Deletion Of MiR-29b2/c Leads To Accelerated Aging And Neuroprotection in MPTP-induced Parkinson's Disease Mice

Abstract

Studies reveal a linkage between miR-29s in aging and Parkinson’s disease (PD). Here we show that the serum levels of miR-29s in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice exhibited dynamic changes. The role of miR-29b2/c in aging and PD was studied utilizing miR-29b2/c gene knockout mice (miR-29b2/c KO). miR-29b2/c KO mice were characterized by markedly lighter weight, kyphosis, muscle weakness, and abnormal gait when compared with wild-type (WT) mice. The WT also developed apparent dermis thickening and adipose tissue reduction. However, deficiency of miR-29b2/c alleviated MPTP-induced damages of the dopaminergic system and glial activation in the nigrostriatal pathway and consequently improved the motor function of MPTP-treated KO mice. Knockout of miR-29b2/c inhibited the expression of inflammatory factors in 1-methyl-4-phenyl pyridinium (MPP+ )-treated primary cultures of mixed glia, primary astrocytes, or LPS-treated primary microglia. Moreover, miR-29b2/c deficiency enhanced the activity of AMPK but repressed the NF-κB p65 signaling in glial cells. Our results show that miR-29b2/c KO mice display the progeria-like phenotype. Less activated glial cells and repressed neuroinflammation might bring forth dopaminergic neuroprotection in miR- 29b2/c KO mice. Conclusively, miR-29b2/c is involved in the regulation of aging and plays a detrimental role in Parkinson’s disease.

Keywords

MiR-29b2/c; neuroprotection; MPTP-induced Parkinson’s disease; Cistanche benefits.

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Introduction

Parkinson’s disease (PD) is the second most frequent neurodegenerative disease, characterized by the progressive loss of dopaminergic neurons in the substantia nigra par compacta (SNpc) of the midbrain, and increased glial activation and neuroinflammation [1–3]. MicroRNAs are short noncoding RNA molecules that regulate gene expression at the post-transcriptional level [4, 5]. MicroRNAs are involved in the regulation of nervous system development, neuronal plasticity, and neurodegenerative diseases. MicroRNA29 family (miR- 29s) are composed of two gene clusters: miR-29a/b1 and miR-29b2/c, which are located on chromosome 6 and chromosome1, respectively, of the mouse genome. miR-29a and miR-29c only have one nucleotide difference, whereas miR-29b1 and miR-29b2 are identical in sequence [6]. miR-29s are involved in multiple biological processes. Many miR-29s target genes have been experimentally verified, including Procell survival genes Bcl-2, Mcl-1, Cdc42, and p85-α; proapoptotic genes Puma, Bim, Bak, and Bmf; and proinflammatory cytokines IFN-γ and IL-12β. In the peripheral system, studies have shown that miR-29s are involved in tissue fibrosis [7–9], metabolism, and immune regulation [10–13].

miR-29s express widely in the central nervous system, and their transcripts exist both in neurons and glial cells [14, 15]. The association between miR-29s and neurological diseases has been increasingly illustrated. miR-29s are involved in the regulation of β-amyloid production, and they are down-regulated in the brain of AD patients [16, 17]; miR-29s levels also decrease in Huntington’s disease (HD) patients and HD model mice [18, 19], whereas they increase in patients with amyotrophic lateral sclerosis (ALS) and ALS mouse models [16, 20]. Both miR-29a/b-1-deficient mice and miR-29s down-regulated mice display an Ataxia-like phenotype [6, 21]. Regarding the effects of miR-29s expression on neuroprotection and the promotion of neuronal death in ischemic rodent models opposing results were reported [22, 23]. In previous studies, we observed that miR-29s levels were markedly decreased in the serum of PD patients with a decreasing trend related to more severe Parkinsonism [24]. Further, the miR-29s levels correlated with memory performance in PD patients [25]. We concluded that miR-29b2/c is linked closely to PD, but its physiological functions and pathological mechanisms involved in PD are largely unknown.

In this study, mouse serum levels of miR-29s in response to MPTP administration were measured up to 120 days post-injection. The effects of miR-29b2/c deficiencies on the peripheral tissues were studied utilizing miR-29b2/c KO mice. The dopaminergic neurotoxin MPTP was further used to induce a mouse model of PD. Injuries of the nigrostriatal dopaminergic system, behavioral performance, and potential mechanisms were then investigated. We observed that the miR-29s levels in the mouse serum displayed dynamic changes after MPTP administration. miR- 29b2/c KO mice displayed accelerated aging, indicated by the lighter body weight, adipose tissue reduction, kyphosis, skin thickening, muscle weakness, and gait abnormality. However, deficiency of miR-29b2/c led to the alleviated dopaminergic damage and glial activation, and consequently to improved behavioral performance in a PD-like animal model.

