TBI induced acute upregulation of mtCB1 receptors
At first, we investigated the expression of mtCB1 receptors at acute stage on ipsilateral cortex and cultured neuron post lesion. WB analysis revealed TBI caused great upregulation of CB1 receptors on mitochondria (2.5 times higher than sham in cortex of wild type mice and 3.5 times higher than sham in cultured neurons) 24 h post injury (Fig. 1a and b). Only relatively small CB1 increase was observed on plasma membranes (1.16 times higher than sham in cortex of wild type mice and 1.25 times higher than sham in cultured neurons) 24 h post injury (Fig. 1c, d). CB1 expression could hardly be detected in CB1 knockout mice (Fig. 1a, c).
Activation of mtCB1 aggravated metabolic defects following TBI
Next, we investigated the effects of CB1 on aerobic metabolism after TBI in vivo. The cell-permeable CB1 agonist tetrahydrocannabinol (THC, 5 mg/kg) and antagonist/inverse agonist AM251 (0.5 mg/kg) were used to control the activity of intracellular CB1 receptors. Microdialysis in wild type brains from 22 to 27 h post injury showed lower glucose and pyruvate levels, and increased lactate level as well as lactate/pyruvate ratio (LPR) (Fig. 2A1–A4). Metabolic defects were also demonstrated by the changed levels of metabolites in injured CB−/− brains (Fig. 2B1–B4). It was noticeable that CB1 knockout promoted metabolic defects after TBI as demonstrated by microdialysis of metabolite levels in CB−/− and wild type mice (p < 0.05).
In wild type mice, intraperitoneal administration of THC (5 mg/kg) resulted in decreased glucose and pyruvate levels, and increased lactate level as well as LPR suggesting an aggravated metabolic condition while AM251 showed opposite effects (Fig. 2A1–A4). Metabolite analysis was also made in CB1−/− mice treated with THC (5 mg/kg) or AM251 (0.5 mg/kg) after injury and results showed metabolite levels were not significantly differ from those of vehicle (Fig. 2B1–B4) indicating the CB1 receptors were the specific targets of THC and AM251. Single inhibition of plasma membrane CB1 receptors by cell-impermeable CB1 antagonist/inverse agonist hemopressin (0.5 mg/kg) did not show any effects on aerobic metabolism after TBI suggesting the effects were due to the intracellular CB1 receptors (Fig. 2C1–C4).
To prove the effects were due to neuronal mtCB1 receptors but not CB1 receptors on other cells, we further tested the metabolites in neuronal models treated with cell-permeable CB1 ligands. In vivo after injury, as a result of the mitochondrial impairment, pyruvate no longer enters the tricarboxylic acid cycle and the need for lactate decrease. On the other hand, pyruvate and glycogen are heavily metabolized into lactate in astrocytes. Therefore, lactate accumulates and pyruvate decreases in the extracellular space, leading to the elevation of LPR [8]. However, in neuronal model of injury without astrocytes, the neuronal need for pyruvate reduces and the transformation of pyruvate into lactate greatly decreases due to the lack of astrocytes. Therefore, pyruvate and lactate both accumulate in the extracellular space [9].
Neuronal traumatic injury induced metabolic defects demonstrated by higher extracellular pyruvate and lactate levels, and reduced cellular oxygen consumption and mitochondrial ATP concentration as compared to sham group (Fig. 2D1–D4). In cultured neurons after injury, the cell-permeable CB1 agonist HU210 (0.5 μM) resulted in increased extracellular pyruvate (1.23 folds higher than vehicle) and lactate (1.22 folds higher than vehicle) levels, and reduced cellular oxygen consumption (83.3 % of vehicle) and mitochondrial ATP concentration (95.3 % of vehicle) (Fig. 2D1–D4). The AM251 (5 μM) decreased extracellular pyruvate (82.6 % of vehicle) and lactate (84.1 % of vehicle) levels, and increased cellular oxygen consumption (1.36 folds higher than vehicle) and mitochondrial ATP concentration (1.06 folds higher than vehicle) (Fig. 2D1–D4). HU210 (0.5 μM) and AM251 (5 μM) were also administrated to primary neuronal cultures prepared from CB1−/− mouse (CB1−/− neurons). HU210 or AM251 did not change brain metabolism in injured CB1−/− neurons (Fig. 2E1–E4).
Then we selectively controlled the plasma CB1 receptors by hemopressin (5 μM) or cell-impermeable CB1 agonist HU210-biotin (0.5 μM) in cultured neurons. No changes were found in extracellular metabolite levels, oxygen consumption or mitochondrial ATP concentration after treatment (Fig. 2F1–F4).
