The mitochondrial permeability transition pore regulates Parkinson's disease development in mutant...

lable at ScienceDirect

Neurobiology of Aging 35 (2014) 1132e1152

Contents lists avai

Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

The mitochondrial permeability transition pore regulatesParkinson’s disease development in mutant a-synuclein transgenicmice

Lee J. Martin a,b,c,*, Samantha Semenkowa,b, Allison Hanaford a,b, Margaret Wong a

aDepartment of Pathology, Division of Neuropathology, Johns Hopkins University School of Medicine, Baltimore, MD, USAb Pathobiology Graduate Program, Johns Hopkins University School of Medicine, Baltimore, MD, USAcDepartment of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

a r t i c l e i n f o

Article history:Received 17 September 2013Received in revised form 7 November 2013Accepted 10 November 2013Available online 16 November 2013

Keywords:Adenine nucleotide translocaseInterneuronPorinPpifCerebellumVoltage-dependent anion channel 1

* Corresponding author at: Johns Hopkins UnivDepartment of Pathology, 558 Ross Building, 720 Rut21205-2196, USA. Tel.: þ1 410 502 5170; fax: þ1 410

E-mail address: [email protected] (L.J. Martin).

0197-4580/$ e see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2013.11.008

a b s t r a c t

Parkinson’s disease (PD) is a movement disorder caused by neurodegeneration in neocortex, substantianigra and brainstem, and synucleinopathy. Some inherited PD is caused by mutations in a-synuclein(aSyn), and inherited and idiopathic PD is associated with mitochondrial perturbations. However, themechanisms of pathogenesis are unresolved. We characterized a human aSyn transgenic mouse modeland tested the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in thedisease mechanisms. C57BL/6 mice expressing human A53T-mutant aSyn driven by a thymic antigen-1promoter develop a severe, age-related, fatal movement disorder involving ataxia, rigidity, and posturalinstability. These mice develop synucleinopathy and neocortical, substantia nigra, and cerebello-rubro-thalamic degeneration involving mitochondriopathy and apoptotic and non-apoptotic neuro-degeneration. Interneurons undergo apoptotic degeneration in young mice. Mutant aSyn associated withdysmorphic neuronal mitochondria and bound voltage-dependent anion channels. Genetic ablation ofcyclophilin D, an mPTP modulator, delayed disease onset, and extended lifespans of mutant aSyn mice.Thus, mutant aSyn transgenic mice on a C57BL/6 background develop PD-like phenotypes, and the mPTPis involved in their disease mechanisms.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Parkinson’s disease (PD) is a chronically progressive, age-related, fatally incapacitating movement disorder in humans. Esti-mates indicate that 4e6 million people are diagnosed with PD, andthis disease affects about 2% of the population at some time in life(Dorsey et al., 2007; Van Den Eeden et al., 2003). The greatestprevalence of PD occurs in the USA, with 100e250 cases per100,000 (Van Den Eeden et al., 2003), placing PD as the secondmost common neurodegenerative diseasewith an adult onset (afterAlzheimer’s disease). Progressive resting tremor, rigidity, brady-kinesia/akinesia, gait disturbance, and postural instability charac-terize PD clinically (Jankovic, 2008; Olanow and Tatton, 1999).Cardinal neuropathological features of PD are degeneration andelimination of dopamine neurons in substantia nigra (SN) and inother brainstem regions, dopamine depletion in striatum, and

ersity School of Medicine,land Avenue, Baltimore, MD955 9777.

ll rights reserved.

a-synuclein (aSyn) pathology (Dickson, 2012; Giasson et al., 2000a;Jellinger, 2012; Lowe et al., 1997; Olanow and Tatton, 1999). Themolecular pathogenesis of PD is not understood. At least 2 forms ofPD exist: idiopathic (sporadic) and heritable (familial) (Klein andSchlossmacher, 2007; Olanow and Tatton, 1999; Schapira, 2006a).Most PD cases are sporadic with no known genetic component.Epidemiologic studies reveal several risk factors for developingidiopathic PD. Aging and aSyn are the most common risk factors foridiopathic PD (Klein and Schlossmacher, 2007). Pesticides are alsolinked to the origin of PD (Ascherio et al., 2006; Tanner et al., 2011).Herbicides, well water (contaminated with pesticides), and indus-trial chemicals are possible environmental agents related to thedevelopment of PD (Schapira, 2006b).

Mitochondrial mechanisms are believed to be involved in PDpathogenesis (Coskun et al., 2012; Olanow and Tatton, 1999; Pilsland Winklhofer, 2012). Mitochondria became suspects in PD eti-ology when heroine abusers presented with PD after exposure to1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (Langston andBallard, 1983) which is converted by monoamine oxidase to acomplex I inhibitor and free radical generator (Cleeter et al., 1992;Nicklas et al., 1985; Ramsay et al., 1986). Mitochondrial

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_surname

mailto:[email protected]

www.sciencedirect.com/science/journal/01974580

http://www.elsevier.com/locate/neuaging

http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.008



L.J. Martin et al. / Neurobiology of Aging 35 (2014) 1132e1152 1133

involvement in idiopathic PD etiology became more compelling bythe discovery that complex I activity (NAPH dehydrogenase) isreduced in the SN (Schapira et al., 1989) and skeletal muscle(Bindoff et al., 1989) of PD cases. Complex I inhibitors, notably 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine and the pesticiderotenone, cause damage to dopaminergic neurons and are the basisof several animal and cell models of PD (Shimohama et al., 2003).Although mitochondrial mechanisms have been implicated in PDpathogenesis for over 2 decades, direct cause-effect relationshipsbetween mitochondrial damage and disease initiation and pro-gression are still unclear. Mutations in nuclear genes encodingdefinite mitochondrial proteins such as complex I, mitofusin 2,frataxin, and optic atrophy protein 1, as well as DNA polymerase d,adenine nucleotide translocase 1, and twinkle, have been linked toLeigh’s syndrome, Charcot-Marie-Tooth disease type 2, Friedreich’sataxia, autosomal dominant optic atrophy, and chronic progressiveexternal opthalmoplegia, respectively (Schapira, 2012), but linkageof these mutations to PD has not been seen. Furthermore, muta-tions in the mitochondrial DNA encoded complex I subunits causeLeber’s hereditary optic neuropathy (Vilkki et al., 1989) but not PD(Olanow and Tatton, 1999). About 5%e10% of people with PD havefamilial inheritance (Schapira, 2006a, 2006b). Most identified mu-tations occur in genes encoding proteins that have both non-mitochondrial and putative mitochondrial functions, includingaSyn (Polymeropoulos et al., 1997; Singleton et al., 2003), DJ-1(Bonifati et al., 2003), parkin (Kitada et al., 1998; Leroy et al.,1998), and leucine-rich repeat kinase-2 (Paisán-Ruíz et al., 2004;Zimprich et al., 2004), confounding the interpretation of theintracellular mechanisms leading to PD. However, mutations inphosphatase and tensin homolog-induced putative kinase-1(PINK1) do directly link mitochondrial dysfunction to the etiologyof some early-onset recessive forms of PD (Hatano et al., 2004; Pilsland Winklhofer, 2012; Valente et al., 2004), but PINK1 null mice donot develop neurodegeneration, despite mitochondrial abnormal-ities (Gispert et al., 2009). Thus, further interrogation of mito-chondrial dysfunction as a general cause of PD is still needed.

Because aSyn andmitochondria both seem to have roles in PD, itis possible that interplay between these 2 entities participates in PDpathogenesis (Hsu et al., 2000). aSyn is largely a cytoplasmic andnuclear protein that is enriched in nervous tissue axon terminals(Maroteaux et al., 1988; Murphy et al., 2000; Lesuisse and Martin,2002) and functions in presynaptic vesicle dynamics duringactivity-dependent neurotransmitter release (Chandra et al., 2005;Fortin et al., 2005); yet, it also has actions at mitochondria. aSynassociates with mitochondrial membranes in neurons (Nakamuraet al., 2011) and has been found to be imported into mitochondriato cause complex I impairment (Devi et al., 2008). Human aSyn(haSyn) with an alanine-53/threonine mutation (A53T) directlyinteracts with mitochondria in transgenic (tg) mice, and these micedevelop mitochondrial abnormalities in association with a severemovement disorder, synucleinopathy, and a shortened lifespan (Leeet al., 2002; Martin et al., 2006) but no loss of dopaminergic neu-rons in substantia nigra (Daher et al., 2012). Unexpectedly, themotor abnormality and neuropathology in mice with prion proteinpromoter-driven haSyn-A53T transgene expression is moreconsistent with a motor neuron disease, rather than a PD pheno-type, because of the profound loss of spinal motor neurons (Martinet al., 2006). Tg mice expressing haSyn-A53T driven by the humanneuron-specific thymic antigen-1 (Thy1)-promoter have also beengenerated, but their neurologic and neuropathological character-ization is limited (Chandra et al., 2005) and relevance to PD as adisease model is uncertain. The brain distribution of pathology andpossible mitochondrial mechanisms of disease in these mice havenot been studied. In this study we characterized the neuropa-thology in Thy1-haSyn-A53T tg mice and tested the hypothesis that

mitochondrial abnormalities are related causally to the diseaseprocess in PD-linked mutant aSyn tg mice through the mitochon-drial matrix protein cyclophilin D (CyPD) that modulates themitochondrial permeability transition pore (Baines et al., 2005;Bernardi et al., 2006; Crompton, 2004; Halestrap, 2009;Nakagawa et al., 2005).

2. Methods

2.1. Transgenic mice

The PD mice studied here were Thy1-haSyn-A53T tg mice. Theoriginal foundermouse of line B6.Cg-Tg[Thy1-SNCA*A53T]M53Sud/J (stock #008135) was purchased from The Jackson Laboratory (BarHarbor, ME, USA). There is no characterization of the brain neuro-pathology in these mice. We bred this line, a hybrid of SV129 andC57BL/6 strains, with pure C57BL/6 mice and then progeny werebackcrossed at least 7 generations into a pure C57BL/6 strainbackground with the goal of eliminating the SV129 background.The SV129 genetic background is known to increase susceptibilityto excitotoxic and necrotic neurodegeneration (Kofler et al., 2004;Schauwecker and Steward, 1997), and thus we wanted to mini-mize this prominent strain effect. All mice were genotyped at theage of 1 month to identify individuals with the A53T transgene. Theprimer pair used for polymerase chain reaction was: 50-GGCACCTAGAGGATCTCGACTAGTGG-30 (forward) and 50-GGACCTCGACGCTTAAGGCTTCAGG-30 (reverse). The agouti fur coat was eventuallyeliminated and studies were conducted exclusively on Thy1-A53Ttg mice on a C57BL/6 background (black fur coats). We evaluatedA53T mice at presymptomatic stages of disease (n ¼ 10), early- tomid-stages of disease (n ¼ 10), defined by presence of bradykinesiaand ataxia, and at near endstage disease (n ¼ 20), defined bypostural stiffness and immobility (Supplementary Data Videos 1and 2). Age-matched non-tg littermates served as controls. TheCyPD null mice are described elsewhere (Basso et al., 2005; Martinet al., 2011). The GlyT2-eGFP tg mice, with expression of eGFP onlyin glycinergic interneurons, are described elsewhere (Martin, 2011;Zeilhofer et al., 2005). The institutional Animal Care and UseCommittee approved the animal protocols.

2.2. Brain harvesting and processing for histology

Mice were anesthetized with an overdose of sodium pentobar-bital and perfused through the heart with ice-cold phosphate buffer-saline (100 mM, pH 7.4) followed by ice-cold 4% paraformaldehyde.After perfusion-fixation, the brain was removed after 2 hours, post-fixed overnight in 4% paraformaldehyde, and cryoprotected 24 hoursin 20% glycerol-phosphate buffer-saline. The brains were frozen andserially sectioned from frontal pole to posterior cerebellum in thecoronal plane at 40 mm on a sliding microtome with every sectionbeing saved individually in 96-well plates containing antifreezebuffer. The sections were stored at �20 �C. For histologic analyses,sections were selected systematically and stained using cresyl violet(CV) for cell morphology and counting, FD-silver for neuro-degeneration, the terminal transferase-mediated deoxyuridinetriphosphate-biotin nick end labeling (TUNEL)method (Martin,1999;Martin et al., 2006; Portera-Cailliau et al., 1997), as an assay for celldeath based on the detection of DNA double-strand breaks, andimmunohistochemistry.

2.3. Immunohistochemistry

We evaluated the localizations of haSyn, tyrosine hydroxylase(TH), the interneuron marker parvalbumin (Kita et al., 1990), sel-ected cell death proteins, and putative mitochondrial permeability

L.J. Martin et al. / Neurobiology of Aging 35 (2014) 1132e11521134

transition pore (mPTP) proteins in the brains of A53T mice. Immu-noperoxidase histochemistry with diaminobenzidine as chromogenor immunofluorescence were used as before (Martin et al., 2005,2006, 2007) to detect haSyn protein with a haSyn-specific mono-clonal Syn211 antibody (Giasson et al., 2000b; Sigma-Aldrich, St.Louis, MO, USA), TH with a rabbit polyclonal antibody (Novus Bi-ologicals, Littleton, CO, USA), cleaved caspase-3 with a rabbitmonoclonal antibody (R&D Systems, Minneapolis, MN, USA), andphospho-p53 with a rabbit polyclonal antibody (R&D systems).Autophagy was assessed using an antibody to LC3A (Cell Signaling

Fig. 1. Thy1-A53T haSyn C57BL/6 tg mice develop a Parkinson’s disease (PD)-likeneurologic phenotype. (A) Thy1-A53T mice develop over the first year of life an age-dependent loss of motor activity in the running wheel. Values are mean � standarddeviation (SD) (n ¼ 6 mice/group). Significant differences from age-matched controlare indicated: * p < 0.05, ** p < 0.01, and *** p < 0.001 (n ¼ 6). (B) Photograph of anendstage Thy1-A53T mouse in an akinetic, rigid, and stooped posture (see Video 2). (C)Thy1-A53T mice develop fatal disease and have a shortened lifespan compared withnon-tg littermate controls. Abbreviations: PD, Parkinson’s disease; SD, standarddeviation.

Technology, Beverly, MA, USA). In some instances the immunoper-oxidase sections were counterstained with CV before viewing.

We studied, as done for other mitochondrial proteins (Martinand Liu, 2002; Martin et al., 2007, 2009), voltage-dependentanion channel (VDAC), adenine nucleotide translocator (ANT), andCyPD. Antibodies to these proteins were characterized previously(Martin et al., 2009) by western blotting to determine specificity inbrain extracts. Additional negative controls for antibody specificitywere incubating sections in the primary antibody diluent withoutprimary antibody with all other steps similar. Immunofluorescencewas used for dual labeling to colocalize putative mPTP proteinregulators with the mitochondrial marker manganese SOD (orSOD2) using a rabbit polyclonal antibody (Assay Designs-Enzo LifeSciences, Farmingdale, NY, USA). Immunoreactivity was visualizedwith species-specific secondary antibodies (all raised in goat)conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen Cor-poration, Carlsbad, CA, USA).

