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Castration Induces Parkinson Disease Pathologies in Young Male Mice via Inducible Nitric-oxide Synthase*

Open AccessPublished:June 06, 2013DOI:https://doi.org/10.1074/jbc.M112.443556
      Although Parkinson disease (PD) is a progressive neurodegenerative disorder, available animal models do not exhibit irreversible neurodegeneration, and this is a major obstacle in finding out an effective drug against this disease. Here we delineate a new irreversible model to study PD pathogenesis. The model is based on simple castration of young male mice. Levels of inducible nitric-oxide synthase (iNOS), glial markers (glial fibrillary acidic protein and CD11b), and α-synuclein were higher in nigra of castrated male mice than normal male mice. On the other hand, after castration, the level of glial-derived neurotrophic factor (GDNF) markedly decreased in the nigra of male mice. Accordingly, castration also induced the loss of tyrosine hydroxylase-positive neurons in the nigra and decrease in tyrosine hydroxylase-positive fibers and neurotransmitters in the striatum. Reversal of nigrostriatal pathologies in castrated male mice by subcutaneous implantation of 5α-dihydrotestosterone pellets validates an important role of male sex hormone in castration-induced nigrostriatal pathology. Interestingly, castration was unable to cause glial activation, decrease nigral GDNF, augment the death of nigral dopaminergic neurons, induce the loss of striatal fibers, and impair neurotransmitters in iNOS−/− male mice. Furthermore, we demonstrate that iNOS-derived NO is responsible for decreased expression of GDNF in activated astrocytes. Together, our results suggest that castration induces nigrostriatal pathologies via iNOS-mediated decrease in GDNF. These results are important because castrated young male mice may be used as a simple, toxin-free, and nontransgenic animal model to study PD-related nigrostriatal pathologies, paving the way for easy drug screening against PD.
      Background: Developing a simple irreversible animal model to study nigrostriatal pathologies is important for Parkinson disease (PD).
      Results: Castration induces glial activation and death of dopaminergic neurons in wild type, but not iNOS−/−, young male mice.
      Conclusion: Castration induces nigrostriatal pathologies via iNOS.
      Significance: Castrated male mice may be used as a simple, toxin-free, nontransgenic, and irreversible animal model for PD.

      Introduction

      Parkinson disease (PD)
      The abbreviations used are: PD
      Parkinson disease
      DA
      dopamine
      DHT
      5α-dihydrotestosterone
      DOPAC
      3,4-dihydroxyphenylacetic acid
      GDNF
      glial-derived neuotrophic factor
      GFAP
      glial fibrillary acidic protein
      HVA
      homovanillic acid
      iNOS
      inducible NOS
      MPTP
      1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
      SNpc
      substantia nigra pars compacta
      α-syn
      α-synuclein
      TH
      tyrosine hydroxylase.
      is a progressive neurodegenerative disease with unclear etiology. PD may appear at any age, but it is uncommon in people younger than 30. The usual age of onset is between 50 and 70. The actual cause of PD is not known. It is believed that a complex interaction between environmental and genetic factors plays the major role in causing the disease (
      • Olanow C.W.
      • Tatton W.G.
      Etiology and pathogenesis of Parkinson's disease.
      ,
      • Vila M.
      • Przedborski S.
      Genetic clues to the pathogenesis of Parkinson's disease.
      ,
      • Dauer W.
      • Przedborski S.
      Parkinson's disease: mechanisms and models.
      ). PD is characterized by a severe shortage of dopamine (DA), an important neurotransmitter. It is this deficiency that causes the symptoms of PD. The deficiency of DA is caused by the gradual loss of dopaminergic neurons that being present in the substantia nigra pars compacta (SNpc) synthesize DA from tyrosine via tyrosine hydroxylase. Once patients are diagnosed with PD, there is no drug available to halt its progression.
      One of the major roadblocks for discovering drugs against PD is the unavailability of a true chronic persistent animal model for PD (
      • Dauer W.
      • Przedborski S.
      Parkinson's disease: mechanisms and models.
      ). One neurotoxin, MPTP, has been being used since the 1980s to model PD. However, for most MPTP models, the loss of dopamine is rapid and reversible. The extent of nigrostriatal damage also decreases in chronic MPTP models with time (
      • Kurz M.J.