To determine the effects were due to the direct modulation of mtCB1 or to an indirect function of CB1 receptors located on other intracellular organelles such as lysosomes [10], mitochondria were purified from injured neurons, wild type and CB1−/− mice after injury then treated with HU210 (0.5 μM) or AM251 (5 μM). The drugs still apparently controlled pyruvate and lactate levels, oxygen consumption and ATP concentration in mitochondria separated from cultured neurons or wild-type mice after injury, but not in mitochondria separated from CB1−/− mice after injury (Fig. 2G1–G4).
MtCB1 activation protected neurons from apoptosis following injury
In wild type model of TBI treated with vehicle, about 8.9 % cells in vivo and 20.1 % in vitro showed apoptosis 24 h post injury (Fig. 3a, b). TUNEL study 24 h post injury showed THC (5 mg/kg in vivo and 10 μM in vitro) or HU-210 (0.1 mg/kg in vivo and 0.5 μM in vitro) significantly mitigated cellular apoptosis (4.1 % in vivo and 9.7 % in vitro in THC, 6.7 % in vivo and 13.7 % in vitro in HU-210), while the AM251 (0.5 mg/kg in vivo and 5 μM in vitro) significantly promoted apoptosis (11.6 % in vivo and 24.8 % in vitro) (Fig. 3a, b). In CB1−/− models, the same dose of THC, HU-210 or AM251 did not affect cellular apoptosis both in vivo and vitro compared with the vehicle indicating the effects were due to CB1 receptors (Fig. 3c, d). It was noticeable that CB1 knockout promoted neuronal apoptosis both in vivo and vitro as demonstrated by the number of TUNEL positive cells in CB1−/− and wild type treated with vehicle (p < 0.05) (Fig. 3a, b, c and d). In vitro of wild type hemopressin or HU210-biotin did not show any effects suggesting the neuronal plasma membrane CB1 receptors were not involved, however in vivo of wild type hemopressin promoted apoptosis and HU210-biotin inhibited apoptosis (Fig. 3e, f).
The mitochondrion serve as a crucial role in regulating neuronal apoptosis in TBI, both as an amplifier of extrinsic pro-apoptotic signal and the source of the activation of intrinsic apoptotic pathway [4]. The mitochondrial apoptotic pathway is classified as caspase dependent or caspase independent. In a caspase dependent pathway, cyt c is the necessary participant, whereas apoptosis inducing factor (AIF) is involved in the caspase independent pathway. Under pro-apoptotic stimulation such as traumatic injury, the release of cyt c from mitochondria into the cytosol triggers apoptosome assembly and the subsequent caspase activation and apoptosis, while the AIF is released and translocated into the nucleus to promote caspase independent DNA degradation [11].
To determine the effects were due to the direct modulation of mtCB1 or to an indirect function of CB1 receptors located on other intracellular organelles which could also be modulated by cell-permeable ligands, mitochondria were purified from injured neurons, wild type and CB1−/− mice 24 h after injury and treated with HU210 (0.5 μM) or AM251 (5 μM). Cyt c and AIF release were measured in supernatants following pelleting of the mitochondrial suspensions. Immunoblots of supernatants revealed HU-210 significantly inhibited cyt c (36.5 % of vehicle in mitochondria of neurons and 73.2 % of vehicle in mitochondria of wild types) and AIF (41.0 % of vehicle in mitochondria of neurons and 76.0 % of vehicle in mitochondria of wild types) release, whereas AM251 promoted cyt c (2.84 folds higher than vehicle in mitochondria of neurons and 1.79 folds higher than vehicle in mitochondria of wild types) and AIF (2.49 folds higher than vehicle in mitochondria of neurons and 1.59 folds higher than vehicle in mitochondria of wild types) release (Fig. 3g, h).
