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The Solution Structure and Dynamics of Full-length Human Cerebral Dopamine Neurotrophic Factor and Its Neuroprotective Role against α-Synuclein Oligomers*

Open AccessPublished:July 06, 2015DOI:https://doi.org/10.1074/jbc.M115.662254
      Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9–107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10–100 and 111–157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1–9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.

      Introduction

      Parkinson disease (PD)
      The abbreviations used are: PD
      Parkinson disease
      NTF
      neurotrophic factor
      CDNF
      cerebral dopamine neurotrophic factor
      6-OHDA
      6-hydroxydopamine
      MANF
      mesencephalic astrocyte-derived NTF
      HSQC
      heteronuclear single quantum coherence
      SAXS
      small angle x-ray scattering
      ThT
      thioflavin T
      AFM
      atomic force microscopy
      TH
      tyrosine hydroxylase
      PDB
      Protein Data Bank
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      r.m.s.
      root mean square.
      is a degenerative disorder of the central nervous system that presents with characteristic motor symptoms that are a result of the death of dopamine-producing neurons in the substantia nigra (
      • Fahn S.
      Description of Parkinson's disease as a clinical syndrome.
      ). A hallmark of this disease is the occurrence of Lewy bodies, which are abnormal aggregates inside of neurons and are primarily composed of α-synuclein (
      • Baba M.
      • Nakajo S.
      • Tu P.H.
      • Tomita T.
      • Nakaya K.
      • Lee V.M.
      • Trojanowski J.Q.
      • Iwatsubo T.
      Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies.
      ). The α-synuclein protein is soluble and is abundant within pre-synaptic terminals in the brain. It can also form oligomers, which are believed to be the toxic species that result in the development of PD (
      • Winner B.
      • Jappelli R.
      • Maji S.K.
      • Desplats P.A.
      • Boyer L.
      • Aigner S.
      • Hetzer C.
      • Loher T.
      • Vilar M.
      • Campioni S.
      • Tzitzilonis C.
      • Soragni A.
      • Jessberger S.
      • Mira H.
      • Consiglio A.
      • Pham E.
      • Masliah E.
      • Gage F.H.
      • Riek R.
      In vivo demonstration that α-synuclein oligomers are toxic.
      ).
      Several compounds, including naturally occurring neurotrophic factors (NTFs), have been predicted to control the symptoms of and even lead to recovery in patients afflicted by PD (
      • Lindsay R.M.
      • Altar C.A.
      • Cedarbaum J.M.
      • Hyman C.
      • Wiegand S.J.
      The therapeutic potential of neurotrophic factors in the treatment of Parkinson's disease.
      ).
      Cerebral dopamine neurotrophic factor (CDNF) is considered one of the most potent NTFs. It can protect or repair rat dopaminergic neurons that have been exposed to 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, both of which are synthetic drugs that induce Parkinsonism. Initially described in 2007, CDNF has been used in therapeutic trials conducted on animal models in which PD was induced by 6-OHDA or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (
      • Airavaara M.
      • Harvey B.K.
      • Voutilainen M.H.
      • Shen H.
      • Chou J.
      • Lindholm P.
      • Lindahl M.
      • Tuominen R.K.
      • Saarma M.
      • Hoffer B.
      • Wang Y.
      CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice.
      ,
      • Voutilainen M.H.
      • Bäck S.
      • Peränen J.
      • Lindholm P.
      • Raasmaja A.
      • Männistö P.T.
      • Saarma M.
      • Tuominen R.K.
      Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease.
      • Lindholm P.
      • Voutilainen M.H.
      • Laurén J.
      • Peränen J.
      • Leppänen V.-M.
      • Andressoo J.-O.
      • Lindahl M.
      • Janhunen S.
      • Kalkkinen N.
      • Timmusk T.
      • Tuominen R.K.
      • Saarma M.
      Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo 2.
      ). Moreover, the overexpression of CDNF in rat striatum that had been lesioned using 6-OHDA was found to reduce neuroinflammation and repair parkinsonian behavior (
      • Nadella R.
      • Voutilainen M.H.
      • Saarma M.
      • Gonzalez-Barrios J.A.
      • Leon-Chavez B.A.
      • Jiménez J.M.
      • Jiménez S.H.
      • Escobedo L.
      • Martinez-Fong D.
      Transient transfection of human CDNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat substantia nigra.
      ,
      • Bäck S.
      • Peränen J.
      • Galli E.
      • Pulkkila P.
      • Lonka-Nevalaita L.
      • Tamminen T.
      • Voutilainen M.H.
      • Raasmaja A.
      • Saarma M.
      • Männistö P.T.
      • Tuominen R.K.
      Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease.
      • Ren X.
      • Zhang T.
      • Gong X.
      • Hu G.
      • Ding W.
      • Wang X.
      AAV2-mediated striatum delivery of human CDNF prevents the deterioration of midbrain dopamine neurons in a 6-hydroxydopamine induced parkinsonian rat model.
      ).
      In its mature form, human CDNF is a protein comprised of 163 amino acids and is a paralog of mesencephalic astrocyte-derived NTF (MANF) (61% amino acid sequence identity and 82% similarity) (
      • Petrova P.
      • Raibekas A.
      • Pevsner J.
      • Vigo N.
      • Anafi M.
      • Moore M.K.
