Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.

Psychological stress and traumatic brain injury (TBI) can both result in lasting neurobehavioral abnormalities. Post-traumatic stress disorder and blast induced TBI (bTBI) have become the most significant health issues in current military conflicts. Importantly, military bTBI virtually never occurs without stress. In this experiment, we assessed anxiety and spatial memory of rats at different time points after repeated exposure to stress alone or in combination with a single mild blast. At 2 months after injury or sham we analyzed the serum, prefrontal cortex (PFC), and hippocampus (HC) of all animals by proteomics and immunohistochemistry. Stressed sham animals showed an early increase in anxiety but no memory impairment at any measured time point. They had elevated levels of serum corticosterone (CORT) and hippocampal IL-6 but no other cellular or protein changes. Stressed injured animals had increased anxiety that returned to normal at 2 months and significant spatial memory impairment that lasted up to 2 months. They had elevated serum levels of CORT, CK-BB, NF-H, NSE, GFAP, and VEGF. Moreover, all of the measured protein markers were elevated in the HC and the PFC; rats had an increased number of TUNEL-positive cells in the HC and elevated GFAP and Iba1 immunoreactivity in the HC and the PFC. Our findings suggest that exposure to repeated stress alone causes a transient increase in anxiety and no significant memory impairment or cellular and molecular changes. In contrast, repeated stress and blast results in lasting behavioral, molecular, and cellular abnormalities characterized by memory impairment, neuronal and glial cell loss, inflammation, and gliosis. These findings may have implications in the development of diagnostic and therapeutic measures for conditions caused by stress or a combination of stress and bTBI.

During our search for developmental regulators of neuronal differentiation, we identified special AT-rich sequence-binding protein (SATB)2 that is specifically expressed in the developing rat neocortex and binds to AT-rich DNA elements. Here we investigated whether the regulatory function of SATB2 involves chromatin remodeling at the AT-rich DNA site. In-vitro and in-vivo assays using a DNA affinity pre-incubation specificity test of recognition and chromatin immunoprecipitation showed that SATB2 specifically binds to histone deacetylase 1 and metastasis-associated protein 2, members of the nucleosome-remodeling and histone deacetylase complex. Double immunohistochemistry showed that, in the developing rat neocortex, SATB2 is coexpressed with both proteins. Using a cell culture model, we showed that trichostatin A treatment, which blocks the activities of histone deacetylases, reverses the AT-rich dsDNA-dependent repressor effect of SATB2. These findings suggested that the molecular regulatory function of SATB2 involves modification of the chromatin structure. Semi-quantitative chromatin immunoprecipitation analysis of cortices from SATB2 mutant and wild-type animals indicated that, in the knock-out brains, SATB2 is replaced in the chromatin-remodeling complex by AU-rich element RNA binding protein 1, another AT-rich DNA binding protein also expressed in differentiating cortical neurons. These results suggested that an altered chromatin structure, due to the presence of different AT-rich DNA binding proteins in the chromatin-remodeling complex, may contribute to the developmental abnormalities observed in the SATB2 mutant animals. These findings also raised the interesting possibility that SATB2, along with other AT-rich DNA binding proteins, is involved in mediating epigenetic influences during cortical development.

De novo hippocampal neurogenesis contributes to functional recovery following traumatic brain injury (TBI). Enriched environment (EEN) can improve the outcome of TBI by positively affecting neurogenesis. Blast induced traumatic brain injury (bTBI) characterized by memory impairment and increased anxiety levels, is a leading cause of chronic disability among soldiers. Using a rodent model of bTBI we asked: (a) whether long-term exposure to EEN after injury can ameliorate behavioral abnormalities and (b) what the effects of EEN are at the molecular and cellular levels and on de novo neurogenesis. We found that housing injured animals in EEN resulted in significantly improved spatial memory while animals in normal housing (NH) showed persistent memory impairment. VEGF and Tau protein but not Interleukin-6 (IL-6) levels were normalized in the dorsal hippocampus (DHC) of EEN rats while all three markers remained elevated in NH rats. Interestingly, after peaking at 6 weeks post-injury, anxiety returned to normal levels at 2 months independent of housing conditions. Housing animals in EEN had no significant effect on VEGF and Tau protein levels in the ventral hippocampus (VHC) and the amygdala (AD). We also found that EEN reduced IL-6 and IFNγ levels in the VHC; these markers remained elevated following NH. We observed an increase in GFAP and DCX immunoreactivities in the VHC of NH animals at 2 months post-injury. Conversely, injured animals housed in EEN showed no increase in GFAP or DCX immunoreactivity in their VHC. In summary, long-term exposure of injured animals to EEN appears to play a positive role in the restoration of memory functions but not on anxiety, which returned to normal levels after a significant period of time. Cellular and molecular changes in response to EEN appear to be a part of neurogenesis-independent as well as dependent recovery processes triggered by bTBI.

AT-rich DNA elements play an important role in regulating cell-specific gene expression. One of the AT-rich DNA binding proteins, SATB1 is a novel type of transcription factor that regulates gene expression in the hematopoietic lineage through chromatin modification. Using DNA-affinity purification followed by mass spectrometry we identified and isolated a related protein, SATB2 from the developing rat cerebral cortex. SATB2 shows homology to SATB1 and the rat protein is practically identical to the mouse and human SATB2. Using competitive EMSA, we show that recombinant SATB2 protein binds with high affinity and specificity to AT-rich dsDNA. Using RT-PCR, Western analysis and immunohistochemistry we demonstrate that SATB2 expression is restricted to a subset of postmitotic, differentiating neurons in the rat neocortex at ages E16 and P4. We suggest that similar to its homologue SATB1, SATB2 is also involved in regulating gene expression through altering chromatin structure in differentiating cortical neurons.