Serum SNTF, a Surrogate Marker of Axonal Injury, Is Prognostic for Lasting Brain Dysfunction in Mild TBI Treated in the Emergency Department.

Mild traumatic brain injury (mTBI) causes persisting post-concussion syndrome for many patients without abnormalities on conventional neuroimaging. Currently, there is no method for identifying at-risk cases at an early stage for directing concussion management and treatment. SNTF is a calpain-derived N-terminal proteolytic fragment of spectrin (αII-spectrin1-1176) generated in damaged axons following mTBI. Preliminary human studies suggest that elevated blood SNTF on the day of mTBI correlates with white matter disruption and lasting brain dysfunction. Here, we further evaluated serum SNTF as a prognostic marker for persistent brain dysfunction in uncomplicated mTBI patients treated in a Level I trauma center emergency department. Compared with healthy controls (n = 40), serum SNTF increased by 92% within 24 h of mTBI (n = 95; p < 0.0001), and as a diagnostic marker exhibited 100% specificity and 37% sensitivity (AUC = 0.87). To determine whether the subset of mTBI cases positive for SNTF preferentially developed lasting brain dysfunction, serum levels on the day of mTBI were compared with multiple measures of brain performance at 90 days post-injury. Elevated serum SNTF correlated significantly with persistent impairments in cognition and sensory-motor integration, and predicted worse performance in each test on a case by case basis (AUC = 0.68 and 0.76, respectively). SNTF also predicted poorer recovery of cognitive stress function from 30 to 90 days (AUC = 0.79-0.90). These results suggest that serum SNTF, a surrogate marker for axonal injury after mTBI, may have potential for the rapid prognosis of lasting post-concussion syndrome and impaired functional recovery following CT-negative mTBI. They provide further evidence linking axonal injury to persisting brain dysfunction after uncomplicated mTBI. A SNTF blood test, either alone or combined with other markers of axonal injury, may have important utilities for research, prognosis, management and treatment of concussion.

Reframing the Biological Basis of Neuroprotection Using Functional Genomics: Differentially Weighted, Time-Dependent Multifactor Pathogenesis of Human Ischemic Brain Damage.

Background: Neuroprotection studies are generally unable to demonstrate efficacy in humans. Our specific hypothesis is that multiple pathophysiologic pathways, of variable importance, contribute to ischemic brain damage. As a corollary to this, we discuss the broad hypothesis that a multifaceted approach will improve the probability of efficacious neuroprotection. But to properly test this hypothesis the nature and importance of the multiple contributing pathways needs elucidation. Our aim is to demonstrate, using functional genomics, in human cardiac surgery procedures associated with cerebral ischemia, that the pathogenesis of perioperative human ischemic brain damage involves the function of multiple variably weighted proteins involving several pathways. We then use these data and literature to develop a proposal for rational design of human neuroprotection protocols. Methods: Ninety-four patients undergoing deep hypothermic circulatory arrest (DHCA) and/or aortic valve replacement surgery had brain damage biomarkers, S100ß and neurofilament H (NFH), assessed at baseline, 1 and 24 h post-cardiopulmonary bypass (CPB) with analysis for association with 92 single nucleotide polymorphisms (SNPs) (selected by co-author WAK) related to important proteins involved in pathogenesis of cerebral ischemia. Results: At the nominal significance level of 0.05, changes in S100ß and in NFH at 1 and 24 h post-CPB were associated with multiple SNPs involving several prospectively determined pathophysiologic pathways, but were not individually significant after multiple comparison adjustments. Variable weights for the several evaluated SNPs are apparent on regression analysis and, notably, are dissimilar related to the two biomarkers and over time post CPB. Based on our step-wise regression model, at 1 h post-CPB, SOD2, SUMO4, and GP6 are related to relative change of NFH while TNF, CAPN10, NPPB, and SERPINE1 are related to the relative change of S100B. At 24 h post-CPB, ADRA2A, SELE, and BAX are related to the relative change of NFH while SLC4A7, HSPA1B, and FGA are related to S100B. Conclusions: In support of the proposed hypothesis, association SNP data suggest function of specific disparate proteins, as reflected by genetic variation, may be more important than others with variation at different post-insult times after human brain ischemia. Such information may support rational design of post-insult time-sensitive multifaceted neuroprotective therapies.