2.4. Regional cell counting and neocortical measurements

Profile counting of Nissl-stained sections was done to estimate thenumbers of neurons in the substantia nigra pars compacta (SNc), rednucleus magnocellular division (RN), thalamic ventrolateral nucleus(VL), and cerebellar deep interposed nucleus (DCN) in Thy1-A53T tgand littermate non-tg mice at the age of 1 and 12 months (n ¼ 4e6mice/age/genotype). Neurons in these regions were counted inanatomic level-matched sections (2e3 sections/region) at 1000�magnification. Corresponding to the mouse brain stereotaxic atlas(Franklin and Paxinos, 1997), SNc neurons were counted atbregma �292, �3.16, and �3.52; RN neurons were counted atbregma�3.52 and�3.80; VL neurons were counted at bregma�0.94and �1.22; and DCN neurons were counted at bregma �5,88and �6.12. Strict morphologi criteria were applied when classifyingnormal appearing neurons, including a round, open, euchromaticnucleus (not condensed and darkly stained), globular Nissl staining ofthe cytoplasm, clear vacuole-free cytoplasm, and a cell body diameterof w10e20 mm. With these criteria, degenerating neurons withnecrotic, apoptotic, and necrotic-apoptotic hybrids as well as astro-cytes, oligodendrocytes, andmicrogliawere excluded from the counts.

Dying cells in TUNEL preparations were counted in SNc andcerebral cortex of A53T and control mice in 6 nonoverlappingmicroscopic fields at 1000� magnification. Four to five sectionsthrough the brain regions of interest were assessed. Only cells withclearly discernible TUNELþ nuclei were counted throughout thedepth of the section.

Immunopositive cell bodies in immunoperoxidase- and immuno-fluorescence- stained sections were also counted. Cleaved caspase-3þ

cells were counted in 6 nonoverlapping, microscopic fields of striatumand cerebellar cortex at 400�magnification. Phospho-p53þ cellswerecounted in 6 nonoverlapping, microscopic fields of striatum at 1000�magnification. Parvalbuminþ cells were counted in 4 nonoverlapping,microscopic fields of striatum at 200� magnification.

Neocortical gray mantle and subcortical white matter thick-nesses were measured by ocular filar micrometry in brain sectionsat a level of bregma �0.58. Somatosensory (S1) cortex and thesubjacent external capsule were analyzed in 5 different sections ineach mouse.

2.5. Silver staining

Silver staining was used to visualize degenerating neuronalelements in brain sections of Thy1-A53T a-Syn mice and non-tgage-matched control mice. Sections were processed using the FDNeuroSilver kit (FD Neurotechnologies Inc, Baltimore, MD, USA).

Fig. 2. Expression, aggregation, and localization of human a-synuclein (haSyn) in Thy1-A53T haSyn C57BL/6 tg mouse central nervous system (CNS). (A) Western blot demon-strating the specificity of monoclonal Syn211 antibody for haSyn. In young tg mouse whole brain extracts subjected to SDS-PAGE, haSyn was detected as a monomer at w14e16 kDa(arrow) and, at lower amounts, as stable high molecular forms greater than 112 kDa, consistent with the size and in vivo aggregation propensity of haSyn (Giasson et al., 2002; Souzaet al., 2000). These immunoreactive proteins were not detected in non-tg mouse brain (right lane). Ponceau S stained membrane shows protein loading. (B) Thy1-A53T haSyn tgmice at early symptomatic stages of disease expressed monomeric haSyn (arrow) in all CNS regions analyzed as well as apparent stable dimeric and oligomeric forms in manyregions, except olfactory bulb and cerebral cortex. Stable higher molecular weight aggregates were detected robustly in hindbrain and spinal cord (and also in hippocampus).



2.6. Western blotting

The expression of haSyn and themitochondrial proteins CyPD, ANT,and VDAC in mouse brain regions were evaluated by immunoblotting.HaSyn A53T tg mice at different stages of disease, including pre-symptomatic, early- to mid-symptomatic, and end-stage (n ¼ 8/dis-ease stage), were deeply anesthetized, decapitated, and the brain andspinal cord were removed quickly and rinsed in ice-cold Hanks bufferbefore snap freezing. Before freezing, the brain was microdissected inice-cold Hanks buffer into olfactory bulb, cerebral cortex, hippocam-pus, striatum, diencephalon, brainstem, and cerebellum. Crude tissueextracts were prepared and protein fractions were subjected to SDS-PAGE and immunoblotting using enhanced chemilumesence detec-tion as described (Martin et al., 2003). The reliability of sample loadingand electroblotting in each experiment was evaluated by stainingnitrocellulose membranes with Ponceau S before immunoblotting. Iftransfer was not uniform, blots were discarded and gels were runagain. Monoclonal antibody Syn211 (Giasson et al., 2000b)was used todetect haSyn. For CyPD, a mouse monoclonal antibody (cloneE11AE12BD4, MitoSciences, Eugene, OR, USA) was used. For ANT, amouse monoclonal antibody (MitoSciences, clone 5F51BB5AG7) and arabbit polyclonal antibody (Santa Cruz, H-188) were used. For VDAC, 2mouse monoclonal antibodies (MitoSciences, clone 20B12AF2 andCalbiochem, clone 89-173/016) were used. ANT and VDAC antibodieswere not isoform specific. The antibodies were used at concentrationsfor visualizing protein immunoreactivity within the linear range. Toquantify protein immunoreactivity, films were scanned and densi-tometry was performed as described (Martin et al., 2003). Proteinlevels were expressed as relative optical density measurements.Immunodensities were normalized to Ponceau S stained proteins.

2.7. Immunoprecipitation

Immunoprecipitation and western blot analysis of centarl ner-vous system (CNS) region homogenates from A53T mice and age-matched non-tg mice were used to identify interactions betweenhaSyn protein and proteins putatively involved in mPTP operation.Protein (100 mg) from mitochondria-enriched membrane fractionsfrom non-tg and A53T mouse brain was immunoprecipitated using5 mg of monoclonal Syn211 haSyn antibody. After immunocapture,the samples were subjected to SDS-PAGE and electroelution forwestern blot detection of CyPD and VDAC using enhanced chem-iluminescence detection.

2.8. Mouse crossing experiments

Mice homozygous for the CyPD targeted mutation on a C57/BL6background are viable, with normal growth and appearance, and

Ponceau S stained membrane shows protein loading. (C) In Thy1-A53T haSyn tg mice at endweight forms in most brain regions, while monomer levels (arrow) tended to be lower thanLocalization of haSyn in Thy1-A53T tg mouse somatosensory cerebral cortex by immunohisviolet counterstaining (blue) at low (D) and higher magnifications (E, F). HaSyn immunoreacbut no immunoreactivity was detected in non-tg mice (D, inset). Scale bars ¼ 120 mm (Dcytoplasm and nucleus of subsets of neurons in superficial layers (E, arrows) and deeper layeand intranuclear inclusions positive for haSyn (F, left inset, arrow). Axons in subcortical whinset), 62 mm (F right inset). GeI Localization of haSyn in Thy1-A53T tg mouse basal gangliaarrows), while midbrain sections in non-tg mice were completely blank after probing with Syand globus pallidus (I upper inset). Some neuronal cell bodies in striatum were haSynþ

ventrolateral nucleus (VL) of motor thalamus were enriched in haSyn immunoreactivity. Sca(J) Localization of haSyn in Thy1-A53T tg mouse cerebellum. HaSyn immunoreactivity wacerebellar cortex. Purkinje cell bodies in the Purkinje cell layer (PCL) were not immunoreacwith haSyn antibody (inset). Scale bars ¼ 23 mm, 47 mm (inset). (K) In the granule cell layer ofcorresponding to mossy fiber rosettes, were seen in the neuropil, and granule cell bodies, alt(inset). Cell bodies in the granule cell layer that were much larger than granule cells and lessmm (inset). (L) Numerous large neuronal cell bodies in the brainstem reticular formatiominobenzidine; CNS, central nervous system; GCL, granule cell layer; haSyn, human a-synu

are fertile (Basso et al., 2005; Martin et al., 2011). Mitochondriaisolated from heart and CNS of CyPD�/� (ppif�/�) mice are devoid ofCyPD, resistant to mitochondria swelling and mitochondrialpermeability transition, and are protected frommitochondrial Ca2þ

overload and oxidative stress (Baines et al., 2005; Martin et al.,2009, 2011). Adult mouse neurons without CyPD are protectedfrom necrosis and apoptosis (Martin, 2011; Martin et al., 2009,2011). We crossed A53T tg mice to ppif�/� mice (Basso et al.,2005) to test the hypothesis that CyPD, and possibly the mPTP,has a role in neurodegeneration cell death caused by haSyn-A53T.Mice with targeted deletions of both CyPD alleles were crossed toA53T mice and F1 offspring positive for the haSyn-A53T transgenewere crossed with F1 siblings lacking the transgene. F2 generationsyielded progeny carrying all polymerase chain reaction-confirmedCyPD genotypes (ppifþ/þ, ppifþ/�, and ppif�/�) with or without thehaSyn-A53T transgene. These mice were maintained for 3 genera-tions and followed longitudinally.

2.9. Statistical analyses

For histologic and western blot measurements groupmeans andvariances were evaluated statistically by 1-way analysis of variancefollowed by a Newman-Keuls post hoc test.

2.10. Photography and figure construction

Marker comparisons between tg and non-tg mice were madefrom sections that were imaged under identical conditions andanalyzed using identical parameters. Original images used forfigure construction were generated using digital photography.Digital images were captured as TiF files using a SPOT digitalcamera and SPOT Advanced software (Diagnostic Instruments,Sterling Heights, MI, USA) or a Nikon digital camera (DXM1200) andACT-1 software. Images were altered slightly for brightness andcontrast using ArcSoft PhotoStudio 2000 or Adobe Photoshopsoftware without changing the content and actual result.Figure composition was done using CorelDraw software with finalfigures being converted to TiF files. Files of composite figures wereadjusted for brightness and contrast in Adobe Photoshop.

3. Results

3.1. Thy1-A53T tg mice develop profound fatal age-related diseasemimicking neurologic aspects of PD

Thy1-A53T tg C57BL/6 mice were born in normal-sized littersand had no overt phenotype at birth. Juvenile mice also appearedovertly normal, as did young adult mice. Thy1-A53T tg mice at the

stage disease, detection of haSyn aggregates was shifted to the much higher molecularin mice at earlier disease. Ponceau S stained membrane shows protein loading. (D, F)tochemistry (with diaminobenzidine [DAB] as chromogen, brown staining) and cresyltivity was seen throughout the neuropil in all 6 layers of cerebral cortex in tg mice (D),), 62 mm (D inset). At higher magnification haSyn immunoreactivity was seen in thers (F, arrows) as well as in the neuropil. Cerebrocortical neurons developed cytoplasmicite matter were also haSynþ (F, right inset). Scale bars ¼ 25 mm (E and F), 14 mm (F leftcircuit regions. HaSyn immunoreactivity was enriched in subsets of nigral neurons (G,n211 antibody (H). HaSyn immunoreactivity was present in the neuropil of striatum (I)(I, arrows), but most neuronal cell bodes were not positive. Neuronal cell bodies inle bars ¼ 33 mm (G), 21 mm (H), 24 mm (I), 125 mm (I upper inset), 44 mm (I lower inset).s found in the neuropil of the molecular layer (ML) and granule cell layer (GCL) oftive for haSyn. Cerebellar sections in non-tg mice were completely blank after probingThy1-A53T tg mouse cerebellum, numerous clusters of haSyn immunoreactivity, likelyhough not haSynþ, had their surface contours decorated with haSyn immunoreactivitynumerous were haSynþ and were likely Golgi cells (inset arrow). Scale bar ¼ 25 mm, 20n (arrows) were positive for haSyn. Scale bar ¼ 30 mm. Abbreviations: DAB, dia-clein; ML, molecular layer; PCL, Purkinje cell layer; VL, ventrolateral nucleus.

Fig. 3. The substantia nigra compacta (SNc) undergoes age-related degeneration in Thy1-A53T haSyn tg mice. (A, B) Low magnification images of cresyl violet (Nissl) stainedmidbrain hemisections of age-matched non-tg (A) and Thy1-A53T (B) mice suggesting neuronal dropout in the SNc of tg mice. The nearby red nucleus (RN) also appears affected intg mice. Scale bar ¼ 400 mm. (C, D) Nissl staining of age-matched mouse midbrain sections shows the SNc of non-tg mice (C) populated with large darkly staining neurons and theSNc of A53T tg mice (D) depopulated of neurons and having reactive changes. Remaining SNc neurons in A53T tg mice contained round cytoplasmic inclusions (D left inset) ordisplayed chromatolytic changes in the cell body (D right inset). Scale bars: C (same for D) ¼ 20 mm, D left inset (same for right inset) ¼ 12 mm. (E) Counts of SNc neurons in Nissl-stained sections revealed a significant loss (asterisk, p < 0.01) in Thy1-A53T tg mice at the age of 12 months but not in tg mice at the age of 1 month. Values are mean � standarddeviation (SD) (n ¼ 6 mice/group). (F, G) Low magnification images of tyrosine hydroxylase (TH) immunohistochemically-stained midbrain hemisections of age-matched non-tg (F)and Thy1-A53T (G) mice showing attenuation of TH immunoreactivity (brown labeling) in the substantia nigra (SN) of A53T mice. Scale bar in F (same for G) ¼ 400 mm. (H, I) THimmunoreactivity was enriched in SNc neuronal cell bodies (H arrows), processes, and neuropil in non-tg mice. TH immunoreactivity was attenuated in SNc neuronal cell bodies,processes, and neuropil in Thy1-A53T tg mice (I) compared with the staining in non-tg mice (H). Scale bar in H (same for I) ¼ 25 mm. (J, K) Low magnification images of THimmunohistochemically-stained forebrain hemisections of age-matched non-tg (J) and Thy1-A53T (K) mice showing attenuation of TH immunoreactivity (brown labeling) in thestriatum of tg mice. TH immunoreactivity was highly enriched in the striatal neuropil of non-tg mice (J inset) compared with the striatum of tg mice (K). The loss of striatal neuropilTH immunoreactivity in A53T mice was accompanied by accumulation of numerous dystrophic axons (K inset). The olfactory tubercle (OT) in tg mice was mostly spared comparedwith the dorsal striatum, consistent with its dopaminergic innervation derived from the ventral tegmental area instead of the SNc. Scale bars: J (same for K) ¼ 800 mm, J inset (samefor K inset) ¼ 50 mm. Abbreviations: OT, olfactory tubercle; RN, red nucleus; SD, standard deviation; SN, substantia nigra; SNc, substantia nigra compacta; TH, tyrosine hydroxylase.



age of 2 and 4 months had normal spontaneous motor activity(Fig. 1A). However tg mice at the age of 6 months showed deficitsin motor activity compared with non-tg littermate controls andThy1-A53T tg mice at the age of 2 and 4 months (Fig. 1A). Motoractivity progressively deteriorated to complete immobility by theage of 12 months (Fig. 1A and B, Supplementary videos 1 and 2).This period of progressive neurologic deterioration was charac-terized by prominent motor phenotypes. A53T mice began toshow spasticity and tremors at the age of about 6 months. Rigidityemerged at the age of about 8 months coinciding with thedevelopment of bradykinesia and gait abnormalities such as jerk-ing movements (Supplementary video 1). Mice that were about 1-year-old showed 100% penetrance in the development of rigidity,postural instability, and immobility (Fig. 1B, Supplementary video2). There was no indication of flaccid paralysis at any stage in thedisease process. Thy1-A53T tg mice had a much shortened life-span. Most mice died between the age of 10 and 12 months(Fig. 1C).

