Degradation of alpha-synuclein by dendritic cell factor 1 delays neurodegeneration and extends lifespan in Drosophila

Neurobiology of Aging 67 (2018) 67e74

Contents lists available at ScienceDirect

Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging

Degradation of alpha-synuclein by dendritic cell factor 1 delays neurodegeneration and extends lifespan in Drosophila Shiqing Zhang 1, Ruili Feng 1, Yanhui Li, Linhua Gan, Fangfang Zhou, Shiquan Meng, Qian Li, Tieqiao Wen* Laboratory of Molecular Neural Biology, School of Life Sciences, Shanghai University, Shanghai, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 July 2017 Received in revised form 3 December 2017 Accepted 7 March 2018 Available online 13 March 2018

Parkinson’s disease (PD) is a common neurodegenerative disease associated with the progressive loss of dopaminergic neurons in the substantia nigra. Proteinaceous depositions of alpha-synuclein (a-syn) and its mutations, A30P and A53T, are one important characteristic of PD. However, little is known about their aggregation and degradation mechanisms. Dendritic cell factor 1 (DCF1) is a membrane protein that plays important roles in nerve development in mouse. In this study, we aimed to show that DCF1 overexpression in a PD Drosophila model significantly ameliorates impaired locomotor behavior in third instar larvae and normalizes neuromuscular junction growth. Furthermore, climbing ability also significantly increased in adult PD Drosophila. More importantly, the lifespan dramatically extended by an average of approximately 23%, and surprisingly, DCF1 could prevent a-syneinduced dopaminergic neuron loss by aggregating a-syn in the dorsomedial region of Drosophila. Mechanistically, we confirmed that DCF1 could degrade a-syn both in vivo and in vitro. Our findings revealed an important role of DCF1 in PD process and may provide new potential strategies for developing drugs to treat neurodegenerative diseases. Ó 2018 Elsevier Inc. All rights reserved.

Keywords: Parkinson’s disease DCF1 Alpha-synuclein Aging Neurodegeneration

1. Introduction Parkinson’s disease (PD), which affects approximately 4% of the world population over 80 years of age (Twelves et al., 2003; de Lau and Breteler, 2006), is the second most common age-associated neurodegenerative disease after Alzheimer’s disease. It is characterized by dopaminergic (DA) neuron loss in the substantia nigra and by the accumulation of proteinaceous intraneuronal inclusions known as Lewy bodies, which mainly consist of the protein alpha-synuclein (asyn) (Jiang et al., 2013; Karuppagounder et al., 2014). The clinical and pathological symptoms of familial PD are resting tremor, muscular rigidity, bradykinesia, hypokinesia/akinesia, small handwriting, flexed posture, and postural instability (Wolters, 2008). a-syn is a soluble, natively unfolded protein that is highly enriched in the presynaptic terminals of neurons in the central nervous system (Iwai et al., 1995). Two missense mutations in a-syn, A30P and A53T, have been identified as genetic lesions in familial PD (Polymeropoulos et al., 1997), which increase the potential for protein misfolding, oligomerization, and aggregation (Li et al., 2001; Olanow and McNaught, 2006). Both * Corresponding author at: School of Life Sciences, Shanghai University, 333 Nanchen Road, Shanghai 200444, China. Tel./fax: 86-021-66132512. E-mail address: [email protected] (T. Wen). 1 These authors contributed equally to this work. 0197-4580/$ e see front matter Ó 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.neurobiolaging.2018.03.010

mutations oligomerize more rapidly than the wild-type a-syn (Dev et al., 2003). The mechanisms for this may be related to the phosphorylation of a-syn at serine 129 (Arawaka et al., 2006; Basso et al., 2013; Smith et al., 2005), tyrosine 125(Chen et al., 2009), and other gene regulation pathways, such as Rab11 (Breda et al., 2015), GRKs (GRK2, GRK3, GRK5, and GRK6), (Pronin et al., 2000) and LRRK2 (Qing et al., 2009). Therefore, blocking a-syn aggregation would represent a breakthrough in the prevention and treatment of PD. Dendritic cell factor 1 (DCF1), also known as TMEM59, is a membrane protein that plays a role in selective autophagy (Boada-Romero et al., 2013). It also affects amyloid precursor protein shedding and Ab generation in Alzheimer’s disease (Ullrich et al., 2010). In a previous study, we showed that DCF1 was involved in neural stem cell differentiation (Wen et al., 2002), brain development, and dendritic spine formation (Liu et al., 2018). More recently, we showed dopamine system dysfunction in DCF1 gene knockout mice, (Liu et al., 2017) which suggested an association with PD. Here, we used a PD Drosophila model to explore the effect of DCF1 expression on PD prevention and processes. Results suggested that DCF1 significantly ameliorates PD-related phenotypes including extending lifespan via degradation of a-syn, which suggested a pivotal role of DCF1 in the onset and development of neurodegenerative diseases.