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Result

1. The progeria-like phenotype in miR-29b2/c KO mice

miR-29b2/c knockout mice (miR-29b2/c KO) were generated using the CRISPR-Cas9 technique. The strategy of gene targeting and mutant mouse genotyping were presented in Supplementary Figure 1. Four- and 16- month-old miR-29b2/c KO mice had decreased body weights (Figure 1A). At three months of age, the features of miR-29b2/c KO mice remained unaltered, as proven by an X-ray micro-computed tomography (micro-CT) scan (Supplementary Figure 2A). There were no differences in bone mineral density (BMD), trabeculae mean BMD, trabecular separation, trabecular thickness, and structural model index (SMI) between miR-29b2/c KO mice and their WT counterpart at 13 months old (Supplementary Figure 2B). By hematoxylin and eosin (H&E) staining, we found that 13-month-old miR-29b2/c KO mice displayed thickened dermis with increased and deepened wrinkles (Figure 1B, 1C). And kyphosis was apparent in 16-month-old miR-29b2/c KO mice (Figure 1C). miR-29b2/c KO mice at a young age (3 months old) had normal adipose tissues (Supplementary Figure 2C, 2D). However, abdominal adipose tissue (subcutaneous fat and visceral fat combined) and brown adipose tissue were dramatically reduced in miR-29b2/c KO mice compared to WT mice at the age of 16 months (Figure 1D). Further, the transcriptional levels of senescence markers p21 and p53 in the brain were analyzed. They increased markedly in the hippocampus, but not in the cortex of miR-29b2/c KO mice at the age of six months. p53 and p16 protein levels in the miR-29b2/c-deficient hippocampus did not differ from the WT controls. In addition, β-galactosidase activity, a known characteristic of cellular senescence, did not differ between the brains of three-month-old WT and miR-29b2/c KO mice.

Muscle weakness and gait abnormality in miR- 29b2/c KO mice

The behaviors of miR-29b2/c-deficient mice were assessed. Muscle strength was measured by the Wire hanging and the Grid hanging tests. The miR-29b2/c KO mice scored lower than the control mice in the Wire hanging test (Figure 2A). And the latency to fall was dramatically shorter in miR-29b2/c KO mice when compared with wild-type counterparts in the Grid hanging test (Figure 2B), but the Rotarod test performance of WT and miR-29b2/c KO mice did not differ significantly (Figure 2C). Animal gait was detected by the Catwalk XT analysis system. Surprisingly, both the speed and stride length of miR-29b2/c KO mice were higher than those of their counterparts, whereas the step cycle, stand, and swing time were shorter, and the duty cycle reduced, in miR-29 b2/c-deficient mice (Figure 2D).

Changes of miR-29s in the MPTP-induced PD mouse serum

miR-29s levels decreased in the serum of patients with PD compared to healthy controls [24]. In this study, miR-29s levels in the serum of PD mice were quantified at 3, 30, and 120 days after administration of a subacute regimen of MPTP. miR-29a and miR-29b levels decreased at 3 days after injection and recovered to baseline values at 30 days, and decreased again at 120 days. However, the miR-29c level did not change significantly (Supplementary Figure 5).

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miR-29b2/c deficiency mitigates MPTP-induced nigrostriatal injuries and motor deficits in mice