      • Peaire A.E.
      • Shridhar V.
      • Smith D.I.
      • Kelly J.
      • Durocher Y.
      • Commissiong J.W.
      MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons.
      ); there are also homologous genes in invertebrates (
      • Palgi M.
      • Lindström R.
      • Peränen J.
      • Piepponen T.P.
      • Saarma M.
      • Heino T.I.
      Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons.
      ). As with other NTFs, human CDNF can be secreted from transiently transfected neurosecretory cells (
      • Sun Z.-P.
      • Gong L.
      • Huang S.-H.
      • Geng Z.
      • Cheng L.
      • Chen Z.-Y.
      Intracellular trafficking and secretion of cerebral dopamine neurotrophic factor in neurosecretory cells.
      ). It has been demonstrated that CDNF is widely expressed by neurons in the brain cortex, cerebellum, hippocampus, midbrain, striatum, and substantia nigra, a distribution that corroborates the important neurotrophic activity of this protein (
      • Lindholm P.
      • Voutilainen M.H.
      • Laurén J.
      • Peränen J.
      • Leppänen V.-M.
      • Andressoo J.-O.
      • Lindahl M.
      • Janhunen S.
      • Kalkkinen N.
      • Timmusk T.
      • Tuominen R.K.
      • Saarma M.
      Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo 2.
      ).
      The crystal structures of the N-terminal domain of CDNF (PDB code 2W50), encompassing residues 1–107, and of full-length MANF (PDB codes 2W51 and 2KVD) have revealed homologous N-terminal domains that are each composed of five α-helices and a 310 helix (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      ). The structure of the CDNF N-terminal domain is conserved among saposins, a class of proteins that interact with lipids and membranes. At the time of this writing, such activity has not been reported for CDNF or for MANF.
      Although the structure of the C-terminal portion of CDNF has never been determined, MANF has been found to have a well folded C-terminal domain. The two domains of MANF, an N-terminal domain (residues 7–91) and a C-terminal domain (residues 112–147), appear to have distinct activities (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      ,
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      • Hoseki J.
      • Sasakawa H.
      • Yamaguchi Y.
      • Maeda M.
      • Kubota H.
      • Kato K.
      • Nagata K.
      Solution structure and dynamics of mouse ARMET.
      ).
      The C-terminal domain of MANF has three-dimensional structural homology to the SAP (SAF-A/B Acinus and PIAS) domain of Ku70 (
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      ), an intracellular inhibitor of Bax (Bcl-2-associated X protein) (
      • Cohen H.Y.
      • Lavu S.
      • Bitterman K.J.
      • Hekking B.
      • Imahiyerobo T.A.
      • Miller C.
      • Frye R.
      • Ploegh H.
      • Kessler B.M.
      • Sinclair D.A.
      Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis.
      ). Accordingly, cytoplasmic injection of either full-length MANF or its C-terminal domain alone leads to an inhibition of Bax-dependent apoptosis in mouse superior cervical ganglion neurons, which suggests that this NTF possesses an intracellular mode of action (
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      ). Indeed, exogenously applied CDNF is able to prevent apoptosis in PC12 cells by modulating Bcl-2/Bax and caspase-3 activation (
      • Mei J.-M.
      • Niu C.-S.
      Effects of CDNF on 6-OHDA-induced apoptosis in PC12 cells via modulation of Bcl-2/Bax and caspase-3 activation.
      ). MANF also contains a functional KDEL endoplasmic reticulum retention signal at its C terminus, which binds to KDEL receptors both intracellularly and on the cell membrane (
      • Henderson M.J.
      • Richie C.T.
      • Airavaara M.
      • Wang Y.
      • Harvey B.K.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.
      ). The removal of this C-terminal tetrapeptide motif can increase the secretion of MANF, which contributes to the hypothesis that MANF has at least two distinct targets: an intracellular target that is associated with the endoplasmic reticulum and an extracellular target.
      It is notable that although MANF possesses two domains that are distinct in their activities, the intact, full-length protein is biologically necessary. For example, only mature MANF (and none of its individual domains) has been shown to rescue larval lethality in Drosophila, as determined by a transgenic rescue approach (
      • Lindström R.
      • Lindholm P.
      • Kallijärvi J.
      • Yu L.-Y.
      • Piepponen T.P.
      • Arumäe U.
      • Saarma M.
      • Heino T.I.
      Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.
      ). More striking is the fact that, in the same study, CDNF was not able to rescue the lethal phenotype, which suggests that these paralogs perform distinct actions. The synergic effect of MANF and CDNF intranigral overexpression in a rat model for PD supports this hypothesis (
      • Cordero-Llana Ó.
      • Houghton B.C.
      • Rinaldi F.
      • Taylor H.
      • Yáñez-Muñoz R.J.
      • Uney J.B.
      • Wong L.F.
      • Caldwell M.A.
      Enhanced efficacy of the CDNF/MANF family by combined intranigral overexpression in the 6-OHDA rat model of Parkinson's disease.
      ).
      It is still not clear why the crystallized structure of CDNF lacks a 6-kDa C-terminal domain, which was intriguingly cleaved either during or after purification even when the solution of the protein appeared to be pure (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      ,
      • Latgé C.
      • Cabral K.M.
      • Almeida M.S.
      • Foguel D.