68

S. Zhang et al. / Neurobiology of Aging 67 (2018) 67e74

2. Materials and methods 2.1. Flies and a-syn mutants The tyrosine hydroxylase-GAL4 driver, elav-GAL4 driver, UAS-asyn, UAS-a-syn-A30P, and UAS-a-syn-A53T were donated by Dr Aike Guo from the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences. UAS-DCF1 was created in our lab. The F1 generation was collected and subjected to further experiments. Drosophila raised at 25
C were cultured in standard cornmeal medium and harvested at the same time.

2.2. Immunocytochemistry Larva or adult fly brains were dissected in 0.01 M phosphatebuffered saline (PBS) and fixed in 4% formaldehyde for 30 minutes followed by 30 minutes in 0.4% PBS with 0.4% Triton X-100. The brains were washed for 3  5 minutes in 0.01 M PBS and then incubated in a blocking solution of 10% goat serum for 1 hour. Samples were incubated overnight at 4
C with horseradish peroxidase (1:100, Santa Cruz), and small size synaptic boutons were defined <1.5 mm, rabbit tyrosine hydroxylase antibody (1:200, Millipore Bioscience Research Reagents), and mouse a-syn antibody (1:200, Santa Cruz LB509).

Unbound antibodies were removed by 3  5 minute washes in 0.01 M PBS before samples were incubated for 2 hours at room temperature with secondary antibody (1:200, antirabbit and antimouse; Santa Cruz). After 3  5 minute washes with 0.01 M PBS, brains were mounted in 3% n-propylgallate þ 80% glycerol PBS solution. 2.3. Electrophysiology At 30 days after eclosion, giant fibers (GF) system neurons in the brains of adult flies (n  10) were directly stimulated and recordings were obtained from the output muscles of the GF system (dorsal longitudinal muscles [DLMs]). Flies were anesthetized with CO2. The fly’s legs and wings were removed, and the rest of the body was fixed in wax. A ground glass electrode was placed into the posterior end of the abdomen, and 2 glass stimulating electrodes were placed through the eyes into the brain. A glass recording electrode was placed into the left (or right) DLM fiber. During the 100 Hz frequency stimulation, the stimulation intensity was 30e60 V with duration of 0.1 ms, and 150% of the threshold stimulation intensity was at 0.5 Hz. A sudden potential drop of 20e60 mV indicated intracellular penetration into muscle. The muscle identity (DLM) was determined by electrode placement. Signals were amplified by Multiple Clamp 700B (Molecular Devices) and digitized at 20 kHz by Digidata 1440A (Molecular

Fig. 1. DCF1 overexpression intensifies NMJ expansion in PD Drosophila larvae. Confocal image of Drosophila NMJ 6/7 stained with neuronal marker, HRP. (A) elav-GAL4/þ; þ/þ heterozygous larvae. (B) elav-GAL4/DCF1; þ/þ heterozygous larvae. (C, E, and G) elav-GAL4/þ; a-syn/þ, elav-GAL4/þ; A30P/þ and elav-GAL4/þ; A53T/þ display a decrease in the total number of synaptic boutons. (D, F, and H) Nervous system overexpression of DCF1 in PD Drosophila larvae partially restores the number of synaptic boutons. (I) Total number of boutons. (J) Number of type Ib boutons. (K) Number of type Is boutons. Data were analyzed by multivariant ANOVA with supplementary NewmaneKeuls test and presented as mean  SEM (n ¼ 15) with * p < 0.05, ** p < 0.01. Scale bar ¼ 50 mm. Abbreviations: DCF1, dendritic cell factor 1; HRP, horseradish peroxidase; NMJ, neuromuscular junction; PD, Parkinson’s disease; type Ib, type I big; type Is, type I small.