3.2. Expression and localization of haSyn protein in Thy1-A53T tgmice

Monoclonal Syn211 antibody with putative specificity for haSyn(with no detection of endogenous mouse aSyn) (Giasson et al.,2000b) was used to map by western blotting the regional expres-sion of haSyn-A53T transgene in the CNS of tg mice. Syn211 anti-body had confirmed specificity for haSyn because it did not detectendogenous a-Syn in non-tg mouse brain (Fig. 2A). In contrast, inA53T mouse brain extracts, Syn211 antibody detected monomerichaSyn at w16 kDa and higher molecular weight forms at w60 kDaand larger (Fig. 2A). Microdissected brain regions and spinal cordsamples showed robust expression of A53T-haSyn throughout theCNS in A53Tmice at the age of 6 months with early disease (Fig. 2B)and at the age of 12 months with endstage disease (Fig. 2C). HaSynwas detected in cerebral cortex, olfactory bulb, striatum, dien-cephalon, brainstem, cerebellum, and spinal cord. The individualCNS regions showed different propensities for the development ofhigher molecular weight species of haSyn early in the disease(Fig. 2B). High molecular weight (aggregated) forms of A53T-haSynwere seen most prominently in spinal cord, brainstem, cerebellum,and hippocampus early in disease. At the endstage of the diseasehigh molecular weight forms of A53T-haSyn were detected in allCNS regions that were examined (Fig. 2C).

Immunohistochemistry was used to localize haSyn protein indifferent brain regions in Thy1-A53T tg mice. Monoclonal Syn211antibody showed immunohistochemical specificity for haSynbecause it did not detect endogenous aSyn in perfusion-fixed non-tgmouse brain sections, including the cerebral cortex (Fig. 2D inset), SN(Fig. 2H), and cerebellar cortex (Fig. 2J inset). In contrast, in A53T tgmouse cerebral cortex, haSyn immunoreactivity was localized toneurons and the neuropil (Fig. 2DeF). Glial cells did not appearimmunoreactive for A53T-haSyn during the course of disease (datanot shown). haSynþ neuronal cell bodies were observed in cerebralcortical superficial (Fig. 2E) and deep (Fig. 2F) layers where it waslocalized to the cytoplasm and nucleus in a subset (about 35%) ofcortical neurons. Cortical neurons showed prominent cytoplasmicand intranuclear aggregates of haSyn (Fig. 2F left inset). Axons insubcortical white matter were also positive for haSyn (Fig. 2F rightinset). Basal ganglia and related compartments of the brain wereenriched in haSyn. Neurons in the SNc were strongly immunoreac-tive for haSyn (Fig. 2G). The striatum had robust haSyn immuor-eactivity in the neuropil (Fig. 2I), consistent with expression of haSynin nigrostriatal SNc projection neurons (Fig. 2G), but most striatalneuron cell bodieswere negative (Fig. 2I). However, aminor subset ofneuronal cell bodies was positive for haSyn (Fig. 2I), which were

subsequently identified as interneurons (see below). The globuspallidus was enriched in punctate immunoreactivity for haSyn thatwas found in the neuropil and decorating the surfaces of pallidalneurons (Fig. 2I upper inset). The ventral anterior-ventral lateralnuclei of thalamus contained numerous haSynþ neurons (Fig. 2Ilower inset). The cerebellum and brainstem also had prominenthaSyn immunoreactivity. The cerebellar cortex showed fine haSynpunctate labeling in the molecular layer (Fig. 2J), but Purkinje cellswere generally negative for haSyn (Fig. 2J). The granule cell layer hadprominent large clusters of immunoreactivity for haSyn (Fig. 2K).These clusters were consistent with the appearance of mossy fiberterminals (Weissman et al., 2011). Granule cell bodieswere uniformlynegative for haSyn (Fig. 2K), but their surfaces were decorated withhaSyn immunoreactivity (Fig. 2K inset). In contrast, larger cells nearthe granule cell layer-Purkinje cell layer border were haSynþ (Fig. 2Kinset). These cells were subsequently identified as Golgi in-terneurons, consistent with the known size and locations of theseneurons (Crook et al., 2006; Sillitoe et al., 2008; Simat et al., 2007). Inbrainstem, numerous neurons in the pontine andmedullary reticularformation, including the pontine nuclei and gigantocellular reticularnucleus, were haSynþ (Fig. 2L). These neuronal cell bodies are knownto be the origin of mossy fiber terminals in the cerebellar granule celllayer (Voogd et al., 1985) and would be congruent with the presenceof haSynþ mossy fiber endings in this layer (Fig. 2K).

3.3. Nigral neuron degeneration in Thy1-A53T tg mice

Thy1-A53T tg C57BL/6 mice were assessed for degeneration ofneurons in the SNc using Nissl staining and immunohistochemistryfor TH (Fig. 3). CV-stained sections of midbrain suggested loss ofneurons in the SNc in A53T tg mice at endstage disease comparedwith age-matchnon-tg littermate controls (Fig. 3A). Closer inspectionsupported this impressionnoted at lowmagnification as therewas anapparent dropout of larger neurons in the SNc in of A53T tg micewhen compared with controls (Fig. 3C and D). Counts of SNc neuronsin CV-stained sections of midbrain revealed a major (w60%) loss ofneurons (Fig. 3E). The loss of SNc neurons was age-related because 1-month-old A53T tg mice had a normal complement of SNc neurons(Fig. 3E). Immunohistochemical staining for TH in midbrain andforebrain sections corroborated the loss of SNc neurons in A53T tgmice. Mice at endstage showed diminished TH immunoreactivity inthe SNc compared with age-matched non-tg littermate controls(Fig. 3FeI). This finding was reflected by an attenuation of TH im-munostaining of neuronal cell bodies and the neuropil (Fig. 3H and I).The loss of TH immunoreactivity in the SNc was corroborated bydissipation of TH immunoreactivity in the striatum of A53T tg mice(Fig. 3J and K). TH immunoreactivity in the striatal neuropil of tgmice(Fig. 3K) was weak compared with the rich TH staining of controls(Fig. 3J). Moreover, dystrophic THþ axonswere seen in the striatumoftg mice (Fig. 3K inset). The dissipation of TH in the forebrain of A53Tmice was more dramatic in the dorsal striatum compared with theolfactory tubercle of ventral striatum (Fig. 3J and K) that receivesdopaminergic innervation from the ventral tegmental area instead ofthe SNc (Fallon and Loughlin, 1985).

3.4. Nigral neurons in Thy1-A53T tg mice undergo apoptotic celldeath

Additional evidence for the degeneration of the ventralmidbrain in Thy1-A53T tg C57BL/6 mice was the presence of con-spicuous argyrophilia within the SNc and surrounding gray matterandwhite matter regions in silver-stained sections (Fig. 4A). Nearbyregions that showed marked argyrophilic degeneration were theRN, superior cerebellar peduncle, medial lemniscus, and cerebralpeduncle, but the adjacent substantia nigra reticulata appeared

Fig. 4. Substantia nigra compacta (SNc) neurons undergo apoptotic cell death in Thy1-A53T haSyn tg mice. (A) Low magnification image of a silver-stained midbrain section of aThy1-A53T mouse demonstrating degeneration (dark gray/black staining) of the SNc and nearby structures including the red nucleus (RN) and major white matter axonal pathwayssuch as the superior cerebellar peduncle (scp), medical lemniscus (ml), and cerebral peduncle (cp). The substantia nigra reticulata (SNr) appeared unaffected as evidenced by thenegligible argyrophilia. Scale bar ¼ 312 mm. (B, C) At higher magnification, silver staining revealed ubiquitous degeneration (black staining) of axons and neuronal cell bodies in theSNc, RN, and scp. Silver impregnated profiles of degenerating SNc neuronal cell bodies (arrow) and processes could be seen detail in tg mice (B inset). Age-matched non-tg mousemidbrain sections were essentially blank for argyrophilia (C). Scale bars ¼ 25 mm (B), 14 mm (B inset), 17 mm (C). (D) Immunohistochemistry for haSyn (brown) and Nissl coun-terstaining (blue) showed that subsets of SNc neurons had strong sustained expression of haSyn (hatched arrows), but nearby neurons were not haSynþ (open arrows) insymptomatic Thy1-A53T haSyn tg mice. Inset shows a degenerating nigral neuron with a condensed nucleus, a cytoplasmic haSynþ inclusion (hatched arrow), and apparentinnervation from haSynþ axon terminals (solid arrow) in a tg mouse at endstage disease. Scale bar ¼ 7 mm, 10 mm (inset). (E) Immunohistochemistry for cleaved caspase-3 (brown)



mostly free of degeneration (Fig. 4A). This degeneration was evi-denced by silver-positive axons, cell bodies, and puncta (Fig. 4B),while age-matched non-tg control sections were blank for argyro-philia (Fig. 4C). Some silverþ degenerating neurons (Fig. 4B inset)had cell body and dendrite morphologies typical of dopaminergicSNc neurons (Fallon and Loughlin, 1985). The presence of robustdegeneration in the SNc is consistent with the high expression ofmutant haSyn in neurons in this region (Fig. 2G and Fig. 4D). Somedegenerating SNc neurons in Thy1-A53T tg showed cytoplasmicLewy-body-like inclusions that were haSynþ (Fig. 4D inset). Thesedegenerating SNc neurons also appeared to receive axosomaticinnervation from haSynþ neurons (Fig. 4D inset).

Thy1-A53T tg mice showed evidence for apoptotic cell death inthe SNc. haSynþ neurons had nuclei condensed into massesconsistent with apoptosis (Fig. 4D inset). Immunohistochemistryrevealed subsets of attritional SNc cells positive for cleavedcaspase-3. Some cleaved caspase-3þ SNc cells were round and hada distinct halo of separation from the extracellular matrix (Fig. 4E),indicative of apoptosis (Martin et al., 1998). Nissl stainingconfirmed the presence of apoptotic cells within the SNc of Thy1-A53T tg mice (Fig. 4E inset). TUNEL was used to demonstrate thepresence of cell death in the SNc (Fig. 4F). TUNELþ cell numberswere increased significantly in the SNc of Thy1-A53T tg mice at theage of 8 and 12 months compared with age-matched non-tglittermate controls (Fig. 4G). No difference between tg and non-tgmouse SNc were seen with TUNEL at the age of 1 month (Fig. 4G).A robust activation of p53, identified by immunohistochemistry forphospho-p53, was observed in subsets of SNc neurons in Thy1-A53T tg mice (Fig. 4H), but not in littermate non-tg controls(Fig. 4I).

3.5. The cerebral cortex degenerates in Thy1-A53T tg mice

Nissl-stained sections through the forebrain of Thy1-A53T tgC57BL/6 mice suggested pathology in cerebral cortex (Fig. 5Aand B). The ventricular system appeared dilated in mice at endstagedisease (Fig. 5B) compared with age matched non-tg controls(Fig. 5A). These sections also suggested thinning of the corticalmantle in tg mice (Fig. 5A and B). Ocular filar measurementsrevealed significant reductions in the thicknesses of the sensori-motor cortical gray matter (Fig. 5C) and subcortical white matter(Fig. 5D) in tg mice. Silver staining demonstrated axonal degener-ation in the cortical gray matter and subcortical white matter(Fig. 5F) and in the cerebral peduncle (Fig. 4A), which containscorticobulbar and corticospinal axons. TUNEL was used to assay forcell death in the cerebral cortex (Fig. 5F). TUNELþ cell numbers wereincreased significantly in layer V of Thy1-A53T tg mice at the age of8 and 12 months compared with non-tg littermate controls(Fig. 5G). Cleaved caspase-3 immunostaining was present in thecerebral cortex of symptomatic tg mice (Fig. 5H), while being un-detectable in age-matched non-tg control cerebral cortex (Fig. 5Hupper right inset). In tg mice, cleaved caspase-3 immunoreactivitywas found throughout the cortical neuropil and in pyramidal neu-rons in layer V (Fig. 5H lower right inset). Some cortical neuronsthat were cleaved caspase-3þ were found to have intranuclearhaSyn aggregates (Fig. 5H left inset). Nissl-staining revealed

showed that subsets of SNc cells were positive for cleaved caspase-3 (hatched arrows) in symand rounded-up and were separating from the extracellular matrix (as seen by the white peret al., 1998). Nissl staining revealed shrunken cells with apoptotically condensed chromatindetection by DNA fragmentation demonstrated TUNELþ cells (open arrows) and apoptotic frbar ¼ 7 mm. (G) Counting of TUNELþ cells in SNc revealed significant elevations above age-mmice at the age of 1 month (* p < 0.05; ** p < 0.01). Values are mean � standard deviation (SDshowed that subsets of SNc neurons accumulated phospho-p53-immunoreactivity in their cgenerally blank for activated p53 immunoreactivity, except for occasional phospho-p53þ intrpeduncle; ml, medical lemniscus; RN, red nucleus; scp, superior cerebellar peduncle; SNc,

morphologic evidence for apoptosis in cortical neurons of Thy1-A53T tg mice (Fig. 5J), and LC3A immunohistochemistry indicatedenhanced autophagy in cortical pyramidal neurons of tg mice(Fig. 5J) compared with age-matched non-tg mice (Fig. 5K).

In comparison to neocortex, the hippocampus displayed lessremarkable neuropathology in Thy1-A53T tg mice. Nissl stainingand cleaved caspase-3 immunohistochemistry yielded scant indi-cation of disease, but silver staining showed modest neuropilpunctate degeneration in the stratum radiatum of CA3 and stratumlacunosum-moleculare of CA1 (data not shown).

3.6. Striatal interneurons degenerate in Thy1-A53T tg mice

In the course of surveying tg mouse forebrain sections stainedimmunohistochemically for haSyn we found occasional cell bodiesin striatum that were positive for haSyn (Fig. 2I). These cells wereidentified as striatal interneurons because they were positive forthe interneuron marker parvalbumin (Fig. 6A). Most medium-sizedstriatal neuronswere not positive for haSyn, although they receivedextensive innervation from apparent presynaptic boutons that werepositive for haSyn (Fig. 6A). In the course of the disease in Thy1-A53T tg mice a small subset of striatal neurons was positive forphospho-p53 (Fig. 6B and F) and cleaved caspase-3 (Fig. 6CeDand F) as visualized by immunoperoxidase histochemistry andimmunofluorescence. Some of the cleaved caspase-3þ neuronswere large compared with most medium sized neurons (Fig. 6C).Immunoreactivity for cleaved caspese-3 was undetectable inparallel-processed sections from non-tg littermates (Fig. 6E). Cellcounting revealed an age-related increase in the number of striatalneurons that were positive for cleaved caspase-3 or phospho-p53(Fig. 6F), along with a corresponding decrease in the number ofparvalbuminþ interneurons in striatum of tg mice (Fig. 6G).