      • Pothakos K.
      • Jamaluddin S.
      • Scott-Pandorf M.
      • Arellano C.
      • Lau Y.S.
      A chronic mouse model of Parkinson's disease has a reduced gait pattern certainty.
      ,
      • Meredith G.E.
      • Totterdell S.
      • Potashkin J.A.
      • Surmeier D.J.
      Modeling PD pathogenesis in mice: advantages of a chronic MPTP protocol.
      ). Because familial PD is associated with mutations of different genes, such as α-synuclein, parkin, PINK1, and DJ1 (
      • Tan E.K.
      • Skipper L.M.
      Pathogenic mutations in Parkinson disease.
      ), efforts have been made to generate genetically engineered mice that express these mutated genes (
      • Masliah E.
      • Rockenstein E.
      • Veinbergs I.
      • Mallory M.
      • Hashimoto M.
      • Takeda A.
      • Sagara Y.
      • Sisk A.
      • Mucke L.
      Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders.
      ). Transgenic animals that overexpress α-syn or mutated human α-syn A53T have been used to study the role of this protein in dopaminergic degeneration. Although some transgenic mice expressing mutated α-syn display motor neuron pathology, these mice do not exhibit death of dopaminergic neurons (
      • Maries E.
      • Dass B.
      • Collier T.J.
      • Kordower J.H.
      • Steece-Collier K.
      The role of α-synuclein in Parkinson's disease: insights from animal models.
      ). Many mutations in the gene encoding parkin cause a significant portion of early onset familial PD (
      • Maries E.
      • Dass B.
      • Collier T.J.
      • Kordower J.H.
      • Steece-Collier K.
      The role of α-synuclein in Parkinson's disease: insights from animal models.
      ). Most of these mutations likely cause a loss of function in parkin, an E3 ubiquitin ligase, probably leading to proteasomal dysfunction. However, mice with mutated parkin exhibit progressive sensorimotor dysfunction without any DA cell loss (
      • Goldberg M.S.
      • Fleming S.M.
      • Palacino J.J.
      • Cepeda C.
      • Lam H.A.
      • Bhatnagar A.
      • Meloni E.G.
      • Wu N.
      • Ackerson L.C.
      • Klapstein G.J.
      • Gajendiran M.
      • Roth B.L.
      • Chesselet M.F.
      • Maidment N.T.
      • Levine M.S.
      • Shen J.
      Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons.
      ). DJ1 mutations cause decreased resistance to oxidative stress in cells, flies, and mice (
      • Goldberg M.S.
      • Fleming S.M.
      • Palacino J.J.
      • Cepeda C.
      • Lam H.A.
      • Bhatnagar A.
      • Meloni E.G.
      • Wu N.
      • Ackerson L.C.
      • Klapstein G.J.
      • Gajendiran M.
      • Roth B.L.
      • Chesselet M.F.
      • Maidment N.T.
      • Levine M.S.
      • Shen J.
      Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons.
      ). DJ1 KO mice, however, have little phenotype and do not develop DA cell loss. Furthermore, almost all of these transgenic mice exhibit any pathology at a much later age (>10 months), making studies more expensive and difficult.
      Therefore, developing a chronic irreversible animal model to study the pathogenesis of PD is of paramount importance, and here we describe a simple, toxin-free and nontransgenic mouse model for PD. Castration induces nigral glial activation, decrease in nigral GDNF, demise of tyrosine hydroxylase (TH)-positive neurons in the nigra, loss of TH fibers and neurotransmitters in the striatum, and impairment of locomotor activities in young male mice. However, castration is unable to induce these nigrostriatal pathologies in male inducible nitric-oxide synthase (iNOS)-null mice. Furthermore, we demonstrate that the expression of GDNF is suppressed by NO. Taken together, these results suggest that castration induces PD-like pathologies in male mice via iNOS-mediated suppression of GDNF.

