and lowered the Aβ42/Aβ40 ratio. Moreover, they found that in 1-month-old APP/PS1 mice with complete loss of CD2AP, the Aβ42/Aβ40 ratio was decreased. While, CD2AP haploinsufficiency had no effect on Aβ deposition up to 7 months of age in APP/PS1 mice (60). One recent study also reported that in APP/PS1 mice with neuron-specific CD2AP knockout, Aβ levels were not altered at 4.5 months of age (61). Due to the inconsistency in different studies, the specific role of CD2AP in APP processing and Aβ requires further in-depth investigation. Table 1 summarizes reported studies on the effects of CD2AP on APP trafficking/Aβ generation and other AD-related processes.

CD2AP Modulates Tau-mediated Neurotoxicity

Neurofibrillary tangles formed by hyperphosphorylated Tau protein represent another important pathological feature of AD. In one study using immunohistochemical assays to explore the association between CD2AP expression and AD pathologies in postmortem human brain samples, CD2AP was found not associated with Aβ deposits in vessels or parenchymal plaques. Instead, CD2AP immunodetection in neurons was positively associated with Braak neurofibrillary stage (62). Furthermore, the AD-associated CD2AP SNP rs10948363 was found to associate with NFTs (63), the AD-associated CD2AP SNP rs9349407 was found to associate with an increase in total Tau protein levels in cerebrospinal fluid, and the rs9381563 variant in CD2AP was correlated with changes in phosphorylated Tau levels in cerebrospinal fluid (64).

CD2AP also affects the neurotoxicity caused by Tau. In a fruit fly AD model expressing human Tau, researchers found that knockdown of the cindr gene (the human homolog of CD2AP) robustly enhanced Tau toxicity (65). Xue et al. also found that the specific knockout of CD2AP in neurons in APP/PS1 mice resulted in a significant increase in the phosphorylation levels of endogenous Tau protein. In contrast, the total Tau protein levels remained unchanged (61). Additionally, the deficiency of CD2AP led to elevated P38 phosphorylation levels in these mice. Treatment with P38 phosphorylation inhibitors attenuated the increased phosphorylation of endogenous Tau protein caused by the loss of CD2AP (61). It is known that P38 can phosphorylate Tau (66, 67). However, although some studies consistently reported that high glucose and TGFβ treatments decreased CD2AP while increased P38 phosphorylation (68, 69) and that CD2AP deficiency enhanced TGFβ-induced P38 activation (70), the precise mechanism by which CD2AP influences P38 phosphorylation remains unclear and deserves further scrutiny.

CD2AP Regulates Synaptic Growth and Development

Synapses are the core structures for signal transmission between neurons, and abnormalities in synaptic function are closely linked to cognitive impairment. In the early stages of AD, synaptic dysfunction appears, although the number of synapses does not significantly change at this stage. However, in the mid to late stages of AD, there is a significant loss of synapses and neuronal death, leading to a sharp decline in cognitive abilities (71–76). Ojelade et al. found that the cindr gene was involved in the development and maturation of synapses during the growth of fruit flies. Mutations in cindr disrupted the release and recycling of synaptic vesicles, impairing synaptic plasticity (38). They also noticed decreased levels of some synaptic proteins in CD2AP knockout mice (38). Additionally, they showed that cindr interacted with 14-3-3ζ to regulate the ubiquitin proteasome system, thereby affecting the conversion of synaptophysin and plasma membrane calcium ATPase (PMCA); and the absence of cindr increased PMCA levels and decreased cytosolic calcium (38). Research by Mirfakhar et al. found that CD2AP knockdown in neurons resulted in decreased spine density and size and decreased neuronal activity (37). They also proposed that CD2AP controls the formation and growth of dendritic spines by regulating the balance of F-actin polymerization and depolymerization (37). Pavešković et al. also observed a marked reduction in the number of synapses and impaired synaptic plasticity in the hippocampus of CD2AP-deficient mice (36). In addition, CD2AP was suggested to regulate the length and complexity of neuronal axons and the number of filopodia at growth cones through coordinating nerve growth factor signaling (77). Moreover, we recently found that specific deletion of CD2AP in microglia attenuated synaptic damage in 5xFAD mice at pathological stages, though this is attributed to reduced synapse phagocytosis by CD2AP-deficient microglia (39). These findings collectively suggest that CD2AP plays a crucial role in synaptic development in neurons, and its alteration may contribute to synaptic dysfunction in AD. A summary of the regulation of APP, Tau, and synapses in neurons by CD2AP is illustrated in Figure 2.

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CD2AP Regulates Microglial Activity

In addition to Aβ plaques and Tau tangles, AD is also characterized by neuroinflammation. Microglia are primary immune cells of the brain and are activated in response to toxicity to exert protective function during the early stages of AD. However, in the later stages of AD, excessively activated microglia can exacerbate disease progression by releasing toxic proinflammatory factors and phagocytosing functional synapses (78–81). CD2AP was initially discovered as a ligand for CD2 in T cells, implicating its significant role in immune cells (12). We recently found that CD2AP deficiency reduced microglial response to Aβ and microglial phagocytosis ability. Notably, we showed that in contrast to the deteriorating effect of neuronal knockout of CD2AP in APP/PS1 mice (61), microglial CD2AP deficiency attenuated cognitive defects, synaptic damage, and disease-associated microglia (DAM) in 5xFAD mice at pathological stages (39). We further suggested that one possibility for such a rescuing effect was that CD2AP could interact with the critical microglial survival factor, colony stimulating factor 1 receptor (CSF1R), so that CD2AP deficiency ameliorated the CSF1R signaling–mediated microglial activation and downstream expression of the C1q complement, which is crucial for synapse phagocytosis, and the formation of DAMs triggered by toxic Aβ (Figure 3) (39). The seemingly contradictory data regarding the effects of microglial CD2AP and neuronal CD2AP on Aβ pathologies are likely stem from distinct action mechanisms in each investigated cell type. However, it is also possible that they are caused by different experimental models used in these studies. However, so far, our understanding of the role of CD2AP in microglia and other cell types lags behind that in neurons. There are urgent requirements for elucidating the pathophysiological functions of CD2AP in different cell types and parallelly comparing their contributions to AD.

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CD2AP Participates in the Integrity of the Blood–Brain Barrier

The blood–brain barrier (BBB) provides a physical barrier to protect the brain from harmful materials in the peripheral environment. The disruption of the BBB can lead to the influx of neurotoxic bloodborne debris, cells, and microbial pathogens into the brain, triggering inflammatory responses and related immune reactions, which may initiate various pathways leading to neurodegenerative diseases such as AD (82–84). MRI studies have shown that the BBB function is impaired in patients with early AD and other neurodegenerative diseases. Analyses of postmortem tissues of AD patient brain tissues also support this conclusion (82). Brain microvascular endothelial cells, which express high levels of CD2AP, are key components of the BBB (85). CD2AP homozygous knockout mice with CD2AP transgene expression in the kidney attenuated their mortality rate. Such mice showed overall normal behaviors. However, they had compromised the integrity of the BBB so intraperitoneally administered pentylenetetrazol increasingly penetrated into the brain to induce seizures in much shorter latency periods in these mice than in controls (85). This finding implicates that abnormal expression of CD2AP in brain microvascular endothelial cells, if any, may lead to BBB dysfunction and facilitate AD progression.