miR-29b2/c knockout mice and the WT littermates were injected with MPTP to induce the PD model. The expression levels of p21, p53, and Pai1 in the striatum of both miR-29b2/c KO mice and WT controls did not change after MPTP administration. MPTP exposure caused significant reductions of the TH-positive dopaminergic neurons in the SNpc [F(1, 20) = 5.441, P=0.0302], and the TH-positive nerve terminals, TH proteins [F(1, 20) = 10.5, P=0.0041] and the levels of dopamine, DOPAC and HVA in the striatum (Figure 3A–3D). However, the nigrostriatal injuries in MPTP-treated miR-29b2/c KO mice were dramatically alleviated when compared with MPTPtreated WT controls as the numbers of dopaminergic neurons, the densities of dopaminergic nerve terminals, and the striatal TH protein levels and dopamine concentration were significantly higher; In addition, changes in the ratios of DOPAC to DA and HVA to DA [F(1, 19) = 12.64, P=0.0021] were markedly mitigated (Figure 3A–3D). In normal saline-injected miR-29b2/c KO mice, the striatal concentrations of 5-HT and its metabolite 5-HIAA increased compared to their WT counterparts, and NE concentrations were close between the wild-type and miR-29b2/c KO mice (Figure 3D). The Rearing and the Pole tests were used to evaluate spontaneous vertical activity [26, 27] and locomotor activity of mice [28], respectively. Two days after the last MPTP injection, rearing frequency decreased in WT mice during the last 2 min when compared with normal saline-treated WT controls. Similar experiments did not reveal an effect of miR- 29b2/c deficiency on MPTP-induced changes in rearing frequency (Figure 3E). Similarly, the total time spent on turning and climbing down increased in WT mice after MPTP administration in the Pole test, whereas the total time was close between normal saline- and MPTPtreated miR-29b2/c KO mice (Figure 3F).

miR-29b2/c deficiency attenuates MPTP-induced glial activation in mice

Glial cell activation and glial cell-mediated neuroinflammation are involved in PD pathology [29]. Astrocytes increased markedly in the nigrostriatal pathway of WT mice, whereas GFAP+ astrocytes increased in the striatum but not in the SNpc [F(1, 8) = 5.412, P=0.0484] of miR-29b2/c KO mice three days after MPTP injection. Notably, astrocytic densities did not differ in the two regions of WT and miR-29b2/c KO mice treated with MPTP (Figure 4A, 4B). Iba 1+ microglia increased in the substantia nigra of WT mice, and in the striatum [F(1, 8) = 12.74, P=0.0073] of both WT and miR-29b2/c KO mice after MPTP administration. Moreover, in miR-29b2/c KO mice MPTP injection reduced microglial densities significantly (Figure 4C, 4D).

2. The effects of miR-29b2/c deficiency in primary cultured astrocytes

A scratch assay was utilized to test if a deficiency of miR-29b2/c affected astrocytic proliferation and migration. Primary miR-29b2/c KO astrocytes showed no difference in the ability of proliferation and migration. The cell viability of primary WT and miR-29b2/c KO astrocytes manifested no difference at baseline. After MPP+ intoxication, the cell viability of WT astrocytes was higher compared to the controls, whereas the viability of mutant astrocytes did not change [F(1, 18) = 5.727, P=0.0278]. Enhanced ROS product and glucose uptake were observed in MPP+ -treated WT and miR-29b2/c KO astrocytes.

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The expression levels of neurotrophic factors, inflammation-related molecules, astrocytic A1 type, and A2 type marker genes in MPP+ -treated astrocytes were measured. At 12 h after the challenge, the expression of BDNF was elevated in the WT and miR-29b2/c KO astrocytes (Figure 5A). The TGF-β1 expression level was significantly upregulated in WT astrocytes after the exposure to MPP+, while it was downregulated in miR- 29b2/c KO astrocytes when compared with wild-type astrocytes both at baseline and after the intoxication (Figure 5B). The base level of TNF transcript was lower in miR-29b2/c KO astrocytes when compared with WT astrocytes. After 12 h-treatment, the expression levels of IL-1β, IL-6, and COX-2 increased significantly in WT and miR-29b2/c astrocytes, whereas TNF transcript was elevated only in WT astrocytes, and IL-1β, TNF, and COX-2 transcripts decreased in miR-29b2/c KO astrocytic cells when compared with wild-type counterparts (Figure 5C). A1 marker H2-T23 and A2 marker CD14 were markedly lower in PBS-treated miR- 29b2/c KO astrocytes when compared with WT controls. After 12 h-treatment, A1 markers H2-D1, Ggta1, and C3, and A2 markers Clcf1 and S100α10 were upregulated both in WT and miR-29b2/c KO astrocytes; H2-T23 increased only in WT astrocytes; Gbp2 and CD14 did not change in the two genotypes of astrocytes. Moreover, transcripts of H2-T23, H2-D1, and CD14 were downregulated in miR-29b2/c KO astrocytes when compared with WT controls (Figure 5D, 5E). 12 h and 24 h after the treatment, the levels of AMPK proteins and phosphorylated-AMPK proteins did not alter in WT astrocytes, whereas, at 24 h after MPP+ challenge, the AMPK protein level decreased in miR-29b2/c KO astrocytes, and the ratio of p-AMPK to AMPK in mutant astrocytes increased in comparison with WT astrocytes and PBS-treated mutant astrocytes as well (Figure 5F). Additionally, the conditioned medium from BV2 cells treated with PBS or LPS (shortly named CM and LCM) was used to stimulate primary astrocytes of WT and miR-29b2/c KO mice. Though the expression of p-AMPK protein was not changed after 12 h-treatment of LCM, it was significantly higher in miR-29b2/c KO astrocytes compared with WT astrocytes. We observed that the pAMPK proteins increased in both genotypes of astrocytes after 24 treatments of LCM. LCM stimulation markedly increased the expression of COX- 2 proteins [F(2, 18) = 10.29, P=0.0010], however, the COX-2 protein level in miR-29b2/c KO astrocytes decreased compared with WT astrocytes (Figure 5G). Nitrite concentration in LCM-treated miR-29b2/c KO astrocytes was also significantly lower compared with LCM-treated WT astrocytes [F(2, 30) = 17, P<0.0001] (Figure 5H). Further, the expression of senescence marker genes was evaluated. 12 h-treatment of MPP+ increased p53 transcript levels in both WT and miR- 29b2/c KO astrocytes. p19 and Pai1 transcripts were elevated only in WT astrocytes, and Pai1 expression levels in miR-29b2/c KO astrocytes were decreased compared to WT astrocytes after the treatment. Moreover, Bcl-2, Bax protein levels, and the ratio of Bcl-2 to Bax did not change in WT and miR-29b2/c KO astrocytes.