      1H-, 13C- and 15N-NMR assignment of the N-terminal domain of human cerebral dopamine neurotrophic factor (CDNF).
      ). It has been speculated that CDNF requires proteolytic cleavage of the linker region that connects its two domains to produce a mature protein that can function through these domains separately (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      ). Additionally, it has been suggested that the C-terminal domain of CDNF, which spans residues 108–161, is intrinsically unfolded (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      ).
      In this paper, we addressed two eminent uncertainties regarding the structure and function of CDNF by addressing the following questions. 1) Does the three-dimensional structure of CDNF differ from that of MANF, specifically with respect to the structure of the C-terminal domain? 2) Can CDNF actively protect or rescue neurons against a physiological effector of neuronal injury that is associated with PD, namely, oligomers of α-synuclein?
      To address the first question, we determined the NMR solution structure of recombinant, full-length CDNF (amino acids 1–161) in solution at pH 6. This task was made possible because of an improved purification protocol that prevents the proteolysis of CDNF. The three-dimensional structure of CDNF revealed a monomeric protein with two well folded domains: an N-terminal domain of ∼100 amino acids and a C-terminal domain of ∼51 amino acids. More strikingly, we found two dynamic surface patches on the N-terminal domain that could represent active sites. The structure of the C-terminal domain resembles the structure of MANF, and the intracellular effects of these paralogs, including those associated with organelle distribution and anti-apoptotic effects, are therefore most likely conserved (
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      ). In answering the second question above, we demonstrated that recombinant CDNF protects and rescues both primary cultures of dopaminergic neurons, which were isolated from E14 mouse mesencephalon, and cultures of differentiated neuron 2a (N2a) cells from toxicity induced by α-synuclein oligomers.

      Discussion

      In addition to the present work, four other studies have been performed to analyze the functions of different regions found within the three-dimensional structures of CDNF and its paralog MANF (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      ,
      • Hoseki J.
      • Sasakawa H.
      • Yamaguchi Y.
      • Maeda M.
      • Kubota H.
      • Kato K.
      • Nagata K.
      Solution structure and dynamics of mouse ARMET.
      ,
      • Cohen H.Y.
      • Lavu S.
      • Bitterman K.J.
      • Hekking B.
      • Imahiyerobo T.A.
      • Miller C.
      • Frye R.
      • Ploegh H.
      • Kessler B.M.
      • Sinclair D.A.
      Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis.
      ,
      • Mei J.-M.
      • Niu C.-S.
      Effects of CDNF on 6-OHDA-induced apoptosis in PC12 cells via modulation of Bcl-2/Bax and caspase-3 activation.
      ,
      • Henderson M.J.
      • Richie C.T.
      • Airavaara M.
      • Wang Y.
      • Harvey B.K.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.
      • Lindström R.
      • Lindholm P.
      • Kallijärvi J.
      • Yu L.-Y.
      • Piepponen T.P.
      • Arumäe U.
      • Saarma M.
      • Heino T.I.
      Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.
      ). In 2011 Hellman et al. (
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      ) indicated that the C-terminal domain of MANF contains an active site similar to that of the anti-apoptotic protein Ku-70; this site in MANF was found to have anti-apoptotic activity when expressed within or injected into mouse superior cervical ganglion neurons. MANF also possesses an active endoplasmic reticulum retention signal at its C terminus, corroborating its anti-apoptotic activity (
      • Henderson M.J.
      • Richie C.T.
      • Airavaara M.
      • Wang Y.
      • Harvey B.K.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.
      ).
      The N-terminal domains of CDNF and MANF remain mysterious. They are structurally homologous to saposin-like proteins (SAPLIPS), a group of proteins with varied activities that include membrane binding, however, there is very little conservation of amino acids between these structural homologs (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      ,
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      • Hoseki J.
      • Sasakawa H.
      • Yamaguchi Y.
      • Maeda M.
      • Kubota H.
      • Kato K.
      • Nagata K.
      Solution structure and dynamics of mouse ARMET.
      ,
      • Bruhn H.
      A short guided tour through functional and structural features of saposin-like proteins.
      ). No membrane binding activity has been detected for CDNF, and exogenous MANF does not bind to superior cervical ganglion neurons in vitro (
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      ). Additionally, mutational analysis of basic residues on the surface of MANF, which are conserved on the surface of CDNF and might enable these proteins to interact with negatively charged phospholipids on cell membranes in a similar manner as SAPLIPS, revealed that they are not essential for its activity (
      • Lindström R.
      • Lindholm P.
      • Kallijärvi J.
      • Yu L.-Y.
      • Piepponen T.P.
      • Arumäe U.
      • Saarma M.
      • Heino T.I.
      Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.
      ). This finding can be added to the recurrent cases of an ancient fold being used by proteins possessing very different activities.
      Our NMR data on CDNF dynamics indicated two patches on the surface of CDNF that possessed dynamic properties equivalent to active sites, which may participate in either protein-protein interactions or catalysis (Fig. 2). The abundance of water-exposed hydrophobic residues (Leu-34, Leu-70, Ile-69, and Ala-66) in one of these sites indicates that it might serve as a protein-protein interaction interface (
      • Chakrabarti P.
      • Janin J.
      Dissecting protein-protein recognition sites.
      ,
      • Bordner A.J.
      • Abagyan R.