3.7. Cerebellar interneurons degenerate early in the course of thedisease in Thy1-A53T tg mice

Silver-stained sections of the cerebellum of Thy1-A53T tg miceat endstage disease displayed remarkable argyrophilic degenera-tion (Fig. 7A), while age-matched non-tg mouse sections had scantargyrophilic degeneration (Fig. 7B). Prominent axonal and terminaldegeneration was observed in cerebellar cortex of tg mice (Fig. 7A).Degenerating axons and putative terminals were concentrated inthe white matter and the granule cell layer, respectively (Fig. 7A).The molecular layer, although overall less argyrophilic than deeperlayers, also contained discernable degenerating axons and cellbodies (Fig. 7A). Immunohistochemical staining for cleavedcaspase-3 disclosed several salient findings in cerebellar cortex(Fig. 7C), while non-tg control sections were blank for cleavedcaspase-3 immunoreactivity (Fig. 7C inset). The molecular layerneuropil was enriched in cleaved caspase-3 immunoreactivity, butthe Purkinje cell bodies were not obviously labeled (Fig. 7C). In thegranule cell layer, subsets of cells larger than granule cells werecleaved caspase-3þ (Fig. 7C and D). These latter cells were oftenfound at the granule cell layer-Purkinje cell layer border (Fig. 7C andD) and were likely to be Golgi cells (Sillitoe et al., 2008; Simat et al.,2007; Zeilhofer et al., 2005). The dendritic arbors of Golgi cells

ptomatic Thy1-A53T haSyn tg mice. Some cleaved caspase-3þ cells appeared shrunkenicellular halo, open arrows), features typical of apoptosis (Martin and Liu, 2002; Martin(E inset arrow) in the SNc of A53T mice. Scale bar ¼ 10 mm, 7 mm (inset). (F) Cell deathagments (hatched arrows) in the SNc of symptomatic Thy1-A53T haSyn tg mice. Scaleatched non-tg control levels in Thy1-A53T tg mice at the age of 8 and 12 but not in tg) (n ¼ 6 mice/group). (H, I) Immunohistochemistry for activated phospho-p53 (brown)ell bodies and nuclei (H, arrow). Midbrain sections of age-matched non-tg mice wereavascular cells (I, arrow). Scale bar (in I same for H) ¼ 10 mm. Abbreviations: cp, cerebralsubstantia nigra compacta; SNr, substantia nigra reticulata.

Fig. 5. The cerebral cortex degenerates in Thy1-A53T haSyn tg mice. (A, B) Lowmagnification images of cresyl violet (Nissl) stained forebrain sections of age-matched non-tg (A) andThy1-A53T (B) mice suggesting atrophy of the neocortex in tg mice. The lateral ventricle (lv) was dilated and the cortical mantle appeared thinned in A53T mice at endstage disease(B) compared with age-matched non-tg mice (A). Scale bar (in A, same for B) ¼ 273 mm. (C) Ocular filar micrometer measurements revealed a significant reduction (asterisk,p < 0.05) in gray matter cortical thickness in endstage Thy1-A53T haSyn tg mice compared with age-matched non-tg littermate mice (n ¼ 6/group). Values are mean � standarddeviation (SD). (D) Ocular filar micrometer measurements revealed a significant reduction (asterisk, p < 0.01) in subcortical white matter thickness in endstage Thy1-A53T haSyn tgmice compared with age-matched non-tg littermate mice (n ¼ 6/group). Values are mean � SD. (E) Silver staining revealed degeneration axons (arrows) in cortical gray matter andsubcortical white matter in A53T tg mice. Scale bar ¼ 35 mm. (F) Cells with TUNELþ nuclei (arrows) were observed in the neocortex of symptomatic Thy1-A53T haSyn tg mice. Scalebar ¼ 10 mm. (G) Counting of TUNELþ cells in motor-sensory cortex revealed significant elevations above age-matched non-tg control levels in Thy1-A53T tg mice at the age of 8 and12 months but not in tg mice at the age of 1 month (* p < 0.01; ** p < 0.001). Values are mean � SD (n ¼ 6 mice/group). (H) Immunohistochemistry for cleaved caspase-3 (brown)showed positive staining throughout the cortical neuropil and in some neuronal cell bodies in symptomatic Thy1-A53T haSyn tg mice, while the cerebral cortex of age-matched


Fig. 6. Striatal interneurons degenerate in Thy1-A53T tg mice. (A) Immunofluorescence showing that parvalbuminþ striatal interneurons express haSyn in Thy1-A53T tg mice(arrow). Human a-synuclein (haSyn) was present in the cytoplasm and nucleus of parvalbuminþ neurons and was enriched in the neuropil, but most striatal neurons were haSyn�

(asterisks). Scale bar ¼ 6 mm. (B) Immunoperoxidase staining shows that a minority of striatal neurons in Thy1-A53T tg mice accumulates phospho-p53 (pp53) immunoreactivity(arrow). Scale bar ¼ 8 mm. (C) Immunoperoxidase staining (brown) identifies some large striatal neurons that are cleaved caspase-3þ in A53T tg mice. Scale bar ¼ 5 mm. (D, E)Immunofluorescence confirms the cleaved caspase-3 staining in subsets of striatal neurons (D, arrows) in tg mice, while striatal neurons in littermate non-tg mice were negative forcleaved caspase-3 (E). Scale bar (in A, same for E) ¼ 12 mm. (F) Graph showing the numbers of cleaved caspase-3þ and phospho-p53 (pp53)þ cells in the striatum of Thy1-A53T tgmice at the age of 1 and 12 months. Values are mean � SD. Non-tg littermates were negative. (G) Graph showing the numbers of parvalbuminþ cells in the striatum of Thy1-A53T tgmice and non-tg littermates at the age of 1 and 12 months. Values are mean � standard deviation (SD). Asterisk denotes significantly different from control (p< 0.01). Abbreviations:Ha-Syn, human a-synuclein; pp53, phospho-p53 cells; SD, standard deviation.


immunoreactive for cleaved caspase-3 would contribute to theobserved immunostaining (Fig. 7C) in the molecular layer neuropil(Sillitoe et al., 2008). Counts of cleaved caspase-3þ putative Golgicells revealed significant increases above non-tg age-matchedcontrols as early as the age of 1 month, and progressive increases inthe number of positive cells were seen thereafter (Fig. 7E). Silver-stained sections of cerebellum from 1-month-old Thy1-A53T tgmice showed isolated degenerating cell bodies in the granule celllayer, apoptotic cells in the molecular layers, and terminal degen-eration at the granule cell layer-Purkinje cell layer border (Fig. 7F).Neurons thought to be Golgi cells in cerebellar cortex were haSynþ

(Fig. 7F inset). To identify that early-degenerating cells in the cer-ebellumwere Golgi cells, Thy1-A53T tgmicewere crossed to GlyT2-eGFP tg which, among other cells throughout the CNS (Zeilhoferet al., 2005), express eGFP in cerebellar Golgi cells (Sillitoe et al.,2008; Simat et al., 2007). Subsets of eGFP-positive Golgi cells that

non-tg littermates was blank (H upper right inset). Layer V pyramidal neurons were positive3þ cortical neurons contained prominent intranuclear haSynþ inclusions (left inset) as seenright inset), 8 mm (lower right inset). (I) Nissl staining identified layer V pyramidal neuroncellular matrix (arrow). Scale bar ¼ 4 mm. (J, K) Immunohistochemistry for autophagy markeA53T tg mice compared with age-matched non-tg littermates. Scale bar (in J, same for K) ¼

expressed haSyn aggregates in their cytoplasm were found to beapoptotic in Thy1-A53T tg mice (Fig. 7G).

Thy1-A53T tg C57BL/6 mice at endstage disease also hadprominent degeneration of the deep cerebellar nuclei. Nissl andsilver staining gave indications of pathology in this region. Theinterposed nucleus contained neurons with prominent large vac-uoles, apparent cell elimination, and nests of small inflammatory-like cells (Fig. 7H). The vacuolar type of neuronal pathology hasbeen seen with chronic deafferentation (Ginsberg and Martin,1998). Age-matched non-tg controls had normal appearing neu-rons in cerebellar deep nuclei (Fig. 7I). Counts of neurons in theinterposed nucleus confirmed the impression of cell dropout bydemonstrating severe (w80%) loss of neurons in endstage mice buta normal complement of neurons in young adult tg mice (Fig. 7J).The interposed and lateral deep cerebellar nuclei also had degen-eration in silver-stained preparations (data not shown).

for cleaved caspase-3 (arrows) in A53T mice (lower right inset). Some cleaved caspase-by immunofluorescence. Scale bars ¼ 75 mm, 100 mm (upper right inset), 25 mm (lowercell bodies undergoing shrinkage, chromatin clumping, and separation for the extra-r LC3A (brown) showed enhanced LC3A immunostaining in layer V cortical neurons in35 mm. Abbreviations: lv, lateral ventricle; SD, standard deviation.

Fig. 7. Cerebellar interneurons and deep nuclei degenerate in Thy1-A53T tg mice. (A, B) Silver-stained sections of cerebellum from endstage Thy1-A53T tg mice displayed robustdegeneration (black staining) of axons in white matter (wm, arrows) and ubiquitous puncta in the granule cell layer (GL). Occasional degeneration of cell bodies was seen in themolecular layer (ML, open arrows). Age-matched non-tg mouse cerebellar sections were blank for argyrophilia (B). Scale bars ¼ 35 mm (A), 25 mm (B). (C, D) Immunohistochemistryfor cleaved caspase-3 (brown) showed positive staining throughout the molecular layer (ML) neuropil and in some cell bodies (C, arrows) in the granule cell layer (near the Purkinjecell layer border) in symptomatic Thy1-A53T haSyn tg mice, while the cerebellar cortex of age-matched non-tg littermates was blank for immunoreactivity (C inset). Cleavedcaspase-3þ cells in the granule cell layer were large cells (D, arrows) compared with the predominating granule cells that were negative. Scale bars ¼ 25 mm (C), 50 mm (C inset), 15mm (D). (E) Counts of cleaved caspase-3þ cells in the granule cell layer of Thy1-A53T tg mice at the age of 1, 8, and 12 months revealed significant elevations above age-matched non-tg control levels (* p < 0.05; ** p < 0.01, *** p < 0.001). Values are mean � standard deviation (SD) (n ¼ 6 mice/group). (F) Silver-stained sections of cerebellum from 1-month-oldThy1-A53T tg mice confirmed the isolated degeneration of cells in the granule cell layer (GL, arrow) and demonstrated punctate degeneration at the GL-Purkinje cell layer (PL)border. Occasional apoptotic cells were also seen in the molecular layer (ML, arrow). haSyn immunohistochemistry identified haSynþ cells in the GL that were attritional andseparating from the extracellular matrix (inset arrow), as well as haSyn immunoreactivity enriched in putative mossy fibers rosettes and decorating the surfaces of granule cells


Fig. 8. Red nucleus (RN) degeneration in Thy1-A53T haSyn tg mice. (AeC) Nissl staining of midbrain sections from age-matched non-tg littermates (A) and endstage Thy1-A53T tgmice (B, C) revealed degenerating neurons in the RN in tg mice, including apparent cell dropout and reactive cell changes (B, C). Many remaining neurons showed chromatolyticchanges (C, arrows). Scale bar ¼ 42 mm (A, same for B), 19 mm (C). (D) Counts of RN neurons revealed a significant loss (asterisk, p < 0.01) in Thy1-A53T tg mice at the age of 12months but not in tg mice at the age of 1 month. Values are mean � standard deviation (SD) (n ¼ 6 mice/group). (E) Some residual RN neurons (arrow) accumulated granulovacuole-like inclusions in their cytoplasm. Scale bar ¼ 11 mm. (F) Apoptotic profiles (arrow) were present in the RN of tg mice. Scale bar ¼ 8 mm. (G) Silver staining revealed severedegeneration of axons and puncta (black) in the red nucleus neuropil, while the RN in age-matched non-tg littermates was blank for argyrophilia (inset). Scale bar ¼ 33 mm (same forinset). (H, I) Immunohistochemistry for autophagy marker LC3A (brown) showed enhanced LC3A immunostaining of granules in the cytoplasm of RN neurons (I, arrows) in A53T tgmice compared with neurons in age-matched non-tg littermates (H). Scale bar (in H, same for I) ¼ 17 mm. Abbreviations: RN, red nucleus; SD, standard deviation.


3.8. Red nucleus degeneration in Thy1-A53T tg mice

Attention was directed to the RN in Thy1-A53T tg C57BL/6 micebecause of the degeneration of the cerebellar interpositus nucleus(Fig. 7H), which projects to the contralateral RN through the

(inset). Scale bars ¼ 8 mm (F), 25 mm (F inset). (G) Double tg mice expressing Thy1-A53Tapoptosis. Scale bar ¼ 6 mm. (H, I). Nissl staining of cerebellar sections from endstage Thy1-Ain the interpositus deep nucleus in tg mice including apparent cell dropout, reactive cell neopen arrow). Scale bars ¼ 20 mm (H, same for I), 10 mm (H inset). (J) Counts of nucleus interpoage of 12 months but not in tg mice at the age of 1 month. Values are mean � SD (n ¼ 6 mlayer; SD, standard deviation.

superior cerebellar peduncle (Voogd et al., 1985), and becausedamage within the RN was suggested while examining midbrainsections for SNc pathology (Figs. 3A, B and 4A, B). Nissl stainingindicated a loss of magnocellular neurons (Figs. 3A, B and 8A, B) inthe RN at endstage disease. Cell counting showed a significant loss

haSyn and GlyT2-eGFP showed that cerebellar Golgi cells expressing haSyn undergo53T tg mice (H) and age-matched non-tg littermates (I) revealed degeneration neuronssts (H inset hatched arrow), and cytoplasmic vacuolation of residual neurons (H insetsitus neurons revealed a significant loss (asterisk, p < 0.01) in Thy1-A53T tg mice at theice/group). Abbreviations: GL, granule cell layer; ML, molecular layer; PL, Purkinje cell


(w60%) of RN neurons in mice at endstage disease but not in youngadult tg mice (Fig. 8D). Many of the remaining neurons in tg micehad marked chromatolytic changes (Fig. 8C) indicative of axonalinjury (Martin and Liu, 2002) or accumulation of granulovacuole-like inclusions (Fig. 8E). Apoptotic cells were observed in the RNin tg mice (Fig. 8F). Silver staining demonstrated prominent axonaland punctate degeneration in the RN and intermingling superiorcerebellar peduncle of tg mice (Fig. 4A, B and Fig. 8G) while the RNin age-matched non-tg mice was blank for argyrophilic degenera-tion (Fig. 4C and Fig. 8G inset). Immunohistochemistry for LC3Awasdone to examine whether the granulovacuole-like inclusions re-flected autophagy. LC3A-positive granule inclusions accumulated insubsets of remaining RN neurons in A53T mice (Fig. 8I) but not incontrol mouse RN neurons (Fig. 8H).