S. Zhang et al. / Neurobiology of Aging 67 (2018) 67e74

69

Fig. 2. DCF1 overexpression relieves TH neuron degeneration and extends lifespan in Drosophila. (A) Confocal images of dopaminergic neurons immunostained with anti-TH antibody in 1-, 30-, and 40-day-old fly brains, with boxes indicating DM clusters and arrows indicating reduced DM neurons. Loss of dopaminergic neurons was observed in 30- and 40-day a-syn, A30P, and A53 T transgenic flies, whereas overexpression of DCF1 rescued the loss of dopaminergic neurons in the DM region. Scale bar ¼ 100 mm. (B) Quantitative analysis of TH immunoreactive DM cluster dopamine neurons in transgenic flies. Data were analyzed by multivariant ANOVA with supplementary NewmaneKeuls test and presented as mean  SEM (n ¼ 20) with * p < 0.05, ** p < 0.01, *** p < 0.001. Log-rank analysis revealed that adult pan-neuronal overexpression of DCF1 extends lifespan in a-syn expressing male flies (C) and female flies (D). The dotted line represents the age of the fly when half of the population has died. Survival curves for respective genotypes of flies are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, n ¼ 100. Abbreviations: DCF1, dendritic cell factor 1; DM, dorsomedial; TH, tyrosine hydroxylase.

70

S. Zhang et al. / Neurobiology of Aging 67 (2018) 67e74

Fig. 3. Overexpression of DCF1 throughout the nervous system improved deficits in neurotransmission in the disynaptic DLM pathway of PD Drosophila (elav-GAL4 driver). (A) Representative 100 Hz brain stimulation-evoked electrophysiological recordings from the Drosophila GF system of GAL4 (control flies), PD flies, and PD flies co-expressing DCF1. (B) The percentage of successful DLM responses to 100 Hz brain stimulation was quantified in each genotype at 30 days after eclosion. Data were analyzed by multivariant ANOVA with supplementary NewmaneKeuls test and presented as mean  SEM (n ¼ 10) with * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: DCF1, dendritic cell factor 1; DLM, dorsal longitudinal muscle; GF, giant fibers; PD, Parkinson’s disease.

Devices). Data were collected and analyzed using the pClamp software (version 10.0; Molecular Devices). All glass electrodes were filled with 3 M KCl. The recording environment temperature was 25
C. 2.4. Expression of a-syn and DCF1 in HEK293T cells HEK293T cells were co-transfected with plasmids containing asyn, DCF1, and DCF1gly mu. After 48 hours, the cells were lysed in RIPA-50 buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.1% SDS, 0.01% NaN3, and 1 mM PMSF, pH 7.4). Equal amounts of protein were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels and electroblotted onto nitrocellulose membranes. Membranes were blocked for 1 hour in Odyssey blocking buffer with gentle shaking and simultaneously incubated overnight at 4
C with mouse a-syn antibody (1:200, Santa Cruz LB509). This was followed by incubation with mouse glyceraldehyde-3-phosphate dehydrogenase antibody and rabbit DCF1 antibody for 2 hours at room temperature. Next, infrared dye 700 and 800 conjugated affinitypurified goat antimouse IgG (Zemed, USA) secondary antibodies were added and allowed to incubate for 1 hour at room temperature. Immunoblotting bands were detected and quantified using the Odyssey infrared imaging system and software (LI-COR Biosciences). 2.5. Protein purification and degradation experiments with a-syn Human DCF1 fragments were ligated with a prokaryotic expression vector pET28a, transformed into Escherichia coli strain

BL21 (DE3), and cultured in broth medium. The expression of recombinant protein was induced by isopropyl-b-D-thiogalactoside and detected by SDS-PAGE. The recombinant protein was purified, collected by Ni-NTA affinity chromatography, and prepared for SDSPAGE and Western blotting analysis. Recombinant human a-syn was purchased from ProSpec-Tany. a-syn (2 mg) and DCF1(10 mg) were incubated in reaction buffer (calpain buffer, 50 mM Tris-HCl, 100 mM NaCl, 2 mM DTT, 1 mM EDTA, and 3 mM CaCl2, pH 7.5) in a total volume of 20 mL and agitated at 300 rpm at 37
C in a Mini-Micro 980140 shaker (VWR) for 2 hours and 24 hours. 3. Results 3.1. DCF1 normalizes neuromuscular junction growth in larvae expressing a-syn and its mutations, A30P and A53T Changes in neuromuscular junctions (NMJs) are one of the major pathologic indicators often used to examine synaptic development and function in the PD Drosophila model (Amorim et al., 2017; Collins and DiAntonio, 2007). Drosophila larval muscles 6 and 7 are innervated exclusively by type I boutons, which are further subdivided into type I small (Is) and type I big (Ib) boutons (Atwood et al., 1997). To investigate the effect of DCF1 expression on NMJ development in PD larvae, we coexpressed DCF1 with a-syn and its mutations, A30P and A53T, in a pan-neural pattern; we assessed the overall morphology of synapses by confocal microscopy. As background controls, GAL4