Mitochondrial cAMP/PKA/complex I inhibition promoted metabolic defects and neuronal apoptosis
As mitochondrial cAMP/PKA/complex I pathway has been found to be the downstream target of mtCB1 and is involved in mtCB1 mediated aerobic metabolism of normal neurons [7]. We tested the possible effects of mitochondrial cAMP/PKA/complex I pathway on metabolic defects and neuronal apoptosis after TBI. Mitochondria were purified from injured neurons and wild type mice then treated with HU-210 (0.5 μM), AM251 (5 μM), H89 (a well-known pharmacological inhibitor of PKA, 1.0 mM), rotenone (specific and potent mitochondrial complex I inhibitor, 2.5 μM) or forskolin (a selective activator of adenylate cyclase, 1.5 mM). The results showed HU-210 significantly decreased mitochondrial cAMP concentration (59.7 % of vehicle in mitochondria of neurons and 68.0 % of vehicle in mitochondria of wild types), PKA activity (70.8 % of vehicle in mitochondria of neurons and 79.2 % of vehicle in mitochondria of wild types) and complex I activity (77.8 % of vehicle in mitochondria of neurons and 86.2 % of vehicle in mitochondria of wild types), while AM251 showed opposite effects on them (1.47 folds higher than vehicle in mitochondria of neurons and 1.38 folds higher than vehicle in mitochondria of wild types to cAMP, 1.37 folds higher than vehicle in mitochondria of neurons and 1.29 folds higher than vehicle in mitochondria of wild types to PKA, 1.26 flods higher than vehicle in mitochondria of neurons and 1.17 folds higher than vehicle in mitochondria of wild types to complex I) (Fig. 4A1, B1).
The inhibition of mitochondrial cAMP/PKA/complex I activity by H89 significantly increased extramitochondrial pyruvate (1.15 folds higher than vehicle in mitochondria of neurons and 1.13 folds higher than vehicle in mitochondria of wild types) and lactate levels (1.13 folds higher than vehicle in mitochondria of neurons and 1.11 folds higher than vehicle in mitochondria of wild types), and decreased oxygen consumption (88.5 % of vehicle in mitochondria of neurons and 90.5 % of vehicle in mitochondria of wild types) accompanied with ATP insufficiency (90.8 % of vehicle in mitochondria of neurons and 93.0 % of vehicle in mitochondria of wild types) (Fig. 4A3, B3). The ratio of ATP decrease to oxygen consumption reduction (percentage of ATP reduction/percentage of oxygen consumption reduction = 12.7 %/13.8 % in mitochondria of cultured neurons, 7.0 %/9.5 % in mitochondria of wild type mice) is higher than that of mtCB1 activation (percentage of ATP reduction/percentage of oxygen consumption reduction = 4.7 %/16.7 % in cultured neurons, 7.2 %/27.2 % in mitochondria of cultured neurons, 5.3 %/16.2 % in mitochondria of wild type mice) (p < 0.05). Parallel supernatant cyt c and AIF analysis demonstrated a significant promotion of mitochondrial cyt c (1.71 folds higher than vehicle in mitochondria of neurons and 1.68 folds higher than vehicle in mitochondria of wild types) and AIF (1.64 folds higher than vehicle in mitochondria of neurons and 1.61 folds higher than vehicle in mitochondria of wild types) release was induced by H89 (1.0 mM) treatment (Fig. 4A2, A3, B2 and B3). Similar results (1.19 folds higher than vehicle to pyruvate, 1.17 folds higher than vehicle to lactate, 86.2 % of vehicle to oxygen consumption, 87.3 % of vehicle to ATP production, 1.60 folds higher than vehicle to cyt c and 1.44 folds higher than vehicle to AIF in mitochondria of neurons/1.17 folds higher than vehicle to pyruvate, 1.15 folds higher than vehicle to lactate, 88.2 % of vehicle to oxygen consumption, 89.3 % of vehicle to ATP production, 1.58 folds higher than vehicle to cyt c and 1.40 folds higher than vehicle to AIF in mitochondria of wild types) were also seen in rotenone treatment groups (Fig. 4A2, A3, B2 and B3). The forskolin treatment showed opposite effects in mitochondria separated from cultured neurons and wild type mice (Fig. 4 A4, B4). Same dose of HU-210 and AM251 were also administrated to mitochondria separated from CB1−/− mice. Results showed no changes were found in mitochondrial cAMP/PKA/complex activity suggesting CB1 receptors were the specific targets (Fig. 4c).
Protein kinase B (AKT) accumulated in neuronal mitochondria after TBI and mtCB1 activation upregulated mitochondrial AKT/complex V activity
The direct downregulation of mitochondrial cAMP/PKA/complex I resulted in a lower efficiency of ATP synthesis compared with that of mtCB1 activation as demonstrated by higher ratio of ATP decrease to oxygen consumption reduction. In addition, direct mitochondrial PKA/complex I inhibition increased cyt c and AIF release which incurred neuronal apoptosis. This was opposite to the anti-apoptosis effects of direct mtCB1 activation as demonstrated in our experiment. Thus we deduced that there might have another anti-apoptosis pathway involved in the mtCB1 activation.
