PD is a disabling neurodegenerative disorder that is strongly associated with aging [39, 40]. At the cellular level, postmortem tissues from sporadic patients with PD displayed an increased expression of senescent markers, including p16INK4a, and several senescence-associated secretory phenotype (SASP) factors, matrix metalloproteinase-3 (MMP-3), interleukin (IL)-6, IL-1a, and IL-8 [41]. Additionally, elevated p16INK4a and MMP-3 expression have been reported in cortical tissues of patients with AD [42], suggesting that the basic mechanisms of aging may be closely related to the pathogenesis of neurogenerative diseases such as AD and PD.
Reportedly, cav-1 expression has been associated with aging. Several previous studies using in vitro senescent cells [19] and in vivo aged tissues [17, 18] have suggested that cav-1 expression is increased with aging. Additionally, cav-1 overexpression displays senescence phenotypes [21, 43, 44], suggesting that increased cav-1 expression may play an important role in aging at the cellular level, although there have been conflicting observations that cav-1 knockout accelerates premature senescence in MEFs [45] and loss of cav-1 accelerates neurodegeneration and aging [46].
In the present study, we observed that cav-1 expression was increased in the brain in an aging-dependent manner, which is consistent with previous studies [17, 18]. Moreover, cav-1 expression was considerably elevated in the brain of A53T TG mice. In patients with PD, homozygous haplotypes have been observed upstream of human cav-1, which induce increased gene expression [47]. In vitro, overexpression of α-syn upregulates cav-1 expression [48, 49], supporting our data that demonstrates the association of elevated cav-1 expression with PD.
Accumulating evidence suggests that the mechanism of pathogenic α-syn spreading throughout the nervous system underlies the pathogenesis of PD [50], which remains poorly understood. We observed that cav-1 overexpression in neurons accelerated α-syn uptake into neurons and inclusion body formation via the cell-to-cell transmission of α-syn. It is well known that cav-1 regulates caveolae-dependent endocytosis [51]. Classical caveolae-mediated endocytosis may not occur in neurons as neurons lack caveolae [52, 53]. Nevertheless, a previous study has suggested that cav-1 also demonstrates caveolae-independent functions including trafficking of proteins to and from the plasma membrane [54]. Furthermore, α-syn is internalized into cells via various mechanisms in a species-dependent manner [55–57]. Accordingly, neuronal cav-1 may be involved in α-syn uptake via caveolin-dependent endocytosis. Our hypothesis was supported by the observation that cav-1 overexpression accelerates lipid rafts-dependent endocytosis, but not clathrin-dependent endocytosis. Interestingly, exogenously added α-syn fibrils induce lipid rafts-dependent endocytosis [26], with numerous PD-associated gene products, including parkin, DJ-1, and UCH-L1 also regulating lipid rafts-dependent endocytosis [22, 58, 59], indicating that the dysfunction of lipid rafts-dependent endocytosis may be associated with the pathogenesis of PD as a common pathological mechanism.
The activity of the degradation pathways, both autophagy and proteasome-mediated, are reduced during aging [60, 61]. Reportedly, cav-1 overexpression prevents autophagy in human osteosarcoma cells [62] and cav-1 deletion increases basal autophagy in the vascular endothelium [63]. Considering that α-syn is degraded by autophagy [64–66], we further investigated whether our finding that increased α-syn uptake into cav-1 OE cells resulted from enhanced cellular uptake of transferred α-syn or limited degradation of transferred α-syn following bafilomycin A1 treatment. Relative amounts of transferred α-syn detected in recipient cells were increased in the presence of bafilomycin A1. Inhibition of α-syn degradation induces the accumulation of α-syn, further releasing α-syn into the extracellular space via different secretory pathways [67–69]. Accordingly, α-syn accumulation could be attributed to increased α-syn release by inhibiting the autophagic degradation of α-syn in donor cells, and/or increased accumulation of α-syn in recipient cells. Nevertheless, α-syn was highly detected in cav-1 OE cells, suggesting that cav-1 overexpression enhanced the uptake of transferred α-syn into cells, although we were unable to comprehensively elucidate whether cav-1 overexpression affected the autophagy system to degrade transferred α-syn in our experimental condition.
Given that cav-1 expression increases with age [17, 18] and cav-1 overexpression induces cellular senescence [21, 43, 44], and our observation that cav-1 overexpression accelerated α-syn uptake into neurons and inclusion body formation, α-syn propagation may be further accelerated in aging individuals (Fig. 8), which is consistent with a previous study revealing that aging-promoting genetic variations accelerate the rate of cell-to-cell transmission of α-syn aggregates in a C. elegans model [70].
The phosphorylation of cav-1 at the tyrosine 14 residue is related to macromolecular transcytosis as a specific form of the scaffold to recruit and organize multiple molecular components of the transcytotic machinery [71, 72]. Src-mediated phosphorylation of caveolin-1 Tyr-14 is necessary for caveolar endocytosis of EGFR under oxidative stress [73]. Accordingly, tyrosine 14 phosphorylation of cav-1 may regulate endocytosis in neurons. Furthermore, we observed that Y14A cav-1 overexpression failed to accelerate α-syn uptake into neurons, as well as inclusion body formation. Y14A cav-1 overexpression also failed to enhance LacCer uptake as a representative molecule for lipid rafts-dependent endocytosis, suggesting that the phosphorylation of cav-1 at the tyrosine 14 residue in neurons plays an important role in cell-to-cell transmission of α-syn by regulating lipid rafts-dependent endocytosis.
As cav-1 was first identified as a major phosphorylated protein in v-Src-expressing cells [74], it has been well documented that the tyrosine 14 residue of cav-1 is phosphorylated by Src kinases [30, 31, 75]. We observed that the phosphorylation of cav-1 at tyrosine 14 was induced by α-syn fibrils, but not by α-syn monomers. Moreover, the phosphorylation of cav-1 at tyrosine 14 induced by α-syn fibrils was dependent on c-Src kinase activity. Additionally, the inhibition of cav-1 phosphorylation by regulating c-Src activity attenuated the accelerated α-syn uptake by cav-1 overexpression. Previously, we have demonstrated that α-syn fibrils bind to FcγRIIB expressed in neurons and stimulate the cell-to-cell transmission of α-syn via SHP-1/-2 as a downstream mediator of FcγRIIB signaling [26]. Furthermore, the activation of SHP-1/-2 by α-syn fibrils stimulates c-Src phosphorylation, accelerating the cell-to-cell transmission of α-syn [24]. Accordingly, cav-1 may function as a downstream mediator of FcγRIIB-SHP-1/-2-c-Src for α-syn uptake.
Inhibiting cav-1 upregulation with selective cyclooxygenase (COX)-2 inhibitors attenuates the development of cellular senescence in human dermal fibroblasts [76]. Furthermore, an inhibitor of phosphatidylcholine-specific phospholipase C reduces the upregulation of cav-1 expression, as well as the number of replicative senescent bone marrow stromal cells [77]. Daidzein, known to inhibit cav-1 expression, restores memory deficits in an intracerebroventricular streptozotocin (ICV-STZ)-induced neurodegeneration rat model [78]. Additionally, our data support the postulation that inhibiting the upregulation of cav-1 expression or cav-1 phosphorylation can attenuate the progression of PD, as well as the aging process. In agreement with our hypothesis, reduced cav-1 expression reportedly extended lifespan and mitigated toxic protein aggregation by modulating the expression of age regulating and signaling-promoting genes [79].