S. Zhang et al. / Neurobiology of Aging 67 (2018) 67e74

(Fig. 1A) and DCF1 (Fig. 1B) were expressed individually. Normal NMJ growth was inhibited (Fig. 1C, E, and G, arrow), and statistical analysis showed 19%, 25%, and 24% reduction in the total number of boutons in the expression of a-syn, A30P, and A53T, respectively (Fig. 1I). By contrast, DCF1 expression resulted in a significant increase in the number of branches at muscles 6 and 7 in larvae (Fig. 1D, F, and H) compared with their corresponding controls (Fig. 1C, E, and G). Interestingly, compared with type Ib (Fig. 1J), the difference in the total number of boutons in DCF1 expression were mainly in type Is boutons (Fig. 1K), which was less extensively enveloped in Discs large epositive postsynaptic subsynaptic reticulum (Atwood et al., 1993; Lahey et al., 1994) than type Ib boutons, showing 10%, 12%, and 10% increase, respectively. Defects in larval locomotor behavior are often associated with neuronal and synaptic dysfunctions (Folwell et al., 2010; Mudher et al., 2004). Therefore, we investigated the effect of DCF1 expression on NMJ 6/7 growth based on larval crawling experiments (Supplementary Fig. S1). Expression of DCF1 in the larval stages led to a recovery in the total number of synaptic connections (boutons) at their target muscles and normalized the development and morphology of NMJ 6/7. Taken together, these results confirmed that DCF1 is involved in stereotypical presynaptic and postsynaptic development, which regulates larval crawling ability. 3.2. DCF1 relieves dopaminergic neuron degeneration and extends lifespan Owing to the sensitivity to a-syn toxicity, (Auluck et al., 2002; Yang et al., 2003) we investigated the dorsomedial (DM) cluster of DA neurons. We found a normal number of DM DA neurons in 1-dayold transgenic flies in each genotype (Fig. 2A). However, compared with the controls, DM neuron loss in A30P and A53T transgenic flies reared for 30 days was more severe than that in a-syn flies, whereas DCF1 prevented neuron loss in these transgenic flies at the same time point (Fig. 2B) (p < 0.01 for flies co-expressing asyn and DCF1; p < 0.001 for flies co-expressing A30P and DCF1 or A53T and DCF1). However, this ability to prevent loss of neurons is limited. Compared with GAL4 lines, we observed DM neuron loss in 40-day-old PD transgenic lines co-expressing DCF1 (data not shown). Nevertheless, these results strongly displayed that DCF1 slows the loss of DA neurons in the DM region of a-syn, A30P, and A53T transgenic flies, particularly in 30-day-old flies, suggesting that DCF1 expression protects DA neurons from a-syn toxicity and delays neurodegeneration in the nervous system of adult Drosophila. Given the inherent connections between the loss of neurons and lifespan in PD (Ortega-Arellano et al., 2017), we tested the effect of DCF1 expression on longevity in PD Drosophila. The results showed whether the lifespan of male (Fig. 2C) or female (Fig. 2D) PD fruit flies significantly increased with DCF1 expression. Statistical results indicated that the female half of lifespan in a-syn, A30P, and A53T was significantly extended by 27.3%, 33.3%, and 18.2%, respectively. In male flies, half of lifespan was also extended by 14.3%, 27.3%, and 15.4%, respectively. The mean lifespan was extended by approximately 23%. In addition, the climbing ability was significantly enhanced in Drosophila expressing a-syn, A30P, and A53T (Supplementary Fig. S1).

