Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the presence of amyloid plaques mainly composed of Aβ peptide (Glenner and Wong 1984), neurofibrillary tangles (Goedert et al. 1992), and synaptic and neuronal loss in distinct brain areas, including the neocortex and hippocampus (Hyman et al. 1984; Masliah et al. 1991; Terry et al. 1991). These changes are accompanied by severe disruption of both axonal and dendritic cytoskeleton leading to failure in neuritic transport, and consequently impaired traffic of neurotransmitters and neuropeptides essential for neuronal viability and function. Axonal abnormalities precede amyloid deposition in some AD mouse models, suggesting that axonal defects play a crucial role in the earliest stages of AD pathogenesis (Stokin et al. 2005). AD axonal pathology includes atypical axonal accumulations of the amyloid precursor protein (APP) (Cras et al. 1991) and its fragments (Sennvik et al. 2004; Takahashi et al. 2004), suggesting altered APP transport and processing.
Amyloid precursor protein proteolytic processing occurs in specific subcellular compartments and trafficking routes. Cleavage by α-secretase takes place preferentially in the secretory anterograde pathway, while proteolysis by β-secretase is favored in the retrograde endocytic pathway. Thus, alterations in the intracellular transport of APP likely affect it fate and the balance between alternative processing pathways, provoking differences in the proteolytic fragments produced. As APP anterograde transport has been associated with kinesin-1, and thus dependent on microtubule tracks, APP vesicular trafficking is susceptible to cytoskeleton network alterations. Interestingly, Aβ was reported to induce cytoskeleton reorganization and morphological alterations in astrocytes (Salinero et al. 1997). In addition, previous studies from our laboratory in a non-neuronal cell line demonstrated that Aβ affects the release of the neuroprotective sAPPα fragment leading to intracellular sAPP (isAPP) retention in cytoskeleton-associated vesicular-like densities (Henriques et al. 2009b). Consequently, high density clusters are clearly visible in the cytoplasm. We also observed that isAPP retention occurs in non-neuronal, neuronal-like cells and primary cultures, although sAPP secretion was affected to different degrees. Hence, and as APP vesicular trafficking has been associated with kinesin-driven transport along the microtubule network, we characterized Aβ-induced effects on neuronal APP/sAPP vesicular trafficking and relate these to altered cytoskeleton network dynamics.
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- Materials and methods
- Supporting Information
Given its ability to trigger a set of biochemical and cellular alterations, Aβ has been described as a key player in the amyloid cascade hypothesis leading to progressive neurotoxicity and neuronal death observed in AD (Hardy and Higgins 1992; Hardy and Selkoe 2002). However, besides the well described neurotoxic/apoptotic effects, Aβ also provokes alterations in APP metabolism (Davis-Salinas et al. 1995; Schmitt et al. 1997; Carlson et al. 2000). Previous work from our laboratory demonstrated that Aβ exerts an effect on APP trafficking/processing leading to isAPPα accumulation in cytoskeleton-associated vesicular-like structures in a non-neuronal cell line. Noticeably, this isAPP cleavage and retention was likewise observed in primary hippocampal cultures and PC12 cells (Henriques et al. 2009b). We have also detected an increase in APP CTFs in primary neuronal cultures (Henriques et al. 2009a), again suggesting enhanced intracellular hAPP cleavage. Nonetheless, differences could be observed at the level of sAPP secretion between these cell types. An important question regarding the specificity and the origin of this potentially pathological intracellular sAPP retention, as well as the cellular structures mediating this Aβ response in neurons and in PC12 cells arises. In the work here described we show Aβ-induced inhibition of sAPP secretion, suggesting an Abeta blocking effect on vesicular secretion. This effect was considerable in both primary cortical and hippocampal cultures, and was not generalized, as other medium secreted proteins were unaffected. Furthermore, we also observed that isAPP retention was associated with cytoskeleton structures, as previously shown for non-neuronal cells (Henriques et al. 2009b).
It is well established that vesicular motility and exocytosis are intimately associated with the cytoskeleton network (Meyer and Burger 1979; Hamm-Alvarez and Sheetz 1998; Lanzetti 2007; Potokar et al. 2007), and in the light of our data, disruption of this system may explain the observed intracellular sAPP retention and decreased secretion. It appears then, that as a response to Aβ exposure, sAPP can be intracellularly produced and retained in cytoskeleton-associated secretory vesicles. This hypothesis is supported by co-immunocytochemistry studies locating isAPP retention to KLC-positive secretory vesicles in Aβ treated cells. Our data clearly shows that isAPP and KLC localize to the same subcellular structure, in accordance with either a direct or indirect interaction between KLC and APP in neuritic anterograde transport.
As vesicular transport is related to the cytoskeleton network, hindered APP/sAPP vesicular transport induced by Aβ could be a consequence of altered cytoskeleton dynamics. Indeed, we have demonstrated that Aβ affects the dynamics of both actin and tubulin networks, thus providing a mechanism for sAPP retention. Further, our data clearly show that Aβ alters vesicular trafficking by affecting cytoskeleton networks. As observed with Aβ withdrawal, drugs that alter cytoskeleton dynamics were able to partially reverse esAPP secretion, even in a short period (3 h). These data correlate sAPP retention with hindered cystoskeleton dynamics and indicates that Aβ induced-effects can be counteracted. Aβ impacts on actin polymerization, and by reversing the polymerization state with cytD, the Aβ inhibition of esAPP secretion could be reversed, although more efficiently in PC12 cells. In these cells, protein secretion is highly dependent on the actin cytoskeleton, whereas in primary cultures protein/neuritic transport is mainly dependent on the microtubule network. This may explain the different degree to which the cytoskeleton modulating drugs reverse the Aβ effect on esAPP secretion in the different cell lines.