      DISCUSSION

      Developing a simple, toxin-free and nontransgenic model to study PD-related nigrostriatal pathologies is important because it could assist in easy drug screening. Here, we have successfully demonstrated that simple castration induces PD-related nigrostriatal pathologies in male mice in the absence of any toxins. Our conclusion is based on the following. First, castration led to the loss of dopaminergic neurons in the nigra and fibers in the striatum. This loss was irreversible; even at 4 months after castration, the extent of nigrostriatal damage did not decrease. Second, the DA level markedly decreased 30 days after castration; and even 120 days after castration, we found no improvement in striatal DA level in castrated male mice compared with age-matched normal male mice. Third, castration also led to impairment in locomotor activities, suggesting that castration caused not only structural and neurotransmitter damage but also functional deficits. Because simple castration induced these PD-related nigrostriatal and behavioral changes, our results suggest that some forms of PD (e.g. early onset) in males may be due to a sudden decrease in male sex hormone.
      Why did we think of such an unconventional approach? It has been shown that testosterone deficiency is common in the older male population and has an increased prevalence in parkinsonian patients (
      • Kenangil G.
      • Orken D.N.
      • Ur E.
      • Forta H.
      • Celik M.
      The relation of testosterone levels with fatigue and apathy in Parkinson's disease.
      ,
      • Okun M.S.
      • Crucian G.P.
      • Fischer L.
      • Walter B.L.
      • Testa C.M.
      • Vitek J.L.
      • DeLong M.R.
      • Hanfelt J.
      • Huang X.
      Testosterone deficiency in a Parkinson's disease clinic: results of a survey.
      ). In another study, testosterone therapy led to significant improvement in the resting tremor and fine motor control in parkinsonian patients with testosterone deficiency (
      • Mitchell E.
      • Thomas D.
      • Burnet R.
      Testosterone improves motor function in Parkinson's disease.
      ). Testosterone treatment also improved the nonmotor symptoms of PD (
      • Okun M.S.
      • Walter B.L.
      • McDonald W.M.
      • Tenover J.L.
      • Green J.
      • Juncos J.L.
      • DeLong M.R.
      Beneficial effects of testosterone replacement for the nonmotor symptoms of Parkinson disease.
      ). Finally, TH is also localized to the Leydig cells at both the mRNA and protein levels (
      • Davidoff M.S.
      • Ungefroren H.
      • Middendorff R.
      • Koeva Y.
      • Bakalska M.
      • Atanassova N.
      • Holstein A.F.
      • Jezek D.
      • Pusch W.
      • Müller D.
      Catecholamine-synthesizing enzymes in the adult and prenatal human testis.
      ). MPTP intoxication causes marked decrease in serum testosterone level and loss of Leydig cells (
      • Ruffoli R.
      • Giambelluca M.A.
      • Scavuzzo M.C.
      • Pasquali L.
      • Giannessi F.
      • Fornai F.
      MPTP-induced parkinsonism is associated with damage to Leydig cells and testosterone loss.
      ). The loss of Leydig cells is accompanied by a marked decrease in TH protein in the interstitium and significant fall in norepinephrine levels in the testis (
      • Ruffoli R.
      • Giambelluca M.A.
      • Scavuzzo M.C.
      • Pasquali L.
      • Giannessi F.
      • Fornai F.
      MPTP-induced parkinsonism is associated with damage to Leydig cells and testosterone loss.
      ). Therefore, we thought that testosterone could be intimately coupled to the dopaminergic pathway and that castration may induce nigrostriatal pathologies. However, in contrast to our finding, some articles have shown either no effect or an increase in markers of nigrostriatal function (e.g. TH-positive neurons in the SN, striatal dopamine content) following castration (
      • Dluzen D.E.
      • Ramirez V.D.
      Effects of orchidectomy on nigro-striatal dopaminergic function: behavioral and physiological evidence.
      ,
      • Tamás A.
      • Lubics A.
      • Lengvári I.
      • Reglodi D.
      Effects of age, gender, and gonadectomy on neurochemistry and behavior in animal models of Parkinson's disease.
      ). In these cases, older mice or rats were used for castration. We also did not observe glial activation or loss in nigral TH and striatal DA 1 month after castration when 8–9-week-old and 13–14-week-old mice were castrated (Fig. 5), indicating that castration induces nigrostriatal pathologies after certain time only when young male mice are castrated probably when testosterone level is maximum (Fig. 7J). Our results may have implications in females as well. Although we have not examined females mice, Rocca et al. (
      • Rocca W.A.
      • Bower J.H.
      • Maraganore D.M.
      • Ahlskog J.E.
      • Grossardt B.R.
      • de Andrade M.