71

total of 100 stimuli) was used to activate the GF system and to evoke excitatory junction potentials (EJP), which were intracellularly recorded in the DLMs (Fig. 3A). At 30 days after eclosion, when the climbing abilities of PD flies begin to decrease, expression of a-syn and its mutations altered neurotransmission by decreasing EJP in the GF system (44.6%, 36.6%, and 39.0% success rates in flies expressing a-syn, A30P, and A53T, respectively, compared with 95.4% success rate in flies expressing GAL4, Fig. 3B). However, expressing DCF1 altered the decreased EJP in PD flies (58.2%, 48.8%, and 52.2% success rates in DCF1 expressing a-syn, A30P, and A53T flies, respectively, Fig. 3B), improving by 13.6%, 12.2%, and 18.3%, respectively. Thus, the electrophysiology of neural transmission through the GF system revealed deficits consistent with PD pathology, whereas co-expression of DCF1 in PD flies effectively ameliorated this defect, resulting in significant differences (p < 0.01 for flies co-expressing a-syn and DCF1, A53T and DCF1; p < 0.05 for flies co-expressing A30P and DCF1). 3.4. DCF1 co-localizes with a-syn, A30P, and A53T to form inclusions To further determine the protective cellular mechanism mediated by DCF1, we co-expressed DCF1 and a-syn as well as A30P and A53T in HEK293T cells, (Fig. 4). The results showed that DCF1 (red) is co-localized with a-syn (green) (Fig. 4B), A30P (green) (Fig. 4D), and A53T (green) (Fig. 4F). These results prompted us to further investigate the impact of this co-localization in the brain. Immunohistochemical staining in a 30-day-old fly brain, expressing GAL4 and DCF1, revealed no a-synepositive inclusions in the DM region (Fig. 5A and B). By contrast, little immunostaining of a-syn inclusions was detected in a-syn expressing flies (Fig. 5C) and even

3.3. DCF1 ameliorates neural transmission in PD flies To more directly explore the influence of DCF1 on a-syneinduced neuronal changes, we examined neural transmission through GF system in vivo. Repetitive brain stimulation at 100 Hz (a

Fig. 4. DCF1 is co-localized with a-syn in HEK293T cells. (A, C, and E) Representative images of a-syn and its mutant construct expressed alone in HEK293T cells. (B, D, and F) a-syn and its mutant construct were co-expressed with DCF1 in HEK293T cells and found to co-localize. Scale bar ¼ 10 mm. Abbreviation: DCF1, dendritic cell factor 1.

72

S. Zhang et al. / Neurobiology of Aging 67 (2018) 67e74

Fig. 5. The number of a-syn positive inclusion bodies increased in DCF1 overexpressing PD flies. Inclusions in the DM region of a-syn flies at 30 days were labeled by whole-mount immunostaining with an antiea-syn antibody. Genotypes: control flies are (A) w; elav-GAL4/þ; þ/þ and DCF1 expressing flies are (B) w; elav-GAL4/UAS-DCF1; þ/þ. PD flies are (C) w; elav-GAL4/þ; UAS-a-syn/þ, (E) w; elav-GAL4/þ; UAS-a-syn-A30P/þ, and (G) w; elav-GAL4/þ; UAS-a-syn-A53T/þ. PD co-expressing DCF1 flies are (D) w; elav-GAL4/UAS-DCF1; UAS-a-syn/þ, (F) w; elav-GAL4/UAS-DCF1; UAS-a-syn-A30P/þ, and (H) w; elav-GAL4/UAS-DCF1; UAS-a-syn-A53T/þ. Scale bar ¼ 100 mm and 50 mm. Abbreviations: DCF1, dendritic cell factor 1; DM, dorsomedial; PD, Parkinson’s disease.

less in A30P (Fig. 5E) and A53T (Fig. 5G) ones. However, in a-syn and DCF1 co-expressing flies, immunostaining revealed an increase in the number and size of a-syn inclusions (Fig. 5D). Furthermore, a few immunostained a-syn inclusions were present in the same region of fly brains in A30P (Fig. 5F) and A53T (Fig. 5H) co-expressing with DCF1. However, in number and size, these were lesser than the wild-type a-syn and DCF1 co-expressing flies. This phenomenon confused us regarding that the formation of inclusions should have worsened PD symptoms; by contrast, DCF1 expression relieved PD symptoms. Therefore, we assumed that DCF1 induces the degradation of a-syn, A30P, and A53T through the formation of inclusions.

