Cystatin C is released in association with exosomes: A new tool of neuronal communication which is unbalanced in Alzheimer's disease
Introduction
Alzheimer's disease (AD) is the most common form of dementia in humans and is characterized neuropathologically by the extracellular deposition of insoluble amyloid fibrils as amyloid plaques, primarily composed of amyloid-β peptides (Aβ) (Selkoe, 1989). Mutations in the presenilin (PS) genes account for the majority of familial Alzheimer's disease (FAD) cases (http://www.molgen.ua.ac.be/ADmutations). The majority of PS mutations result in increased production of Aβ peptides, which are derived from the larger amyloid-β precursor protein (APP). Although FAD-linked PS mutations cause increased generation of Aβ42, a large and increasing number of FAD-linked PS mutations have been shown to inhibit other PS activities. It has been recently reported that presenilins are essential for regulating neurotransmitter release (Zhang et al., 2009), suggesting that PS mutations have pathological effects beyond those caused by their abnormal proteolytic function.
Some proteins associated with AD lesions may have a role in the pathological processes leading to amyloidogenesis and neuronal degeneration and others may bind secondarily to amyloid deposits. Neuropathological and molecular studies suggest a functional link between Aβ and cystatin C (Kaeser et al., 2007, Levy et al., 2001, Mi et al., 2007, Sastre et al., 2004, Vinters et al., 1990). The physiological high concentration of cystatin C in the cerebrospinal fluid and its proliferative effect on neural rat stem cells (Taupin et al., 2000) strongly suggest that cystatin C could exert a trophic function in the brain. While Aβ is the major amyloid forming peptide in the brains of AD patients, the cysteine protease inhibitor, cystatin C, co-localizes with Aβ predominantly in amyloid-laden vascular walls, and in senile plaque cores of amyloid in brains of patients with amyloidoses (i.e. AD, Down syndrome, cerebral amyloid angiopathy, hereditary cerebral hemorrhage with amyloidosis Dutch type and cerebral infarction) and nondemented aged individuals (Levy et al., 2006).
New evidence shows that neurons and astrocytes have the capacity to secrete membrane proteins by incorporating them into exosomes, which are small vesicles derived from the intralumenal membranes of multivesicular bodies (Fauré et al., 2006). From their original discovery in the removal of unwanted proteins from maturing reticulocytes through their role in immune surveillance, the scope of discovered functions continues to grow (Belting and Wittrup, 2008, Février and Raposo, 2004, Schorey and Bhatnagar, 2008, Vella et al., 2008). It has been recently demonstrated that also proteins and peptides (i.e. APP, APP C-terminal fragments, APP intracellular domain, Aβ, PS) associated with AD are released in association with exosomes (Rajendran et al., 2006, Sharples et al., 2008, Vingtdeux et al., 2007). The identification of Aβ in association with exosomes is an important finding especially since other exosomal proteins, such as alix and flotillin, have been found to accumulate in the plaques of AD brains (Rajendran et al., 2006). These findings could provide potential explanation for extracellular amyloid deposition in the brain. We recently reported that FAD-linked PS2 mutations (PS2 M239I and T122R) (Binetti et al., 2003) alter cystatin C trafficking in mouse primary neurons reducing secretion of its glycosylated form (Ghidoni et al., 2007). Here we investigated the link between cystatin C and exosomes in an in vitro model of FAD.
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Neuronal cultures and transfections
Mouse primary cortical neuronal preparation and transfections were performed as previously described (Benussi et al., 2005): after 4 days in culture, neurons were transfected using pcDNA3 void vector or pcDNA3 constructs containing human PS2 wt, PS2 M239I or T122R mutation cDNAs. Forty-eight hours after transfection, cells were lysed and conditioned media collected.
Isolation of exosomes from cell culture media
Exosomes from 3 to 4 × 107 neurons were prepared as described elsewhere (Théry et al., 2006). Briefly, conditioned media were collected
Cystatin C is released in association with exosomes
To test whether cystatin C is released in association with exosomes, we isolated these vesicles from conditioned media of mouse primary neurons through serial centrifugations and density gradient centrifugation. TSG101 and flotillin were used as marker proteins to identify exosome-containing fractions (Fig. 1A). Western blot analyses with antibodies directed against AD-associated proteins showed the presence, in these vesicles, of PS2 and APP along with cystatin C (Fig. 1A). Electron microscopy
Discussion
Alzheimer's disease is characterized by a continuous loss of neurons that are not replaced and the cause of neuronal death in AD-affected brain regions is still a matter of discussion. It was recently demonstrated that proteins associated with neurodegenerative disorders (such as AD and prion disease) can be selectively incorporated into intraluminal vesicles of multivesicular bodies and subsequently released into the extracellular environment, enriched within exosomes (Vella et al., 2008).
Disclosure statement
The authors declare having no actual or potential conflicts of interest in regards to this research.
Acknowledgments
This work was supported by the grants: ERA-Net NEURON JTC 2008 nEUROsyn; Ricerca Corrente, Italian Ministry of Health; National Institute on Aging (AG017617), and the Alzheimer's Association (IIRG0759699).
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