Emerging evidence for the neuroprotective role of α-synuclein

Experimental Neurology 200 (2006) 1 – 7 www.elsevier.com/locate/yexnr

Emerging evidence for the neuroprotective role of α-synuclein Hyoung-gon Lee a , Xiongwei Zhu a , Atsushi Takeda b , George Perry a,c , Mark A. Smith a,⁎ a

Department of Pathology, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106, USA b Department of Neurology, Tohoku University School of Medicine, Sendai, Miyagi, Japan c College of Sciences, University of Texas at San Antonio, San Antonio, TX 78249, USA Received 13 January 2006; revised 26 April 2006; accepted 28 April 2006 Available online 14 June 2006

Of the many different types of neurodegenerative diseases affecting various regions of the brain, while the pathogenic mechanisms involved are likely different, one clear commonality is the almost invariant presence of insoluble protein aggregates that occur as both intra- and extracellular forms and, in many cases, contribute towards characteristic pathological lesions found during the disease process—the majority of which are pathognomonic for each disease entity. For example, the aggregation of amyloid-β (Aβ) peptide into extracellular senile plaques and the aggregation of intracellular tau as neurofibrillary tangles (NFT) is an obligate feature of Alzheimer disease (AD) (Smith, 1998). In Parkinson disease (PD), aggregates of αsynuclein contribute to the formation of Lewy bodies, pathological lesions found in the cytoplasm. Other diseases, such as prion disease, progressive supranuclear palsy, frontotemporal dementia, and Huntington disease (HD) are likewise associated with lesions containing disease-specific protein aggregates. Regardless of whether such aggregates are localized to the intracellular or extracellular space, it has generally been suggested that such protein aggregates are cytotoxic and the cause of the disease. Such an assertion is supported by the fact that protein aggregates, such as Aβ, tau and α-synuclein, are cytotoxic to neurons in vitro (Pike et al., 1991; Xu et al., 2002; Yankner et al., 1990) and therefore, it is assumed, are likely cytotoxic in the human brain. However, contrary to this, many recent studies support the idea that the aggregates are actually neuroprotective compensatory responses mounted by neurons against neurotoxic stress (Lee et al., 2004, 2005a,b; Smith et al., 2002). For example, in a cell culture model of HD, inclusion body formation is associated with increased cell survival and decreased levels of huntingtin throughout the neuron (Arrasate

⁎ Corresponding author. Fax: +1 216 368 8964. E-mail address: [email protected] (M.A. Smith). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.04.024

et al., 2004). In fact, in this system, cells that failed to form inclusion bodies were more likely to die. Formation of the inclusion body appeared to decrease cell death by reducing the amount of free huntingtin, a protein with abnormal polyglutamine expansion. Along the same lines, in this issue of Experimental Neurology, Quilty et al. (2006) show that the accumulation of α-synuclein is associated with neuroprotection from chronic oxidative stress. Specifically, they found that chronic oxidative stress in their unique cell culture model does not affect the viability of neurons expressing α-synuclein but instead causes a significant increase in the number of neuronal cells expressing increased level of α-synuclein. Moreover, those neuronal cells expressing high levels of α-synuclein are relatively resistant to apoptotic changes compared with neuronal cells lacking α-synuclein. α-Synuclein in Parkinson disease: toxic or protective? Several lines of evidence suggest that, in most forms of PD, protein aggregates within dopaminergic neurons of the substantia nigra are a common feature. For instance, αsynuclein is present in Lewy bodies in sporadic PD (Choi et al., 2001; Mouradian, 2002). Accumulation of α-synuclein in cultured human cells also causes selective degeneration of dopaminergic neurons in the presence of dopamine, but not in non-dopaminergic neurons, suggesting selective toxicity dependent upon aggregate accumulation (Xu et al., 2002). Also, mice expressing the Ala53Thr human α-synuclein mutation exhibit adult onset neurodegeneration and α-synuclein aggregation in the brain (Lee et al., 2002) and mutations in αsynuclein are associated with inherited forms of PD (Shastry, 2001). While the toxicity of α-synuclein is supported as reviewed above, an alternate interpretation for the role of α-synuclein in PD is emerging. Indeed, while the in vitro aggregation of αsynuclein results in the selective degeneration of dopaminergic neurons (Lee et al., 2002; Xu et al., 2002), the toxic