3.5. DCF1 accelerates a-syn degradation in HEK293T cells and in vitro To confirm the hypothesis that DCF1 degrades a-syn, we cotransfected pcDNA3.1-a-syn and pcDNA3.1-DCF1 into HEK293T cells and analyzed the expression of a-syn after a 48-hour culture. Surprisingly, Western blots showed that compared with cells co-transfected with pcDNA3.1-a-syn (control), a-syn expression was significantly decreased in cells co-transfected with pcDNA3.1-DCF1 (Fig. 6A). Similarly, a mutation in the glycosylation site in DCF1 expressing cells maintained this decrease, suggesting that DCF1 glycosylation is not involved in this process. Although DCF1 protein could still be detected (Fig. 6A), statistical analysis showed a significant difference (Fig. 6B). To confirm whether DCF1 directly degrades a-syn, purified a-syn and DCF1 proteins were incubated in calpain buffer. The degree of asyn degradation was detected by Western blotting (Fig. 6C). Results

demonstrated that 75% of the total a-syn protein was degraded by DCF1, providing direct evidence for the degradation of a-syn (Fig. 6D). 4. Discussion Proteinaceous depositions of a-syn and its mutations, A30P and A53T, are the neuropathological hallmark of PD. Therefore, it may be beneficial to identify certain proteins with the potential to degrade a-syn. Our research showed that expressing DCF1 in larvae and adult fruit fly models of PD could relieve symptoms of PD and extend lifespan. Furthermore, in vivo and in vitro expression of DCF1 confirmed its ability to degrade a-syn protein and showed an unprecedented new understanding of the mechanisms regulating a-syn degradation. In previous studies, we showed that DCF1 is associated with neural stem cell differentiation and glioblastoma U251 cell line inhibition. Furthermore, we found abnormalities in neural development in the brain (Liu et al., 2018), and changes in social interactions mediated via the dopamine system (Liu et al., 2017) in DCF1 knockout mice. Naturally, we were very curious about any potential relationship between DCF1 and PD. Because a-syn is one of important pathogenic genes in PD, we constructed a PD Drosophila model co-expressing a-syn and DCF1 to investigate what may happen. We observed that DCF1 expression caused a significant increase in the number of boutons, whereas normal NMJ growth was inhibited without DCF1 in the PD model (Fig. 1). In addition, we observed that DCF1 better prevented DA neuron loss, protected them from a-syn toxicity, and delayed neurodegeneration in the Drosophila nervous system (Fig. 2). More importantly, the half-life of longevity was significantly extended

S. Zhang et al. / Neurobiology of Aging 67 (2018) 67e74

73

Fig. 6. DCF1 accelerates a-syn degradation both in HEK293T cells and in vitro experiments. (A) HEK293T cells were co-transfected with pcDNA3.1-a-syn and pcDNA3.1-DCF1 for 48 hours. Cell lysates were subjected to immunoblotting using antiea-syn and anti-DCF1 antibodies. (B) Quantitative analysis of a-syn expression using densitometry. Double asterisks indicate a significant decrease in a-syn expression when co-transfected with DCF1 and DCF1 with a glycosylation site mutation (** p < 0.01, multivariant ANOVA with supplementary NewmaneKeuls test). (C) DCF1 degradation of a-synWestern blot analysis of a-syn and DCF1 probed with antibodies against a-syn and DCF1 after incubation at 37
C for 24 hours in calpain buffer. (D) Quantitative analysis of a-syn protein residual quantity after incubation with DCF1 using densitometry. Abbreviation: DCF1, dendritic cell factor 1.

(Fig. 2C and D), which is a significant finding in this study. All of these results point to a positive role of DCF1 in alleviating PD symptoms, although further strategic decisions are necessary for providing insights into how this occurs. To explore the mechanisms underlying DCF1 expression, we confirmed the co-localization of DCF1 and a-syn as well as A30P and A53T (Fig. 4). Further observation confirmed the formation of inclusions composed of DCF1 and a-syn proteins as well as A30P and A53T proteins (Fig. 5). We hypothesized that DCF1 may alleviate PD symptoms by degrading a-syn protein. This was confirmed by in vivo experiments. By cotransfecting a-syn and DCF1 in HEK293T cells, we found that asyn protein was significantly decreased. In vitro experiments directly confirmed a-syn degradation by DCF1 (Fig. 6). However, it seems to have contradictory results from Figs. 5 and 6, but it reflects deepening mechanism of a-syn aggregation and degradation by DCF1. An interaction between DCF1 and a-syn promotes the accumulation of a-syn inclusions. We hypothesize that DCF1 increases the accumulation of a-syn possibly as a consequence of DCF1 proteasomal degradation, that is, a-syn aggregation is the prerequisite and basis for delivery, degradation, and clearance by DCF1. And there is research to show that a-syn polymers are more likely to be degraded (Iljina et al., 2016), supporting this speculation. These results suggested the possibility of a new approach to treat PD. Finally, it should be pointed out that a more profound meaning or value lies in the extension of life in this article. It will be a novel information with a great potential for human good. Its significance