SNTF immunostaining reveals previously undetected axonal pathology in traumatic brain injury.

Diffuse axonal injury (DAI) is a common feature of severe traumatic brain injury (TBI) and may also be a predominant pathology in mild TBI or "concussion". The rapid deformation of white matter at the instant of trauma can lead to mechanical failure and calcium-dependent proteolysis of the axonal cytoskeleton in association with axonal transport interruption. Recently, a proteolytic fragment of alpha-II spectrin, "SNTF", was detected in serum acutely following mild TBI in patients and was prognostic for poor clinical outcome. However, direct evidence that this fragment is a marker of DAI has yet to be demonstrated in either humans following TBI or in models of mild TBI. Here, we used immunohistochemistry (IHC) to examine for SNTF in brain tissue following both severe and mild TBI. Human severe TBI cases (survival <7d; n = 18) were compared to age-matched controls (n = 16) from the Glasgow TBI archive. We also examined brains from an established model of mild TBI at 6, 48 and 72 h post-injury versus shams. IHC specific for SNTF was compared to that of amyloid precursor protein (APP), the current standard for DAI diagnosis, and other known markers of axonal pathology including non-phosphorylated neurofilament-H (SMI-32), neurofilament-68 (NF-68) and compacted neurofilament-medium (RMO-14) using double and triple immunofluorescent labeling. Supporting its use as a biomarker of DAI, SNTF immunoreactive axons were observed at all time points following both human severe TBI and in the model of mild TBI. Interestingly, SNTF revealed a subpopulation of degenerating axons, undetected by the gold-standard marker of transport interruption, APP. While there was greater axonal co-localization between SNTF and APP after severe TBI in humans, a subset of SNTF positive axons displayed no APP accumulation. Notably, some co-localization was observed between SNTF and the less abundant neurofilament subtype markers. Other SNTF positive axons, however, did not co-localize with any other markers. Similarly, RMO-14 and NF-68 positive axonal pathology existed independent of SNTF and APP. These data demonstrate that multiple pathological axonal phenotypes exist post-TBI and provide insight into a more comprehensive approach to the neuropathological assessment of DAI.

The mTOR Inhibitor Rapamycin Mitigates Perforant Pathway Neurodegeneration and Synapse Loss in a Mouse Model of Early-Stage Alzheimer-Type Tauopathy.

The perforant pathway projection from layer II of the entorhinal cortex to the hippocampal dentate gyrus is especially important for long-term memory formation, and is preferentially vulnerable to developing a degenerative tauopathy early in Alzheimer's disease (AD) that may spread over time trans-synaptically. Despite the importance of the perforant pathway to the clinical onset and progression of AD, a therapeutic has not been identified yet that protects it from tau-mediated toxicity. Here, we used an adeno-associated viral vector-based mouse model of early-stage AD-type tauopathy to investigate effects of the mTOR inhibitor and autophagy stimulator rapamycin on the tau-driven loss of perforant pathway neurons and synapses. Focal expression of human tau carrying a P301L mutation but not eGFP as a control in layer II of the lateral entorhinal cortex triggered rapid degeneration of these neurons, loss of lateral perforant pathway synapses in the dentate gyrus outer molecular layer, and activation of neuroinflammatory microglia and astroglia in the two locations. Chronic systemic rapamycin treatment partially inhibited phosphorylation of a mechanistic target of rapamycin substrate in brain and stimulated LC3 cleavage, a marker of autophagic flux. Compared with vehicle-treated controls, rapamycin protected against the tau-induced neuronal loss, synaptotoxicity, reactive microgliosis and astrogliosis, and activation of innate neuroimmunity. It did not alter human tau mRNA or total protein levels. Finally, rapamycin inhibited trans-synaptic transfer of human tau expression to the dentate granule neuron targets for the perforant pathway, likely by preventing the synaptic spread of the AAV vector in response to pathway degeneration. These results identify systemic rapamycin as a treatment that protects the entorhinal cortex and perforant pathway projection from tau-mediated neurodegeneration, axonal and synapse loss, and neuroinflammatory reactive gliosis. The findings support the potential for slowing the progression of AD by abrogating tau-mediated neurotoxicity at its earliest neuropathological stages.