      Statistical analysis and prediction of protein-protein interfaces.
      • De S.
      • Krishnadev O.
      • Srinivasan N.
      • Rekha N.
      Interaction preferences across protein-protein interfaces of obligatory and non-obligatory components are different.
      ). The N-terminal region of α-helix 4 (residues Ala-65 to Val-73), which is the central portion of this active site, was found to have intermediate dynamics and therefore might experience conformations ranging from the solvent exposure of residues Ala-66 and Leu-70 to a conformation that resembles or is identical to the crystallographic structure, which has a well defined helix with buried Ala-66 and Leu-70 side chains (Fig. 3).
      The C-terminal domain of CDNF, which was absent in the constructs used for the crystallization, has been presumed to be unstructured and to therefore introduce a long, disordered segment into CDNF (
      • Parkash V.
      • Lindholm P.
      • Peränen J.
      • Kalkkinen N.
      • Oksanen E.
      • Saarma M.
      • Leppänen V.M.
      • Goldman A.
      The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
      ); however, in this study, we show that this domain is well folded and possesses high structural similarity to the MANF C-terminal domain, including to its two active sites (Fig. 4).
      We demonstrate that CDNF prevents the toxic effects produced by α-synuclein oligomers from affecting dopaminergic neurons (FIGURE 6., FIGURE 7.). It has already been reported that CDNF protects dopaminergic neurons against lesions induced by 6-OHDA and that this protection is dose-dependent (
      • Lindholm P.
      • Voutilainen M.H.
      • Laurén J.
      • Peränen J.
      • Leppänen V.-M.
      • Andressoo J.-O.
      • Lindahl M.
      • Janhunen S.
      • Kalkkinen N.
      • Timmusk T.
      • Tuominen R.K.
      • Saarma M.
      Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo 2.
      ). We also observed that CDNF protects against 6-OHDA-induced injury in primary cell cultures prepared from mouse mesencephalon, which supports the relevance of using this model to assess the neurotrophic properties of this protein.
      The toxicity of α-synuclein has primarily been related to its propensity to form prefibrillar oligomers. The lethal effect observed in our experimental setup is consistent with the most accepted model for the toxicity of α-synuclein, which is the gain of toxic function by the oligomers. It is worth noting that the neurotoxicity can be caused by exogenous α-synuclein that is taken up through endocytotic mechanism (
      • Lashuel H.A.
      • Overk C.R.
      • Oueslati A.
      • Masliah E.
      The many faces of α-synuclein: from structure and toxicity to therapeutic target.
      ). Interestingly, the formation of toxic aggregates seems to be triggered by factors such as the dopamine (
      • Conway K.A.
      • Rochet J.C.
      • Bieganski R.M.
      • Lansbury Jr., P.T.
      Kinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adduct.
      ). In fact, we tested α-synuclein against undifferentiated N2a cells and it did not exert any toxic effect (data not shown). The downstream mechanism of toxicity induced by the oligomers is still not well known, and may involve proteasomal dysfunction, down-regulation of mitochondrial complex I activity, Ca2+ influx across the plasma membrane, apoptosis, and necrosis (
      • Lashuel H.A.
      • Overk C.R.
      • Oueslati A.
      • Masliah E.
      The many faces of α-synuclein: from structure and toxicity to therapeutic target.
      ). The cellular clearance of α-synuclein is performed by the ubiquitin-proteasome system and chaperone-mediated autophagy (
      • Webb J.L.
      • Ravikumar B.
      • Atkins J.
      • Skepper J.N.
      • Rubinsztein D.C.
      α-Synuclein is degraded by both autophagy and the proteasome.
      ), and interventions that stimulate chaperone-mediated autophagy can prevent α-synucleinopathy (
      • Spencer B.
      • Potkar R.
      • Trejo M.
      • Rockenstein E.
      • Patrick C.
      • Gindi R.
      • Adame A.
      • Wyss-Coray T.
      • Masliah E.
      Beclin 1 gene transfer activates autophagy and ameliorates the neurodegenerative pathology in α-synuclein models of Parkinson's and Lewy body diseases.
      ,
      • Decressac M.
      • Mattsson B.
      • Weikop P.
      • Lundblad M.
      • Jakobsson J.
      • Björklund A.
      TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity.
      • Xilouri M.
      • Brekk O.R.
      • Landeck N.
      • Pitychoutis P.M.
      • Papasilekas T.
      • Papadopoulou-Daifoti Z.
      • Kirik D.
      • Stefanis L.
      Boosting chaperone-mediated autophagy in vivo mitigates α-synuclein-induced neurodegeneration.
      ). Furthermore, aberrant α-synuclein inhibits the chaperone-mediated autophagy, which adds to the multitude of proposed mechanisms for its toxicity (
      • Xilouri M.
      • Vogiatzi T.
      • Vekrellis K.
      • Park D.
      • Stefanis L.
      Abberant α-Synuclein confers toxicity to neurons in part through inhibition of chaperone-mediated autophagy.
      ).
      Because CNDF contains putative protein-protein interaction sites, we first hypothesize that the protective effect of CDNF could arise by direct binding to α-synuclein, which could avoid the formation of toxic oligomers or block their uptake by neurons. Using steady-state tryptophan intrinsic fluorescence polarization (
      • Jameson D.M.
      • Croney J.C.