is not only a simple lifespan extension but also a healthy longevity without neurodegenerative diseases. Disclosure statement The authors declare no conflicts of interest. Acknowledgements This work was funded by the National Science Foundation of China (81271253, 81471162), the Science and Technology Commission of Shanghai (14JC1402400), and the Key Innovation Project of Shanghai Municipal Education Commission (Grant No.14ZZ090). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.neurobiolaging.2018. 02.021. References Amorim, I.S., Graham, L.C., Carter, R.N., Morton, N.M., Hammachi, F., Kunath, T., Pennetta, G., Carpanini, S.M., Manson, J.C., Lamont, D.J., Wishart, T.M., Gillingwater, T.H., 2017. Sideroflexin 3 is an alpha-synuclein-dependent mitochondrial protein that regulates synaptic morphology. J. Cell Sci. 130, 325e331. Arawaka, S., Wada, M., Goto, S., Karube, H., Sakamoto, M., Ren, C.H., Koyama, S., Nagasawa, H., Kimura, H., Kawanami, T., Kurita, K., Tajima, K., Daimon, M.,

74

S. Zhang et al. / Neurobiology of Aging 67 (2018) 67e74

Baba, M., Kido, T., Saino, S., Goto, K., Asao, H., Kitanaka, C., Takashita, E., Hongo, S., Nakamura, T., Kayama, T., Suzuki, Y., Kobayashi, K., Katagiri, T., Kurokawa, K., Kurimura, M., Toyoshima, I., Niizato, K., Tsuchiya, K., Iwatsubo, T., Muramatsu, M., Matsumine, H., Kato, T., 2006. The role of G-protein-coupled receptor kinase 5 in pathogenesis of sporadic Parkinson’s disease. J. Neurosci. 26, 9227e9238. Atwood, H.L., Govind, C.K., Wu, C.F., 1993. Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae. J. Neurobiol. 24, 1008e1024. Atwood, H.L., Karunanithi, S., Georgiou, J., Charlton, M.P., 1997. Strength of synaptic transmission at neuromuscular junctions of crustaceans and insects in relation to calcium entry. Invert. Neurosci. 3, 81e87. Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., Bonini, N.M., 2002. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science 295, 865e868. Basso, E., Antas, P., Marijanovic, Z., Goncalves, S., Tenreiro, S., Outeiro, T.F., 2013. PLK2 modulates alpha-synuclein aggregation in yeast and mammalian cells. Mol. Neurobiol. 48, 854e862. Boada-Romero, E., Letek, M., Fleischer, A., Pallauf, K., Ramon-Barros, C., PimentelMuinos, F.X., 2013. TMEM59 defines a novel ATG16L1-binding motif that promotes local activation of LC3. EMBO J. 32, 566e582. Breda, C., Nugent, M.L., Estranero, J.G., Kyriacou, C.P., Outeiro, T.F., Steinert, J.R., Giorgini, F., 2015. Rab11 modulates alpha-synuclein-mediated defects in synaptic transmission and behaviour. Hum. Mol. Genet. 24, 1077e1091. Chen, L., Periquet, M., Wang, X., Negro, A., McLean, P.J., Hyman, B.T., Feany, M.B., 2009. Tyrosine and serine phosphorylation of alpha-synuclein have opposing effects on neurotoxicity and soluble oligomer formation. J. Clin. Invest. 119, 3257e3265. Collins, C.A., DiAntonio, A., 2007. Synaptic development: insights from Drosophila. Curr. Opin. Neurobiol. 17, 35e42. de Lau, L.M., Breteler, M.M., 2006. Epidemiology of Parkinson’s disease. Lancet Neurol. 5, 525e535. Dev, K.K., Hofele, K., Barbieri, S., Buchman, V.L., van der Putten, H., 2003. Part II: alpha-synuclein and its molecular pathophysiological role in neurodegenerative disease. Neuropharmacology 45, 14e44. Folwell, J., Cowan, C.M., Ubhi, K.K., Shiabh, H., Newman, T.A., Shepherd, D., Mudher, A., 2010. Abeta exacerbates the neuronal dysfunction caused by human tau expression in a Drosophila model of Alzheimer’s disease. Exp. Neurol. 223, 401e409. Iljina, M., Tosatto, L., Choi, M.L., Sang, J.C., Ye, Y., Hughes, C.D., Bryant, C.E., Gandhi, S., Klenerman, D., 2016. Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein. Sci. Rep. 6, 33928. Iwai, A., Masliah, E., Yoshimoto, M., Ge, N., Flanagan, L., de Silva, H.A., Kittel, A., Saitoh, T., 1995. The precursor protein of non-A beta component of Alzheimer’s disease amyloid is a presynaptic protein of the central nervous system. Neuron 14, 467e475. Jiang, P., Gan, M., Ebrahim, A.S., Castanedes-Casey, M., Dickson, D.W., Yen, S.H., 2013. Adenosine monophosphate-activated protein kinase overactivation leads to accumulation of alpha-synuclein oligomers and decrease of neurites. Neurobiol. Aging 34, 1504e1515. Karuppagounder, S.S., Brahmachari, S., Lee, Y., Dawson, V.L., Dawson, T.M., Ko, H.S., 2014. The c-Abl inhibitor, nilotinib, protects dopaminergic neurons in a preclinical animal model of Parkinson’s disease. Sci. Rep. 4, 4874.

