Protective effects of EUK4010 on beta-amyloid(1-42) induced degeneration of neuronal cells

EUK4010 has been identified to exhibit an inhibitory effect on beta-amyloid (Abeta)(1-42)-induced loss of neuronal cell viability. Further studies demonstrated that EUK4010 attenuated the Abeta(1-42)-induced degeneration in both cultured rat hippocampal neurons and human neuroblastoma cells, as demonstrated by typical morphological changes, cell viability and the chip-based flow cytometric assay. Gene expression analysis using DNA microarray showed that the senescence marker calcium-binding protein, regucalcin (Rgn), GABA-A receptor pi subunit (Gabrp), the huntingtin binding protein, optineurin (Optn) and a semaphorin family plexin A3 similar protein (Plex-similar) changed their expression levels significantly in cultured neurons after Abeta(1-42) treatment. In this report, we have undertaken a chemical genetic approach to study the molecular basis of Abeta(1-42) effects on the neuronal degeneration. Our results demonstrate that EUK4010 completely blocked the Abeta(1-42)-induced up-regulation of GABA-A receptor pi subunit and the semaphorin family plexin A3 similar protein, and partially attenuated the down-regulation of senescence marker calcium-binding protein, regucalcin. These observations suggest that EUK4010 may prevent or reduce the Abeta toxicity by regulating the expression of genes involved in the Abeta induced neuronal degeneration. These genes may represent a promising target for the therapeutic drug development for Alzheimer's disease (AD) and other neurological disorders. Furthermore, EUK4010 and its analogues could potentially be developed as neuronal protective agents for the treatment of these diseases.     

Differential cellular expression of tumor necrosis factor-alpha and Type I tumor necrosis factor receptor after transient global forebrain ischemia.

We examined the expression of tumor necrosis factor-alpha (TNF-alpha) and the Type I tumor necrosis factor receptor, (TNFR1), in relation to c-fos, a known regulator gene of immediate cellular responses, after an extended period of global ischemia. The number of TNF-alpha mRNA expressing cells peaked in most brain areas after 8 h of reperfusion. Significant increases in TNFR1 mRNA expression were evident in the cortex at 2 and 8 h of reperfusion and after 8 h of reperfusion in the CA3/CA4 region of the hippocampus. Transient neuronal c-fos mRNA expression preceded these responses. TNF-alpha immunoreactivity was seen in neurons>oligodendrocytes=perivascular cells=ependymal cells=vessel wall structures. After ischemia/reperfusion, increased TNF-alpha immunoreactivity was evident only in oligodendrocytes. TNFR1 immunoreactivity in sham brains manifested in bundles of cellular fibers of variable length and thickness. In post-ischemic brains, immunoreactivity in these cellular processes representing mainly astroglial extensions was suppressed at 2 h but recovered partially by 8 and 24 h of reperfusion. In contradiction, transient ischemia-induced TNFR1 immunoreactivity was observed in somas of large cortical neurons, in activated microglia/macrophages, perivascular and endothelial cells.Taken together, the increase in neuronal TNF-alpha mRNA appeared not to be followed by substantial translation to protein in the cerebral tissue after an extended period of global ischemia. However, there was increased neuronal TNFR1 immunostaining in conjunction with increased immunostaining for TNF-alpha in oligoglial elements, which suggests signaling to neurons by enhanced oligoglial TNF-alpha.     

Fas and Fas ligand are associated with neuritic degeneration in the AD brain and participate in beta-amyloid-induced neuronal death

