Transgenic supplementation of SIRT1 fails to alleviate acute loss of nigrostriatal dopamine neurons and gliosis in a mouse model of MPTP-induced parkinsonism

Background Dopamine (DA) neuron-selective uptake and toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in humans. Loss of DA neurons via mitochondrial damage and oxidative stress is reproduced by systemic injection of MPTP in animals, which serves as models of parkinsonism and Parkinson’s disease (PD). This study aimed to test whether pan-neural supplementation of the longevity-related, pleiotropic deacetylase SIRT1, which confers partial tolerance to at least three models of stroke and neurodegeneration, could also alleviate MPTP-induced acute pathological changes in nigrostriatal DA neurons and neighboring glia. Results We employed a line of prion promoter-driven Sirt1-transgenic (Sirt1Tg) mice that chronically overexpress murine SIRT1 in the brain and spinal cord. Sirt1Tg and wild-type (WT) male littermates (3‒4 months old) were subjected to intraperitoneal injection of MPTP. Acute histopathological changes in the midbrain and striatum (caudoputamen) were assessed with serial coronal sections triply labeled for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and nuclear DNA. In the substantia nigra pars compacta (SNpc) of the midbrain, the number of TH-positive neurons and the reactive gliosis were comparable between the Sirt1Tg and WT littermates. In the striatum, the relative fluorescence intensity of TH-positive nerve terminals and the level of gliosis did not differ by the genotypes. Conclusions Sirt1Tg and WT littermate mice exhibited comparable acute histopathological reactions to the systemic injection of MPTP, loss of TH-positive neurons and reactive gliosis. Thus, the genetic supplementation of SIRT1 does not confer histologically recognizable protection on nigrostriatal DA neurons against acute toxicity of MPTP.

We have established a distinct line of transgenic mice that overexpress SIRT1 in wider neuronal lineages and additionally in glial and vascular endothelial cells via the murine prion gene promoter (Prp) 11,12 . Unlike the NSE-SIRT1 mice, our Prp-SIRT1 mice are resistant to cerebral hypoperfusion by bilateral common carotid artery stenosis, due to vascular dilatation which is potentiated by SIRT1-mediated deacetylation of endothelial nitric oxide synthase (eNOS) 12 . Further, Prp-SIRT1 mice are resistant to proteotoxic stress by an ALS-linked mutant of superoxide dismutase 1 (SOD1), due partly to SIRT1-mediated deacetylation of the heat shock factor 1 (HSF1) and the resulting upregulation of HSP70i 11 . On the basis of the SIRT1-HSF1 axis, and the protective effects of HSF1/HSPs against neurodegenerative insults such as MPTP and α-synuclein 9,13-16 , we assessed whether Prp-SIRT1 mice is resistant to acute loss of DA neurons and gliosis by MPTP.

Methods
Ethics, consent and permissions on animals and experimental design All animal procedures were done in accordance with the guidelines of the Animal Use and Care Committees of Kyoto University (Med-Kyo08097), Nagoya University (#13151), and Kanazawa University (AP-101606). A line of transgenic mice with a C57BL/6J background harboring the PrP-Sirt1cDNA transgene had been generated and deposited at RIKEN Bioresource Center (RBRC06467) as described elsewhere in detail (Watanabe et al., 2014). Mice were reared in a specific pathogen-free environment at 23 ± 2°C, and identified by PCR using a pair of primers, 5′-CAAGAGGTTGTTAATGAAGC-3′ and 5′-TTTCCTGTTGCCTTCAATCAGCTATCG-3′. All comparisons were made between 3-4-month-old, wild-type (WT) and transgenic (Tg) male littermates. Eight mice were subjected to intraperitoneal injection of MPTP (20 μg/g body weight) in saline or saline alone, each 4 times with 2 h-intervals 17 , followed by histological analysis 4 days later 3 .

Histological analysis and quantification
Mice were deeply anesthetized with sodium pentobarbital (50 μg/g, i.p.), fixed with transcardial perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Frozen-sectioned 10 μm-thick coronal brain sections were reacted with antibodies for tyrosine hydroxylase (TH, rabbit IgG, Chemicon) and glial fibrillary acidic protein (GFAP, mouse IgG, Sigma) 17,19 . The sections were reacted with Cy3-conjugated anti-rabbit IgG and FITC-conjugated anti-goat IgG (Jackson ImmunoResearch), and observed with a laser scanning confocal microscope (Eclipse TE2000U, Nikon) with the Nikon EZ-C1 software. We counted TH-positive neurons in the SNpc in three planes (−3.08, −3.16, and −3.40 mm from the bregma), and measured immunofluorescence intensity for TH in the striatum (caudoputamen; CPu) as described previously 17,19 . Detailed information can be found in the text file provided ('Raw data description').