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mechanism remains relatively obscure and, more importantly, studies by other groups have failed to show consistent results for the neurotoxicity of α-synuclein (Hashimoto et al., 2002; Masliah et al., 2000; Matsuoka et al., 2001; Ostrerova-Golts et al., 2000). Furthermore, there are several lines of evidence suggesting that α-synuclein may play a protective role (Hashimoto et al., 2002; Manning-Bog et al., 2003). For example, the oxidative stress caused by the herbicide paraquat causes α-synuclein aggregation in the brains of experimental animals and this increased expression and aggregation of αsynuclein is neuroprotective (Manning-Bog et al., 2003). In our own studies, using an α-synuclein overexpressing neuronal cell line, it appears that an intracellular retrograde transport system via microtubules plays a crucial role in the aggregate formation and that, once formed, the aggregates may represent a cytoprotective response against noxious stimuli (Hasegawa et al., 2004; Matsuzaki et al., 2004). Given that many different types of neurotoxins such as MPTP and rotenone increase αsynuclein expression in the brain (Betarbet et al., 2000; Vila et al., 2000), it is quite possible, if not highly likely, that the increase of α-synuclein represents an adaptive homeostatic regulatory response to toxic stimuli. Indeed, overexpression of α-synuclein in transgenic mice does not consistently result in neuronal damage (Masliah et al., 2000; Matsuoka et al., 2001) nor does it exacerbate neurodegeneration caused by MPTP (Rathke-Hartlieb et al., 2001). Therefore, α-synuclein itself may possess properties that counteract toxic injury, and its expression could be associated with cell survival strategies (Manning-Bog et al., 2003). Lessons from the observations in Alzheimer disease. Study of the AD brain demonstrated that the Aβ peptide is the major constituent in two of the hallmark pathologies, namely senile plaques and cerebral amyloid angiopathy (Glenner and Wong, 1984a,b; Masters et al., 1985). Aβ is derived by proteolytic cleavage of the amyloid-β precursor protein (AβPP), a protein of unknown cellular function that has the general properties of a cell surface receptor (Kang et al., 1987; Nishimoto et al., 1993). Regardless of the fact that AβPP is a transmembrane protein in the neuronal plasma membrane, the large majority of the protein is processed in the Golgi apparatus into a secreted form before it ever reaches the cell surface (Caporaso et al., 1992; Citron et al., 1995; Kuentzel et al., 1993). While it was originally thought that Aβ was an abnormal cleavage product, it has since been established that Aβ is in fact a normal metabolic product of neuronal AβPP and is found in the cerebral spinal fluid (CSF) and serum of healthy individuals (Haass et al., 1992; Shoji et al., 1992). The regulatory activity of three different proteolytic enzymes, α, β and γ-secretases, at their specific cleavage sites, yields a number of different products, including Aβ1–40 and Aβ1–42 (Sisodia, 1992). While Aβ1–40 is the predominant product of this proteolytic pathway, Aβ1–42 is far more fibrillogenic in vitro and is the major Aβ species present in the core of senile plaques (both AD and non-AD related) (Burdick et al., 1992; Jarrett and Lansbury, 1993). The deposition of Aβ1–40 and Aβ1–42 into senile plaques likely begins with the nucleation of soluble Aβ1–42 into fibrils followed by the accumulation of normally soluble Aβ1–40 (Jarrett and Lans-