It has recently been suggested that neuronal cell death in response to many brain insults may be mediated by the upregulation of tumor necrosis factor receptor (TNFR) family members and their ligands. In the present study, we investigated whether the expression of the TNFR family death domain receptor, Fas, and its ligand, FasL, is altered in association with neuropathology and activated caspase markers in Alzheimer disease (AD) brain, and Abeta-induced neuronal cell death in vitro. To evaluate this hypothesis, we examined Fas and FasL expression in AD and control brain, and Abeta-treated primary neurons, using immunocytochemistry and Western blots. Neurons in both AD brain and Abeta-treated cultures exhibited FasL upregulation and changes in immunoreactivity for Fas receptor. Further, FasL expression was remarkably elevated in senile plaques and neurofilament-positive dystrophic neurites, and in association with caspase activation and neuritic apoptosis in AD brain. Based on these and previous data regarding protection of primary neuronal cultures from Abeta(1-42)-induced apoptosis by blockade of Fas-associated death domain signaling, we also tested the hypothesis that dynamic regulation of Fas and FasL may contribute to Abeta-mediated neuronal cell death. Accordingly, neuronal cultures derived from mice carrying inactivating mutations in Fas (Faslpr) or FasL (Fasgld) exhibited protection from Abeta(1-42)-induced cell death. These findings suggest that Fas-FasL interactions may contribute to mechanisms of neuronal loss and neuritic degeneration in AD.     

Age-related changes in GluR2 and NMDAR1 glutamate receptor subunit protein immunoreactivity in corticocortically projecting neurons in macaque and patas monkeys

A distinct subpopulation of neurons forming long corticocortical projections in the association neocortex is highly vulnerable to the degenerative process in Alzheimer's disease. However, the degree to which age-related molecular and morphologic alterations of identifiable neuronal populations reflects early cellular degeneration leading to functional deficits has not yet been fully investigated in the aging brain. We performed an immunohistochemical analysis of neurons forming short and long corticocortical projections in young and old monkeys using antibodies to the GluR2 and NMDAR1 glutamate receptor subunit proteins. Projection neurons differed in their expression of these receptor subunits, as GluR2 was less prevalent than NMDAR1 among retrogradely labeled neurons. Long and short corticocortical pathways in old animals demonstrated a considerable decrease in the proportions of projection neurons containing GluR2 and NMDAR1, an observation that was particularly consistent in the case of GluR2. No age-related differences were observed in distribution of neurofilament protein in either type of projection neurons. These data suggest that cortical neurons furnishing long and short corticocortical projections display consistent neurochemical changes during aging and that a differential decrease in cellular expression of glutamate receptor subunit proteins occurs. The fact that in aging these neurons have lower levels of GluR2 than in young individuals, but comparatively higher levels of NMDAR1 than GluR2, may render them prone to calcium-mediated excitotoxicity, which in humans may be related to the selective vulnerability of such neurons during the course of Alzheimer's disease. Also, it is apparent that age-related neuronal changes are quite subtle and involve subcellular components of the cortical circuits rather than major morphologic alterations.     

TNFR1 upregulation mediates tolerance after brain ischemic preconditioning.

A short ischemic event (ischemic preconditioning (IPC)) can result in subsequent resistance to severe ischemic injury (ischemic tolerance (IT)). The expression and neuroprotective role of tumor necrosis factor (TNF-alpha) have been described in models of IPC and we have showed the participation of its processing enzyme, the TNF-alpha convertase enzyme (TACE) in this process. We have now decided to explore the expression and localization of TNF receptors (TNFR) as well as other signalling mechanisms involved in IT. A period of 10 mins of temporary middle cerebral artery occlusion (tMCAO) was used for focal IPC. To evaluate the ability of IPC to produce IT, permanent MCAO was performed 48 hours after IPC. Ischemic preconditioning produced a reduction in infarct volume, as we showed previously. Ischemic preconditioning caused upregulation of neuronal TNFR1 that was reduced by the selective TACE inhibitor BB1101. Intracerebral administration of TNFR1 antisense oligodeoxynucleotide, which caused a reduction in TNFR1 expression, inhibited the IPC-induced protective effect, showing that TNFR1 upregulation is implicated in IT. Moreover, treatment with BB1101, TNFR1 antisense and lactacystin-a specific proteasome inhibitor-blocked IPC-induced NF-kappaB. Immunohistochemical studies showed the expression of TACE and TNFR1 in neurons. In summary, these data show that IPC produces neuronal upregulation of TACE and TNFR1, and that the pathway TACE/TNF-alpha/TNFR1/NF-kappaB is involved in IT.     