Serum SNTF Increases in Concussed Professional Ice Hockey Players and Relates to the Severity of Postconcussion Symptoms.

Biomarkers for diffuse axonal injury could have utilities for the acute diagnosis and clinical care of concussion, including those related to sports. The calpain-derived αII-spectrin N-terminal fragment (SNTF) accumulates in axons after traumatic injury and increases in human blood after mild traumatic brain injury (mTBI) in relation to white matter abnormalities and persistent cognitive dysfunction. However, SNTF has never been evaluated as a biomarker for sports-related concussion. Here, we conducted longitudinal analysis of serum SNTF in professional ice hockey players, 28 of whom had a concussion, along with 45 players evaluated during the preseason, 17 of whom were also tested after a concussion-free training game. Compared with preseason levels, serum SNTF increased at 1 h after concussion and remained significantly elevated from 12 h to 6 days, before declining to preseason baseline. In contrast, serum SNTF levels were unchanged after training. In 8 players, postconcussion symptoms resolved within a few days, and in these cases serum SNTF levels were at baseline. On the other hand, for the 20 players withheld from play for 6 days or longer, serum SNTF levels rose from 1 h to 6 days postconcussion, and at 12-36 h differed significantly from the less-severe concussions (p=0.004). Serum SNTF exhibited diagnostic accuracy for concussion, especially so with delayed return to play (area under the curve=0.87). Multi-variate analyses of serum SNTF and tau improved the diagnostic accuracy, the relationship with the delay in return to play, and the temporal window beyond tau alone. These results provide evidence that blood SNTF, a biomarker for axonal injury after mTBI, may be useful for diagnosis and prognosis of sports-related concussion, as well as for guiding neurobiologically informed decisions on return to play.

Evidence That the Blood Biomarker SNTF Predicts Brain Imaging Changes and Persistent Cognitive Dysfunction in Mild TBI Patients.

Although mild traumatic brain injury (mTBI), or concussion, is not typically associated with abnormalities on computed tomography (CT), it nevertheless causes persistent cognitive dysfunction for many patients. Consequently, new prognostic methods for mTBI are needed to identify at risk cases, especially at an early and potentially treatable stage. Here, we quantified plasma levels of the neurodegeneration biomarker calpain-cleaved αII-spectrin N-terminal fragment (SNTF) from 38 participants with CT-negative mTBI, orthopedic injury (OI), and normal uninjured controls (UCs) (age range 12-30 years), and compared them with findings from diffusion tensor imaging (DTI) and long-term cognitive assessment. SNTF levels were at least twice the lower limit of detection in 7 of 17 mTBI cases and in 3 of 13 OI cases, but in none of the UCs. An elevation in plasma SNTF corresponded with significant differences in fractional anisotropy and the apparent diffusion coefficient in the corpus callosum and uncinate fasciculus measured by DTI. Furthermore, increased plasma SNTF on the day of injury correlated significantly with cognitive impairment that persisted for at least 3 months, both across all study participants and also among the mTBI cases by themselves. The elevation in plasma SNTF in the subset of OI cases, accompanied by corresponding white matter and cognitive abnormalities, raises the possibility of identifying undiagnosed cases of mTBI. These data suggest that the blood level of SNTF on the day of a CT-negative mTBI may identify a subset of patients at risk of white matter damage and persistent disability. SNTF could have prognostic and diagnostic utilities in the assessment and treatment of mTBI.

