The Journal of Neuroscience, July 27, 2016 • 36(30):7971–7984 • 7971 Neurobiology of Disease Reducing Endogenous ␣-Synuclein Mitigates the Degeneration of Selective Neuronal Populations in an Alzheimer’s Disease Transgenic Mouse Model X Brian Spencer,1 Paula A. Desplats,1,2 Cassia R. Overk,1 Elvira Valera-Martin,1 Robert A. Rissman,1 Chengbiao Wu,1 Michael Mante,1 Anthony Adame,1 Jazmin Florio,1 Edward Rockenstein,1 and Eliezer Masliah1,2 1Department of Neurosciences and 2Department of Pathology, University of California–San Diego, La Jolla, California 92093 Alzheimer’s disease (AD) is characterized by the progressive accumulation of amyloid ␤ (A␤) and microtubule associate protein tau, leading to the selective degeneration of neurons in the neocortex, limbic system, and nucleus basalis, among others. Recent studies have shown that ␣-synuclein (␣-syn) also accumulates in the brains of patients with AD and interacts with A␤ and tau, forming toxic hetero-oligomers. Although the involvement of ␣-syn has been investigated extensively in Lewy body disease, less is known about the role of this synaptic protein in AD. Here, we found that reducing endogenous ␣-syn in an APP transgenic mouse model of AD prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. Together, these results suggest that ␣-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing ␣-syn might be a potential approach to protecting these populations from the toxic effects of A␤. Key words: ␣-synuclein; Alzheimer’s disease; amyloid ␤ oligomer; cholinergic neuron; selective vulnerability; transgenic animal model Significance Statement Reducing endogenous ␣-synuclein (␣-syn) in an APP transgenic mouse model of Alzheimer’s disease (AD) prevented the degen- eration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. These results suggest that ␣-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing ␣-syn might be a potential approach to protecting these populations from the toxic effects of amyloid ␤. Introduction among others (Morrison and Hof, 2002; Saxena and Caroni, Alzheimer’s disease (AD) is an heterogeneous neurodegenerative 2011; Mattsson et al., 2016). In addition to A␤ and Tau, recent disorder (Schellenberg, 1995; Komarova and Thalhauser, 2011; studies have shown that ␣-synuclein (␣-syn) (Hansen et al., 1990; Lam et al., 2013) that affects Ͼ5 million in the United States alone McKeith, 2006; Lippa et al., 2007; Larson et al., 2012; Winslow et (Alzheimer’s Association, 2015) and is characterized by the pro- al., 2014; Walker et al., 2015) and TAR DNA-binding protein gressive accumulation of amyloid ␤ (A␤) and microtubule asso- (TDP)-43 (Wilson et al., 2011; Colom-Cadena et al., 2013; ciated protein tau (Tau), leading to the selective degeneration of Borchelt et al., 2014) might also contribute to the pathogenesis of neurons in the neocortex, limbic system, and nucleus basalis, AD. The mechanisms leading to the accumulation of multiple proteins in AD are not completely understood, but defective pro- tein clearance, including alterations in autophagy (Lucin and Received March 8, 2016; revised June 8, 2016; accepted June 13, 2016. Wyss-Coray, 2013; Nixon, 2013; Peric and Annaert, 2015; Zare- Author contributions: B.S., P.A.D., R.A.R., C.W., and E.M. designed research; B.S., P.A.D., C.R.O., E.V.-M., R.A.R., Shahabadi et al., 2015) and proteolysis of aggregates (Borchelt et C.W., M.M., A.A., J.F., E.R., and E.M. performed research; R.A.R., C.W., and E.M. analyzed data; B.S., P.A.D., C.R.O., R.A.R., C.W., and E.M. wrote the paper. al., 2014), might be centrally involved. This work was supported by the National Institutes of Health (Grants AG18840, AG022074, and AG10435). ␣-syn has been investigated extensively in other neurodegen- The authors declare no competing financial interests. erative disorders such as Parkinson’s disease (PD) and dementia Correspondence should be addressed to Dr. E. Masliah, Department of Neurosciences, University of California, with Lewy bodies (DLB) (Takeda et al., 1998; Piao et al., 2000; 9500 Gilman Drive, La Jolla, CA 92093-0624. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.0775-16.2016 Spillantini and Goedert, 2016); however, less is known about its Copyright © 2016 the authors 0270-6474/16/367971-14$15.00/0 role as a synaptic protein in AD. Previous studies have shown that 7972 • J. Neurosci., July 27, 2016 • 36(30):7971–7984 Spencer et al. • Reducing ␣-Synuclein Improves AD Mouse Model the pathology of AD and PD overlap (Hansen et al., 1990; McK- entrances into target quadrant were recorded with a Noldus Instruments eith, 2006; Lippa et al., 2007; Winslow et al., 2014; Walker et al., EthoVision video tracking system set to analyze two samples per second. 