Protein kinase B (AKT) which is another well-known downstream target of CB1 was studied. At first, we tested whether there was mitochondrial accumulation of AKT under the stimulation of TBI. Western blots were performed on mitochondria separated from cultured neurons, wild type and CB−/− mice. Western blots analysis showed approximately 5.3–6.3 folds increase to AKT protein expression in mitochondria separated from cultured neurons (5.33 folds higher than sham), wild type (6.26 folds higher than sham) and CB1−/− mice (6.07 folds higher than sham) 24 h after injury (Fig. 5A1, A2). Although the AKT value in mitochondria from wild type TBI model was higher than that of CB1−/− mice, the difference was statistically nonsignificant (p > 0.05) indicating the CB1 knockout did not influence mitochondrial AKT accumulation following TBI. Then, phospho-Ser473-AKT and phospho-Thr308-AKT, two main kind of phosphorylated AKT, were analyzed and results showed only a minor increase of phosphorylated AKT (1.32, 1.29 and 1.32 folds higher than sham in mitochondria of wild types, CB−/− and neurons respectively to pSer473-AKT/1.20, 1.19 and 1.29 folds higher than sham in mitochondria of wild types, CB−/− and neurons respectively to pThr308-AKT) was found on mitochondria suggesting the TBI-induced AKT accumulation on mitochondria might largely be unphosphorylated (Fig. 5A1).
Then we tested the effects of mtCB1 activation on mitochondrial AKT and complex V activity. The results showed HU-210 (0.5 μM) significantly increased mitochondrial AKT (1.76 folds higher than vehicle in mitochondria of neurons and 1.71 folds higher than vehicle in mitochondria of wild type) and complex V (1.63 folds higher than vehicle in mitochondria of neurons and 1.59 higher than vehicle in mitochondria of wild types) activities, while AM251 (5 μM) showed opposite effects (56.3 % of vehicle in mitochondria of neurons and 60.8 % of vehicle in mitochondria of wild type to AKT/65.0 % of vehicle in mitochondria of neurons and 67.2 % of vehicle in mitochondria of wild types to complex V) on them (Fig. 5A3, A4). Same dose of HU-210 and AM251 did not show any effects on mitochondria from CB1−/− mice indicating the effects were due to CB1 reporters (Fig. 5b).
Mitochondrial AKT activation alleviated neuronal metabolic defects and apoptosis
Then we investigated if the activation of mitochondrial AKT/complex V was directly involved in the protection effects on metabolic defects and apoptosis after TBI. Mitochondria were purified from injured neurons or wild type mice then treated with sodium valproate (VPA, 1.0 mM) which could increase the activation dependent phosphorylation of Ser-473 of AKT through inhibitory effect on histone deacetylase [12].
The results showed VPA resulted in decreased pyruvate (89.5 % of vehicle in mitochondria of neurons and 91.2 % of vehicle in mitochondria of wild types) and lactate (90.3 % of vehicle in mitochondria of neurons and 91.5 % of vehicle in mitochondria of wild types), and increased oxygen consumption (1.10 folds higher than vehicle in mitochondria of neurons and 1.09 folds higher than vehicle in mitochondria of wild types) accompanied with raised ATP supply (1.33 folds higher than vehicle in mitochondria of neurons and 1.21 folds higher than vehicle in mitochondria of wild types) (Fig. 6A1, B1). The ratio of ATP rise to oxygen consumption increase (percentage of ATP increase/percentage of oxygen consumption increase = 133 %/110 % in mitochondria from neuron, 121 %/109 % in mitochondria from wild type mice) was higher than that of mtCB1 activation. Parallel supernatants cyt c and AIF analysis demonstrated VPA significantly inhibited the mitochondrial cyt c (31.2 % of vehicle in mitochondria of neurons and 34.0 % of vehicle in mitochondria of wild types) and AIF (49.3 % of vehicle in mitochondria of neurons and 45.2 % of vehicle in mitochondria of wild types) release (Fig. 6A2, A3, B2 and B3). Preincubation 30 min with AKT specific inhibitor API-2 (1 mM) significantly counteracted the VPA (1 mM) induced cyt c (83.0 % of vehicle in mitochondria of neurons and 64.8 % of vehicle in mitochondria of wild types) and AIF release (66.8 % of vehicle in mitochondria of neurons and 69.0 % of vehicle in mitochondria of wild types) (Fig. 6A2, A3, B2 and B3). Examination of the concentration dependent effects revealed that greater responses were induced by 2.0 and 4.0 mM VPA (Fig. 6A2, A3, B2 and B3).