A rapid gene delivery-based mouse model for early-stage Alzheimer disease-type tauopathy.

The perforant pathway projection from the entorhinal cortex (EC) to the hippocampal dentate gyrus is critically important for long-term memory and develops tau and amyloid pathologies and progressive degeneration starting in the early stages of Alzheimer disease (AD). However, perforant pathway function has not been assessed in experimental models of AD, and a therapeutic agent that protects its structure and function has not yet been identified. Therefore, we developed a new adeno-associated virus-based mouse model for perforant pathway tauopathy. Microinjection into the lateral EC of vectors designed to express either human tau bearing a pathogenic P301L mutation or enhanced green fluorescent protein as a control selectively drove transgene expression in lateral EC layer II perikarya and along the entire rostrocaudal extent of the lateral perforant pathway afferents and dentate terminal field. After human tau expression, hyperphosphorylated tau accumulated only within EC layer II perikarya, thereby modeling Braak stage I of transentorhinal AD tauopathy. Expression of pathologic human tau but not enhanced green fluorescent protein led to specific dose-dependent apoptotic death of perforant pathway neurons and loss of synapses in as little as 2 weeks. This novel adeno-associated virus-based method elicits rapid tauopathy and tau-mediated neurodegeneration localized to the mouse perforant pathway and represents a new experimental approach for studying tau-driven pathogenic processes and tau-based treatment strategies in a highly vulnerable neural circuit.

Amyloid deposition and advanced age fails to induce Alzheimer's type progression in a double knock-in mouse model.

It has been challenging to develop transgenic and gene-targeted mouse models that recapitulate all of the neuropathological features of Alzheimer's disease (AD). For example, in the APP/PS-1 double knock-in mutant mouse (DKI), frank neurodegeneration is not observed at middle age and synapse loss is pronounced only within amyloid plaques. Here, we investigated whether continued amyloid deposition and advanced age of 24-27 months lead to loss of neurons and synapses, tau hyperphosphorylation, and other pathological features of AD. We focused on the perforant pathway projection from entorhinal cortex to hippocampal dentate gyrus, since it is preferentially impacted by plaques, tangles, and neuronal loss early in the course of AD. Compared with wild type controls matched for age and gender, expression of neither reelin nor NeuN was altered in the entorhinal layer 2 neurons of origin. Retrograde labeling of the perforant pathway with Fluorogold indicated no cell loss, axonal atrophy, or nerve terminal degeneration. The lack of neuronal loss or atrophy was confirmed by volumetric analysis of the ventral dentate gyrus and immunostaining for a synaptic marker. We also searched for other hallmarks of AD neuropathology by labeling for hyperphosphorylated pre-tangle tau, accumulation of cathepsin D-containing autolysosomes, and cyclin A-positive neurons aberrantly re-entering the cell cycle. None of these AD pathologies were observed in the entorhinal cortex, dentate gyrus, or any other forebrain region. Our results indicate that the DKI mouse does not show appreciable Alzheimer-type disease progression, even at advanced age and in the phase of over 18 months of robust cerebral amyloid deposition. The insufficiency of amyloid deposition to induce other AD-type neuropathologies and neurodegeneration in the aging mouse brain suggests an important role for tauopathy or other factors for triggering the pathogenesis of AD.

Mechanisms of calpain mediated proteolysis of voltage gated sodium channel α-subunits following in vitro dynamic stretch injury.

Although enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) α-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of α-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh α-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.

Combination therapy prevents amyloid-dependent and -independent structural changes.

