Bioluminescence imaging of Smad signaling in living mice shows correlation with excitotoxic neurodegeneration

Abstract

The TGF-␤ signaling pathway is a key organizer of injury and immune responses, and recent studies suggest it fulfills critical roles in CNS function and maintenance. TGF-␤ receptor activation results in phosphorylation of Smad proteins, which subsequently translocate to the nucleus to regulate gene transcription by binding to Smad binding elements (SBE). Using SBE-luciferase reporter mice, we recently discovered that the brain has the highest Smad baseline activity of any major organ in the mouse, and we now demonstrate that this signal is primarily localized to pyramidal neurons of the hippocampus. In vivo excitatory stimulation with kainic acid (KA) resulted in an increase in luciferase activity and phosphorylated Smad2 (Smad2P), and nuclear translocation of Smad2P in hippocampal CA3 neurons correlated significantly with luciferase activity. Although this activation was most prominent at 24 h after KA administration in neurons, Smad2P immunoreactivity gradually increased in astrocytes and microglial cells at 3 and 5 days, consistent with reactive gliosis. Bioluminescence measured over the skull in living mice peaked at 12-72 h and correlated with the extent of microglial activation and pathological markers of neurodegeneration 5 days after injury. Treatment with the glutamate receptor antagonist MK-801 strongly reduced bioluminescence and pathology. These results show that Smad2 signaling is a sensitive marker of neuronal activation and CNS injury that can be used to monitor KA-induced neuronal degeneration. This and related mouse models may provide valuable tools to study mechanisms and treatments for neurodegeneration.

Smad2 Protein Disruption in the Central Nervous System Leads to Aberrant Cerebellar Development and Early Postnatal Ataxia in Mice

“…As shown in Fig. 3, Smad2 is preferentially expressed in neuronal cells such as PCs and GCs in the cerebellum, which is consistent with previous studies showing neuron-specific expression of Smad2 (8,9,10). Together, apoptotic cells induced in Smad2-CNS-KO mice are likely to be neuronal cells.…”

Differential TGF-β Signaling in Glial Subsets Underlies IL-6–Mediated Epileptogenesis in Mice

“…Furthermore, TGF-b1 pathway blockers efficiently blocked epileptogenesis in BBB breakdown and albumin models of epilepsy in mice and rats (11,16). Although TGF-b1 is a well-characterized immunoregulatory cytokine (19,20,25,(31)(32)(33)(34), it can also exert autoimmunity, glial activation, and brain inflammation (22,23,35,36). In the present study, we demonstrate that glial exposure to TGF-b1 induces a rapid and robust IL-6 upregulation at both the mRNA and protein levels.…”

A Unique TGFB1-Driven Genomic Program Links Astrocytosis, Low-Grade Inflammation and Partial Demyelination in Periplaques of Spinal Cords from Progressive Multiple Sclerosis Patients

“…Regarding astrocytes, genes that were found to co-upregulate with GJA1/Cx43 in periplaques formed a pro-gliotic signature which composition is compatible with a combined TGFB/SAMD1/SMAD2-and SOX2-driven program. While TGFB and SOX2 were both previously identified as astrocytosis-promoting factors [22][23][24][25], TGFB was further shown to induce an astrocyte-specific developmental program that, in turn, prevents effective remyelination in MS plaques and periplaques [53]. Superimposing to the TGFB/SMAD1/SMAD2 and SOX2 progliotic signatures, we also found that the GJA1/Cx43 module was characterized by a highly significant enrichment in genes that are regulated by the AR.…”

“…Interestingly, the TFs that co-expressed with Cx43/GJA1 comprised SMAD1 and SMAD2, two major signaling components of the TGF-beta and Bone Morphogenetic Proteins (BMPs) pro-gliotic pathways [22][23][24][25]. Moreover, TFs that co-expressed with Cx43/GJA1 also comprised SOX2 a stem cell-related gene expressed by proliferating astrocytes [26,27] and NFAI, a positive regulator of GFAP transcription [28].…”