Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model

Abstract

Background: The acupuncture or electroacupuncture (EA) shows the therapeutic effect on various neurodegenerative diseases. This effect was thought to be partially achieved by its ability to alleviate existing neuroinflammation and glial dysfunction. In this study, we systematically investigated the effect of EA on abnormal neurochemical changes and motor symptoms in a mouse neurodegenerative disease model.

Methods: The transgenic mouse which expresses a mutant α-synuclein (α-syn) protein, A53T α-syn, in brain astrocytic cells was used. These mice exhibit extensive neuroinflammatory and motor phenotypes of neurodegenerative disorders. In this study, the effects of EA on these phenotypic changes were examined in these mice.

Results: EA improved the movement detected in multiple motor tests in A53T mutant mice. At the cellular level, EA significantly reduced the activation of microglia and prevented the loss of dopaminergic neurons in the midbrain and motor neurons in the spinal cord. At the molecular level, EA suppressed the abnormal elevation of proinflammatory factors (tumor necrosis factor-α and interleukin-1β) in the striatum and midbrain of A53T mice. In contrast, EA increased striatal and midbrain expression of a transcription factor, nuclear factor E2-related factor 2, and its downstream antioxidants (heme oxygenase-1 and glutamate-cysteine ligase modifier subunits).

Conclusions: These results suggest that EA possesses the ability to ameliorate mutant α-syn-induced motor abnormalities. This ability may be due to that EA enhances both anti-inflammatory and antioxidant activities and suppresses aberrant glial activation in the diseased sites of brains.

Fig. 1

EA reduced loss of body weight, delayed paralysis onset, and increased lifespan in A53T mice. a Schematic diagram illustrating the experimental timeline for behavioral tests and EA treatments. b Effects of EA on the loss of body weight in A53T mice (n = 10 per group). Note a significant loss of body weight starting at 70 days in A53T mice. EA was able to reverse the loss at three testing days (80, 90, and 100 days). c Effects of EA on the onset of paralysis observed in A53T mice (n = 15 per group). d Effects of EA on survival rate of A53T mice (n = 15 per group). Data in Fig. 1b are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. nTg group. # P < 0.05, ## P < 0.01 vs. A53T group

Fig. 2

EA improved motor function in A53T mice. a Effects of EA on total distance in A53T and control mice. Mice in each group were recorded for 30 min (n = 9 per group). b Effects of EA on the duration of A53T and control mice stayed on the rotating rod (n = 9 per group). c Effects of EA on the grip strength of forelimbs and hindlimbs in A53T and control mice (n = 9 per group). d and e Effects of EA on the stride length of four limbs in A53T and control mice (n = 6 per group). f Effects of EA on the regularity index of A53T and control (n = 6 per group). Note that EA stimulation generally improved all motor activities surveyed. Data are expressed as means ± SEM. ***P < 0.001 vs. nTg group. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. A53T group

Fig. 3

Expression of α-syn in astrocytes in the SN and striatum of A53T mice. a Representative immunofluorescent photomicrographs showing α-syn (red) and GFAP (green) costaining in the SN of A53T mice. The α-syn immunoreactivity was restricted to GFAP-expressing astrocytes. b and c Immunoblot analysis of expression of α-syn proteins in the midbrain and striatum of A53T mice. Representative immunoblots are shown above the quantified data. A53T mice were sacrificed for subsequent immunoblot analysis at 1 or 2 months after the birth or at the paralysis development in the symptomatic A53T mice. Data are expressed as means ± SEM (n = 4 per group). ***P < 0.001 vs. 1 or 2 months

Fig. 4

EA decreased exogenous α-syn expression in the midbrain and striatum of A53T mice. a and b Effects of EA on α-syn protein expression in the midbrain (a) and striatum (b) of A53T and nTg control mice. Representative immunoblots are shown above the quantified data. Note that expression level of α-syn proteins in both the midbrain and striatum was markedly reduced by EA. c and d Effects of EA on α-syn mRNA expression in the midbrain (c) and striatum (d) of A53T and nTg control mice. The α-syn mRNA level in midbrain and striatal tissue was detected by real-time PCR. Data are expressed as means ± SEM (n = 4 per group). #P < 0.05, ##P < 0.01 vs. A53T group

Fig. 5

EA reduced the astrogliosis in the midbrain of A53T mice. a Immunofluorescent images illustrating effects of EA on GFAP immunostaining in the SN of A53T and control nTg mice. GFAP-containing astrocytes were visualized by GFAP immunostaining. b Immunoblots illustrating the effects of EA on GFAP protein expression in the midbrain. Representative immunoblots are shown above the quantified data. c and d Effects of EA on GFAP mRNA expression in the midbrain (c) and striatum (d) of A53T and control nTg mice. α-syn mRNAs were detected determined by quantitative real-time PCR. Data are expressed by means ± SEM (n = 4 per group). **P < 0.01, ***P < 0.001 vs. nTg group. #P < 0.05, ###P < 0.001 vs. A53T group

Fig. 6

EA inhibited microglia activation and inflammatory responses in A53T mice. a Immunofluorescent images illustrating effects of EA on Iba1 immunostaining in the SN of A53T and nTg control mice. Iba1-containing microglia was visualized by Iba-1 immunostaining. b and c Effects of EA on TNF-α (b) and IL-1β (c) expression in the midbrain (upper panels) and striatum (lower panels) of A53T mice and nTg control mice. TNF-α and IL-1β protein levels were assessed by ELISA. Note that EA significantly reduced TNF-α expression in the midbrain and striatum and IL-1β expression in the striatum of A53T mice. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01 vs. nTg group. #P < 0.05, ###P < 0.001 vs. A53T group

Fig. 7

EA protected DA neurons in the SNpc and motor neurons in the spinal cord in A53T mice. a Representative immunohistochemical photomicrographs showing TH immunostainings in the SNpc of A53T and nTg control mice following EA treatment. Scale bar, 200 μm. b Quantification of TH-positive dopaminergic neurons in the SNpc of A53T and nTg control mice following EA treatment. The TH neurons were counted by stereological analysis. c Effects of EA on the striatal DA content. Striatal DA levels were assessed by HPLC. d Effects of EA on TH protein levels in the midbrain. TH proteins were detected by immunoblot analysis. Representative immunoblots are shown above the quantified data. e Representative images show NeuN staining in the ventral horn of lumbar spinal cord of control nTg mice, EA+ nTg mice, A53T mice, and EA+ A53T mice. Scale bar, 200 μm. f These images are the enlarged views from areas indicated in the (e). Scale bar, 200 μm. Data are expressed by means ± SEM (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. nTg group. #P < 0.05, ##P < 0.01 vs. A53T group

Fig. 8

EA increased expression of Nrf2 and its downstream factors in the midbrain and striatum of A53T mice. a and b Effects of EA on Nrf2, HO-1 and GCLM protein expression in the midbrain (a) and striatum (b). Representative immunoblots are shown above the quantified data. Note that EA significantly reversed the reduction of three proteins in A53T mice. c Effects of EA on Nrf2, HO-1, and GCLM mRNA expression in the midbrain. EA produced the similar recovery of reduced Nrf2 and HO-1 mRNA expression in A53T mice. Data are expressed by means ± SEM (n = 4 per group). *P < 0.05 vs. nTg group. ##P < 0.01, ###P < 0.001vs. A53T group