N-docosahexaenoylethanolamine regulates Hedgehog signaling and promotes growth of cortical axons

Axonogenesis, a process for the establishment of neuron connectivity, is central to brain function. The role of metabolites derived from docosahexaenoic acid (DHA, 22:6n-3) that is specifically enriched in the brain, has not been addressed in axon development. In this study, we tested if synaptamide (N-docosahexaenoylethanolamine), an endogenous metabolite of DHA, affects axon growth in cultured cortical neurons. We found that synaptamide increased the average axon length, inhibited GLI family zinc finger 1 (GLI1) transcription and sonic hedgehog (Shh) target gene expression while inducing cAMP elevation. Similar effects were produced by cyclopamine, a regulator of the Shh pathway. Conversely, Shh antagonized elevation of cAMP and blocked synaptamide-mediated increase in axon length. Activation of Shh pathway by a smoothened (SMO) agonist (SAG) or overexpression of SMO did not inhibit axon growth mediated by synaptamide or cyclopamine. Instead, adenylate cyclase inhibitor SQ22536 abolished synaptamide-mediated axon growth indicating requirement of cAMP elevation for this process. Our findings establish that synaptamide promotes axon growth while Shh antagonizes synaptamide-mediated cAMP elevation and axon growth by a SMO-independent, non-canonical pathway

Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

Apoptosis of post-mitotic neurons plays a significant role in secondary tissue damage following traumatic spinal cord injury (SCI). Activation of E2F1-dependent transcription promotes expression of pro-apoptotic factors, including CDK1; this signal transduction pathway is believed to represent an important mechanism for the physiological or pathological neuronal cell death. However, a specific role for this pathway in neuronal apoptosis induced by SCI has not yet been reported. Here we demonstrate up-regulation of the E2F1/CDK1 pathway that is associated with neuronal apoptosis following impact SCI in rats. Expression of E2F1 and CDK1 were robustly up-regulated as early as 15 min after injury and sustained until 3 days post-injury. CDK1 activity and E2F1 downstream targets bim and c-Myb were significantly increased after SCI. Activation of E2F1/CDK1 signaling also was associated with death of neurons in vitro; this was attenuated by shRNA knockdown or pharmacological inhibition of the E2F1/CDK1 pathway. CR8, a novel and potent CDK1 inhibitor, blocked apoptosis of primary cortical neurons at low-micromolar concentrations. Moreover, SCI-induced up-regulation of E2F1/CDK1 and associated neuronal apoptosis was significantly attenuated by systemic injection of CR8 (1 mg/kg, i.p.) at 5 min after injury. CR8 significantly decreased posttraumatic elevation of biochemical markers of apoptosis, such as products of caspase-3 and α-fodrin cleavage, as well as neuronal cell death, as indicated by TUNEL staining. Importantly, CR8 treatment also increased the number of surviving neurons at 5 weeks after injury. Together, these findings indicate that activation of the E2F1/CDK1 pathway contributes to the pathophysiology of SCI and that selective inhibition of this signaling cascade may represent an attractive therapeutic strategy

Pharmacological inhibition of CDK1 blocks neuronal apoptosis.

<p>Cortical neurons were pre-treated with Roscovitine or CR8 (CDK1 inhibitors) or vehicle and then exposed to TD-or campthotecin induced apoptosis. <b>A</b>. Representative photomicrographs of control and trophic deprived neurons treated with the indicated concentrations Roscovitine and CR8 are shown. Upper row presents phase contrast images (Healthy neurons are indicated by larger cell bodies and abundant processes; Apoptotic neurons display shrunken cell bodies and sparse or lost processes). Lower row shows chromatin staining with Hoechst 33258. Arrows and arrowheads indicate surviving and apoptotic neurons, respectively suggesting an attenuation of TD-induced neuronal death in neurons pre-treated with Roscovinine or CR8. <b>B</b>. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons pre-treated with Roscovitine (10 µM; ^*p<0.05, vs. TD vehicle) whereas CR8 at concentrations as low as 1 µM (^***p<0.001, vs. TD vehicle) almost completely blocked development of apoptotic features in neuronal nuclei. <b>C</b>. Significant attenuation of campthotecin-induced apoptosis in neurons pre-treated with Roscovitine (50 µM; ^*p<0.001, vs. vehicle) and CR8 at concentrations as low as 1 µM (^***p<0.001, vs. vehicle).</p

The temporal profile and cell specificity of E2F1 and CDK1 expression after SCI.