3.9. Motor thalamus degeneration in Thy1-A53T tg mice

The thalamus receives cerebellar input (Bostan and Strick, 2010;Voogd et al., 1985), and because of the degeneration of the nucleusinterpositus of cerebellum and superior cerebellar peduncle nearthe RN, our attention was directed to the motor thalamus. Silverstaining offered the first indication of degeneration in this region(Fig. 9). Highly selective nuclear-specific degeneration was seen inthe ventral anterior (VA) and VL thalamic nuclei in Thy1-A53T tgmice at endstage disease (Fig. 9B), while the thalamus in age-matched non-tg mice was negative for argyrophilia (Fig. 9A). Thecentral medial, central lateral, and paracentral thalamic nuclei werealso affected (data not shown). Closer examination showed thepresence of degeneration cell bodies, axons, and puncta in theseregions (Fig. 9C and D). Nissl staining suggested reactive changes inresponse to neurodegeneration in the VA and/or VL of Thy1-A53T tgmice (Fig. 9F and H), while controls gave no indication of neuro-degeneration and small cell changes (Fig. 9E and G). Apoptoticprofiles were encountered in the VA and/or VL of Thy1-A53T tgmice at endstage disease (Fig. 9H). Neuronal cell counting revealeda significant loss of neurons (w40%) in the VA and/or VL of 12-month-old tg mice but not in 1 month old tg mice (Fig. 9I).

3.10. mPTP involvement in disease mechanisms in Thy1-A53T tgmice

Mitochondria have been implicated in the pathogenesis of PD inhumans (Beal, 2005; Coskun et al., 2012; Martin, 2006, 2010;Olanow and Tatton, 1999; Pilsl and Winklhofer, 2012; Reddy andReddy, 2011) and in A53T-haSyn tg mice (Martin et al., 2006). ThemPTPmay have particular importance in this regard (Martin, 2010).We attempted to establish direct cause-effect relationships be-tween the mPTP and disease mechanisms in PD using tg mice.Putative components or regulators of the mPTP are expressed inmouse SNc neurons (Fig. 10A and B). CyPD (Fig. 10A) and the ANT(Fig. 10B) are present in mitochondria marked by SOD2. Westernblotting for mPTP components revealed a modest increase in CyPDlevels in brainstem, striatum, and cortex of early and late symp-tomatic Thy1-A53T tg mice, but ANT and VDAC levels were notdifferent from age-matched non-tg mice (Fig. 10C and D). Co-

Fig. 9. Motor thalamus degeneration in Thy1-A53T haSyn tg mice. (AeD) Silver-stained sectidegeneration (black staining) of the ventrolateral nucleus (VL) of thalamus (B, C), while the tterritory designated as VL might include the ventral anterior (VA) thalamic nucleus. Reticula(D, open arrow), cell bodies (D, hatched arrow), and puncta were abundant in the VL of A53Tsections through diencephalon viewed at low magnification (E, F) revealed dilation of the ldark-staining enhancement in VL of Thy1-A53T haSyn tg mice. At higher magnification, thneurons (H, open arrows), and apparent small cell infiltrates. Scale bars ¼ 200 mm (E, same fop < 0.01) in Thy1-A53T tg mice at the age of 12 months but not in tg mice at the age of 1 moglobus pallidus; ic, internal capsule; lv, lateral ventricle; Rt, Reticular thalamic nucleus; SD,

immunoprecipitation showed that A53T-haSyn avidly binds VDACin brainstem, striatum, and cortex of early and late symptomaticThy1-A53T tg mice, but only slightly binds CyPD in cortex in vivo(Fig. 10E). A53T-haSyn associates with neuronal mitochondria thatappear aggregated and swollen in Thy1-A53T tg mouse SNc neu-rons (Fig. 10F). Reducing the levels of CyPD by genetic ablationsignificantly delayed disease onset and extended the lifespan ofThy1-A53T tg mice (Fig. 10G).

4. Discussion

This study provides new information on the pathogenesis of PDusing a tg mouse model. This mouse model expresses the humanA53Tmutant variant of aSyn driven bya Thy1 promoter. The originaltg mouse line was generated in the Sudhof laboratory on a hybridbackground (Chandra et al., 2005), and we backcrossed this originaltg mouse line over many generations into a pure C57BL/6 back-ground. These Thy1-A53T-haSyn C57BL/6 tg mice develop robustneurologic and neuropathological phenotypes and a fully penetrantfatal disease consistent with PD. They show age-related ataxia,bradykinesia, postural instability, and rigidity (see supplementalVideos 1 and 2) and have motor system-preferential neuropa-thology involving synucleinopathy and interneuron degenerationearly in the disease. HaSyn-A53T mutant protein associates withputative components of the mPTP in these mice and genetic inacti-vation of the ppif gene encoding CyPD, amajor regulator of themPTP(Baines et al., 2005; Basso et al., 2005), in Thy1-A53T-a-Syn C57BL/6tg mice delays disease onset and extends lifespan. These resultsdemonstrate for the first time that Thy1-A53T-a-Syn C57BL/6 tgmice are a useful model that faithfully simulates neurologic andneuropathological aspects of human PD and that the mPTP isinvolved directly in the disease mechanisms in these mice.

Many tg mouse lines have been engineered to express haSyncontrolled by different promoters. These mice have varying neuro-logic andpathological characteristics, someofwhichmay be presentin human PD, but most haSyn tg mouse models are imperfect formodeling human PD (Antony et al., 2011; Chesselet and Richter,2011). Mice expressing wildtype haSyn driven by a humanplatelet-derived growth factor-b promoter displayed a mild motorphenotype, developed amorphous nonfilamentous intranuclear in-clusions, and reduced striatal TH immunoreactivity, but did not haveSNc neuron loss or fatal disease (Masliah et al., 2000). Miceexpressing wildtype or mutant (A53T or A30P) haSyn driven by therat TH promoter did not develop clinical or neuropathological phe-notypes (Matsuoka et al., 2001), but,mice expressing doublymutant(G88C and G209A) haSyn driven by a rat TH promoter showedreduced locomotor activity, mild loss of striatal dopamine, andmoderate reduction of TH immunostained SNc neurons, but no fataldisease (Richfield et al., 2002; Thiruchelvam et al., 2004). Tg miceexpressing truncated haSyn driven bya rat THpromoter developed amild motor phenotype, mild loss of striatal dopamine, and non-progressive loss (w50%) of SNc neurons, but no fatal disease(Wakamatsu et al., 2008). Tgmice expressing haSyn (wildtype, A53Tand A30P variants) under the control of the murine Thy-1 promoterdeveloped pronounced motor deficits on the rotating rod task,

ons through the diencephalon of endstage Thy1-A53T tg mice displayed selective robusthalamus in age-matched non-tg littermates was blank for argyrophilia (A). Some of ther thalamic nucleus (Rt), globus pallidus (GP), internal capsule (ic). Degenerating axonstg mice. Scale bars ¼ 150 mm (A), 200 mm (B), 100 mm (C), 40 mm (D). (EeH) Nissl-stainedateral ventricle (lv) and third ventricular cavities of the cerebroventricular system ande VL thalamus contained numerous apoptotic profiles (H, arrows) and chromatolyticr F), 25 mm (G, same for H). (I) Counts of VL neurons revealed a significant loss (asterisk,nth. Values are mean � standard deviation (SD) (n ¼ 6 mice/group). Abbreviations: GP,standard deviation; VA, ventral anterior thalamic nucleus; VL, ventrolateral nucleus.

Fig. 10. The mitochondrial permeability transition pore (mPTP) contributes to disease mechanisms in Thy1-A53T haSyn tg mice. (A, B) Putative components of the mPTP areenriched in substantia nigra compacta (SNc) neurons compared with substantia nigra reticulata (SNr) neurons as shown by immunofluorescence for cyclophilin D (CyPD, A, red) andadenine nucleotide translocator (ANT, B, red) and co-labeling with the mitochondrial marker superoxide dismutase-2 (SOD2, A and B, green). Co-localization is seen as yellow. Nucleiwere stained with DAPI (blue). Scale bars ¼ 12.5 mm (A), 4 mm (A inset), 10 mm (B), 5 mm (B inset). (C) Western blots for putative components of the mPTP, including ANT, voltage-dependent anion channel (VDAC), and CyPD, in the brainstem, striatum, and cerebral cortex of early and late symptomatic Thy1-A53T haSyn tg mice and non-tg mice. A



intraneuronal haSyn accumulation, axonal degeneration in spinalroots, and evidence for muscle denervation, but no reported loss ofSNc neurons or fatal disease (van der Putten et al., 2000). In contrast,using the mouse prion protein (PrP) promoter, Giasson et alengineered mice expressing A53T mutant haSyn and found thatthese mice become paralyzed and develop haSyn inclusions,primarily in brainstem and spinal cord, and axonal degeneration inspinal roots (Giasson et al., 2002). Similarly, Lee et al generatedtg mice expressing haSynwildtype, A53T, and A30P variants drivenby a murine PrP promoter and showed A53T mice with a fatalparalytic disease phenotype (Lee et al., 2002). These mice alsoshowed mitochondrially-associated A53T haSyn, mitochondrialmorphologyandbiochemical abnormalities, andneuronal cell deathin neocortex, brainstem, and spinal cord (Martin et al., 2006).However, no evidence of neurodegeneration andneuronal cell deathin the SNc of PrP haSyn tg mice has been reported (Giasson et al.,2002; Lee et al., 2002; Martin et al., 2006). Recently, tg mice withconditional expression of haSyn-A53T in midbrain dopaminergicneurons using a tetracycline-regulated inducible PITX3 promoterfailed todevelopmajor neurologic disease butwere reported tohavedeficits in striatal dopamine release and early onset loss of dopa-minergic neurons in SNc andventral tegmental area (Lin et al., 2012).The distinguishing observations that set apart our study from theseearlier studies are the findings that humanThy1-driven A53T-haSynC57BL/6 tg mice show cardinal neurologic and motor system-preferential pathologic abnormalities and fatal disease consistentwith PD. The pronounced clinical neurologic features of these micewere rigidityand/or spasticity, postural instability, bradykinesia, andakinesia (Supplementary videos 1 and 2). The robust neuropatho-logical features in these mice, that are also cardinal pathologies inPD, were major age-related loss of SNc neurons, denervation ofstriatum, neocortical degeneration, and formation of intraneuronalhaSynþ inclusions. Additional pathologic changes in these mice,which are not examined commonly in human PD, include cerebellarand red nucleus degeneration and loss of striatal and cerebellarcortical inhibitory interneurons.

PD is a multisystem neural circuit disorder (DeLong andWichmann, 2007; Jellinger, 2012). We show that A53T-haSyn tgmice have neuropathology consistent with a neural circuit degen-erative disorder organized by connectivity. This is best illustrated bythe degeneration in the SNc and nigrostriatal pathway and in theknown network formed by the DCN, RN, VA and/or VL thalamus,and motor-sensory neocortex. The striking selective degenerationof VA and/or VL regions of thalamus highlights a probable trans-ynaptic mechanism related to basal ganglia and cerebellar degen-eration in these mice. Most investigations of PD focus on the basalganglia; in contrast, the cerebellum has received little attention, buta cerebellar role in PD is now realized (Helmich et al., 2012;Wu andHallett, 2013). Functional magnetic resonance imaging studies havefound hyperactivation of the cerebellum in human PD (Rascol et al.,1997; Yu et al., 2007). Cerebellar abnormalities may contributedirectly to the manifestation of resting tremor in PD oscillatoryactivity (Bostan and Strick, 2010; Helmich et al., 2012). Our work ona tg mouse model of PD reinforces the idea that be cerebellum mayhave a role in the pathophysiology of PD. However, the cerebellum,red nucleus, globus pallidus, and thalamus in human PD aregenerally thought spared from neuropathology (Dickson, 2012), butthese regions, as well as the SNc and striatum, are affected with

representative Ponceau S-stained membrane shows protein loading. (D) Quantification of ilevels in brainstem of early symptomatic Thy1-A53T haSyn tg mice compared with non-tg coand late symptomatic mice. (E) HaSyn interacts strongly with VDAC (all brain regions) andwith mitochondria as shown by the immunofluorescent co-localization of haSyn (red) and thSome mitochondria positive for haSyn appear swollen (arrow). Scale bar ¼ 4 mm. (G) Genetimice. Abbreviations: ANT, adenine nucleotide translocator; CyPD, cyclophilin D; mPTP, mitonigra reticulata; SOD2, superoxide dismutase-2; VDAC, voltage-dependent anion channel.

human progressive supranuclear palsy (PSP) (Dickson, 2012). LikePD, PSP is characterized clinically by postural instability and rigidity(Dickson, 2012). Thus, it is possible that this A53T-haSyn C57BL/6 tgmouse is a model of PSP, although this interpretation is contingenton a characterization of the tau pathology in these mice.

The early degeneration of cerebellar Golgi interneurons andstriatal parvalbumin interneurons in A53T-haSyn tg mice isinstructive by demonstrating that, (1) interneurons are involved inthe disease process in PD mice, and (2) interneuron diseaseemerges early in life, but the overt clinical manifestations appearmuch later in life. Epidemiologic accounts suggest that individualswith PD may have a preclinical phase spanning decades (Savicaet al., 2010). A clinical study of the orbicularis oculi reflex in PDpatients found indications of brainstem interneuron dysfunction(Kimura, 1973). The 6-hydroxydopamine model of PD has alsorevealed evidence for interneuron abnormalities. Striatal inter-neuron innervation of striatopallidal and striatonigral neuronsundergoes differential structural (Salin et al., 2009) and functional(Gittis et al., 2011) remodeling of synapses after toxin exposure.Electrophysiological studies support the idea that inhibitory in-terneurons may be vulnerable in haSyn tg mice. Patch-clamprecording shows deficits in GABAergic inhibitory transmission infrontal cortex of these mice (Ito et al., 2012). Early presymptomaticdegeneration or dysfunction of interneurons has also been found inmouse models of amyotrophic lateral sclerosis (Martin and Chang,2012) and Huntington’s disease (Dougherty et al., 2014). The dis-ease within the cerebello-rubro-thalamo-cortical circuit in our tgmice possibly starts with the degeneration of haSynþ Golgi cellinterneurons in cerebellar cortex because we found these neuronsdegenerating in 1-month old mice. In human Thy1-driven A53T-haSyn C57BL/6 tg mice, haSyn protein levels are generally higher inhindbrain structures than in forebrain and/or diencephalic struc-tures early in disease, but, later in disease, haSyn protein is presentmore globally throughout the brain. A cerebellum to midbrain todiencephalon to forebrain spread of pathology would be consistentwith a prion-like neuron-to-neuron transmission of haSyn-medi-ated disease (Desplats et al., 2009).

