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The goal of this project was to determine whether biochemical markers of brain damage can be used to diagnose and assess the severity of injury in a rat model of penetrating ballistic-like brain injury (PBBI). To determine the relationship between injury magnitude and biomarker levels, rats underwent three discrete PBBI severity levels defined by the magnitude of the ballistic component of the injury, calibrated to equal 5%, 10%, or 12.5% of total rat brain volume. Cortex, cerebrospinal fluid (CSF), and blood were collected at multiple time points. Levels of three biomarkers (αII-spectrin breakdown product [SBDP150], glial fibrillary acidic protein [GFAP], and ubiquitin C-terminal hydrolase-L1 [UCH-L1]), were measured using quantitative immunoblotting and/or enzyme-linked immunosorbent assays. In injured cortex, SBDP150 and GFAP levels were increased significantly over controls. Cortical SBDP150 was elevated at 1 day but not 7 days, and GFAP at 7 days but not 1 day. At their respective time points, mean levels of SBDP150 and GFAP biomarkers in the cortex rose stepwise as injury magnitude increased. In the CSF, increasing severity of PBBI was associated with increasing concentrations of both neuronal and glial biomarkers acutely at 1 day after injury, but no trends were observed at 7 days. In plasma, SBDP150 was elevated at 5 min after 10% PBBI and at 6 h after 12.5% PBBI. UCH-L1 levels in plasma were elevated acutely at 5 min post-injury reflecting injury severity and rapidly decreased within 2 h. Overall, our results support the conclusion that biomarkers are effective indicators of brain damage after PBBI and may also aid in the assessment of injury magnitude.

The susceptibility of the normal cellular prion protein isoform, cellular prion protein (PrP(C)), to proteolytic digestion has been well documented. In addition, a link between PrP(C) and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP) was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrP(C)-expressing mice were treated with calpain, no appreciable loss of the intact PrP(C), nor the appearance of PrP(C) breakdown products (BDPs) were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC) were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX) and N-methyl-d-aspartate (NMDA), PrP(C)-BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrP(C) is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrP(C). Similarly, the lysosomal protease inhibitor, NH(4)Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrP(C) or its release from the RtCNC. In conclusion, rat neuronal PrP(C) is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrP(C) into the cell culture media suggests an unidentified neuroprotective mechanism for PrP(C).

Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts.

Vertebrate genomes harbor two Atrophin genes, Atrophin-1 (Atn1) and Atrophin-2 (Atn2). The Atn1 locus produces a single polypeptide, whereas two different protein products are expressed from the Atn2 (also known as Rere) locus. A long, or full-length, form contains an amino-terminal MTA-2-homologous domain followed by an Atrophin-1-related domain. A short form, expressed via an internal promoter, consists solely of the Atrophin domain. Atrophin-1 can be co-immunoprecipitated along with Atrophin-2, suggesting that the Atrophins ordinarily function together. Mutations that disrupt the expression of the long form of Atrophin-2 disrupt early embryonic development. To determine the requirement for Atrophin-1 during development we generated a null allele. Somewhat surprisingly we found that Atrophin-1 function is dispensable. To gain a better understanding of the requirement for Atrophin function during development, an analysis of the functional domains of the three different gene products was carried out. Taken together, these data suggest that Atrophins function as bifunctional transcriptional regulators. The long form of Atrophin-2 has a transcriptional repression activity that is not found in the other Atrophin polypeptides and that is required for normal embryogenesis. Atrophin-1 and the short form of Atrophin-2, on the other hand, can act as potent and evolutionarily conserved transcriptional activators.

Atrophins are evolutionarily conserved proteins that are thought to act as transcriptional co-repressors. Mammalian genomes contain two atrophin genes. Dominant polyglutamine-expanded alleles of atrophin 1 have been identified as the cause of dentatorubralpallidoluysian atrophy, an adult-onset human neurodegenerative disease with similarity to Huntington's. In a screen for recessive mutations that disrupt patterning of the early mouse embryo, we identified a line named openmind carrying a mutation in atrophin 2. openmind homozygous embryos exhibit a variety of patterning defects that first appear at E8.0. Defects include a specific failure in ventralization of the anterior neural plate, loss of heart looping and irregular partitioning of somites. In mutant embryos, Shh expression fails to initiate along the anterior midline at E8.0, and Fgf8 is delocalized from the anterior neural ridge at E8.5,revealing a crucial role for atrophin 2 in the formation and function of these two signaling centers. Atrophin 2 is also required for normal organization of the apical ectodermal ridge, a signaling center that directs limb pattern. Elevated expression of atrophin 2 in neurons suggests it may interact with atrophin 1 in neuronal development or function. We further show that atrophin 2 associates with histone deacetylase 1 in mouse embryos, providing a biochemical link between Atr2 and a chromatin-modifying enzyme. Based on our results, and on those of others, we propose that atrophin proteins act as transcriptional co-repressors during embryonic development.

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38-50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0-1 days) to late (7-10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.