bury, 1993). Micro-environmental changes in the brain, such as pH, metal ion availability and oxidants likely impact upon Aβ conformation and its subsequent deposition into amyloid plaques (Atwood et al., 1998; Smith et al., 1997). Recently, a great deal of attention has also been focused on the fact that soluble forms of Aβ, so-called ‘pre-fibrillar’ forms, may be involved in the pathogenesis of AD (Lambert et al., 1998). Investigators studying the primary culprit responsible for AD have, as highlighted above, primarily focused on Aβ such that the “Amyloid Cascade Hypothesis” (Hardy and Higgins, 1992; Hardy and Selkoe, 2002) is the predominant hypothesis for neurodegeneration in AD. However, there is accumulating evidence to suggest that Aβ might not be a major bad player in disease pathogenesis but, like α-synuclein in Quilty's study in this issue, is instead a compensatory protective response (Perry et al., 2000; Rottkamp et al., 2002). First of all, the temporal occurrence of amyloid in the chronology of the disease argues against its importance. In cell culture “models”, Aβ can lead to oxidative stress, yet it is apparent from cell (Frederikse et al., 1996; Yan et al., 1994), animal (Drake et al., 2003; Pratico et al., 2001) and human (Nunomura et al., 2001, Nunomura et al., 1999) studies that oxidative stress temporally precedes Aβ deposition. Moreover, such oxidation-mediated increases in Aβ are associated with a decrease in oxidative stress indicating a potential antioxidant action of Aβ (Nunomura et al., 2000, 2001). Therefore, cell culture “models”, which were instrumental in formulating the Amyloid Cascade Hypothesis (Hardy and Higgins, 1992), are clearly neither an accurate reflection of in vivo nor diseased conditions (Rottkamp et al., 2002; Rottkamp et al., 2001). Additionally, it should be noted that the deposition of Aβ into senile plaques is by no means specific to AD patients and, in fact, is characteristic of normal aging (Davies et al., 1988; Joseph et al., 2001). The incidence of amyloid plaques increases with age, as does the incidence of AD, and the number of plaques in cognitively normal individuals can rival those found in advanced AD (Mann et al., 1992). Even considering only those patients with AD, there is a weak correlation between the burden of Aβ and neuronal loss or cognitive impairment (Davies et al., 1988; Neve and Robakis, 1998). While one might argue that pathology does not wait to be counted and simply reflects formation/resolution dynamics, studies following cases from biopsy to autopsy tend to show increases over time (Di Patre et al., 1999). Moreover, increased production and deposition of Aβ in the central nervous system are observed in response to a variety of injuries, including ischemia and head trauma (Geddes et al., 1997; Gentleman et al., 1993; Roberts et al., 1994). Also, despite marked Aβ deposition in the brains of Down syndrome patients by the fourth decade, there is little evidence of further cognitive decline. The evolution of the classical amyloid cascade (Hardy and Higgins, 1992) to implicate oligomeric amyloid (Hardy and Selkoe, 2002), rather than large aggregates, has found considerable support (Klein et al., 2001; Selkoe, 2002) and the development of in situ methods to detect these oligomers (Kayed et al., 2003) shows an increase in AD brain. However, detracting from the importance of oligomeric Aβ species, the