There is little doubt about the loss of SNcdopamineneurons inPD(Rudow et al., 2008), yet an important unresolved basic question ishow SNc neurons degenerate and are eventually eliminated. Cellscan die by different ways through the involvement of differentmolecularmechanisms. Apoptosis, necrosis, autophagy, and hybridsof apoptosis and necrosis are forms of cell death (Klionsky and Emr,2000;Martin, 2010;Martin et al.,1998). The reports on the incidenceof dying cells in the PD SNcwith DNA fragmentation based on in situend labeling methods are conflicting. Some studies report no la-beling (Jellinger, 1999, 2000), but other studies have found nuclearDNA fragmentation (Martin, 2010; Tatton et al., 1998; Tompkinset al., 1997). Some studies report evidence for apoptosis in PD(Anglade et al., 1997; Tatton et al., 1998; Hartmann et al., 2001), butother studies reveal little evidence of apoptosis (Burke andKholodilov, 1998; Wullner et al., 1999). Some of the confusionmight be because of the use ofDNA fragmentationdetection systemsthat do not distinguish between DNA strand breaks associated withapoptosisor necrosis and to arbitrarymorphologic interpretationsofnuclear morphology and chromatin condensation (Jellinger, 1999;Martin et al., 1998; Martin and Liu, 2004). In human PD, despitethe reported presence of TUNELþ and cleaved caspase-3þneurons in

mmunoreactivity by densitometry showed a significant increase (* p < 0.05) in CyPDntrols. Similar elevations in CyPD were seen in brainstem, striatum, and cortex of earlyslightly with CyPD (cortex) as shown by co-immunoprecipitation. (F) HaSyn associatese mitochondrial marker SOD2 (green) in SNc neurons. Co-localization is seen as yellow.c deletion of CyPD delayed disease onset and extended lifespan in Thy1-A53T haSyn tgchondrial permeability transition pore; SNc, substantia nigra compacta; SNr, substantia


the SNc (Hartmann et al., 2001; Martin, 2010), the morphology ofdegenerating SNc neurons appears different fromclassical apoptosis(Martin, 2001, 2010). In contrast, in A53T-haSyn tgmicewefind thatSNc neuron death appears to be consistent with a caspase-3-drivenclassically apoptotic form of degeneration. The activation of p53 inSNc neurons suggested DNA damage induced by mutant haSyn as apossible upstream mechanism of SNc neuron apoptosis. Apoptosisand cleaved caspase-3 immunoreactivity was also observed in theneocortex, striatum, thalamus, red nucleus, and cerebellar cortex inthese mice, while evidence for necrotic-like cell death was found inthe deep cerebellar nuclei. The absence of neurodegenerativephenotype that fully manifests as classical apoptosis has been seenalso in human amyotrophic lateral sclerosis (Martin,1999, 2010). It isunknownwhy rodent experimental systems appear to more readilyengage classically apoptotic forms of neuronal cell death in matureCNS compared with adult human CNS. It is possible that the mo-lecular regulations of neuronal aging, compensation, and cell deathare nuanced in human and rodent neurons because of differences inDNA damage and repair mechanisms, DNA methylation, gene pro-moter and transcriptome activity, senescence, caspase activationand utilization, and mitochondrial biology (Bañuelos et al., 2008;Bell et al., 2012; Horvath et al., 2007; Loerch et al., 2008; Odomet al., 2007; Panov et al., 2007; Westermann, 2010). Therefore, thevalue of this tg mouse as a translational model for testing therapiesfor neuroprotection in PD warrants exploration.

In addition to the newclinical and neuropathological descriptionof this tg mouse model of PD, a novel contribution of this work re-lates to the identification of the role of the mPTP in the diseaseprocess in these mice. Mitochondrial permeability transition is amitochondrial state in which the proton-motive force is disrupted(Bernardi, 2013; Bernardi et al., 2006; Crompton, 1999; Leung andHalestrap, 2008; van Gurp et al., 2003). This disruption involvesthe mPTP that functions as a voltage, thiol, and Ca2þ sensor. Condi-tions ofmitochondrial Ca2þ overload, excessive oxidative stress, anddecreased electrochemical gradient (DP), adenosine diphosphate,and adenosine triphosphate (ATP) can favor mitochondrial perme-ability transition. The mPTP is believed to be a transmembranechannel formedat the contact sites between the innermitochondrialmembrane (IMM) and the outer mitochondrial membrane (OMM).The components of the mPTP are still controversial. Dimers of theF0F1 ATP synthase (complex V) form a channel that appears elec-trophysiologically equivalent to the mPTP (Bernardi, 2013). TheVDAC in the OMM, the ANT in the IMM, and CyPD in the matrix arealso believed to have some involvement (Bernardi et al., 2006;Crompton, 1999; van Gurp et al., 2003), but they are not obligatoryformPTP formation (Bernardi, 2013; Bernardi et al., 2006). TheVDACadoptsanopenconformationat lowor zeromembranepotential anda closed conformation at potentials above 30e40 mV making theOMM permeable to most small hydrophilic molecules up to 1.3 kDafor free exchange of respiratory chain substrates (Rostovtseva et al.,2005). The ANT is a multi-pass membrane protein, with odd-numbered transmembrane helices having kinks because of prolineresidues, which mediates the exchange of cytosolic adenosinediphosphate for mitochondrial ATP (Graham et al., 1997). Duringnormalmitochondrial function theOMMand the IMMare separatedby the intermembrane space, and the VDAC and the ANT do notinteract (Crompton,1999). Permeability transition is activated by theformation of the mPTP; the IMM loses its integrity and the ANTchanges its conformation from its native state into a nonselectivepore (Crompton, 2004). This process is catalyzed by CyPD,which is apeptidylprolyl isomerase that functions in protein cis-trans isomer-ization and chaperoning (Waldmeier et al., 2003). TheANTandCyPDinteract directly (Woodfield et al., 1998). Themolar concentration ofCyPD (in heart mitochondria) is much less (>5%) than ANT; thus,under normal conditions only a minor fraction of the ANT can be in

complex with CyPD (Johnson et al., 1999; Leung and Halestrap,2008). When this occurs, small ions and metabolites permeatefreely across the IMM and oxidation of metabolites by O2 proceedswith electron flux not coupled to proton pumping, resulting incollapse of DP, dissipation of ATP production, production of reactiveoxygen species, equilibrationof ionsbetween thematrix andcytosol,matrix volume increases, and mitochondrial swelling (Rostovtsevaet al., 2005; van Gurp et al., 2003). We demonstrate that putativecomponents or regulators of the mPTP are present in mouse SNcneurons. We also show that A53T-haSyn associates with neuronalmitochondria and interacts with VDAC and CyPD in vivo. Mito-chondria positive for A53T-haSyn undergo prominent morphologicchanges in SNcneurons in thesemice. Other groups have shown thathaSyn is imported into mitochondria in cultured dopaminergicneurons and in human PD brain neurons (Devi et al., 2008) and thatthis impairs complex I activity (Devi et al., 2008; Liu et al., 2009).Mitochondrial membrane associated A53T-haSyn can causemorphologic changes in HeLa cell mitochondria (Nakamura et al.,2011). Moreover, haSyn interacts with the ANT in rat midbrain(Zhu et al., 2011). Finally, we show that the genetic deletion of amajor mPTP regulator, CyPD, has robust effects in PD mice bydelaying disease onset and extending survival. Our finding that themPTP has a role in the development of PD in A53T-haSyn mice isconsistent with observations made in cell culture showing that lossof PINK1 and DJ-1 function mutations result in mPTP opening(Gautier et al., 2012; Giaime et al., 2012). Noteworthy in this regard isthe finding that CyPD deletion protects interneurons fromapoptosisin the adult CNS (Martin, 2011). This work shows for the first time adirect causal role of a mitochondrial mechanism (i.e., the mPTP) inthe pathobiology of a genetic mouse model of PD.

In summary we characterized the neurologic abnormalities,neuropathology, and mechanisms of disease in Thy1-haSyn-A53TC57BL/6 tg mice. We found that these mice develop a profoundmovementdisorderwithmanyPD-like features anddevelopamotorcircuit neuropathology and synucleinopathy involving the nigros-triatal and cerebellar pathways. Interneurons degeneration is aprominent early feature of the disease in these mice suggesting thatinterneuron disease emerging early in life may have neurologicconsequences later in life. Genetic deletion of the mPTP regulatorCyPD had major disease modifying effects. We conclude that themPTP actively participates in themechanisms of neurodegenerationin this PD mouse model. This work defines new cellular and mito-chondrial mechanisms for neurodegeneration in PD that might beuseful for preclinical translation studies and the identification ofmolecular mechanism-based therapies for treating this debilitatingfatal disease.

Disclosure statement

All authors have no actual or potential conflicts of interest.

Acknowledgements

The authors thank Ann Price and Yan Pan for their outstandingtechnical work and Isabella Martin for her editorial assistance. Thiswork was supported by grant NS034100 and NS052098 from theNIH-NINDS and AG016282 from the NIH-NIA.

Appendix A. Supplementary data

Supplementary data related with this article can be found in theonline version, at http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.008.




References

Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M.T., Michel, P.P., Marguez, J., Mouatt-Prigent, A., Ruberg, M., Hirsch, E.C., Agid, Y., 1997. Apoptosis and autophagy innigral neurons of patients with Parkinson’s disease. Histol. Histopathol. 12,25e31.

Antony, P.M.A., Diederich, N.J., Balling, R., 2011. Parkinson’s disease mouse models intranslational research. Mamm. Genome 22, 401e419.

Ascherio, A., Chen, H., Weisskopf, M.G., O’Reilly, E., McCullough, M.L., Calle, E.E.,Schwarzchild, M.A., Thun, M.J., 2006. Pesticide exposure and risk for Parkinson’sdisease. Ann. Neurol. 60, 197e203.

Baines, C.P., Kaiser, R.A., Purcell, N.H., Blair, H.S., Osinska, H., Hambleton, M.A.,Brunskill, E.W., Sayen, M.R., Gottlieb, R.A., Dorn, G.W., Robbins, J.,Molkentin, J.D., 2005. Loss of cyclophilin D reveals a critical rolefor mitochondrial permeability transition in cell death. Nature 434, 658e662.

Bañuelos, C.A., Banáth, J.P., MacPhail, S.H., Zhao, J., Eaves, C.A., O’Connor, M.D.,Lansdorp, P.M., Olive, P.L., 2008. Mouse but not human embryonic stem cells aredeficient in rejoining of ionizing radiation-induced DNA double-strand breaks.DNA Repair 7, 1471e1483.

Basso, E., Fante, L., Fowlkes, J., Petronilli, V., Fortes, M.A., Bernardi, P., 2005. Prop-erties of the permeability transition pore in mitochondria devoid of cyclophilinD. J. Biol. Chem. 280, 18558e18561.

Beal, M.F., 2005. Mitochondria take center stage in aging and neurodegeneration.Ann. Neurol. 58, 495e505.

Bell, C.G., Wilson, G.A., Butcher, L.M., Roos, C., Walter, L., Beck, S., 2012. Human-specific CpG “beacons” identify loci associated with human-specific traits anddisease. Epigenetics 7, 1188e1199.

Bernardi, P., 2013. The mitochondrial permeability transition pore: a mysterysolved? Front. Physiol. 4, 95. http://dx.doi.org/10.3389/fphys.2013.00095.

Bernardi, P., Krauskopf, A., Basso, E., Petronilli, V., Blalchy-Dyson, E., Di Lisa, F.,Forte, M.A., 2006. The mitochondrial permeability transition from in vitroartifact to disease target. FEBS J. 273, 2077e2099.

Bindoff, L.A., Birch-Machin, M., Cartlidge, N.E.F., Parker, W.D., Turnbull, D.M., 1989.Mitochondrial function in Parkinson’s disease. Lancet 2, 49.

Bonifati, V., Rizzu, P., van Baren, M.J., Schaap, O., Breedveld, G.J., Krieger, E.,Dekker, M.C., Squitieri, F., Ibanez, P., Joosse, M., van Dongen, J.W., Vanacore, N.,van Swieten, J.C., Brice, A., Meco, G., van Duijn, C.M., Oostra, B.A., Heutink, P.,2003. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 299, 256e259.

Bostan, A., Strick, P.L., 2010. The cerebellum and basal ganglia are interconnected.Neuropsychol. Rev. 20, 261e270.

Burke, R.E., Kholodilov, N.G., 1998. Programmed cell death: does it play a role inParkinson’s disease? Ann. Neurol. 44 (3 Suppl 1), S1126eS1133.

Chandra, S., Gallardo, G., Fernandez-Chacon, R., Schluter, O.M., Sudholf, T.C., 2005.a-Synuclein cooperates with CSPa in preventing neurodegeneration. Cell 123,383e396.

Chesselet, M.-F., Richter, F., 2011. Modeling of Parkinson’s disease in mice. Lancet 10,1108e1118.

Cleeter, M.W.J., Cooper, J.M., Schapira, A.H.V., 1992. Irreversible inhibition of mito-chondrial complex I by 1-methyl-4-phenylpyridine: evidence for free radicalinvolvement. J. Neurochem. 58, 786e789.

Coskun, P., Wyrembak, J., Schriner, S.E., Chen, H.-W., Marciniack, C., LaFerla, F.,Wallace, D.C., 2012. A mitochondrial etiology of Alzheimer and Parkinson dis-ease. Biochim. Biophys. Acta 1820, 553e564.

Crook, J., Hendrickson, A., Robinson, F.R., 2006. Co-localization of glycine and GABAimmunoreactivity in interneurons in Macaca monkey cerebellar cortex.Neuroscience 141, 1951e1959.

Crompton, M., 1999. The mitochondrial permeability transition pore and its role incell death. Biochem. J. 341, 233e249.

Crompton, M., 2004. Mitochondria and aging: a role for the permeability transition?Aging Cell 3, 3e6.

Daher, J.P.L., Pletnikova, O., Biskup, S., Musso, A., Gellhaar, S., Galter, D., Troncoso, J.C.,Lee, M.K., Dawson, T.M., Dawson, V.L., Moore, D.J., 2012. Neurodegenerativephenotypes in an A53T a-synuclein transgenic mouse model are independent ofLRRK2. Hum. Mol. Genet. 21, 2420e2431.

DeLong, M.R., Wichmann, T., 2007. Circuits and circuit disorders of the basal ganglia.Arch. Neurol. 64, 20e24.

Desplats, P., Lee, H.-J., Bae, E.-J., Patrick, C., Rockenstein, E., Crews, L., Spencer, B.,Masliah, E., Lee, S.-J., 2009. Inclusion formation and neuronal cell death throughneuron-to-neuron transmission of a-synuclein. Proc. Natl. Acad. Sci. U.S.A 106,13010e13015.

Devi, L., Raghavendran, V., Prabhu, B.M., Avadhani, N.G., Anandatheerthavarada, H.K.,2008.Mitochondrial import and accumulation of a-synuclein impair complex I inhuman dopaminergic neuronal cultures and Parkinson disease brain. J. Biol.Chem. 283, 9089e9100.

Dickson, D.W., 2012. Parkinson’s disease and parksonism: neuropathology. ColdSpring Harb. Perspect. Med. 2, a009258.

