Research Article Deletion of Herpud1 Enhances Heme Oxygenase-1 Expression in a Mouse Model of Parkinson's Disease

Hindawi Publishing Corporation Parkinson’s Disease Volume 2016, Article ID 6163934, 9 pages http://dx.doi.org/10.1155/2016/6163934 Research Article Deletion of Herpud1 Enhances Heme Oxygenase-1 Expression in a Mouse Model of Parkinson’s Disease Thuong Manh Le,1 Koji Hashida,1 Hieu Minh Ta,1 Mika Takarada-Iemata,1,2 Koichi Kokame,3 Yasuko Kitao,1,2 and Osamu Hori1,2 1 Department of Neuroanatomy, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa 920-8640, Japan 2CREST, JST (Japan Science and Technology), Tokyo 102-8666, Japan 3Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Osaka 565-8565, Japan Correspondence should be addressed to Osamu Hori; [email protected] Received 21 November 2015; Revised 25 January 2016; Accepted 28 January 2016 Academic Editor: Antonio Pisani Copyright © 2016 Thuong Manh Le et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Herp is an endoplasmic reticulum- (ER-) resident membrane protein that plays a role in ER-associated degradation. We studied the expression of Herp and its effect on neurodegeneration in a mouse model of Parkinson’s disease (PD), in which both the oxidative stress and the ER stress are evoked. Eight hours after administering a PD-related neurotoxin, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), to mice, the expression of Herp increased at both the mRNA and the protein levels. Experiments using +/+ −/− Herpud1 and Herpud1 mice revealed that the status of acute degeneration of nigrostriatal neurons and reactive astrogliosis was comparable between two genotypes after MPTP injection. However, the expression of a potent antioxidant, heme oxygenase-1 −/− +/+ (HO-1), was detected to a higher degree in the astrocytes of Herpud1 micethanintheastrocytesofHerpud1 mice 24 h after + MPTP administration. Further experiments using cultured astrocytes revealed that the stress response against MPP ,anactive form of MPTP, and hydrogen peroxide, both of which cause oxidative stress, was comparable between the two genotypes. These results suggest that deletion of Herpud1 may cause a slightly higher level of initial damage in the nigrastrial neurons after MPTP administration but is compensated for by higher induction of antioxidants such as HO-1 in astrocytes. 1. Introduction SNpc [4–6]. Similarly, overexpression of -synuclein, a familial PD-associated cytoplasmic protein, enhances the Parkinson’s disease (PD) is a common neurodegenerative levels of both oxidative stress and ER stress [7] and affects the disease characterized pathologically by the loss of dopamin- sensitivity of SNpc neurons to MPTP [8–10]. Overexpression ergic neurons and the presence of Lewy bodies (LB), or of Parkin-associated endothelin receptor-like receptor intraneuronal protein aggregates, in the substantia nigra pars (Pael-R) in the knockout mice of Parkin, another familial compacta (SNpc) [1]. Recent studies utilizing genetic and PD-associated protein, also causes both types of stress in environmental approaches have demonstrated that enhanced vitro and in vivo [11, 12]. These lines of evidence suggest the levels of intracellular stress such as oxidative stress, which is existence of cross talk between the different intracellular derived from mitochondrial dysfunction and/or dopamine stresses in the pathogenesis of PD, although the underlying metabolites, and endoplasmic reticulum (ER) stress, a con- mechanisms remain unclear. dition where unfolded proteins accumulate in the ER [2], are Homocysteine-inducible, endoplasmic reticulum protein involved in the pathogenesis of PD [3]. (Herp) is a membrane-bound, ubiquitin- (Ub-) like protein The mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6- that is located in the endoplasmic reticulum (ER) of a variety tetrahydropyridine (MPTP), which is widely used for of cells, including neurons [13–15]. Herpud1, which encodes creating an experimental PD model, causes both oxidative Herp, is strongly induced in response to ER stress [2] and is stress and ER stress, resulting in neurodegeneration in the rapidly degraded by the proteasome [15]. Targeted disruption 2 Parkinson’s Disease of the Herpud1 gene renders F9 embryonic carcinoma cells of which cause oxidative stress. The cells were collected and vulnerable to ER stress, suggesting that Herp plays a protec- subjectedtowesternblotting. tive role against ER stress [15]. The function of Herp is not fully understood, but accumulating evidence suggests that it 2.3. qRT-PCR. Total RNA was extracted from the ventral is involved in ER-associated degradation (ERAD), a system midbrain or caudate putamen (CPu) of each mouse using that clears unfolded or misfolded proteins