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chronological order between oligomeric amyloid and oxidative stress is not clear and even in a transgenic mouse model (Tg2576), driven by amyloid overproduction, oligomeric amyloid and oxidative stress appear at approximately the same age (Kawarabayashi et al., 2001; Pratico et al., 2001) and most importantly does not result in cell death (Urbanc et al., 2002). Therefore, while oligomers have certainly refreshed the amyloid field, their role in disease pathogenesis is uncertain at best and further study is definitely required to dissect the relationship between oxidative stress and oligomer formation. According to our hypothesis (Joseph et al., 2001; Lee et al., 2004; Rottkamp et al., 2001, 2002;, Smith et al., 2002), it is important to remember that Aβ is viewed as a protective response to oxidative stress and this, as we previously reported, is consistent with the production of Aβ being associated with decreased levels of oxidative stress (Nunomura et al., 2000, 2001). Therefore, one could easily imagine a scenario whereby an initial oxidative stress results in an increase in amyloid monomer and oligomeric species (Misonou et al., 2000; Paola et al., 2000; Yan et al., 1995) that dampens the initial cellular stress (Nunomura et al., 2000, 2001) giving the appearance of amyloid in the absence of oxidative stress and resulting in the misinterpretation that amyloid is the initiating event. One thing that both Aβ-philes and -phobes can agree is that Aβ aggregation and deposition are insufficient to develop full-fledged AD. Our view is that Aβ is necessary for disease diagnosis but its major role in disease has more to do with factors that led to its aggregation than consequences of that aggregation. Indeed, supporting this view, Aβ peptides, which are normal physiologic products, have a protective effect and under certain conditions can function as an antioxidant (Hou et al., 2002; Zou et al., 2002). These findings are consistent with the reports showing trophic and neuroprotective actions of Aβ at physiological concentrations (Behl et al., 1992; Kaltschmidt et al., 1999; Koo et al., 1993; Luo et al., 1996; Postuma et al., 2000; Singh et al., 1994; Stephenson et al., 1992; Takenouchi and Munekata, 1995; Whitson et al., 1990; Whitson et al., 1989; Yankner et al., 1990). Another interesting hypothesis is that Aβ may function as a trap or sink (Robinson and Bishop, 2002), such that the accumulation and aggregation of Aβ might serve an analogous function in the brain to that of albumin, which binds metals, drugs, metabolites and proteins, in the systemic circulation (KraghHansen et al., 1990). In viewing Aβ as protective, how can we explain the Aβ toxicity demonstrated in in vitro studies? As mentioned above, while Aβ can be toxic in cell culture “models”, in animals and humans, despite massive amyloid concentrations, there is generally little associated cell death (Mann et al., 1992) and consequently only a weak correlation to cognitive decline (Neve and Robakis, 1998). In fact, Aβ appears to be a response to neuronal injury (Gentleman et al., 1993), rather than the mediator of such an injury and this scenario is consistent with a protective function for Aβ (Atwood et al., 2003; Joseph et al., 2001; Lee et al., 2005a,b; Rottkamp et al., 2002). We suspect that the underlying stress in AD is energetic (Hirai et al., 2001),

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since a depletion of the energy supply induces upregulation of AβPP expression such that ischemia, hypoglycemia and traumatic brain injury, a condition that has been shown to put neurons under metabolic stress (Xiong et al., 1997), all upregulate AβPP and its mRNA in animal models and culture systems (Hall et al., 1995; Jendroska et al., 1995; Murakami et al., 1998; Shi et al., 1997, 1998; Yokota et al., 1996). Not only does energy shortage and calcium dysregulation promote AβPP expression, but they also route the metabolism of AβPP from the non-amyloidogenic to the amyloidogenic pathway. Inhibition of mitochondrial energy production alters the processing of AβPP to generate amyloidogenic derivatives (Frederikse et al., 1996; Gabuzda et al., 1994; Mattson and Pedersen, 1998), while oxidative stress has been shown to specifically increase the generation of Aβ (Frederikse et al., 1996; Misonou et al., 2000; Paola et al., 2000). Consistent with this response, Aβ has been detected in the human brain several days after traumatic brain injury (Gentleman et al., 1993). This fits well with the role of AβPP as an acute phase reactant upregulated in neurons, astrocytes and microglial cells in response to inflammation and a multitude of associated cellular stresses including axonal injury (Blumbergs et al., 1995; Gentleman et al., 1993), loss of innervation (Wallace et al., 1993), excitotoxic stress (Panegyres, 1998; Topper et al., 1995), heat shock (Ciallella et al., 1994), oxidative stress (Frederikse et al., 1996; Yan et al., 1994), aging (Higgins et al., 1990; Nordstedt et al., 1991; van Gool et al., 1994) and inflammatory processes (Brugg et al., 1995). Other pro-inflammatory stimuli that mediate the synthesis and release of AβPP include IL-1β (Buxbaum et al., 1992; Goldgaber et al., 1989) and TNFα converting enzyme (Buxbaum et al., 1998). The increased expression of AβPP under these stress conditions is likely a reaction to a decreased energy supply. The strongest evidence supporting the Aβ hypothesis in AD may be the familial forms of the disease, which involve a mutation in AβPP or polymorphisms in genes that are directly involved in AβPP processing (Hardy and Selkoe, 2002; Selkoe, 1999). Despite the fact that mutations in AβPP have been identified in only 20–30 families worldwide and represent less than 0.1% of the 15 million known cases of AD, mutations in both presenilin 1 and 2 only contribute an additional 120–130 families. A tremendous amount of effort and resources have been dedicated in the past decade to determine the mechanism of AD using models based on these mutations. From these studies, it is clear that the mutations all affect AβPP processing and are capable of inducing amyloid neuropathies and dementia. However, the key question is whether this is through a direct or indirect mechanism. Indeed, since the sensitivity of the neuronal environment to insults increases with advancing age, it is very likely that the most important parameter in the development of AD involves mechanisms that are strongly associated with aging such as oxidative stress. In this regard, it is noteworthy that, even with mutations, AD rarely develops prior to middle/old aged, i.e., an age where oxidative balance is already perturbed. Therefore, Aβ aggregation and deposition may be viewed either as the savior of neurons from such stress or a corpse resulting from the battle to save the neuron. That Aβ and senile plaques might represent a protective homeostatic