Neuropathological features of Alzheimer's disease (AD) are recapitulated in transgenic mice expressing familial AD-causing mutations, but ectopic transgene overexpression makes it difficult to relate these abnormalities to disease pathogenesis. Alternatively, the APP/PS-1 double knock-in (DKI) mouse produces mutant amyloid precursor protein (APP) and presenilin-1 (PS-1) with normal levels and regulatory controls. Here, we investigated effects of amyloid on brain structure and neuroplasticity by vaccinating DKI mice with amyloid-ß starting at 8 months of age. At 14 months, vaccination blocked cerebral amyloid deposition and its attendant microglial activation. Neuropil abnormalities were pronounced only within plaques, and included circumscribed loss and dysmorphology of axons, dendrites, terminals and spines. Blockade of amyloid deposition restored neuropil integrity. Amyloid removal did not rescue reductions in the hippocampal neural progenitor and neuroblast populations, but adding 1 month of voluntary exercise to amyloid-ß vaccination markedly stimulated hippocampal neurogenesis. These results identify amyloid-dependent and -independent structural changes in the DKI mouse model of AD. Combining exercise with amyloid-directed immunotherapy produces greater restoration of brain structure and neuroplasticity than is achieved with either maneuver alone.

Evidence that a panel of neurodegeneration biomarkers predicts vasospasm, infarction, and outcome in aneurysmal subarachnoid hemorrhage.

Biomarkers for neurodegeneration could be early prognostic measures of brain damage and dysfunction in aneurysmal subarachnoid hemorrhage (aSAH) with clinical and medical applications. Recently, we developed a new panel of neurodegeneration biomarkers, and report here on their relationships with pathophysiological complications and outcomes following severe aSAH. Fourteen patients provided serial cerebrospinal fluid samples for up to 10 days and were evaluated by ultrasonography, angiography, magnetic resonance imaging, and clinical examination. Functional outcomes were assessed at hospital discharge and 6-9 months thereafter. Eight biomarkers for acute brain damage were quantified: calpain-derived α-spectrin N- and C-terminal fragments (CCSntf and CCSctf), hypophosphorylated neurofilament H,14-3-3 ß and ζ, ubiquitin C-terminal hydrolase L1, neuron-specific enolase, and S100ß. All 8 biomarkers rose up to 100-fold in a subset of patients. Better than any single biomarker, a set of 6 correlated significantly with cerebral vasospasm, brain infarction, and poor outcome. Furthermore, CSF levels of 14-3-3ß, CCSntf, and NSE were early predictors of subsequent moderate-to-severe vasospasm. These data provide evidence that a panel of neurodegeneration biomarkers may predict lasting brain dysfunction and the pathophysiological processes that lead to it following aSAH. The panel may be valuable as surrogate endpoints for controlled clinical evaluation of treatment interventions and for guiding aSAH patient care.

A panel of neuron-enriched proteins as markers for traumatic brain injury in humans.

Surrogate markers have enormous potential for contributing to the diagnosis, prognosis, and therapeutic evaluation of acute brain damage, but extensive prior study of individual candidates has not yielded a biomarker in widespread clinical practice. We hypothesize that a panel of neuron-enriched proteins measurable in cerebrospinal fluid (CSF) and blood should vastly improve clinical evaluation and therapeutic management of acute brain injuries. Previously, we developed such a panel based initially on the study of protein release from degenerating cultured neurons, and subsequently on rodent models of traumatic brain injury (TBI) and ischemia, consisting of 14-3-3beta, 14-3-3zeta, three distinct phosphoforms of neurofilament H, ubiquitin hydrolase L1, neuron-specific enolase, alpha-spectrin, and three calpain- and caspase-derived fragments of alpha-spectrin. In the present study, this panel of 11 proteins was evaluated as CSF and serum biomarkers for severe TBI in humans. By quantitative Western blotting and sandwich immunoassays, the CSF protein levels were near or below the limit of detection in pre-surgical and most normal pressure hydrocephalus (NPH) controls, but following TBI nine of the 11 were routinely elevated in CSF. Whereas different markers peaked coordinately, the time to peak varied across TBI cases from 24-96 h post-injury. In serum, TBI increased all four members of the marker panel for which sandwich immunoassays are currently available: a calpain-derived NH(2)-terminal alpha-spectrin fragment and the three neurofilament H phosphoforms. Our results identify neuron-enriched proteins that may serve as a panel of CSF and blood surrogate markers for the minimally invasive detection, management, mechanistic, and therapeutic evaluation of human TBI.