in the ER via the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, the ubiquitin-proteasome system and autophagy [2]. Several USA) or from cultured astrocytes using the RNeasy Mini Kit ERAD substrates were stabilized by the deletion of Herp [15– (Qiagen). RT reactions containing 1 g of total RNA were 17] and Herp forms a complex with components of the ERAD performed using PrimeScript (Takara, Shiga, Japan). cDNA machinery [18]. was amplified with THUNDERBIRD™ SYBR qPCR® Mix A study on PD reported that Herp was highly expressed (TOYOBO Co., Ltd., Osaka, Japan) by using specific primers both in neuronal and glial cells in the SNpc from patients with for Herpud1, Hmox1, Nfe2l2, Hspa5,andActb.Thecompara- PD [19]. Gene silencing of Herp rendered PC12 cells vulnera- tive Ct method was used to analyze the data with MxPro 4.10 + ble to 1-methyl-4-phenylpyridinium (MPP ), an active form (Agilent Technologies, Santa Clara, CA, USA). The values for of MPTP [20]. Furthermore, Herp counteracted mutant - each gene were normalized to the Actb expression levels. The synuclein-induced ER stress via the homeostatic regulation of sequences of the primers that were used for qRT-PCR are ER-resident calcium release channel proteins [21]. However, listed in Supplemental Table 1 (in Supplementary Material we recently reported that the deletion of Herp facilitated available online at http://dx.doi.org/10.1155/2016/6163934). the degradation of -synuclein and its binding partner synphilin-1 and improved cell viability during proteasomal 2.4. Western Blotting. SamplesfromtheCPuorfromcultured inhibition [22]. Another recent report also demonstrated that astrocytes were solubilized in buffer containing 1% NP40, Herp depletion protected cells from protein aggregation by 0.1% sodium dodecyl sulfate, and 0.2% deoxycholate and upregulating autophagy [23]. To understand the details of were subjected to western blotting with the following anti- theroleHerpplaysinPD,weanalyzedtheexpressionand bodies: tyrosine hydroxylase (TH; EMD Millipore, Billerica, possible involvement of Herp in an experimental model of MA, USA), glial fibrillary acidic protein (GFAP; Dako, +/+ −/− PD using wild-type (Herpud1 )andHerpud1 mice. Glostrup, Denmark), GRP78 (StressGen, Victoria, British Columbia, Canada), heme oxygenase-1 (HO-1; Abcam, Cam- 2. Materials and Methods bridge, UK), and -actin (Sigma). Primary antibody binding was visualized using the ECL system (GE Healthcare Bio- 2.1. Mice and the MPTP Injection PD Model. All animal Sciences Corp., Piscataway, NJ, USA). care and handling procedures were approved by the Animal Care and Use Committee of Kanazawa University. Her- 2.5. Immunohistochemistry. Immunohistochemical analysis −/− pud1 micewere generated as described previously [24] was performed as previously described [27]. In brief, brains and backcrossed to the C57BL/6 strain over eight times. were removed from mice after perfusion with 4% parafor- +/+ −/− maldehyde and postfixed in the same fixative for 4 hours Herpud1 and Herpud1 male mice (aged 10–14 weeks and ∘ weighing 25–30 g) were used for the experiments. The acute at 4 C. After being cryoprotected in 30% sucrose, brains MPTP injection PD model was created by administering were cut in serial coronal 10 m-thick sections containing the intraperitoneal injections of MPTP (20 mg/kg) four times at CPu(fromBregma+1.34mmtoBregma+0.26mm)andthe 2 h intervals, as described previously [25]. At 0, 8, 24, and midbrain covering the whole SNpc (from Bregma −2.80 mm 72 h after MPTP injection, brain samples were prepared for to Bregma −3.80 mm) on a cryostat. Brain sections were the quantitative real-time reverse transcription polymerase mounted in series on ten slides (around ten sections were chain reaction (qRT-PCR), western blotting, and histological mounted on each slide). One out of these ten slides, represent- analyses. ing a set of sections 100 mapart,wasprocessedforimmuno- histochemistry with the following antibodies: TH, GFAP, 2.2. Cell Culture. Astrocytes were isolated from the cerebral GRP78, HO-1, Ub (StressGen), -synuclein (BD, Franklin cortex of 1- to 3-day-old neonatal mice following a previously Lakes, NJ, USA), and LC3B (Cell Signaling Technology, described method with minor modifications [26]. Briefly, the Danvers, MA, USA). In some cases, the cell nuclei were visu- cerebral hemispheres were harvested from neonatal mice, alized with 4 ,6-diamidino-2-phenylindole (DAPI; Sigma). and the meninges were carefully removed. Then, the brain Alexa488-conjugated (Thermo Fisher Scientific, Rockford, ∘ tissue was digested at 37 C in HEPES-buffered saline con- IL, USA) or Cy3-conjugated (Jackson ImmunoResearch Lab- taining Dispase

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