      • Moens P.D.
      Fluorescence: basic concepts, practical aspects, and some anecdotes.
      ) and far dot blotting (
      • Chambraud B.
      • Sardin E.
      • Giustiniani J.
      • Dounane O.
      • Schumacher M.
      • Goedert M.
      • Baulieu E.E.
      A role for FKBP52 in Tau protein function.
      ) we did not find any evidence of interaction among CDNF and neither monomeric nor oligomeric α-synuclein (data not shown). Notwithstanding, other possible mechanisms of action for CDNF might include: 1) binding to a transmembrane receptor, like other neurotrophic factors, activating survival pathways that surpasses the toxic effect of α-synuclein; 2) stimulating the cellular clearance pathways such as chaperone-mediated autophagy; and 3) inhibiting apoptosis by interaction with BAX via its Ku70-like active site (
      • Hellman M.
      • Arumäe U.
      • Yu L.Y.
      • Lindholm P.
      • Peränen J.
      • Saarma M.
      • Permi P.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
      ,
      • Mei J.-M.
      • Niu C.-S.
      Effects of CDNF on 6-OHDA-induced apoptosis in PC12 cells via modulation of Bcl-2/Bax and caspase-3 activation.
      ). Because CDNF has two domains, it can use at least two mechanisms to protect dopaminergic neurons against toxic prefibrillar oligomers of α-synuclein.
      CDNF was also found to be active in cultures of differentiated TH- and MAP2-positive N2a cells, which is a straightforward model for studying neuroprotective mechanisms. Moreover, this model may be useful in subsequent studies aimed at identifying the active site that resides within the N terminus of CDNF, as well as for the discovery of its receptor.

      Author Contributions

      D. F., F. L. P., and M. S. A. conceived and coordinated the study. D. F., D. P. R., F. L. P., K. M. S. C., and M. S. A. wrote the paper. C. L., M. S. A., and T. H. designed, performed, and analyzed the NMR data. C. L., D. P. R., J. A. F., K. M. S. C., and L. J. prepared the protein samples and provided technical assistance. G. A. P. O. designed, performed, and analyzed the SAXS data. D. P. R., J. A. F., and L. F. R. designed, performed, and analyzed experiments with cell cultures. D. R. P. and J. A. F. designed, performed, and analyzed the dot blot, Western blotting, ThT binding, and AFM. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      The 1 GHz NMR spectra were collected at the RALF-NMR large scale facility for high-field NMR in Lyon was supported by proposal European Union FP7 IRSES Grant 247546 (WW-NMR). SAXS data were collected at the Brazilian Synchrotron Light Laboratory (LNLS) under proposal SAXS1-14200. We thank Dr. Luciana Ferreira Romão for the primary cultures of dopaminergic neurons. The Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas (CNRMN) is gratefully acknowledged for providing access to NMR instrumentation.

      References

        • Fahn S.
        Description of Parkinson's disease as a clinical syndrome.
        Ann. N.Y. Acad. Sci. 2003; 991: 1-14
        • Baba M.
        • Nakajo S.
        • Tu P.H.
        • Tomita T.
        • Nakaya K.
        • Lee V.M.
        • Trojanowski J.Q.
        • Iwatsubo T.
        Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies.
        Am. J. Pathol. 1998; 152: 879-884
        • Winner B.
        • Jappelli R.
        • Maji S.K.
        • Desplats P.A.
        • Boyer L.
        • Aigner S.
        • Hetzer C.
        • Loher T.
        • Vilar M.
        • Campioni S.
        • Tzitzilonis C.
        • Soragni A.
        • Jessberger S.
        • Mira H.
        • Consiglio A.
        • Pham E.
        • Masliah E.
        • Gage F.H.
        • Riek R.
        In vivo demonstration that α-synuclein oligomers are toxic.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 4194-4199
        • Lindsay R.M.
        • Altar C.A.
        • Cedarbaum J.M.
        • Hyman C.
        • Wiegand S.J.
        The therapeutic potential of neurotrophic factors in the treatment of Parkinson's disease.
        Exp. Neurol. 1993; 124: 103-118
        • Airavaara M.
        • Harvey B.K.
        • Voutilainen M.H.
        • Shen H.
        • Chou J.
        • Lindholm P.
        • Lindahl M.
        • Tuominen R.K.
        • Saarma M.
        • Hoffer B.
        • Wang Y.
        CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice.
        Cell Transplant. 2012; 21: 1213-1223
        • Voutilainen M.H.
        • Bäck S.
        • Peränen J.
        • Lindholm P.
        • Raasmaja A.
        • Männistö P.T.
        • Saarma M.
        • Tuominen R.K.
        Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease.
        Exp. Neurol. 2011; 228: 99-108
        • Lindholm P.
        • Voutilainen M.H.
        • Laurén J.
        • Peränen J.
        • Leppänen V.-M.
        • Andressoo J.-O.
        • Lindahl M.
        • Janhunen S.
        • Kalkkinen N.
        • Timmusk T.
        • Tuominen R.K.
        • Saarma M.
        Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo 2.
        Nature. 2007; 448: 73-77
        • Nadella R.
        • Voutilainen M.H.
        • Saarma M.
        • Gonzalez-Barrios J.A.
        • Leon-Chavez B.A.
        • Jiménez J.M.