Dorsey, E.R., Constantinescu, R., Thompson, J.P., Biglan, K.M., Holloway, R.G.,Kieburtz, K., Marshall, F.J., Ravina, B.M., Schifitto, G., Siderow, F.A., Tanner, C.M.,2007. Projected number of people with Parkinson disease in the most populousnation, 2005 through 2030. Neurology 68, 384e386.

Dougherty, S.E., Hollimon, J.J., McMeekin, L.J., Bohannom, A.S., West, A.B., Lesort, M.,Hablitz, J.J., Cowell, R.M., 2014. Hyperactivity and cortical disinhibition in mice

with restricted expression of mutant huntingtin to parvablumin-positive cells.Neurobiol. Dis. 62, 160e171.

Fallon, J.H., Loughlin, S.E., 1985. Substantia nigra. In: Paxinos, G. (Ed.), The RatNervous System, Volume 1Academic Press, Australia, pp. 353e374.

Fortin, D.L., Nemani, V.M., Voglmaier, S.M., Anthony, M.D., Ryan, T.A., Edwards, R.H.,2005. Neural activity control the synaptic accumulation of a-synuclein.J. Neurosci. 25, 10913e10921.

Franklin, K.B.J., Paxinos, G., 1997. The Mouse Brain in Stereotaxic Coordinates. Ac-ademic Press, San Diego.

Gautier, C.A., Giaime, E., Caballero, E., Ny�ñez, L., Song, Z., Chan, D., Villalobos, C.,Shen, J., 2012. Regulation of mitochondrial permeability transitionpore by PINK1. Mol. Neurodegener. 7, 22. http://dx.doi.org/10.1186/1750-1326-7-22.

Giaime, E., Yamaguchi, H., Gautier, C.A., Kitada, T., Shen, J., 2012. Loss of DJ-1 doesnot affect mitochondrial respiration but increases ROS production and mito-chondrial permeability transition pore opening. PLoS One 7, e40501. http://dx.doi.org/10.1371/journal.pone.0040501.

Giasson, B.I., Duda, J.E., Murray, I.V.J., Chen, Q., Souza, J.M., Hurtig, H.I.,Ischiropoulos, H., Trojanowski, J.Q., Lee, V.M.-Y., 2000a. Oxidative damage linkedto neurodegeneration by selective a-synuclein nitration in synucleinopathylesions. Science 290, 985e989.

Giasson, B.I., Jakes, R., Goedert, M., Duda, J.E., Leight, S., Trojanowski, J.Q., Lee, V.M.-Y., 2000b. A panel of epitope-specific antibodies detects protein domainsdistributed throughout human a-synuclein in Lewy bodies of Parkinson’s dis-ease. J. Neurosci. Res. 59, 528e533.

Giasson, B.I., Duda, J.E., Quinn, S.M., Zhang, B., Trojanowski, J.Q., Lee, W.M.-Y., 2002.Neuronal a-synucleinopathy with severe movement disorder in mice express-ing A53T human a-synuclein. Neuron 34, 521e533.

Ginsberg, S.D., Martin, L.J., 1998. Ultrastructural analysis of the progression ofneurodegeneration in the septum following fimbria-fornix transection. Neuro-science 86, 1259e1272.

Gispert, S., Ricciardi, F., Kurz, A., Azizov, M., Hoepken, H.-H., Becker, D.,Voos, W., Leuner, K., Mueller, W.E., Kudin, A.P., Kunz, W.S.,Zimmermann, A., Roeper, J., Wenzel, D., Jendrach, M., Garcia-Arenciba, M.,Fernandez-Ruiz, J., Huber, L., Rohrer, H., Barrera, M., Reichert, A.S., Rub, U.,Chen, A., Nussbaum, R.L., Auburger, G., 2009. Parkinson phenotype in agedPINK1-deficient mice is accompanied by progressive mitochondrialdysfunction in absence of neurodegeneration. PLoS One 4, e5777. http://dx.doi.org/10.1371/journal.pone.0005777.

Gittis, A.H., Hang, G.B., LaDow, E.S., Shoenfeld, L.R., Atallah, B.V., Finkbeiner, S.,Kreitzer, A.C., 2011. Rapid target-specific remodeling of fast-spiking inhibitorycircuits after loss of dopamine. Neuron 71, 858e868.

Graham, B.H., Waymire, K.G., Cotrell, B., Trounce, I.A., MacGregor, G.R., Wallace, D.C.,1997. A mouse model for mitochondrial myopathy and cardiomyopathyresulting from a deficiency in the heart/muscle isoform of the adenine nucle-otide translocator. Nat. Genet. 16, 226e234.

Halestrap, A.P., 2009. What is the mitochondrial permeability transition pore?J. Mol. Cell. Cardiol. 46, 821e831.

Hatano, Y., Li, Y., Sato, K., Asakawa, S., Yamamura, Y., Tomiyama, H., Yoshino, H.,Asahina, M., Kobayashi, S., Hassin-Baer, S., Lu, C.S., Ng, A.R., Rosales, P.L.,Shimizu, N., Toda, T., Mizuno, Y., Hattori, N., 2004. Novel PINK1 mutations inearly-onset parkinsonism. Ann. Neurol. 56, 424e427.

Hartmann, A., Michel, P.P., Troadec, J.-D., Mouatt-Prignet, A., Faucheux, B.A.,Ruberg, M., Agid, Y., Hirsch, E.C., 2001. Is Bax a mitochondrial mediator inapoptotic death of dopaminergic neurons in Parkinson’s disease? J. Neurochem.76, 1785e1793.

Helmich, R.C., Hallett, M., Deuschl, G., Toni, I., Bloem, B.R., 2012. Cerebral causes andconsequences of parkinsonian resting tremor: a tale of two circuits. Brain 135,3206e3226.

Horvath, M.M., Wang, X., Resnick, M.A., Bell, D.A., 2007. Divergent evolution ofhuman p53 binding sites: cell cycle versus apoptosis. PLoS Genet. 3, e127. http://dx.doi.org/10.1371/journal.pgen.0030127.

Hsu, L.J., Sagara, Y., Arroyo, A., Rockenstein, E., Sisk, A., Mallory, M., Wong, J.,Takenouchi, T., Hashimoto, M., Masliah, E., 2000. Alpha-synuclein promotesmitochondrial deficit and oxidative stress. Am. J. Pathol. 157, 401e410.

Ito, H., Nakayama, K., Jin, C., Suzuki, Y., Yazawa, I., 2012. a-Synuclein accumulationreduces GABAergic inhibitory transmission in a model of multiple systematrophy. Biochem. Biophys. Res. Commun. 428, 348e353.

Jankovic, J., 2008. Parkinson’s disease: clinical features and diagnosis. J. Neurol.Neurosurg. Psychiatry 79, 368e376.

Jellinger, K.A., 1999. Is there apoptosis in Lewy body disease? Acta Neuropathol. 97,413e415.

Jellinger, K.A., 2000. Cell death mechanisms in Parkinson’s disease. J. Neural Transm.107, 1e29.

Jellinger, K.A., 2012. Neuropathology of sporadic Parkinson’s disease: evaluation andchanges of concepts. Mov. Disord. 27, 8e30.

Johnson, N., Khan, A., Virji, S., Ward, J.M., Crompton, M., 1999. Import and processingof heart mitochondrial cyclophilin D. Eur. J. Biochem. 263, 353e359.

Kimura, J., 1973. Disorder of interneurons in parkinsonism. The orbicularis oculireflex to paired stimuli. Brain 96, 87e96.

Kita, H., Kossak, T., Heizmann, C.W., 1990. Parvalbumin-immunoreactive neurons inthe rat neostriatum: a light and electron microscopic study. Brain Res. 536,1e15.

http://refhub.elsevier.com/S0197-4580(13)00576-9/sref1

































http://dx.doi.org/10.3389/fphys.2013.00095



















































































http://dx.doi.org/10.1186/1750-1326-7-22

http://dx.doi.org/10.1186/1750-1326-7-22

http://dx.doi.org/10.1371/journal.pone.0040501
















































http://dx.doi.org/10.1371/journal.pgen.0030127

http://dx.doi.org/10.1371/journal.pgen.0030127
































Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S.,Yokochi, M., Mizuno, Y., Shimizu, N., 1998. Mutations in the parkin gene causeautosomal recessive juvenile parkinsonism. Nature 392, 605e608.

Klein, C., Schlossmacher, M.G., 2007. Parkinson disease, 10 years after its geneticrevolution. Neurology 69, 2093e2104.

Klionsky, D.J., Emr, S.D., 2000. Autophagy as a regulated pathway of cellulardegeneration. Science 290, 1717e1721.

Kofler, J., Hattori, K., Sawada, M., DeVries, A.C., Martin, L.J., Hurn, P.D., Traystman, R.J.,2004. Histopathological and behavioral characterization of a novel model ofcardiac arrest and cardiopulmonary resuscitation in mice. J. Neurosci. Methods136, 33e44.

Langston, J.M., Ballard, P., 1983. Chronic parkinsonism in humans due to a product ofmeperidine-analog synthesis. Science 219, 979e980.

Lee, M.K., Stirling, W., Xu, Y., Xu, X., Qui, D., Mandir, A.S., Dawson, T.M.,Copeland, N.G., Jenkins, N.A., Price, D.L., 2002. Human a-synuclein-harboringfamilial Parkinson’s disease-linked Ala-53 / Thr mutation causes neurode-generative disease with a-synuclein aggregation in transgenic mice. Proc. Natl.Acad. Sci. U.S.A 99, 8968e8973.

Leroy, E., Boyer, R., Auburger, G., Leube, B., Ulm, G., Mezey, E., Harta, G.,Browstein, M.J., Jonnalagada, S., Chernova, T., Dehejia, A., Lavedan, C., Gasser, T.,Steinbach, P.J., Wilkinson, K.D., Polymeropoulos, M.H., 1998. The ubiquitinpathway in Parkinson’s disease. Nature 395, 451e452.

Lesuisse, C., Martin, L.J., 2002. Long-term culture of mouse cortical neurons as amodel for neuronal development, aging, and death. J. Neurobiol. 51, 9e23.

Leung, A.W.C., Halestrap, A.P., 2008. Recent progress in elucidating the molecularmechanism of the mitochondrial permeability transition pore. Biochim. Bio-phys. Acta 1777, 946e952.

Lin, X., Parisiadou, L., Sgobio, C., Liu, G., Yu, J., Sun, L., Shim,.H, Gu, X.-L., Luo, J.,Long, C.-X., Ding, J., Mateo, Y., Sullivan, P.H., Wu, L.-G., Goldstein, D.S.,Lovinger, D., Cai, H., 2012. Conditional expression of Parkinson’s disease-relatedmutant a-synuclein in the midbrain dopaminergic neurons causes progressiveneurodegeneration and degradation of transcription factor nuclear receptorrelated 1. J. Neurosci. 32, 9248e9264.

Liu, G., Zhang, C., Yin, J., Li, X., Cheng, F., Li, Y., Yang, H., Ueda, K., Chan, P., Yu, S., 2009.a-Synuclein is differentially expressed in mitochondria from different rat brainregions and dose-dependently down-regulates complex I activity. Neurosci.Lett. 454, 187e192.

Loerch, P.M., Lu, T., Dakin, K.A., Vann, J.M., Issacs, A., Geula, C., Wang, J., Pan, Y.,Gabuzda, D.H., Li, C., Prolla, T.A., Yankner, B.A., 2008. Evolution of the aging braintranscriptome and synaptic regulation. PLoS One 3, e3329. http://dx.doi.org/10.1371/journal.pone.0003329.

Lowe, J., Lennox, G., Leigh, P.N., 1997. Disorders of movement and system degen-eration. In: Graham, D.I., Lantos, P.L. (Eds.), Greenfield’s Neuropathology. Arnold,London, pp. 281e366.

Maroteaux, L., Campanelli, J.T., Scheller, R.H., 1988. Synuclein: a neuron-specificprotein localized to the nucleus and presynaptic nerve terminals. J. Neurosci.8, 2804e2815.

Martin, L.J., 1999. Neuronal death in amyotrophic lateral sclerosis is apoptosis:possible contribution of a programmed cell death mechanism. J. Neuropathol.Exp. Neurol. 58, 459e471.

Martin, L.J., 2001. Neuronal cell death in nervous system development, disease, andinjury. Int. J. Mol. Med. 7, 455e478.

Martin, L.J., 2006. Mitochondriopathy in Parkinson disease and amyotrophic lateralsclerosis. J. Neuropathol. Exp. Neurol. 65, 1103e1110.

Martin, L.J., 2010. Mitochondrial and cell death mechanisms in neurodegenerativediseases. Pharmaceuticals 3, 839e915.

Martin, L.J., 2011. An approach to experimental synaptic pathology using greenfluorescent protein-transgenic mice and gene knockout mice to show mito-chondrial permeability transition pore-driven excitotoxicity in interneuronsand motoneurons. Toxicol. Pathol. 39, 220e233.

Martin, L.J., Chang, Q., 2012. Inhibitory synaptic regulation of motoneurons: a newtarget of disease mechanisms in amyotrophic lateral sclerosis. Mol. Neurobiol.45, 30e42.

Martin, L.J., Liu, Z., 2002. Injury-induced spinal motor neuron apoptosis is precededby DNA single-strand breaks and is p53- and bax-dependent. J. Neurobiol. 5,181e197.

Martin, L.J., Liu, Z., 2004. Opportunities for neuroprotection in ALS using cell deathmechanism rationales. Drug Discov. Today 1, 135e143.

Martin, L.J., Chen, K., Liu, Z., 2005. Adult motor neuron apoptosis is mediated bynitric oxide and Fas death receptor linked by DNA damage and p53 activation.J. Neurosci. 25, 6449e6459.

Martin, L.J., Adams, N.A., Pan, Y., Price, A., Wong, M., 2011. The mitochondrialpermeability transition pore regulates nitric oxide-mediated apoptosis of neu-rons induced by target deprivation. J. Neurosci. 31, 359e370.

Martin, L.J., Al-Abdulla, N.A., Brambrink, A.M., Kirsch, J.R., Sieber, F.E., Portera-Cailliau, C., 1998. Neurodegeneration in excitotoxicity, global cerebral ischemia,and target deprivation: a perspective on the contributions of apoptosis andnecrosis. Brain Res. Bull. 46, 281e309.

Martin, L.J., Gertz, B., Pan, Y., Price, A.C., Molkentin, J.D., Chang, Q., 2009. Themitochondrial permeability transition pore in motor neurons: involvement inthe pathobiology of ALS mice. Exp. Neurol. 218, 333e346.

Martin, L.J., Price, A.C., McClendon, K.B., Al-Abdulla, N.A., Subramaniam, J.R.,Wong, P.C., Liu, Z., 2003. Early events of target deprivation/axotomy-inducedneuronal apoptosis in vivo: oxidative stress, DNA damage, p53

phosphorylation and subcellular redistribution of death proteins. J. Neurochem.85, 234e247.

Martin, L.J., Pan, Y., Price, A.C., Sterling, W., Copeland, N.G., Jenkins, N.A., Price, D.L.,Lee, M.K., 2006. Parkinson’s disease a-synuclein transgenic mice developneuronal mitochondrial degeneration and cell death. J. Neurosci. 26, 41e50.