Calpain mediates proteolysis of the voltage-gated sodium channel alpha-subunit.

Alterations in the expression, molecular composition, and localization of voltage-gated sodium channels play major roles in a broad range of neurological disorders. Recent evidence identifies sodium channel proteolysis as a key early event after ischemia and traumatic brain injury, further expanding the role of the sodium channel in neurological diseases. In this study, we investigate the protease responsible for proteolytic cleavage of voltage-gated sodium channels (NaChs). NaCh proteolysis occurs after protease activation in rat brain homogenates, pharmacological disruption of ionic homeostasis in cortical cultures, and mechanical injury using an in vitro model of traumatic brain injury. Proteolysis requires Ca(2+) and calpain activation but is not influenced by caspase-3 or cathepsin inhibition. Proteolysis results in loss of the full-length alpha-subunits, and the creation of fragments comprising all domains of the channel that retain interaction even after proteolysis. Cell surface biotinylation after mechanical injury indicates that proteolyzed NaChs remain in the membrane before noticeable evidence of neuronal death, providing a mechanism for altered action potential initiation, propagation, and downstream signaling events after Ca(2+) elevation.

Biomarker evidence for mild central nervous system injury after surgically-induced circulation arrest.

Previously, we identified 14-3-3 beta and zeta isoforms and proteolytic fragments of alpha-spectrin as proteins released from degenerating neurons that also rise markedly in cerebrospinal fluid (CSF) following experimental brain injury or ischemia in rodents, but these proteins have not been studied before as potential biomarkers for ischemic central nervous system injury in humans. Here we describe longitudinal analysis of these proteins along with the neuron-enriched hypophosphorylated neurofilament H (pNFH) and the deubiquitinating enzyme UCH-L1 in lumbar CSF samples from 19 surgical cases of aortic aneurysm repair, 7 involving cardiopulmonary bypass with deep hypothermic circulatory arrest (DHCA). CSF levels of the proteins were near the lower limit of detection by Western blot or enzyme-linked fluorescence immunoassay at the onset of surgical procedures, but increased substantially in a subset of cases, typically within 12-24 h. All cases involving DHCA were characterized by >3-fold elevations in CSF levels of the two 14-3-3 isoforms, UCH-L1, and pNFH. Six of 7 also exhibited marked increases in alpha-spectrin fragments generated by calpain, a protease known to trigger necrotic neurodegeneration. Among cases involving aortic cross-clamping but not DHCA, the proteins rose in CSF preferentially in the subset experiencing acute neurological complications. Our results suggest the neuron-enriched 14-3-3beta, 14-3-3zeta, pNFH, UCH-L1, and calpain-cleaved alpha-spectrin may serve as a panel of biomarkers with clinical potential for the detection and management of ischemic central nervous system injury, including for mild damage associated with surgically-induced circulation arrest.

Temporal profiles of cytoskeletal protein loss following traumatic axonal injury in mice.

To examine the time course and relative extent of proteolysis of neurofilament and tubulin proteins after traumatic axonal injury (TAI), anesthetized mice were subjected to optic nerve stretch injury. Immunohistochemistry confirmed neurofilament accumulation within axonal swellings at 4, 24, and 72 h postinjury (n = 4 injured and 2 sham per time point). Immunoblotting of optic nerve homogenates (n = 5 injured and 1 sham at 0.5, 4, 24 or 72 h) revealed calpain-mediated spectrin proteolytic fragments after injury. Protein levels for NF68 progressively decreased from 0.5 h to 24 h postinjury, while NF200 and alpha-tubulin levels decreased acutely (0.5-4 h), with a secondary decline at 72 h postinjury. These data demonstrate that diffusely distributed TAI is associated not only with a localized accumulation of neurofilament proteins, but also significant decreases in total cytoskeletal protein levels which may be mediated, in part, by calpains. Protection of the axonal cytoskeleton represents a potential therapeutic target for axonal damage associated with injury or neurodegenerative diseases.