      • Melton 3rd, L.J.
      Increased risk of parkinsonism in women who underwent oophorectomy before menopause.
      ) have reported increased risk of parkinsonism in women who underwent oophorectomy before menopause. According to this study (
      • Rocca W.A.
      • Bower J.H.
      • Maraganore D.M.
      • Ahlskog J.E.
      • Grossardt B.R.
      • de Andrade M.
      • Melton 3rd, L.J.
      Increased risk of parkinsonism in women who underwent oophorectomy before menopause.
      ), the risk increased with younger age at oophorectomy. Together, these results suggest that sudden loss of sex hormone in young population may increase the risk of having PD later in life.
      How does castration induce nigrostriatal pathologies? Although the disease mechanisms that cause PD are poorly understood, recent studies strongly support a role of inflammation in nigrostriatal degeneration in PD. It has been found that early intervention with nonsteroidal anti-inflammatory drugs slows disease incidence. Furthermore, significant microglial activation occurs in close proximity to damaged or dying dopaminergic neurons. Several studies have shown that iNOS-NO-ONOO (peroxynitrite) plays important role in the loss of dopaminergic neurons in PD. First, the concentration of NO2 (nitrite), a metabolite of NO, increases in the CSF of patients with PD compared with a group of patients without dopaminergic dysfunction (
      • Qureshi G.A.
      • Baig S.
      • Bednar I.
      • Södersten P.
      • Forsberg G.
      • Siden A.
      Increased cerebrospinal fluid concentration of nitrite in Parkinson's disease.
      ). Second, it has been shown that many cells in the SNpc from postmortem PD samples express considerable amounts of iNOS, whereas those from age-matched controls do not (
      • Hunot S.
      • Boissière F.
      • Faucheux B.
      • Brugg B.
      • Mouatt-Prigent A.
      • Agid Y.
      • Hirsch E.C.
      Nitric-oxide synthase and neuronal vulnerability in Parkinson's disease.
      ). Third, iNOS is not only up-regulated in the SNpc of MPTP-treated mice, but its ablation in mutant mice significantly attenuates MPTP neurotoxicity, thus indicating that iNOS is essential in MPTP-induced SNpc dopaminergic neurodegeneration (
      • Liberatore G.T.
      • Jackson-Lewis V.
      • Vukosavic S.
      • Mandir A.S.
      • Vila M.
      • McAuliffe W.G.
      • Dawson V.L.
      • Dawson T.M.
      • Przedborski S.
      Inducible nitric-oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease.
      ). Therefore, we examined the role of iNOS in castration-induced nigrostriatal pathologies in male mice. Interestingly, castration remained unable to induce the loss of nigral TH-positive neurons and striatal TH fibers and the decrease in striatal neurotransmitters in iNOS−/− mice, clearly describing an important role of iNOS in castration-mediated nigrostriatal pathologies.
      NO, a short lived and diffusible free radical, plays many roles as a signaling and effector molecule in diverse biological systems; it is a neuronal messenger and is involved in vasodilation as well as in antimicrobial and antitumor activities (
      • Nathan C.
      Nitric oxide as a secretory product of mammalian cells.
      ,
      • Saha R.N.
      • Pahan K.
      Regulation of inducible nitric-oxide synthase gene in glial cells.
      ). On the other hand, NO has also been implicated in several CNS disorders, including inflammatory, infectious, traumatic, and degenerative diseases (
      • Liberatore G.T.
      • Jackson-Lewis V.
      • Vukosavic S.
      • Mandir A.S.
      • Vila M.
      • McAuliffe W.G.
      • Dawson V.L.
      • Dawson T.M.
      • Przedborski S.
      Inducible nitric-oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease.
      ,
      • Akama K.T.
      • Albanese C.
      • Pestell R.G.
      • Van Eldik L.J.
      Amyloid β-peptide stimulates nitric oxide production in astrocytes through an NFκB-dependent mechanism.
      ,
      • Merrill J.E.
      • Ignarro L.J.
      • Sherman M.P.
      • Melinek J.
      • Lane T.E.
      Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide.
      ,
      • Liu X.
      • Jana M.
      • Dasgupta S.
      • Koka S.
      • He J.
      • Wood C.
      • Pahan K.