        • Jiménez S.H.
        • Escobedo L.
        • Martinez-Fong D.
        Transient transfection of human CDNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat substantia nigra.
        J. Neuroinflammation. 2014; 11: 209
        • Bäck S.
        • Peränen J.
        • Galli E.
        • Pulkkila P.
        • Lonka-Nevalaita L.
        • Tamminen T.
        • Voutilainen M.H.
        • Raasmaja A.
        • Saarma M.
        • Männistö P.T.
        • Tuominen R.K.
        Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease.
        Brain Behav. 2013; 3: 75-88
        • Ren X.
        • Zhang T.
        • Gong X.
        • Hu G.
        • Ding W.
        • Wang X.
        AAV2-mediated striatum delivery of human CDNF prevents the deterioration of midbrain dopamine neurons in a 6-hydroxydopamine induced parkinsonian rat model.
        Exp. Neurol. 2013; 248: 148-156
        • Petrova P.
        • Raibekas A.
        • Pevsner J.
        • Vigo N.
        • Anafi M.
        • Moore M.K.
        • Peaire A.E.
        • Shridhar V.
        • Smith D.I.
        • Kelly J.
        • Durocher Y.
        • Commissiong J.W.
        MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons.
        J. Mol. Neurosci. 2003; 20: 173-188
        • Palgi M.
        • Lindström R.
        • Peränen J.
        • Piepponen T.P.
        • Saarma M.
        • Heino T.I.
        Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2429-2434
        • Sun Z.-P.
        • Gong L.
        • Huang S.-H.
        • Geng Z.
        • Cheng L.
        • Chen Z.-Y.
        Intracellular trafficking and secretion of cerebral dopamine neurotrophic factor in neurosecretory cells.
        J. Neurochem. 2011; 117: 121-132
        • Parkash V.
        • Lindholm P.
        • Peränen J.
        • Kalkkinen N.
        • Oksanen E.
        • Saarma M.
        • Leppänen V.M.
        • Goldman A.
        The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional.
        Protein Eng. Des. Sel. 2009; 22: 233-241
        • Hellman M.
        • Arumäe U.
        • Yu L.Y.
        • Lindholm P.
        • Peränen J.
        • Saarma M.
        • Permi P.
        Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.
        J. Biol. Chem. 2011; 286: 2675-2680
        • Hoseki J.
        • Sasakawa H.
        • Yamaguchi Y.
        • Maeda M.
        • Kubota H.
        • Kato K.
        • Nagata K.
        Solution structure and dynamics of mouse ARMET.
        FEBS Lett. 2010; 584: 1536-1542
        • Cohen H.Y.
        • Lavu S.
        • Bitterman K.J.
        • Hekking B.
        • Imahiyerobo T.A.
        • Miller C.
        • Frye R.
        • Ploegh H.
        • Kessler B.M.
        • Sinclair D.A.
        Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis.
        Mol. Cell. 2004; 13: 627-638
        • Mei J.-M.
        • Niu C.-S.
        Effects of CDNF on 6-OHDA-induced apoptosis in PC12 cells via modulation of Bcl-2/Bax and caspase-3 activation.
        Neurol. Sci. 2014; 35: 1275-1280
        • Henderson M.J.
        • Richie C.T.
        • Airavaara M.
        • Wang Y.
        • Harvey B.K.
        Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.
        J. Biol. Chem. 2013; 288: 4209-4225
        • Lindström R.
        • Lindholm P.
        • Kallijärvi J.
        • Yu L.-Y.
        • Piepponen T.P.
        • Arumäe U.
        • Saarma M.
        • Heino T.I.
        Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.
        PLoS ONE. 2013; 8e73928
        • Cordero-Llana Ó.
        • Houghton B.C.
        • Rinaldi F.
        • Taylor H.
        • Yáñez-Muñoz R.J.
        • Uney J.B.
        • Wong L.F.
        • Caldwell M.A.
        Enhanced efficacy of the CDNF/MANF family by combined intranigral overexpression in the 6-OHDA rat model of Parkinson's disease.
        Mol. Ther. 2015; 23: 244-254
        • Latgé C.
        • Cabral K.M.
        • Almeida M.S.
        • Foguel D.
        1H-, 13C- and 15N-NMR assignment of the N-terminal domain of human cerebral dopamine neurotrophic factor (CDNF).
        Biomol. NMR Assign. 2013; 7: 101-103
        • Braga C.A.
        • Follmer C.
        • Palhano F.L.
        • Khattar E.
        • Freitas M.S.
        • Romão L.
        • Di Giovanni S.
        • Lashuel H.A.
        • Silva J.L.
        • Foguel D.
        The anti-Parkinsonian drug selegiline delays the nucleation phase of α-synuclein aggregation leading to the formation of nontoxic species.
        J. Mol. Biol. 2011; 405: 254-273
        • Bax A.D.
        • Grzesiek S.
        Methodological advances in protein NMR.
        Acc. Chem. Res. 1993; 26: 131-138
        • Guerry P.
        • Herrmann T.
        Comprehensive automation for NMR structure determination of proteins.
        Methods Mol. Biol. 2012; 831: 429-451
        • Serrano P.
        • Pedrini B.
        • Mohanty B.
        • Geralt M.
        • Herrmann T.
        • Wüthrich K.