Long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of familial Alzheimer's disease.

Neurogenesis in the adult hippocampus has been implicated in regulating long-term memory and mood, but its integrity in Alzheimer's disease (AD) is uncertain. Studies of neurogenesis in transgenic mouse models of familial AD are complicated by ectopic overexpression restricted to terminally differentiated neurons, while AD cases have been studied only at the pre-senile or end-stage of disease. To investigate further the fidelity of adult neurogenesis, we examined mice carrying targeted mutations in amyloid precursor protein (APP), presenilin-1 (PS-1), or both APP and PS-1, in which FAD-causing mutations have been inserted into their endogenous genes. The latter "double knock-in" mice developed aging- and region-dependent amyloid deposition starting around 6 months, and by 9 months exhibited microglial activation associated with the amyloid. In the 9-month-old dentate gyrus, the double knock-in mutations reduced the numbers of MCM2-positive neural stem and progenitor cells by 3-fold and doublecortin-positive neuroblasts by 2-fold. The reduction in dentate neuroblasts persisted at 18 months of age. The impairment in neurogenesis was confirmed by quantitative Western blot analysis of doublecortin content and was restricted to the hippocampal but not the olfactory bulb neurogenic system. In contrast, neither mutant PS-1 nor APP alone led to amyloid deposition or significant alterations in the two markers. These results demonstrate long-lasting and selective impairment in adult hippocampal neurogenesis in a knock-in mutant mouse model of FAD and suggest a novel mechanism by which amyloid and its attendant microglia-mediated neuroinflammation could contribute to the cognitive and behavioral abnormalities of AD.

Traumatic mechanical injury to the hippocampus in vitro causes regional caspase-3 and calpain activation that is influenced by NMDA receptor subunit composition.

Apoptotic or necrotic cell death in the hippocampus is a major factor underlying the cognitive impairments following traumatic brain injury. In this study, we examined if traumatic mechanical injury would produce regional activation of calpain and caspase-3 in the in vitro hippocampus and studied how the mechanically induced activation of NR2A and NR2B containing N-methyl-d-aspartate receptors (NMDARs) affects the activation of these proteases following mechanical injury. Following a 75% stretch, significant levels of activated caspase-3 and calpain-mediated spectrin breakdown products were evident only in cells within the dentate gyrus, and little co-localization of the markers was identified within individual cells. After 100% stretch, only calpain activation was observed, localized to the CA3 subregion 24 h after stretch. At moderate injury levels, both caspase-3 and calpain activation was attenuated by blocking NR2B containing NMDARs prior to stretch or by blocking all NMDARs prior to stretch injury. Treatment with an NR2A selective NMDAR antagonist had little effect on either activated caspase-3 or Ab38 immunoreactivity following moderate injury but resulted in the appearance of activated caspase-3 in the dentate gyrus following severe mechanical stretch. Together, these studies suggest that the injury induced activation of NR2A containing NMDARs functions as a pro-survival signal, while the activation of NR2B containing NMDARs is a competing, anti-survival, signal following mechanical injury to the hippocampus.

Caspase-mediated cell death predominates following engraftment of neural progenitor cells into traumatically injured rat brain.

Neural progenitor cells (NPCs) have been shown to be a promising therapy for cell replacement and gene transfer in neurological diseases including traumatic brain injury (TBI). However, NPCs often survive poorly after transplantation despite immunosuppression, and the mechanisms of graft cell death are unknown. In this study, we evaluated caspase- and calpain-mediated mechanisms of cell death of neonatal mouse C17.2 progenitor cells, transplanted at 24 h following lateral fluid percussion brain injury (FP) in rats. Adult Male Sprague-Dawley rats (n = 30) were subjected to lateral FP injury (n = 18) or sham surgery (n = 12). C17.2 cells labeled with green fluorescent dye (CMFDA) were engrafted in the perilesional deep cortex, and animals were sacrificed at 24 h, 72 h and 1 week post-transplantation. Pro-apoptotic caspase-mediated cleavage products (Ab246) and calpain-mediated cleavage products (Ab38) were detected in the engrafted cells using immunohistochemistry. Only 2 to 4.5% of grafted NPCs were found to survive at 24 h post-transplantation, regardless of injury status of the host brain, although brain-injured animals had significantly fewer graft cells than sham-injured animals. Limited caspase and calpain-mediated graft cell death was observed in both sham- and brain-injured animals, and caspase-mediated graft cell death was significantly greater than calpain-mediated graft cell death in all animals. Brain-injured animals had significantly increased caspase-mediated graft cell death compared to sham-injured animals. These results suggest that both the caspase and calpain family of proteases are involved in graft cell death, and that caspase-mediated apoptotic graft cell death predominates in the acute post-traumatic period following TBI.

E2F1 induces cell death, calpain activation, and MDMX degradation in a transcription independent manner implicating a novel role for E2F1 in neuronal loss in SIV encephalitis.

The E2F1 transcription factor can initiate proliferation or apoptosis, the latter by both transcription-dependent and -independent mechanisms. Recently, an E2F1 mutant lacking the DNA binding domain, E2F1(180-437), has been implicated in degradation of MDMX and MDM2 proteins via lysosomal proteases. MDM proteins block p53 dependent apoptosis by directly inhibiting p53 stability and function. Here we demonstrate E2F1(180-437) induces death in HEK293 cells independent of E2F1 transcriptional activation and p53 stabilization. E2F1(180-437) elevates the activity of the calcium-activated protease, calpain, which is required for E2F1 induced proteolysis of MDMX and E2F1 induced cell loss. To determine if E2F1 could be activating proteolysis via calpains in neurodegeneration, we examined MDMX immunofluorescence in simian immunodeficiency virus encephalitis (SIVE). We found a reciprocal relationship between E2F1 and MDMX staining: in SIVE where E2F1 immunostaining is increased, MDMX is decreased, while in controls where E2F1 immunostaining is low, MDMX is high. Together these experiments support a new function for E2F1 in the activation of calpain proteases and suggest a role for this pathway in SIVE.

Novel surrogate markers for acute brain damage: cerebrospinal fluid levels corrrelate with severity of ischemic neurodegeneration in the rat.

Previously, we identified proteins released from degenerating cultured cortical neurons as novel cerebrospinal fluid (CSF) markers for acute brain injury in the rat. Here, we investigate relationships between CSF changes in these novel markers and the severity of acute ischemic brain injury. Rats underwent sham surgery or 3,6,8, or 10 mins of transient global forebrain ischemia. At 48 h after insult, CSF levels of 14-3-3beta, 14-3-3zeta, and calpain cleavage products of alpha-spectrin and tau were quantified. Regional acute neurodegeneration was assessed by Fluoro-Jade and silver impregnation staining, and confirmed by immunohistochemical detection of the activation of calpain and caspase, cysteine proteases involved in neurodegenerative signaling. Ischemic neurodegeneration and activation of at least one cysteine protease were observed in the hippocampal CA1 sector, dentate hilus, caudate nucleus, parietal cortex, thalamus, and inferior colliculus. As expected, the total number of degenerating cells increased as a function of ischemia duration. Cerebrospinal fluid levels of the four marker proteins increased markedly after ischemia, and rose in proportion with its duration. Irrespective of the length of ischemia, CSF levels of the neuron-enriched proteins 14-3-3beta and calpain-cleaved tau correlated significantly with the magnitude of acute ischemic neurodegeneration. Additionally, CSF levels of the two proteins correlated with one another. These results show that certain proteins released from degenerating neurons are CSF markers for brain injury in the rat whose levels reflect the severity of acute ischemic neurodegeneration. Measurement of 14-3-3beta and calpain-cleaved tau may be useful for the minimally invasive diagnosis, prognosis, and therapeutic evaluation of acute brain damage.