Lahey, T., Gorczyca, M., Jia, X.X., Budnik, V., 1994. The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure. Neuron 13, 823e835. Li, J., Uversky, V.N., Fink, A.L., 2001. Effect of familial Parkinson’s disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human alpha-synuclein. Biochemistry 40, 11604e11613. Liu, Q., Feng, R., Chen, Y., Luo, G., Yan, H., Chen, L., Lin, R., Ding, Y., Wen, T., 2018. Dcf1 Triggers dendritic spine formation and Facilitates memory Acquisition. Mol. Neurobiol. 55, 763e775. Liu, Q., Shi, J., Lin, R., Wen, T., 2017. Dopamine and dopamine receptor D1 associated with decreased social interaction. Behav. Brain Res. 324, 51e57. Mudher, A., Shepherd, D., Newman, T.A., Mildren, P., Jukes, J.P., Squire, A., Mears, A., Drummond, J.A., Berg, S., MacKay, D., Asuni, A.A., Bhat, R., Lovestone, S., 2004. GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila. Mol. Psychiatry 9, 522e530. Olanow, C.W., McNaught, K.S., 2006. Ubiquitin-proteasome system and Parkinson’s disease. Mov. Dis. 21, 1806e1823. Ortega-Arellano, H.F., Jimenez-Del-Rio, M., Velez-Pardo, C., 2017. Minocycline protects, rescues and prevents knockdown transgenic parkin Drosophila against paraquat/iron toxicity: implications for autosomic recessive juvenile parkinsonism. Neurotoxicology 60, 42e53. Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E.S., Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, W.G., Lazzarini, A.M., Duvoisin, R.C., Di Iorio, G., Golbe, L.I., Nussbaum, R.L., 1997. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045e2047. Pronin, A.N., Morris, A.J., Surguchov, A., Benovic, J.L., 2000. Synucleins are a novel class of substrates for G protein-coupled receptor kinases. J. Biol. Chem. 275, 26515e26522. Qing, H., Wong, W., McGeer, E.G., McGeer, P.L., 2009. Lrrk2 phosphorylates alpha synuclein at serine 129: Parkinson disease implications. Biochem. Biophys. Res. Commun. 387, 149e152. Smith, W.W., Margolis, R.L., Li, X., Troncoso, J.C., Lee, M.K., Dawson, V.L., Dawson, T.M., Iwatsubo, T., Ross, C.A., 2005. Alpha-synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells. J. Neurosci. 25, 5544e5552. Twelves, D., Perkins, K.S., Counsell, C., 2003. Systematic review of incidence studies of Parkinson’s disease. Mov. Dis. 18, 19e31. Ullrich, S., Munch, A., Neumann, S., Kremmer, E., Tatzelt, J., Lichtenthaler, S.F., 2010. The novel membrane protein TMEM59 modulates complex glycosylation, cell surface expression, and secretion of the amyloid precursor protein. J. Biol. Chem. 285, 20664e20674. Wen, T., Gu, P., Chen, F., 2002. Discovery of two novel functional genes from differentiation of neural stem cells in the striatum of the fetal rat. Neurosci. Lett. 329, 101e105. Wolters, E., 2008. Variability in the clinical expression of Parkinson’s disease. J. Neurol. Sci. 266, 197e203. Yang, Y., Nishimura, I., Imai, Y., Takahashi, R., Lu, B., 2003. Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila. Neuron 37, 911e924.