      Human immunodeficiency virus type 1 (HIV-1) tat induces nitric-oxide synthase in human astroglia.
      ). There is considerable evidence for the transcriptional induction of iNOS (the high output isoform of NOS) in the CNS that is associated with degenerative brain injury (
      • Liberatore G.T.
      • Jackson-Lewis V.
      • Vukosavic S.
      • Mandir A.S.
      • Vila M.
      • McAuliffe W.G.
      • Dawson V.L.
      • Dawson T.M.
      • Przedborski S.
      Inducible nitric-oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease.
      ,
      • Saha R.N.
      • Pahan K.
      Regulation of inducible nitric-oxide synthase gene in glial cells.
      ,
      • Merrill J.E.
      • Ignarro L.J.
      • Sherman M.P.
      • Melinek J.
      • Lane T.E.
      Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide.
      ,
      • Liu X.
      • Jana M.
      • Dasgupta S.
      • Koka S.
      • He J.
      • Wood C.
      • Pahan K.
      Human immunodeficiency virus type 1 (HIV-1) tat induces nitric-oxide synthase in human astroglia.
      ). NO is potentially toxic to neurons and oligodendrocytes that may mediate toxicity through the formation of iron-NO complexes of iron-containing enzyme systems (
      • Drapier J.C.
      • Hibbs Jr., J.B.
      Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in l-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells.
      ), oxidation of protein sulfhydryl groups (
      • Radi R.
      • Beckman J.S.
      • Bush K.M.
      • Freeman B.A.
      Peroxynitrite oxidation of sulfhydryls: the cytotoxic potential of superoxide and nitric oxide.
      ), nitration of proteins, and nitrosylation of nucleic acids and DNA strand breaks (
      • Wink D.A.
      • Kasprzak K.S.
      • Maragos C.M.
      • Elespuru R.K.
      • Misra M.
      • Dunams T.M.
      • Cebula T.A.
      • Koch W.H.
      • Andrews A.W.
      • Allen J.S.
      DNA deaminating ability and genotoxicity of nitric oxide and its progenitors.
      ). Here, we have described a new mechanism by which iNOS-derived NO may couple nigrostriatal degeneration (summarized in Fig. 12I). Whereas castration increased the expression of iNOS in the nigra, the level of GDNF went down drastically in the nigra after castration. Interestingly, castration remained unable to decrease the expression of GDNF in nigra and striatum in iNOS−/− mice. This also paralleled with the inability of castration of inducing nigrostriatal pathologies in iNOS−/− mice, suggesting that castration causes the loss of dopaminergic neurons in the nigra via iNOS-mediated down-regulation of GDNF (Fig. 12I). GDNF is a particularly potent factor for survival and axonal growth of mesencephalic dopaminergic neurons (
      • Burke R.E.
      GDNF as a candidate striatal target-derived neurotrophic factor for the development of substantia nigra dopamine neurons.
      ). Kordower et al. (
      • Kordower J.H.
      • Emborg M.E.
      • Bloch J.
      • Ma S.Y.
      • Chu Y.
      • Leventhal L.
      • McBride J.
      • Chen E.Y.
      • Palfi S.
      • Roitberg B.Z.
      • Brown W.D.
      • Holden J.E.
      • Pyzalski R.
      • Taylor M.D.
      • Carvey P.
      • Ling Z.
      • Trono D.
      • Hantraye P.
      • Déglon N.
      • Aebischer P.
      Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease.
      ) used a lentiviral vector to increase GDNF production in the striatum and demonstrated functional and structural recovery when initiating treatment 1 week after a systemic MPTP lesion in monkeys. Although GDNF gene therapy does not work well in PD patients, intraventricular administration of GDNF protein exhibits protection in PD patients (
      • Hoffer B.J.
      • Harvey B.K.
      Is GDNF beneficial in Parkinson disease?.
      ). Therefore, increasing the levels of these trophic factors and/or maintaining their physiological levels in the CNS of patients with neurodegenerative disorders is/are an important area of research. However, such mechanisms are poorly understood. Here, we demonstrate that NO is a negative regulator of GDNF, suggesting that scavenging of NO may help in restoring and/or increasing the level of GDNF in the degenerative brain.

      Acknowledgments

      We thank Dr. Jeffrey H. Kordower for help in the dihydrotestosterone study.

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