Neuroprotective and anti-inflammatory roles of the phosphatase and tensin homolog deleted on chromosome Ten (PTEN) Inhibition in a Mouse Model of Temporal Lobe Epilepsy

Abstract

Excitotoxic damage represents the major mechanism leading to cell death in many human neurodegenerative diseases such as ischemia, trauma and epilepsy. Caused by an excess of glutamate that acts on metabotropic and ionotropic excitatory receptors, excitotoxicity activates several death signaling pathways leading to an extensive neuronal loss and a consequent strong activation of astrogliosis. Currently, the search for a neuroprotective strategy is aimed to identify the level in the signaling pathways to block excitotoxicity avoiding the loss of important physiological functions and side effects. To this aim, PTEN can be considered an ideal candidate: downstream the excitatory receptors activated in excitotoxicity (whose inhibition was shown to be not clinically viable), it is involved in neuronal damage and in the first stage of the reactive astrogliosis in vivo. In this study, we demonstrated the involvement of PTEN in excitotoxicity through its pharmacological inhibition by dipotassium bisperoxo (picolinato) oxovanadate [bpv(pic)] in a model of temporal lobe epilepsy, obtained by intraperitoneal injection of kainate in 2-month-old C57BL/6J male mice. We have demonstrated that inhibition of PTEN by bpv(pic) rescues neuronal death and decreases the reactive astrogliosis in the CA3 area of the hippocampus caused by systemic administration of kainate. Moreover, the neurotoxin administration increases significantly the scanty presence of mitochondrial PTEN that is significantly decreased by the administration of the inhibitor 6 hr after the injection of kainate, suggesting a role of PTEN in mitochondrial apoptosis. Taken together, our results confirm the key role played by PTEN in the excitotoxic damage and the strong anti-inflammatory and neuroprotective potential of its inhibition.

Figure 1. PTEN inhibition protects CA3 neurons from kainate-induced excitotoxicity.

Nissl-staining of hippocampus sections of mice treated with saline (A, control), with kainate (B, KA) or with both kainate and bpv(pic) (C, KA+I), killed one day after treatment (scale bar: 1 mm). A1. CA3 area of control animals (ctr) treated with saline only (scale bar: 50 µm). B1. CA3 area of animals treated with kainate (KA). Arrowheads point to apoptotic bodies and condensed nuclei as a consequence of kainate treatment. At higher magnification, a neuron that is degenerating. C1. CA3 area of animals treated with both kainate and PTEN inhibitor (KA+I). D. Immunostaining showing cleaved caspase-3 (in red) in neurons labeled with MAP-2 (in green) in the CA3 area of the hippocampus, one day after kainate administration. At higher magnification, arrowheads indicating neurons with a clear expression of cleaved caspase-3. Scale bar: 50 µm (30 µm at higher magnification). E. Surviving neurons in the hippocampal CA3 region. Histogram showing the linear density (cells/mm) of surviving neurons in the CA3 area of the hippocampus one day following kainic (KA) acid injection or kainic (KA) acid injection and PTEN inhibition (I) by bpv(pic) (KA+I). **P<0.01, §§P<0.01.

Figure 2. Astrogliosis in the CA3 area of the hippocampus following kainic acid injection.

Glial fibrillary acidic protein (GFAP)-positive profiles in the hippocampi of control (A), kainic acid (KA)-treated, 1 day survival mice (B) and KA/bpv(pic)-treated, 1 day survival mice (C) (scale bar: 50 µm). D. Histogram showing the % of GFAP staining in the hippocampal CA3 area one day after kainic acid injection and PTEN inhibition by bpv(pic). The significant increase in GFAP immunoreactivity after kainate administration, expressed as a percentage of the whole area occupied by positive profiles, is significantly prevented by bpv(pic) treatment. *P<0.05, §<0.05.

Figure 3. Activation of c-Jun after kainate stimulation in the CA3 area of the hippocampus.

The P-c-Jun immunoreactivity, absent in control mice (A), can be observed in neurons of animals treated with kainate (B) or with both kainate and PTEN inhibitor (C), sacrificed one day after the treatment. At higher magnification, nuclei of neurons positive for P-c-Jun after kainate treatment. D. Immunostaining showing that P-c-Jun (red) is revealed in the nucleus of neurons labeled with NeuN (green) in the CA3 area of the hippocampus, one day after the kainate stimulation. Scale bar: 50 µm. E. Histogram showing the % of P-c-Jun staining in hippocampal CA3 subfield one day following KA injection and PTEN inhibition by bpv(pic). As it can be observed, bpv(pic) is unable to prevent the significant increase in the P-c-Jun immunoreactivity caused by kainate treatment. *P<0.05.

Figure 4. PTEN expression in the CA3 area of the hippocampus.

Immunofluorescence showing PTEN expression in the CA3 area 1 day after kainic acid i.p. injection (B) and in control mice treated only by saline (A). Nuclei were stained with nuclear marker DAPI. Scale bar: 50 µm.

Figure 5. PTEN expression in mitochondrial and cytosolic fraction after the excitotoxic stimulus.

Representative Western blot (A) and relative quantification (B) showing mitochondrial PTEN translocation in control (ctr), kainic treated group (KA) and kainic/bpv(pic) group (KA+I) 3, 6 and 12 h after the kainate treatment. As it can be observed, the excitotoxic stimulus leads to a significant increase in mitochondrial PTEN expression, significantly decreased 6 h after kainic acid injection in animals treated with bpv(pic). On the contrary, in the cytosolic fraction kainic acid or PTEN inhibitor does not lead to significant differences in PTEN levels in any time points considered as it can be observed from representative Western blots (C) and relative quantification (D). Data are expressed as mean ± S.E.M., *P<0.05, #P<0.05, **P<0.01 with Two way ANOVA and Bonferroni post hoc test. Experiments were repeated three times.