        The J-UNIO protocol for automated protein structure determination by NMR in solution.
        J. Biomol. NMR. 2012; 53: 341-354
        • Zhu G.
        • Xia Y.
        • Nicholson L.K.
        • Sze K.H.
        Protein dynamics measurements by TROSY-based NMR experiments.
        J. Magn. Reson. 2000; 143: 423-426
        • Renner C.
        • Schleicher M.
        • Moroder L.
        • Holak T.A.
        Practical aspects of the 2D 15N-[1H]-NOE experiment.
        J. Biomol. NMR. 2002; 23: 23-33
        • Meiboom S.
        • Gill D.
        Modified spin-echo method for measuring nuclear relaxation times.
        Rev. Sci. Instrum. 1958; 29: 688-691
        • Vold R.L.
        • Waugh J.S.
        • Klein M.P.
        • Phelps D.E.
        Measurement of spin relaxation in complex systems.
        J. Chem. Phys. 1968; 48: 3831-3832
        • Peng J.W.
        • Wagner G.
        Investigation of protein motions via relaxation measurements.
        Methods Enzymol. 1994; 239: 563-596
        • Nederveen A.J.
        • Doreleijers J.F.
        • Vranken W.
        • Miller Z.
        • Spronk C.A.
        • Nabuurs S.B.
        • Güntert P.
        • Livny M.
        • Markley J.L.
        • Nilges M.
        • Ulrich E.L.
        • Kaptein R.
        • Bonvin A.M.
        RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank.
        Proteins. 2005; 59: 662-672
        • Brunger A.T.
        Version 1.2 of the Crystallography and NMR system.
        Nat. Protoc. 2007; 2: 2728-2733
        • Spera S.
        • Bax A.
        Empirical correlation between protein backbone conformation and C.α and C.β 13C nuclear magnetic resonance chemical shifts.
        J. Am. Chem. Soc. 1991; 113: 5490-5492
        • Luginbühl P.
        • Szyperski T.
        • Wüthrich K.
        Statistical basis for the use of 13Cα chemical shifts in protein structure determination.
        J. Magn. Reson. B. 1995; 109: 229-233
        • Lin D.
        • Manning N.O.
        • Jiang J.
        • Abola E.E.
        • Stampf D.
        • Prilusky J.
        • Sussman J.L.
        AutoDep: a web-based system for deposition and validation of macromolecular structural information.
        Acta Crystallogr. D Biol. Crystallogr. 2000; 56: 828-841
        • Koradi R.
        • Billeter M.
        • Wüthrich K.
        MOLMOL: a program for display and analysis of macromolecular structures.
        J. Mol. Graph. 1996; 14 (29–32.): 51-55
        • Svergun D.I.
        Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing.
        Biophys. J. 1999; 76: 2879-2886
        • Volkov V.V.
        • Svergun D.I.
        Uniqueness of ab initio shape determination in small-angle scattering.
        J. Appl. Cryst. 2003; 36: 860-864
        • Kozin M.B.
        • Svergun D.I.
        Automated matching of high- and low-resolution structural models.
        J. Appl. Cryst. 2001; 34: 33-41
        • Krüger R.
        • Kuhn W.
        • Müller T.
        • Woitalla D.
        • Graeber M.
        • Kösel S.
        • Przuntek H.
        • Epplen J.T.
        • Schöls L.
        • Riess O.
        Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease.
        Nat. Genet. 1998; 18: 106-108
        • Shaltiel-Karyo R.
        • Frenkel-Pinter M.
        • Rockenstein E.
        • Patrick C.
        • Levy-Sakin M.
        • Schiller A.
        • Egoz-Matia N.
        • Masliah E.
        • Segal D.
        • Gazit E.
        A blood-brain barrier (BBB) disrupter is also a potent α-synuclein (α-syn) aggregation inhibitor: a novel dual mechanism of mannitol for the treatment of Parkinson disease (PD).
        J. Biol. Chem. 2013; 288: 17579-17588
        • LeVine 3rd, H.
        Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides: detection of amyloid aggregation in solution.
        Protein Sci. 1993; 2: 404-410
        • Romão L.F.
        • Sousa Vde O.
        • Neto V.M.
        • Gomes F.C.
        Glutamate activates GFAP gene promoter from cultured astrocytes through TGF-β1 pathways.
        J. Neurochem. 2008; 106: 746-756
        • Mosmann T.
        Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
        J. Immunol. Methods. 1983; 65: 55-63
        • Laemmli U.K.
        Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
        Nature. 1970; 227: 680-685
        • Jarymowycz V.A.
        • Stone M.J.
        Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences.
        Chem. Rev. 2006; 106: 1624-1671
        • Boehr D.D.
        • Wright P.E.
        How do proteins interact?.
        Science. 2008; 320: 1429-1430
        • Csermely P.
        • Palotai R.
        • Nussinov R.
        Induced fit, conformational selection and independent dynamic segments: an extended view of binding events.
        Trends Biochem. Sci. 2010; 35: 539-546
        • Sawada M.
        • Hayes P.
        • Matsuyama S.
        Cytoprotective membrane-permeable peptides designed from the Bax-binding domain of Ku70.
        Nat. Cell. Biol. 2003; 5: 352-357
        • Iwai A.
        • Masliah E.
        • Yoshimoto M.
        • Ge N.
        • Flanagan L.