3. The effects of miR-29b2/c deficiency in primary cultured microglia

To measure the response of miR-29b2/c-deficient microglia to inflammatory stimuli, LPS was used. BDNF expression level was lower in miR-29b2/c KO microglia at baseline. Four and eight hours after LPS treatment, the amount of IL-1β, IL-6, TNF, and COX-2 transcripts were upregulated, while IGF-1 expression was downregulated in WT and miR-29b2/c KO microglial cells, the expression of BDNF only decreased in WT microglia [F(2, 27) = 41.5, P<0.0001]. IL-10 transcript increased in WT and miR-29b2/c KO microglia [F(2, 27) = 3.753, P=0.0365], and TGF-β1 transcript decreased in WT microglia at eight hours after the LPS challenge. BDNF and TGF-β1 transcripts were not changed in miR-29b2/c KO microglia. In addition, IL-6 transcripts were significantly reduced in miR-29b2/c KO microglia after the challenge compared to WT controls, IGF-1 and IL-10 transcripts were markedly higher in miR-29b2/c KO microglia after four and eight hours of intoxication, respectively (Figure 6A–6C). At baseline, the p-AMPK protein level and pAMPK to AMPK ratio were dramatically elevated in miR-29b2/c KO microglia when compared with WT microglia. 24 h after the treatment of LPS, p-AMPK level [F(1, 17) = 5.066, P=0.0379] and the ratio [F(1, 17) = 5.537, P=0.0309] were elevated only in WT microglia (Figure 6D). At one hour after the treatment, the phosphorylated-NF-κB p65 (p-p65) proteins and pp65 to p65 ratio, but not p65 proteins, increased in wildtype and miR-29b2/c KO microglial cells, yet the ratio decreased modestly in miR-29b2/c KO microglial cells when compared with WT microglia (p=0.071) (Figure 6E). The expression of COX-2 was enhanced in the two genotypes of microglial cells; whereas, the level of COX-2 in mutant microglia decreased when compared with WT controls after 24 h-treatment of LPS [F(1, 17) = 5.33, P=0.0338] (Figure 6D). Nitrite product was induced by the treatment of LPS in both genotypes of microglia, however, it was dramatically lower in miR- 29b2/c KO microglia at baseline and after the treatment of LPS [F(1, 20) = 14.03, P=0.0013] (Figure 6F).

Discussion

The expression of miR-29 family members is upregulated in multiple tissues when individuals are getting older [30–32]. Works from our lab and others have shown a linkage between miR-29s and Parkinson’s disease [33]. In the present study, the roles of miR- 29b2/c in aging and Parkinson’s disease were studied. miR-29s exert pro- and anti-aging effects depending on tissues, species, and stages of development. We found that 3-month-old miR-29b2/c KO mice had a normal skeleton and adipose tissue, and 4-month-old miR- 29b2/c KO mice displayed lighter body weight compared with their WT counterpart, which is in agreement with other studies [11, 34]. Muscle weakness and unstable gait were detected in 3-month-old miR- 29b2/c KO mice. Thirteen-month-old miR-29b2/c KO mice showed dermis thickening. However, they had the normal fine structure of the femur bone. The reduction of body weight existed in 16-month-old miR-29b2/c KO mice. At this age, miR-29b2/c KO mice had less abdominal fat and brown fat and exhibited apparent kyphosis. miR-29s are involved in p53-mediated cell cycle arrest [35, 36] and p16/Rb-driven cellular senescence [37]. However, cellular senescence was not evident in the miR-29b2/c KO mouse brain. Therefore, though miR-29b2/c contributes to the regulation of aging, its roles in the peripheral tissues and the brain might be different.

