Smad3 deficiency increases cortical and hippocampal neuronal loss following traumatic brain injury

Transforming growth factor-β (TGF-β) signaling is involved in pathological processes following brain injury. TGF-β signaling through Smad3 contributes significantly to the immune response and glial scar formation after brain injury. However, TGF-β is also neuroprotective, suggesting that Smad3 signaling may also be involved in neuroprotection after injury. We found expression of the TGF-β type II receptor (TβRII) and Smad3 protein to be strongly and rapidly induced in neurons in the ipsilateral cortex and CA1 region of the hippocampus after stab wound injury. In contrast, astrocytic expression of TβRII and Smad3 was induced more slowly. Comparison of the response of wild-type and Smad3 null mice to cortical stab wound injury showed a more pronounced loss of neuronal viability inSmad3 null mice. Neuronal density was more strongly reduced in Smad3 null mice than in wild-type mice at 1 and 3days post lesion in both the ipsilateral cortex and hippocampal CA1 region. Fluoro-Jade B, TUNEL staining, and cleaved caspase-3 staining also demonstrated increased neuronal degeneration at early time points after injury in the ipsilateral hemisphere in Smad3 null mice. Taken together, our results suggest that TGF-β cytokine family signaling through Smad3 protects neurons in the damaged cortex and hippocampus at early time points after injury

TGF-β Superfamily Gene Expression and Induction of the Runx1 Transcription Factor in Adult Neurogenic Regions after Brain Injury

<div><p>Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-β (TGF-β) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.</p> </div

Adult hippocampal radial glia-like cells and mature neurons express Runx1.

<p>Confocal microscopy images of adult hippocampal mouse sections showing Runx1 (red) colocalized with Nestin+ (green) radial glia-like cells projecting radially from the dentate gyrus subgranular zone (SGZ) after injury (A, inset a′, a′′, DAPI in blue). These Runx1+ cells (red) often expressed GFAP (green) shown at 3 (B, b, b′, b′′, DAPI in blue) and 7 days post-injury (dpi) (C, c, c′, c′′, Nestin in blue), and extended processes through the granule cell layer (GCL) (an example is highlighted by the arrowheads in c′′). Some of these Runx1+/Nestin+ cells in the SGZ incorporated BrdU (D, d, d′, d′′). Nuclear Runx1 was also found to colocalize with Sox2 (green) expression in radial glia-like cells of the SGZ after injury (E, e, e′). At late time points (30 and 60 dpi), Runx1 was rarely expressed in the SGZ, but widely expressed in mature neurons (NeuN - green) of the dentate gyrus hilus (F, inset in f, f′, f′′, f′′′). Scale bars for A, a′′, b′′, c′′, e′, f′′′ = 20 µm; for d′′ and F = 50 µm. Abbreviations: molecular layer (ML).</p

Colocalization of Runx1 with cell-type markers in the neurogenic regions.

<p>The degree of colocalization is graded as;+++(high),++(moderate),+(occasional), +/− (in rare cases), and – (non existing).</p

Cortical brain injury increases mRNA expression of TGF-β and BMP related molecules in the adult neurogenic areas.

<p>(A) Brain diagrams show the approximate area of the lesion penumbra at −1.82 mm from bregma for DG and +0.26 mm for SVZ, (adapted from Paxinos & Franklin, 2007). (B, C) QPCR analysis with TGF-β related PCR arrays indicates changes in RNA expression of specific genes at different times after CCI injury in either the DG (B) or the SVZ (C). Graphs (log₁₀ scale) show genes whose expression was significantly altered by injury (n = 3± s.e.m.). (D, E) Runx1 mRNA expression is significantly increased at 1 and 7 dpi in both regions and is specifically upregulated in the DG (D) and SVZ (E) at 1 dpi when measured by P1–P2 primers, but not by P1 primers (n = 3± s.e.m.). Gene expression is displayed as the relative fold change in mRNA levels as compared to the level in control animals. *p<0.05, ◊ p<0.01, Φ p<0.001. Abbreviations: dentate gyrus (DG), subventricular zone (SVZ), cortex (CX), corpus callosum (CC), hippocampus (HP), lateral ventricle (LV), and days post-injury (dpi).</p

Runx1 expression in the two neurogenic areas of the adult brain.

<p>Runx1 is present at low levels in control animals in the DG (A, inset a) and SVZ (D, inset d). Runx1 expression increases in both regions after injury (DG; B, inset b and SVZ; E, inset e). (C, F) The number of cells expressing Runx1 is significantly increased in the DG at 3, 7, 14, and 30 days after injury and significantly increased in the SVZ at 7 days after injury compared with control animals. The peak of Runx1 expression in both regions was seen at 7 dpi. *p<0.05, **p<0.01 ***p<0.001, n = 5± s.e.m. Scale bars for A, B, D, E = 50 µm; for a, b, d, e = 20 µm. Abbreviations: subventricular zone (SVZ), dentate gyrus (DG), molecular layer (ML), granule cell layer (GCL), subgranular zone (SGZ), lateral ventricle (LV), and days post-injury (dpi).</p

Primary (1°) and secondary (2°) antibodies used.

<p>Primary (1°) and secondary (2°) antibodies used.</p

Runx1 is expressed predominantly in microglia after CCI injury in the neurogenic regions of the adult mouse.

<p>(A, B) The number of Runx1+ cells/mm² identified as Iba1+ microglia (blue bars), Iba1+ microglia incorporating BrdU (green bars), or Iba1− cells (red bars) are shown in the DG and SVZ. No Iba1+ cell proliferation was observed in control animals. Asterisk indicates statistically significant difference as compared to control Runx1+/Iba1+ cell counts and number symbol indicates difference as compared to control Runx1+/Iba1− cell counts. *p<0.05, **p<0.01 ***p<0.001, and #p<0.01. n = 5± s.e.m. (C, D) Confocal micrographs of triple labeling for Runx1 (red), Iba1 (blue), and BrdU (green) positive hypertrophic microglia in the DG (C and insets c, c′, c′′) and intermediate microglia in the SVZ (D and insets d, d′, d′′) at 1 dpi. Arrowheads denote Runx1+ microglia (C, D), and an asterisk (C) denotes a non-microglial, Runx1+ cell. Scale bars for C and D = 50 µm; for c and d = 20 µm. (E, F) Graphs show the mean area of Iba1+ cell body (total pixels) and mean Runx1 immunoreactivity (pixel intensity) at different time points post-injury and in control in the DG (E) and SVZ (F). Each point represents combined data from one animal. Abbreviations: subventricular zone (SVZ), dentate gyrus (DG), granule cell layer (GCL), subgranular zone (SGZ), molecular layer (ML), lateral ventricle (LV), and days post-injury (dpi).</p