<p><b>A–B</b>. Coronal section in intact spinal cord (A) showed that E2F1 is relatively weak and detected mainly in neurons of the gray matter. At 24 h after SCI, E2F1 immunoreactivity was upregulated not only in gray matter but also in lesion area (B). C. E2F1⁺ cells were also co-labelled with NeuN in the dorsal horn of the gray matter at 1 day after SCI. D–E. Only a small subset of E2F1⁺ cells in the lesion area were positive for OX42 at 24 h (D) and 7 d (E) after SCI. F–G. In the intact spinal cord (F), CDK1 immunoreactivity is relatively weak and detected mainly in motor neurons in the ventral horn and CC1⁺ oligodendrocytes. At 24 h after SCI (G), CDK1 immunoreactivity was upregulated not only in the ventral horn but also in the spared white matter, colocalized with CC1⁺ oligodendrocytes. CDK1⁺ cells also appeared in the lesion area. <b>H–I</b>. CDK1 was expressed by CC1⁺ oligodendrocytes in the white matter in the intact spinal cord (H) and at 1 day after SCI. <b>J</b>. Only a small subset of CDK1⁺ cells in the lesion area were positive for OX42 at 24 h after SCI. <b>K</b>. Coronal section in intact spinal cord (a) shows that E2F1 was expressed in the motor neurons in the ventral horn. Immunoreactivity of E2F1 (b–d) was increased at 5 h, and 1–3 days post injury, and highly expressed by motor neurons. L. In intact spinal cord (a), CDK1/NeuN was detected in the motor neurons in the ventral horn. At 5 h after injury, immunoreactivity of CDK1 (b) was increased and sustained until 3 days post injury (c–d), and highly expressed by motor neurons. All images were taken at 2 mm rostral to epicenter. Scale bar = 500 µm for A–B, F–G. Scale bar = 100 µm for C–E, H–J, K–L.</p

CDK1 activity is increased after spinal cord injury.

<p>Western blotting analysis of common CDK substrates, CDK1 co-activator cyclin B1 and phosphorylations of specific CDK1 substrate (Ser54)-n-myc was performed in homogenates obtained from intact and injured spinal cord. <b>A</b>. Cyclin B1 expression was upregulated at all time points tested. Phosphorylation (Ser54) of n-myc and phospho-CDK substrate motif signal levels were increased from 5 h to day 7. <b>B–D</b>. Quantification of respective western blots in panel A. n = 4 rats/time point. *P<0.05 vs sham group.</p

E2F1 gene silencing down-regulates endogenous CDK1 expression <i>in vitro</i>.

<p><b>A</b>. 27 mer siRNA duplexes for human E2F1 or trilencer-27 universal scrambled negative control siRNA duplex was transfected in the human neuroblastoma SH-SY5Y cells. Two days after transfection, the cells were harvested and subjected to western blotting using mouse monoclonal antibodies to E2F1 and CDK1. Transfection with shRNA against E2F1 resulted in reduction of E2F1 expression (58% to 66% of control), accompanied by 50% of reduction of CDK1 expression. <b>B</b>. Primary rat cerebral cortical neurons were transfected with shRNA against rat E2F1. E2F1 protein expression was robust reduced to 47% or 59% for shRNAs 1 and 2 respectively, and E2F1 knockdown resulted in reduction of CDK1 expression from 47% to 64% for shRNAs 1 and 2 respectively. N = 4 dishes from 3 independent culture. *P<0.05 vs sham group.</p