Martin, L.J., Liu, Z., Chen, K., Price, A.C., Pan, Y., Swaby, J.A., Golden, W.C., 2007. Motorneuron degeneration in amyotrophic lateral sclerosis mutant superoxidedismutase-1 transgenic mice: mechanisms of mitochondriopathy and celldeath. J. Comp. Neurol. 500, 20e46.

Masliah, E., Rockenstein, E., Veinbergs, I., Mallory, M., Hashimoto, M., Takeda, A.,Sagara, Y., Sisk, A., Mucke, L., 2000. Dopaminergic loss and inclusion body for-mation in a-synuclein mice: implications for neurodegenerative disorders.Science 287, 1265e1269.

Matsuoka, Y., Vila, M., Lincoln, S., McCormack, A., Picciano, M., LaFrancois, J., Yu, X.,Dickson, D., Langston, W.J., McGowen, E., Farrer, M., Hardy, J., Duff, K.,Przedborski, S., Di Monte, D.A., 2001. Lack of nigral pathology in transgenic miceexpressing alpha-synuclein driven by the tyrosine hydroxylase promoter.Neurobiol. Dis. 8, 535e539.

Murphy, D.D., Rueter, S.M., Trojanowski, J.Q., Lee, V.M.-Y., 2000. Synucleins aredevelopmentally expressed, and a-synuclein regulates the size of the presyn-aptic vesicular pool in primary hippocampal neurons. J. Neurosci. 20,3214e3220.

Nakagawa, T., Shimizu, S., Watanabe, T., Yamaguchi, O., Otsu, K., Yamagata, H.,Inohara, H., Kubo, T., Tsujimoto, Y., 2005. Cyclophilin D-dependent mitochon-drial permeability transition regulates some necrotic but not apoptotic celldeath. Nature 434, 652e658.

Nakamura, K., Nemani, V.M., Azarbal, F., Skibinski, G., Levy, J.M., Egami, K.,Munishkina, L., Zhang, J., Gardner, B., Wakabayashi, J., Sesaki, H., Cheng, Y.,Finkbeiner, S., Nussbaum, P.L., Masliah, E., Edwards, R.H., 2011. Direct membraneassociation drives mitochondrial fission by the Parkinson disease-associatedprotein a-synuclein. J. Biol. Chem. 286, 20710e20726.

Nicklas,W.J., Vyas, I., Heikkila, R.E.,1985. Inhibition of NADH-linked oxidation in brainmitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin,1-methyl-4-phenyl-1,2,5,6,-tetrahydropyridine. Life Sci. 36, 2503e2508.

Odom, D.T., Dowell, R.D., Jacobsen, E.S., Gordon, W., Danford, T.W., MacIssac, K.D.,Rolfe, P.A., Conboy, C.M., Gifford, D.K., Fraenkel, E., 2007. Tissue-specific tran-scriptional regulation has diverged significantly between human and mouse.Nat. Genet. 39, 730e732.

Olanow, C.W., Tatton, W.G., 1999. Etiology and pathogenesis of Parkinson’s disease.Annu. Rev. Neurosci. 22, 123e144.

Paisán-Ruíz, C., Jain, S., Whitney Evans, E., Gilks, W.P., Simón, J., van der Brug, M.,López de Munain, A., Aparicio, S., Martínez-Gil, A., Khan, N., Johnson, J., RuizMartinez, J., Nicholl, D., Marti Carrera, I., Saénz Pe�na, A., de Silva, R., Lees, A.,Martí-Massó, J.F., Pérez-Tur, J., Wood, N.W., Singleton, A.B., 2004. Cloning of thegene containing mutations that cause PARK8-linked Parkinson’s disease.Neuron 44, 595e600.

Panov, A., Dikalov, S., Shalbuyeva, N., Hemendinger, R., Greenamyre, J.T.,Rosenfeld, J., 2007. Species- and tissue-specific relationships between mito-chondrial permeability transition and generation of ROS in brain and livermitochondria of rats and mice. Am. J. Physiol. Cell Physiol. 292, C708eC718.

Pilsl, A., Winklhofer, K.F., 2012. Parkin, PINK1 and mitochondrial integrity: emergingconcepts of mitochondrial dysfunction in Parkinson’s disease. Acta Neuropathol.123, 173e188.

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 a-synuclein geneidentified in families with Parkinson’s disease. Science 276, 2045e2047.

Portera-Cailliau, C.P., Price, D.L., Martin, L.J., 1997. Non-NMDA and NMDA receptor-mediated excitotoxic neuronal death in adult brain are morphologicallydistinct: further evidence for an apoptosis necrosis continuum. J. Comp. Neurol.378, 88e104.

Ramsay, R.R., Salach, J.I., Dadgar, J., Singer, T.P.,1986. Inhibition ofmitochondrialNADHdehydrogenase by pyridine derivatives and its possible relation to experimentaland idiopathic parkinsonism. Biochem. Biophys. Res. Comm. 135, 269e275.

Rascol, O., Fabre, S.N., Brefel, C., Loubinoux, I., Celsis, P., Senard, J.M., Montastruc, J.L.,Chollet, F., 1997. The ipsilateral cerebellar hemisphere is overactive during handmovements in akinetic parkinsonian patients. Brain 120, 103e110.

Reddy, P.H., Reddy, T.P., 2011. Mitochondria as a therapeutic target for aging andneurodegenerative diseases. Cur. Alzheimer Res. 8, 393e409.

Richfield, E.K., Thiruchelvam, M.J., Cory-Slechta, D.A., Wuetzer, C., Gainetdinov, R.R.,Caron, M.G., Di Monte, D.A., Federoff, H.J., 2002. Behavioral and neurochemicaleffects of wild-type and mutated human alpha-synuclein in transgenic mice.Exp. Neurol. 175, 35e48.

Rostovtseva, T.K., Tan, W., Colombini, M., 2005. On the role of VDAC in apoptosis:fact and fiction. J. Bioenerg. Biomembr 37, 129e142.

Rudow, G., O’Brien, R., Savonenko, A.V., Resnick, S.M., Zonderman, A.B.,Pletnikova, O., Marsh, C., Dawson, T.M., Crain, B.T., West, M.T., Troncoso, J.C.,2008. Morphometry of the human substantia nigra in ageing and Parkinson’sdisease. Acta Neuropathol. 115, 461e470.

Salin, P., López, I.P., Kachidian, P., Barroso-Chinea, P., Rico, A.J., Gómez-Bautista, V.,Coulon, P., Kerkerian-Le Goff, L., Lanciego, J.L., 2009. Changes to interneuron-driven striatal microcircuits in a rat model of Parkinson’s disease. Neurobiol.Dis. 34, 545e552.


























































































































































































































Savica, R., Rocca, W.A., Ahlskog, J.E., 2010. When does Parkinson disease start? Arch.Neurol. 67, 798e801.

Schapira, A.H.V., 2006a. Etiology of Parkinson’s disease. Neurology 66 (Suppl 4),S10eS23.

Schapira, A.H.V., 2006b. Mitochondrial disease. Lancet 368, 70e82.Schapira, A.H.V., 2012. Mitochondrial diseases. Lancet 379, 1825e1834.Schapira, A.H.V., Cooper, J.M., Dexter, D., Jenner, P., Clark, J.B., Marsden, C.D., 1989.

Mitochondrial complex I deficiency in Parkinson’s disease. Lancet 1, 1269.Schauwecker, P.E., Steward, O., 1997. Genetic determinants of susceptibility to

excitotoxic cell death: implications for gene targeting approaches. Proc. Natl.Acad. Sci. U.S.A 94, 4103e4108.

Shimohama, S., Swada, H., Kitamura, Y., Taniguchi, T., 2003. Disease model: Par-kinson’s disease. Trends Mol. Med. 9, 360e365.

Sillitoe, R.V., Chung, S.-H., Fritschy, J.-M., Hoy, M., Hawkes, R., 2008. Golgi celldendrites are restricted by Purkinje cell stripe boundaries in the adult mousecerebellar cortex. J. Neurosci. 28, 2820e2826.

Simat, M., Ambrosetti, L., Lardi-Studler, B., Fritschy, J.-M., 2007. GABAergic syn-aptogenesis marks the onset of differentiation of basket and stellate cells inmouse cerebellum. Eur. J. Neurosci. 26, 2239e2256.

Singleton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J.,Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., Lincoln, S., Crawley, A.,Hanson, M., Maraganore, D., Adler, C., Cookson, M.R., Muenter, M., Baptista, M.,Miller, D., Blancato, J., Hardy, J., Gwinn-Hardy, K., 2003. Alpha-synuclein locustriplication causes Parkinson’s disease. Science 302, 841.

Souza, J.M., Giasson, B.I., Chen, Q., Lee, V.M.-Y., Ischiropoulos, H., 2000. Dityrosinecross-linking promotes formation of stable a-synuclein polymers. J. Biol. Chem.265, 18344e18349.

Tanner, C.M., Kamel, F., Ross, G.W., Hoppin, J.A., Goldman, S.M., Korell, M., Marras, C.,Bhudhikanok, G.S., Kasten, M., Chade, A.R., Comyns, K., Batber Richards, M.,Meng, C., Priestly, B., Fernandez, H.H., Cambi, F., Umbach, D.M., Blair, A.,Sandler, D.P., Langston, W., 2011. Rotenone, paraquat, and Parkinson’s disease.Environ. Health Perspect. 119, 866e872. http://dx.doi.org/10.1289/ehp.1002839.

Tatton, N.A., MaClean-Fraser, A., Tatton, W.G., Perl, D.P., Olnanow, C.W., 1998.A fluorescent double-labeling method to detect and confirm apoptotic nuclei inParkinson’s disease. Ann. Neurol. 44, S142eS148.

Thiruchelvam, M.J., Powers, J.M., Cory-Slechta, D.A., Richfield, E.K., 2004. Risk factorsfor dopaminergic neuron loss in human a-synuclein transgenic mice. Eur. J.Neurosci. 19, 845e854.

Tompkins, M.M., Basgall, E.J., Zamrini, E., Hill, W.D., 1997. Apoptotic-like changes inLewy-body-associated disorders and normal aging in substantia nigra neurons.Am. J. Pathol. 150, 119e131.

Valente, E.M., Abou-Sleiman, P.M., Caputo, V., Muqit, M.M., Harvey, K., Gispert, A.,Ali, Z., Del Turco, D., Bentivoglio, A.R., Healy, D.G., Albanese, A., Nussbaum, R.,Gonzalez-Maldonado, R., Deller, T., Salvi, S., Cortelli, P., Gilks, W.P.,Latchman, D.S., Harvey, R.J., Dallapiccola, B., Auburger, G., Wood, N.W., 2004.Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Sci-ence 304, 1158e1160.

Van Den Eeden, S.K., Tanner, C.M., Bernstein, A.L., Fross, R.D., Leimpeter, A.,Bloch, D.A., Nelson, L.M., 2003. Incidence of Parkinson’s disease: variations byage, gender and race/ethnicity. Am. J. Epidemiol. 157, 1015e1022.

van der Putten, H., Wiederhold, K.-H., Probst, A., Barbieri, S., Mistl, C., Danner, S.,Kauffmann, S., Hofele, K., Spooren, W.P.J.M., Ruegg, M.A., Lin, S., Caroni, P.,Sommer, B., Tolnay, M., Bilbe, G., 2000. Neuropathology in mice expressinghuman a-synuclein. J. Neurosci. 20, 6021e6029.

van Gurp, M., Festjens, N., van Loo, G., Saeleus, X., Vandenabeele, P., 2003. Mito-chondrial intermembrane proteins in cell death. Biochem. Biophys. Res. Comm.304, 487e497.

Vilkki, J., Savontaus, M.L., Kalimo, H., Nikoskelainen, E.K., 1989. Mitochondrial DNApolymorphism in Finish families with Leber’s hereditary optic neuro-retinopathy. Hum. Genet. 82, 208e212.

Voogd, J., Gerrittis, N.M., Marani, E., 1985. Cerebellum. In: Paxinos, G. (Ed.), The RatNervous System, Volume 2Academic Press, Australia, pp. 251e291.

Wakamatsu, M., Ishii, A., Iwata, S., Sakagami, J., Ukai, Y., Ono, M., Kanbe, D.,Muramatsu, S.-I., Kabayashi, K., Iwatsubo, T., Yoshimoto, M., 2008. Selective lossof nigral dopamine neurons induced by overexpression of truncated humana-synuclein in mice. Neurobiol. Aging 29, 574e585.

Waldmeier, P.C., Zimmermann, K., Qian, T., Tintelnot-Blomley, M., Lemasters, J.J.,2003. Cyclophilin D as a drug target. Curr. Med. Chem. 10, 1485e1506.

Weissman, T.A., Sanes, J.R., Lichtman, J.W., Livet, J., 2011. Generation and imaging ofbrainbow mice. Cold Spring Harb. Protoc. 7, 851e856.

Westermann, B., 2010. Mitochondrial dynamics in model organisms: what yeasts,worms and flies have taught us about fusion and fission of mitochondria. Semin.Cell Dev. Biol. 21, 542e549.

Woodfield, K., Rück, A., Brdiczka, D., Halestrap, A.P., 1998. Direct demonstration of aspecific interaction between cyclophilin-D and the adenine nucleotide trans-locase confirms their role in the mitochondrial permeability transition. Bio-chem. J. 336, 287e290.

Wu, T., Hallett, M., 2013. The cerebellum in Parkinson’s disease. Brain 136,696e709.

Wullner, U., Kornhuber, J., Weller, M., Schulz, J.B., Loschmann, P.A., Riederer, P.,Klockgether, T., 1999. Cell death and apoptosis regulating proteins in Parkinson’sdisease- a cautionary note. Acta Neuropathol. 97, 408e412.

Yu, H., Sternad, D., Corcos, D.M., Vaillancourt, D.E., 2007. Role of hyperactive cere-bellum and motor cortex in Parkinson’s disease. NeuroImage 35, 222e233.

Zeilhofer, H.U., Studler, B., Arabadzisz, D., Schweizer, C., Ahmadi, S., Layh, B.,Bosl, M.R., Fritschy, J.-M., 2005. Glycinergic neurons expressing enhanced greenfluorescent protein in bacterial artificial chromosome transgenic mice. J. Comp.Neurol. 482, 123e141.

Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lincoln, S., 2004. Muta-tions in LRRK2 cause autosomal-dominant parkinsonism with pleomorphicpathology. Neuron 44, 601e607.

Zhu, Y., Duan, C., Li, L., Gao, L.L., Zhao, C., Yu, S., Ueda, K., Chan, P., Yang, H., 2011. a-Synuclein overexpression impairs mitochondrial function by associating withadenylate translocator. Int. J. Biochem. Cell Biol. 43, 732e741.





































http://dx.doi.org/10.1289/ehp.1002839



















































































The mitochondrial permeability transition pore regulates Parkinson's disease development in mutant...

Documents

Transcript of The mitochondrial permeability transition pore regulates Parkinson's disease development in mutant...