Results
We used the original Prp-SIRT1 Tg mouse line that chronically expresses murine Sirt1cDNA in the central nervous system (CNS) under control of the murine prion gene promoter 11,18 . The expression levels of SIRT1 in the midbrain and striatum assessed by immunoblot were approximately three times higher in heterozygous Tg mice than in the non-Tg (WT) littermates 12 . Four days after serial administrations of MPTP (20 μg/g body weight), we assessed acute histopathological changes of the nigrostriatal tract in the two genotypes (n = 8).
In the midbrain of Tg and WT mice without MPTP administration, the distribution and appearance of TH-positive cells (presumed DA neuronal somata and dendrites), surrounding GFAP-positive astrocytes, and the nuclei of these and other cells (consist mostly of non-DA neurons and microglia) were comparable ( Figure 1A, top). MPTP administration induced acute, significant loss of TH-positive cells and reactive gliosis at comparable severity between the genotypes ( Figure 1A, bottom). The numbers of TH-positive neuronal somata in the SNpc (identified in three serial sections) did not show statistically significant difference ( Figure 1B). These data indicate that the supplementation of SIRT1 does not suppress the loss of DA neurons and reactive gliosis by acute MPTP toxicity.
In the striatum/caudoputamen without MPTP administration, the staining patterns for TH (mostly axons and axon terminals of DA neurons), GFAP-positive astrocytes, and the nuclei of these and other cells were comparable between the Tg and WT littermates (Figure 2A, left). Loss of TH-positive neuropil and reactive gliosis after MPTP administration were also comparable between the genotypes (Figure 2A, middle; higher magnifications in the right).
Fluorescence intensity for TH in the striatum after MPTP administration did not differ ( Figure 2B), indicating that the supplementation of SIRT1 does not alleviate the loss of DA nerve terminals by acute MPTP toxicity.
Overall, PrP-SIRT1 and WT male littermates exhibited similar responses to MPTP toxicity in terms of acute damages to nigrostriatal DA neurons, and proliferation/remodeling of neighboring astrocytes.
These findings indicate that supplementation of SIRT1 in neurons and glia does not alleviate MPTP-induced DA neuronal damages and reactive gliosis.

Discussion
Acute degeneration of DA neurons in the mouse MPTP model can be rescued by resveratrol pre-administration 4,5 , or by transgenic supplementation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) which controls mitochondrial biogenesis and oxidative phosphorylation 19 . The rescue effects had been attributed at least partly to SIRT1 7,8 on the basis that resveratrol directly or indirectly potentiates SIRT1 20 , and that SIRT1 activates PGC-1α by deacetylation 21 . However, transgenic supplementation of SIRT1 either with the neuron-specific promoter 10 or with the neuron/glia/ vascular endothelial promoter (this study) did not confer tolerance to MPTP-induced pathology. The consistent results indicate that the resveratrol-mediated tolerance to MPTP is due to SIRT1independent mechanisms (e.g., antioxidant activity as a polyphenol.  See Introduction.), and that SIRT1-mediated activation of PGC-1α is insufficient to confer tolerance (i.e., the upregulation of PGC-1α is necessary). Thus, this study has made a case against the unproven notions that health benefits of resveratrol are attributed mostly to SIRT1, and that potentiation of SIRT1 in neurons and glia nonselectively suppresses neurodegeneration and gliosis. Nevertheless, it is worth testing whether Prp-SIRT1 mice are resistant to chronic neurotoxin models or genetic models of PD 22,23 , and whether PGC-1α/SIRT1-double Tg mice are more resistant than the original PGC-1α Tg mice 19 .
Our recent study with Prp-SIRT1 mice has demonstrated their resistance, albeit limited, to spinal cord degeneration caused by chronic overload of a mutant SOD1 11 . The proteotoxic stress by misfolded SOD1 is alleviated at least in part by SIRT1-mediated deacetylation of a master transcription factor HSF1 and the resulting upregulation of HSP70i and perhaps other molecular chaperones 11 . Intriguingly, either transgenic supplementation of HSP70 or its heat shock-mediated upregulation (i.e., preconditioning) confers recognizable resistance to MPTP 13-15 . We therefore hypothesize that, in DA neurons of Prp-SIRT1 mice, the expression levels of the SIRT1 substrate HSF1 and the downstream effectors including HSP70i are insufficient to counter the toxicity of MPTP-as with the aforementioned situation of PGC-1α. Thus, an obvious subject for future studies is to test whether some preconditioning or milder insults (e.g., lower dose of MPTP or rotenone) could differentiate Prp-SIRT1 mice from wild-type mice.

Data availability
All Author contributions YK and OH conducted the toxicological and histological analyses on mice bred by NA-I under the supervision of RT and MK. MK and OH designed the study and wrote the manuscript. All authors read and approved the manuscript.

Competing interests
No competing interests were disclosed.

Grant information
This work was supported in part by CREST (Creation of a novel technology for prevention, diagnosis, and therapy for psychiatric and neurological disorders) from JST, Grant-in-Aid for Scientific Research on Innovative Areas, Comprehensive Brain Science Network, and Grants-in-Aid for Scientific Research from the MEXT of Japan.
We confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
sound qualitative and quantitative analysis. An important point, also noted by the authors in the discussion, is the possibility that SIRT1 contributes only minimally to DA neuron survival -this effect could be unmasked by using a lower dose of MPTP or alternatively model parkinsonism using the neurotoxin 6-Hydroxydopamine. In either case, further analysis is required to completely negate the neuroprotective effects of SIRT1 in Parkinson's models. in vivo I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. Competing Interests: 05  This manuscript deals with the possible alleviative effect of pan-neural supplementation of the longevity-related, pleiotropic deacetylase SIRT1 on a mouse model of MPTP-induced parkinsonism. The present work was totally based on negative data. However, the entire experimental procedures are sound and the anatomical results obtained are well presented. Since the present work has a potential to provide a certain contribution to the progress in Parkinson's disease research, this reviewer recommends that the manuscript may be accepted as it is.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. Competing Interests: