Rotenone dose-dependently impairs cognitive capacity of mice
To determine whether rotenone-treated mice display cognitive dysfunction, the MWM, novel objective recognition and passive avoidance test were performed after 3 weeks of rotenone exposure. In MWZ test, mice in control group exhibited normal spatial leaning ability as indicated by time-dependent decease of escape latency and travelled distance (Fig. 1A). We did not find significant difference in escape latency and travelled distance between 0.75 mg/kg rotenone-treated mice and vehicle controls, although an increased trend was observed (Fig. 1A). However, rotenone at 1.5 mg/kg significantly elevated escape latency by showing longer time of rotenone-treated mice than controls to locate the platform (Fig. 1A). Consistently, 1.5 mg/kg rotenone also markedly increased the travelled distance of mice (Fig. 1B), indicating impaired spatial learning function. Analysis of swimming speed among the groups showed no significant difference (Fig. 1C). The spatial probe test was subsequently accessed to evaluate the spatial memory capacity of mice. Mice received 1.5 mg/kg rotenone displayed increased latency for first platform crossing, as well as reduced total number of platform crossing and time percentage in target quadrant compared with control group (Fig.1D-F), indicating impaired memory ability. While, mice in 0.75 mg/kg rotenone and control group failed to show significant difference in spatial probe test (Fig.1D-F).
To further confirm the impaired cognitive capacity in mice, the novel objective recognition and passive avoidance tests were subsequently performed. Consistently, rotenone at 1.5 mg/kg significantly reduced recognition index and step-through latency as well as increased error times of step-through compared to vehicle controls (Fig 1G-I). No significant difference of recognition index, step-through latency and number was observed between 0.75 mg/kg rotenone-treated mice and control group. These results suggest that 1.5 mg/kg rotenone-induced mouse PD model displayed impaired cognitive performance.
Rotenone dose-dependently induces neuronal damage
The cognitive dysfunction is associated with abnormal neuronal damage in PD, especially in hippocampal and cortical regions [24, 25]. To investigate whether rotenone-induced cognitive deficits was associated with hippocampal and cortical neuron damage, neuronal cells were immunostained with neuron-specific anti-Neu-N antibody. As illustrated in Fig. 2A-C, 1.5 mg/kg rotenone treatment significantly reduced the number of Neu-N+ neurons in the hippocampal and cortical regions of mice compared with vehicle controls. Additionally, synapses are critical for neuron-neuron communication and cognitive function. Reduced expressions of key proteins of synapse, such as postsynaptic density protein 95 (PSD-95) significantly correlated with cognition decline in neurodegenerative disorders [15, 26]. In agreement with neuronal loss, immunohistochemical analysis revealed a reduced density of hippocampal and cortical PSD-95 immunostaining in 1.5 mg/kg rotenone-treated mice (Fig. 2A-C and Supplementary Fig. 1).
To further confirm the damage of neurons in rotenone-treated mice, the levels of Neu-N and PSD95 were determined. As shown in Fig. 2D-F, rotenone at 1.5 mg/kg reduced the expressions of Neu-N and PSD-95 in the hippocampal and cortical regions of mice. The comparison of Neu-N+ cell number, PSD95 immunostaining density and expressions of Neu-N and PSD95 between 0.75 mg/kg rotenone-treated mice and vehicle controls showed no significant difference (Fig. 2A-F).
In addition to neuronal damage, α-synuclein accumulation and phosphorylation, especially at Ser129 site, also contributes to cognitive deficits in PD [27]. Immunofluorescence staining showed an elevated phosphorylation of α-synuclein at Ser129 site in the hippocampus and cortex of 1.5 mg/kg rotenone-treated mice compared with control group (Fig. 2G). Fluorescence density analysis of Ser129-phosphorylated α-synuclein immunostaining confirmed this observation (Fig. 2H).
Microglial activation precedes neuronal damage in rotenone-treated mice
To investigate whether microglial activation contributes to rotenone-induced cognitive deficits in mice, microglia were immunostained with a microglial marker, Iba-1. As seen in Fig. 3A, microglial cells in the hippocampus and cortex of 1.5 mg/kg rotenone-injected mice exhibited hypertrophic morphology and increased Iba-1 expression, indicating microglial activation. Quantitative analysis revealed 45.4% and 39.7% of increase of Iba-1 density in the hippocampus and cortex, respectively, of 1.5 mg/kg rotenone-treated mice compared with controls (Fig. 3B and C). No significant microglial activation was observed in 0.75 mg/kg rotenone-injected mice (Fig. 3A-C).
The time sequence of microglial activation and neuronal damage was further investigated in 1.5 mg/kg rotenone-treated mice. Time course studies revealed that microglial activation in the hippocampal and cortical regions of mice occurred as early as 1 week after rotenone exposure and was sustained up to 3 weeks (Fig. 3D). Quantitative analysis of Iba-1 immunostaining density revealed 21.1%, 32.6% and 54.9% increase in the hippocampus and 22.8%, 42.0% and 65.3% increase in the cortex after 1, 2, and 3 weeks of rotenone injection, respectively (Fig. 3E and F). By Contrast, neuronal damage as evidenced by decreased Neu-N+ cell number and PSD95 immunostaining density was observed after 2 weeks of rotenone injection (Fig. 3D-F). These results suggested that rotenone-induced microglial activation precedes neurodegeneration.
PLX3397 and minocycline attenuates rotenone-induced cognitive deficits in mice
To determine whether microglial activation contributes to cognitive impairments, rotenone-injected mice were administered with PLX3397 or minocycline. PLX3397 is an inhibitor of colony-stimulating factor 1 receptor (CSF1R) and can efficiently reduce the number of microglia in the brain of mice [13]. Minocycline is a widely used antibiotic that could suppress neuroinflammation in a variety of rodent models of neurodegenerative diseases [28]. Consistent with previous reports, PLX3397 or minocycline treatment in mice significantly reduced the number of microglia or degree of microglial activation, respectively, in the hippocampus and cortex (Supplementary Fig 2A-F). Subsequently, cognitive performed was measured in mice treated with rotenone with or without PLX3397 and minocycline. In agreement with that of Fig 1A, impaired learning and memory performance of mice was detected in MWM test after rotenone (1.5 mg/kg) treatment. Interestingly, PLX3397 and minocycline significantly ameliorated rotenone-induced learning and memory impairments by showing reduced escape latency, travelled distance and latency for first platform crossing as well as recovered platform crossing number and time percentage in target quadrant in combined PLX3397 or minocycline and rotenone-treated mice compared with rotenone alone group (Fig. 4A, B, D-F). No statistical difference of swimming speed for mice in each group excluded the possibility that PLX3397 and minocycline-afforded protective effects was attributed to recovered motor activity (Fig. 4C).
To further confirm the protection of PLX3397 and minocycline against rotenone-induced cognitive deficits, the novel objective recognition and passive avoidance tests were performed. In agreement with that of MWZ test, PLX3397 and minocycline significantly elevated the recognition index and step-through latency of rotenone-treated mice in the novel objective recognition and passive avoidance tests, respectively (Fig. 4G and H). PLX3397 and minocycline also decreased the error times of step-through in rotenone-treated mice; however, the difference had not reached statistical significance (Fig. 4I). These results suggested that microglial activation contributed to rotenone-induced cognitive deficits in mice.
PLX3397 and minocycline attenuate rotenone-induced neuronal damage and Ser129-phosphorylation of α-synuclein
The neuroprotective effects of PLX3397 and minocycline were subsequently determined. Consistent with attenuated cognitive decline, PLX3397 and minocycline also mitigated rotenone-induced neuronal damaged in mice. Restored Neu-N+ cell number and increased PSD95 staining density were observed in both hippocampal and cortical regions of PLX3397 or minocycline and rotenone co-treated mice compared with rotenone alone group (Fig. 5A-C and Supplementary Fig. 3), indicating neuroprotection. This conclusion was further supported by analysis of expressions of Neu-N and PSD95 (Fig. 5D-F).
In addition to neuroprotection, PLX3397 and minocycline also mitigated the Ser129-phosphorylation of α-synuclein by showing reduced expressions of Ser129-phosphorylated α-synuclein in PLX3397 or minocycline and rotenone co-treated mice compared with rotenone alone group (Fig. 5 G).
PLX3397 and minocycline attenuate rotenone-induced astroglial activation and proinflammatory factors production
Recent study revealed that microglial activation can induce astrocyte into a neurotoxic activation status in multiple neurological disorders [29]. To determine whether microglial depletion by PLX3397 or inactivation by minocycline could suppress astroglial activation, immunohistochemistry by using anti-GFAP antibody, was performed in mice. As seen in Fig 6A, astroglial cells in the hippocampus and cortex of rotenone-treated mice displayed hypertrophied morphology and intensified GFAP immunostaining, indicating astroglial activation. Densitometric analysis of GFAP staining further supported this conclusion (Fig. 6B and C). Interestingly, astrocytes in combined PLX3397 or minocycline and rotenone-treated mice showed normal morphology and a reduced expression of GFAP compared with rotenone alone group, suggesting that activation of astrocyte is blocked by PLX3397 or minocycline (Fig. 6A-C).
Production of proinflammatory factors is a common executor for activated glial cells to damage neurons in central nervous system. To determine whether microglial depletion by PLX3397 or inactivation by minocycline could dampen the production of proinflammatory factors, the gene expressions of iNOS, TNFα and IL-1b were measured. As seen in Fig 6D and E, rotenone injection increased gene transcripts of iNOS, TNFα and IL-1b in both hippocampus and cortex of mice, which were significantly reduced by PLX3397 and minocycline.
PLX3397 and minocycline attenuate rotenone-induced oxidative stress
Oxidative stress usually co-exists with neuroinflammation. To determine whether microglial depletion by PLX3397 or inactivation by minocycline could dampen oxidative stress, the contents of GSH and MDA were measured. In agreement with reduced production of proinflammatory factors, rotenone-induced lipid peroxidation was also mitigated by PLX3397 and minocycline. Fig. 7C and D depicted the decreased MDA levels and recovered GSH contents in PLX3397 or minocycline and rotenone co-treated mice compared with rotenone alone group.
PLX3397 and minocycline attenuate rotenone-induced neuronal apoptosis
The effects of PLX3397 and minocycline on neuronal apoptosis were further determined in rotenone-treated mice. TUNEL staining revealed a high number of TUNEL+ cells in the hippocampal and cortical regions of rotenone-treated mice compared with control (Fig. 8A and B), indicating apoptosis occurs after rotenone exposure. Immunofluorescence staining against MAP2, Iba-1 and GFAP antibodies with TUNEL labeling was further performed to observe the distribution of apoptosis in neuron, microglia and astrocyte, respectively. Results showed that rotenone-induced increase of TUNEL staining in the hippocampus and cortex was mainly concentrated to MAP2+ cells, indicating neuronal apoptosis (Data not shown). Interestingly, PLX3397 and minocycline treatment markedly decreased rotenone-induced neuronal apoptosis by showing reduced number and percentage of TUNEL+ cells in PLX3397 or minocycline and rotenone co-treated mice compared with rotenone alone group (Fig. 8A and B).
It’s well-documented that apoptosis is controlled by multiple proteins, such as Bcl-2 family and caspase [30]. Bcl-XL and Bax, two key members of Bcl-2 family, display anti-apoptotic and pro-apoptotic function, respectively [31]. Caspase-3, a member caspase family, is the main executor of apoptosis [30]. The effects of PLX3397 and minocycline on expressions of these apoptotic regulators were further determined by using Western blot. Consistent with increase of TUNEL staining, rotenone treatment caused increase of active caspase-3 and Bax expressions and decrease of BcL-XL expression in mice (Fig. 8C-E). In contrast, mice treated with combined PLX3397 or minocycline and rotenone displayed reduced expressions of caspase-3 and Bax as well as elevated expression of BcL-XL compared with rotenone alone group (Fig. 8C-E), indicating attenuated apoptosis.