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adaptation begs the question of whether a similar attribute might be ascribed to tau protein and NFT. In this regard, there is significant evidence that tau phosphorylation is controlled by oxidative stress (Zhu et al., 2000, 2001a,b) and consequently serves as an oxidative sink (Cash et al., 2003; Gomez-Ramos et al., 2003; Wataya et al., 2002) that reduces oxidative damage to key macromolecules (Nunomura et al., 1999, 2001). Therefore, we suspect that tau, like Aβ, is serving a protective antioxidant function in the aging and diseased brain (Lee et al., 2005a,b; Smith et al., 2002). Conclusions. Protein aggregates are the most obvious features of many neurodegenerative diseases and the protein components isolated from these lesions are neurotoxic to cells in culture. It all apparently makes sense: the lesions are bad. However, the brain is not akin to the Petri dish and there is emerging evidence, including that provided by Quilty et al. (2006), that the lesions are not really bad, but in fact they might even be good. Final thought. Mother said do not pick your scabs (incidentally, and likely not coincidentally, another type of protein aggregate). Neither should we choose to eradicate protein aggregates in neurodegenerative diseases. Let us look towards the causes of these diseases rather than the end result. References Arrasate, M., Mitra, S., Schweitzer, E.S., Segal, M.R., Finkbeiner, S., 2004. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431, 805–810. Atwood, C.S., Moir, R.D., Huang, X., Scarpa, R.C., Bacarra, N.M., Romano, D.M., Hartshorn, M.A., Tanzi, R.E., Bush, A.I., 1998. Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J. Biol. Chem. 273, 12817–12826. Atwood, C.S., Perry, G., Smith, M.A., 2003. Cerebral hemorrhage and amyloidbeta. Science 299, 1014 (author reply 1014). Behl, C., Davis, J., Cole, G.M., Schubert, D., 1992. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem. Biophys. Res. Commun. 186, 944–950. Betarbet, R., Sherer, T.B., MacKenzie, G., Garcia-Osuna, M., Panov, A.V., Greenamyre, J.T., 2000. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat. Neurosci. 3, 1301–1306. Blumbergs, P.C., Scott, G., Manavis, J., Wainwright, H., Simpson, D.A., McLean, A.J., 1995. Topography of axonal injury as defined by amyloid precursor protein and the sector scoring method in mild and severe closed head injury. J. Neurotrauma 12, 565–572. Brugg, B., Dubreuil, Y.L., Huber, G., Wollman, E.E., Delhaye-Bouchaud, N., Mariani, J., 1995. Inflammatory processes induce beta-amyloid precursor protein changes in mouse brain. Proc. Natl. Acad. Sci. U. S. A. 92, 3032–3035. Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., Yates, J., Cotman, C., Glabe, C., 1992. Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs. J. Biol. Chem. 267, 546–554. Buxbaum, J.D., Oishi, M., Chen, H.I., Pinkas-Kramarski, R., Jaffe, E.A., Gandy, S.E., Greengard, P., 1992. Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor. Proc. Natl. Acad. Sci. U. S. A. 89, 10075–10078. Buxbaum, J.D., Liu, K.N., Luo, Y., Slack, J.L., Stocking, K.L., Peschon, J.J., Johnson, R.S., Castner, B.J., Cerretti, D.P., Black, R.A., 1998. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J. Biol. Chem. 273, 27765–27767.

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