        • de Silva H.A.
        • Kittel A.
        • Saitoh T.
        The precursor protein of non-Aβ component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system.
        Neuron. 1995; 14: 467-475
        • 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.
        Mutation in the α-synuclein gene identified in families with Parkinson's disease.
        Science. 1997; 276: 2045-2047
        • Narhi L.
        • Wood S.J.
        • Steavenson S.
        • Jiang Y.
        • Wu G.M.
        • Anafi D.
        • Kaufman S.A.
        • Martin F.
        • Sitney K.
        • Denis P.
        • Louis J.C.
        • Wypych J.
        • Biere A.L.
        • Citron M.
        Both familial Parkinson's disease mutations accelerate α-synuclein aggregation.
        J. Biol. Chem. 1999; 274: 9843-9846
        • van Rooijen B.D.
        • van Leijenhorst-Groener K.A.
        • Claessens M.M.
        • Subramaniam V.
        Tryptophan fluorescence reveals structural features of α-synuclein oligomers.
        J. Mol. Biol. 2009; 394: 826-833
        • Wan O.W.
        • Chung K.K.
        The role of alpha-synuclein oligomerization and aggregation in cellular and animal models of Parkinson's disease.
        PLoS ONE. 2012; 7e38545
        • Smeyne R.J.
        • Jackson-Lewis V.
        The MPTP model of Parkinson's disease.
        Mol. Brain Res. 2005; 134: 57-66
        • Fraichard A.
        • Chassande O.
        • Bilbaut G.
        • Dehay C.
        • Savatier P.
        • Samarut J.
        In vitro differentiation of embryonic stem cells into glial cells and functional neurons.
        J. Cell Sci. 1995; 108: 3181-3188
        • Bruhn H.
        A short guided tour through functional and structural features of saposin-like proteins.
        Biochem. J. 2005; 389: 249-257
        • Chakrabarti P.
        • Janin J.
        Dissecting protein-protein recognition sites.
        Proteins. 2002; 47: 334-343
        • Bordner A.J.
        • Abagyan R.
        Statistical analysis and prediction of protein-protein interfaces.
        Proteins. 2005; 60: 353-366
        • De S.
        • Krishnadev O.
        • Srinivasan N.
        • Rekha N.
        Interaction preferences across protein-protein interfaces of obligatory and non-obligatory components are different.
        BMC Struct. Biol. 2005; 5: 15
        • Lashuel H.A.
        • Overk C.R.
        • Oueslati A.
        • Masliah E.
        The many faces of α-synuclein: from structure and toxicity to therapeutic target.
        Nat. Rev. Neurosci. 2013; 14: 38-48
        • Conway K.A.
        • Rochet J.C.
        • Bieganski R.M.
        • Lansbury Jr., P.T.
        Kinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adduct.
        Science. 2001; 294: 1346-1349
        • Webb J.L.
        • Ravikumar B.
        • Atkins J.
        • Skepper J.N.
        • Rubinsztein D.C.
        α-Synuclein is degraded by both autophagy and the proteasome.
        J. Biol. Chem. 2003; 278: 25009-25013
        • Spencer B.
        • Potkar R.
        • Trejo M.
        • Rockenstein E.
        • Patrick C.
        • Gindi R.
        • Adame A.
        • Wyss-Coray T.
        • Masliah E.
        Beclin 1 gene transfer activates autophagy and ameliorates the neurodegenerative pathology in α-synuclein models of Parkinson's and Lewy body diseases.
        J. Neurosci. 2009; 29: 13578-13588
        • Decressac M.
        • Mattsson B.
        • Weikop P.
        • Lundblad M.
        • Jakobsson J.
        • Björklund A.
        TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E1817-E1826
        • Xilouri M.
        • Brekk O.R.
        • Landeck N.
        • Pitychoutis P.M.
        • Papasilekas T.
        • Papadopoulou-Daifoti Z.
        • Kirik D.
        • Stefanis L.
        Boosting chaperone-mediated autophagy in vivo mitigates α-synuclein-induced neurodegeneration.
        Brain. 2013; 136: 2130-2146
        • Xilouri M.
        • Vogiatzi T.
        • Vekrellis K.
        • Park D.
        • Stefanis L.
        Abberant α-Synuclein confers toxicity to neurons in part through inhibition of chaperone-mediated autophagy.
        PLoS ONE. 2009; 4e5515
        • Jameson D.M.
        • Croney J.C.
        • Moens P.D.
        Fluorescence: basic concepts, practical aspects, and some anecdotes.
        Methods Enzymol. 2003; 360: 1-43
        • Chambraud B.
        • Sardin E.
        • Giustiniani J.
        • Dounane O.
        • Schumacher M.
        • Goedert M.
        • Baulieu E.E.
        A role for FKBP52 in Tau protein function.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2658-2663
        • Ulrich E.L.
        • Akutsu H.
        • Doreleijers J.F.
        • Harano Y.
        • Ioannidis Y.E.
        • Lin J.
        • Livny M.
        • Mading S.
        • Maziuk D.
        • Miller Z.
        • Nakatani E.
        • Schulte C.F.
        • Tolmie D.E.
        • Kent Wenger R.
        • Yao H.
        • Markley J.L.
        BioMagResBank.
        Nucleic Acids Res. 2008; 36: D402-D408