2015). In AD and in APP transgenic (Tg) models, ␣-syn interacts All experimental conditions were the same for the probe test except the with A␤ (Tsigelny et al., 2008) and Tau (Giasson et al., 2003; platform was removed and the amount of time spent in the quadrant that Esposito et al., 2007; Clinton et al., 2010) and accumulates in previously contained the platform was recorded. For these experiments, selected regions of the limbic system in patients with AD (Swirski mice were 4–6 months old to avoid widespread amyloid deposition, which occurs in older mice, as a compounding factor (n ϭ 10 mice/ et al., 2014), including dystrophic neurites and synapses around ␤ ␤ ␣ group, half male and half female). the A plaques (Masliah et al., 1996). A promotes -syn aggre- Behavioral testing in the open field. The open field locomotor test was gation and toxicity (Masliah et al., 2001), and they have synergis- used to determine basal activity levels of study subjects (total move time) tic effects leading to synaptic dysfunction (Wang et al., 2015). In during a 15 min session. Spontaneous activity in an open field (25.5 ϫ patients with AD, levels of soluble ␣-syn are elevated in brain and 25.5 cm) was monitored for 15 min using an automated system (Truscan fluids, which differs from PD (Larson et al., 2012; Tateno et al., system for mice; Coulbourn Instruments). Animals were tested within 2012). Moreover, recent studies showed that, in APP Tg mice, the first 2–4 h of the dark cycle after being habituated to the testing room ␣-syn infusion reduces A␤ plaque formation but enhances syn- for 15 min. The open field was illuminated with an anglepoise lamp aptic degeneration (Bachhuber et al., 2015) and that A␤ and equipped with a 25 W red bulb. Animals were tested at 4–6 months of ␣-syn cooperated to block SNARE-vesicle fusion (Choi et al., age. Time spent in motion was automatically collected in 3 ϫ 5 min time 2015). This is of interest given the role of ␣-syn in regulating bins using TruScan software. Data were analyzed for both the entire 15 intracellular vesicle trafficking via interactions with Rab3 and min session and for each of the 5 min time blocks. Rab8 (Cooper et al., 2006). Quantitative PCR. Total RNAs were extracted from mice brains using RNeasy Lipid mini kit (Qiagen) and reverse transcribed using Super- Furthermore, we have shown that coexpression of human ␣ Script VILO cDNA synthesis kit (Life Technologies), respectively, as de- -syn in APP Tg mice results in enhanced degeneration of cho- scribed previously (Kim et al., 2015). Quantitative real-time PCR was linergic neurons in the nucleus basalis (Masliah et al., 2001) and performed using TaqMan Fast Advanced Master Mix (Life Technolo- glutamatergic neurons in the CA3 region of the hippocampus gies) according to the manufacturer’s instructions with gene specific (Overk et al., 2014). Here, we found that reducing endogenous primers for Rab3. Amplification of DNA products was measured by the ␣-syn levels in an APP mouse model of AD prevented the degen- StepOnePlus real-time PCR system (Applied Biosystems). Relative eration of cholinergic neurons, ameliorated functional deficits, mRNA levels were calculated according to the 2-exp (ddCt) method and normalized levels of Rab3a and Rab5 proteins involved in (Kim et al., 2015). All dCT values were normalized to ␤-actin. intracellular transport and sorting of nerve growth factor (NGF) Immunohistochemical analysis. Analysis was performed using free- and brain-derived neurotrophic factor (BDNF). These findings floating, 40 ␮m-thick, vibratome-cut, blind-coded sections, as described also support the notion that ␣-syn is an important contributor to previously (Zwilling et al., 2011; Games et al., 2013). Briefly, sections ␣ AD pathogenesis and that regulating ␣-syn might be of therapeu- were incubated overnight at 4°C with antibodies against total -syn (1: 500, affinity-purified rabbit polyclonal, Millipore; Masliah et al., 2000), tic value in AD. A␤ (6E10, mouse monoclonal; BioLegend), ChAT (1:500, affinity- Materials and Methods purified monoclonal; Millipore), parvalbumin (1:500, affinity-purified monoclonal; Millipore), calbindin (1:500, polyclonal; Millipore), neuro- Crosses between mThy1-APP Tg mice and ␣-syn knock-out (KO) mice. The peptide Y (NPY; 1:500, affinity-purified polyclonal; Millipore), NeuN University of California at San Diego Institutional Animal Care and Use (1:500, affinity-purified monoclonal; Millipore), GFAP (1:500, affinity- Committee approved all animal experiments. For these experiments, purified monoclonal; Millipore), Rab3a (1:500, polyclonal; Abcam), mice expressing human APP751 cDNA containing the London (V717I) Rab5 (1:500, polyclonal; Santa Cruz Biotechnology), Rab7 (1:500, and Swedish (K670M/N671L) mutations under the regulatory control of monoclonal; Abcam), Rab7 (1:500 monoclonal; Millipore), and Rab11 the murine (m)Thy-1 gene (mThy1-hAPP751; line 41) on a C57BL/6 (1:500, polyclonal; Millipore), followed by biotin-tagged anti-rabbit or background were used (Rockenstein et al., 2001).