The serum levels of miR-29s were downregulated in patients with PD compared to healthy controls [24]. Here, we found that miR-29s expression in PD mouse serum fluctuated from 3 to 120 days after MPTP administration. miR-29s are abundant in the brain [6, 30]. Via going through the GEO profile, miR-29c expression is found to be upregulated in the substantia nigra of PD patients (p=0.0059, by Mann-Whitney test), moreover, miR-29c expression in the superior frontal gyrus did not differ (p=0.47, by Student-T-test), when compared with the control subjects (Supplementary Figure 10). Here, miR-29b2/c KO mice were challenged with MPTP to induce PD-like injuries. The mutant mice showed less severe injuries to the nigrostriatal dopaminergic system and milder glial activation, and subsequently behavioral resistance to some extent. Thus, miR-29b2/c has a detrimental role in the pathology of PD. We observed that in the MPTPinduced mouse PD model, the striatal levels of p21, p53, and Pai1 transcripts did not change, suggesting cellular senescence might not occur. Aging is regarded as a high-risk factor for the development of PD [38], however, aging-related changes in the brain, especially the dopaminergic system might be a more relevant factor.

Primary glia from WT and miR-29b2/c KO mice were cultured to investigate the underlying mechanisms. MPP+ treatment increased GDNF expression and decreased IL-1β expression in the miR-29b2/c-deficient mixed glia. MPP+ also increased the expression of proinflammatory genes in WT astrocytes but not in miR- 29b2/c-deficient astrocytes. Likewise, mutant astrocytes produced less NO compared with WT astrocytes after being exposed to the conditioned medium of BV2 cells treated with LPS. In primary microglia culture, compared to LPS-treated WT controls, the transcripts of the anti-inflammation cytokine IL-10 and neurotrophic factor IGF-1 were markedly higher, and the transcripts of pro-inflammation cytokine IL-6 decreased in LPStreated miR-29b2/c KO microglia. NO levels were decreased in miR-29b2/c KO microglia at baseline and after the intoxication of LPS. Studies have shown that miR-29s and many predicted target genes are involved in metabolic processes [34, 39–41]. As an essential regulator, AMPK has been proven to be protective in PD when activated [42]. Activation of AMPK stimulates Sirtuin 1 and inhibits NF-κB activation and downstream inflammatory target genes indirectly [43]. We observed that the phosphorylated-AMPK protein level and the ratio of p-AMPK to AMPK were upregulated in LCM-treated and MPP+ -treated miR- 29b2/c KO astrocytes respectively. The COX-2 protein level was decreased in LCM-treated mutant astrocytes compared to WT astrocytes. At baseline, AMPK activity was elevated in miR-29b2/c KO microglia. The amount of COX-2 protein was significantly reduced in miR-29b2/c KO microglia when compared with WT controls after the treatment of LPS. Under LPS treatment, the p-p65 and p65 ratio was slightly downregulated in the mutant microglia, implying a mitigated NF-κB signaling pathway. Our results indicate that enhanced AMPK activity and reduced inflammatory response in glia protect the nigrostriatal pathway in miR-29b2/c KO mice (Figure 6G). Conclusively, miR-29b2/c plays an important role in aging and damage in the nigrostriatal dopaminergic system.

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Xiaochen Bai 1,2, Xiaoshuang Zhang 1, Rong Fang 1, Jinghui Wang 1, Yuanyuan Ma 1, Zhaolin Liu 1, Hongtian Dong 1, Qing Li 1, Jingyu Ge 1, Mei Yu 1, Jian Fei 3,4, Ruilin Sun 4, Fang Huang 1

1. Department of Translational Neuroscience, Jing’an District Centre Hospital of Shanghai, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai 200032, China

2. Department of Rehabilitation Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China

3. School of Life Science and Technology, Tongji University, Shanghai 200092, China

4. Shanghai Engineering Research Center for Model Organisms, Shanghai Model Organisms Center, INC, Shanghai 201203, China