Aβ25–35 was previously shown to affect axonal transport by inducing neuronal actin polymerization and aggregation (Hiruma et al. 2003), and Abeta induction of local actin polymerization may impair vesicular exocytosis. Induction of F-actin polymerization by fibrillar Aβ1–42 was reported by Mendoza-Naranjo et al. (2007) in hippocampal neurons and related to increased activity of Rac1/Cdc42 Rho GTPases induced by this peptide. In addition, we have previously shown that Aβ can inhibit PP1 (Vintem et al. 2009), a phosphatase involved in many signaling cascades, including regulation of the actin/microtubule dynamics. Cofilin is an actin-binding protein whose regulation is critical to actin polymerization, and is regulated by protein phosphorylation and Protein Phosphatase 1 (PP1) , which as mentioned can be influenced by Aβ.
Presently, we hypothesize that Aβ effects on the microtubule network also has consequences on protein trafficking. The microtubule stabilizing drug, taxol, was able to attenuate the Aβ inhibition of sAPP release in PC12 cells. Even so, the resulting response in terms of the levels of esAPP and taxol reversal, specific to different cell lines, deserves further investigation. More interestingly, tubulin acetylation was also found compromised upon Aβ exposure, which in turn may affect APP microtubule mediated transport (Reed et al. 2006; Gardiner et al. 2007). It is consensual that α-tubulin acetylation is an indirect measure of the amount of the tubulin polymer and of microtubule stability, not conferring stability in itself (Black et al. 1989; Bloom 2004). Of note, Gardiner et al. (2007) suggested that increased α-tubulin acetylation was associated with enhanced transport along microtubules and Reed et al. (2006) showed that α-tubulin acetylation can influence the binding and the motility of the microtubule motor protein kinesin 1. In addition, work by Dompierre et al. (2007) suggested that hyperacetylation of neuronal tubulin leads to the release of neurotrophic factors-containing vesicles, via recruitment of motor proteins to microtubules, highlighting the importance of tubulin acetylation in vesicular secretion. The degree of tubulin acetylation is cell type specific, increasing with cell specialization (Black and Keyser 1987), and results point to a major role for α-tubulin acetylation in APP/sAPP neuritic transport. In fact, when Aβ is withdrawn in the last 3 h, the levels of α-tubulin acetylation recovered to basal levels (Fig. S2), concomitantly with esAPP secretion. Possible underlying mechanisms include Abeta stimulation of histone deacetylase 6 activity, rendering decreased levels of acetylated tubulin and lower rate of vesicular trafficking. In addition, we cannot exclude that Abeta may also be affecting microtubule polymerization and/or stabilization via Tau hyperphosphorylation (Ekinci and Shea 2000; Town et al. 2002), or other microtubule-associated proteins, such as MAP1b, which cross-talk between actin and microtubule dynamics.
In conclusion, in neurons, Aβ impairs APP/sAPP vesicular anterograde transport and exocytosis, in a mechanism mediated by altered cytoskeleton dynamics of both microtubule and actin networks. Aβ-mediated mechanisms leading to cytoskeleton abnormalities and impaired protein vesicular secretion consequently contribute to AD neurodegeneration. Microtubule destabilization has been reported to be associated with neurotoxicity, and Aβ-induced neurodegeneration could be prevented by microtubule stabilization drugs (Michaelis et al. 1998, 2005; Seyb et al. 2006). An important additional consequence of the Aβ effects here reported is altered APP/sAPP neuritic transport and decreased sAPP secretion. As extracellular sAPP has potential neurotrophic and neuroprotective properties, its depletion is of extreme importance in a background of neuronal damage and loss as in AD.
- Top of page
- Materials and methods
- Supporting Information
Figure S1. Levels of F-actin polymerization in response to Aβ peptides. Intracellular levels of F-actin polymers were evaluated in PC12 cells following 24 h exposure to different Aβ peptides. Cells homogenates were resolved by native gel electrophoresis and filamentous actin detected following 2 h gel incubation with AlexaFluor568-conjugated phalloidin. Analysis was achieved using Molecular IMAGE FX and Quantity One densitometry software (Bio-Rad). Fold increase, compared to control levels are also presented. Non-previously aggregated species: Aβ25-35, Aβ1-40, Aβ1-42 and Scrb (control scrambled Aβ25-35 peptide). Aβ1-40 species, which have the lowest aggregation capacity in solution, were also tested after being previously aggregated for 48 h (Aβ1-40 Ag).
Figure S2. Levels of α-tubulin acetylation in response to Aβ. Intracellular levels of α-tubulin acetylation were evaluated in primary neuronal cultures and PC12 cells following Aβ treatment (n = 2). Fold increase when compared to control levels are also presented. C, control cells; Aβ, cells exposed to Aβ for 24 h; Aβ-Aβ, cells exposed to Aβ for 21 h, and further incubated in Aβ-free medium for 3 h.
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