RAGE-Abeta interactions in the pathophysiology of Alzheimer's disease

RAGE is a cell surface molecule primarily identified for its capacity to bind advanced glycation end-products and amphoterin. Immunocytochemical studies demonstrated that in Alzheimer's Disease (AD) the expression of RAGE is elevated in neurons close to neuritic plaque beta-amyloid (Abeta) deposits and in the cells of Abeta containing vessels. Cross-linking of surface bound Abeta 1-40 to endothelial cells, yielded a band of 50 kDa identified as RAGE. Using the soluble extracellular domain of recombinant human RAGE, we found that Abeta binds to RAGE with a Kd = 57 +/- 14 nM, a value close to those found for mouse brain endothelial cells and rat cortical neurons. The interaction of Abeta with RAGE in neuronal, endothelial, and RAGE-transfected COS-1 cells induced oxidative stress, as assessed by the TBARS and MTT assays. ELISA demonstrated a 2.5 times increase of RAGE in AD over control brains. Activated microglia also showed elevated expression of RAGE. In the BV-2 microglial cell line, RAGE bound Abeta in dose dependent manner with a Kd of 25 +/- 9 nM. Soluble Abeta induced the migration of microglia along a concentration gradient, while immobilized Abeta arrested this migration. Abeta-RAGE interaction also activated NF-kappaB, resulting in neuronal up-regulation of macrophage-colony stimulating factor (M-CSF) which also induced microglial migration. Taken together, our data suggest that RAGE-Abeta interactions play an important role in the pathophysiology of Alzheimer's Disease.     

Age- and concentration-dependent neuroprotection and toxicity by TNF in cortical neurons from beta-amyloid

The induction of an inflammatory response and release of cytokines such as TNF may be involved in the age-related etiology of Alzheimer disease (AD). In the brain, microglia have been shown to produce a wide variety of immune mediators, including the pro-inflammatory cytokine tumor necrosis factor (TNF). We hypothesize that with age there is increased ability of microglia to produce TNF or that age decreases the neuroprotective effect of TNF against beta-amyloid (Abeta) toxicity in neurons. We investigated the effects of Abeta(1-40) on TNF secretion from forebrain cultures of microglia from embryonic, middle-age (9-month) and old (36-month) rats. Over the first 12 hr of exposure to 10 microM Abeta (1-40), microglia from embryonic and old rats increase TNF secretion, although microglia from middle-age rats did not produce detectable levels of TNF. When low concentrations of TNF are added to neurons together with Abeta (1-40) in the absence of exogenous antioxidants, neuroprotection for old neurons is significantly less than neuroprotection for middle-age neurons. In neurons from old rats, high levels of TNF together with Abeta are more toxic than in neurons from middle-age or embryonic rats. These results are discussed in relation to neuroprotection and toxicity of the age-related pathology of AD.     

Stimulation of PGE receptors EP2 and EP4 protects cultured neurons against oxidative stress and cell death following beta-amyloid exposure

Alzheimer's disease (AD) is associated with gliosis, neuroinflammation and higher levels of prostaglandins. Conflicting roles for cyclooxygenases and prostaglandins in the etiopathology of AD have been reported. We hypothesized that PGE2 signaling through EP2 and EP4 G-protein-coupled receptors could protect against amyloid beta-peptide (Abeta) neurotoxicity by increasing the cAMP signaling cascade. Using primary neuronal cultures, we investigated the presence of EP receptors (EP1-4) and the action of PGE2 and EP receptor agonists on neuronal susceptibility to Abeta1-42 toxicity. Low concentrations (1 microm) of PGE2, butaprost (EP2 agonist), and 1-hydroxy-PGE1 (EP4/EP3 agonist) were neuroprotective against Abeta1-42 toxicity, while sulprostone (EP3/EP1 agonist) at similar doses had no detectable effects. EP2 and EP4 receptor-mediated neuroprotection would involve changes in cAMP levels, as both EP2 and EP4 agonists increased intracellular cAMP concentration by approximately doubling basal levels, and both exhibited neuroprotective actions against Abeta-induced toxicity. The protein kinase A (PKA) inhibitor RpcAMPS significantly attenuated the neuroprotection by butaprost, but not that by 1-hydroxy-PGE1, implying differences between EP2 and EP4 receptor protective mechanisms. Additionally, the increase in reactive oxygen species generated following exposure to Abeta was reduced by stimulation of both EP2 and EP4 receptors. Together, these results indicate that PGE2 can protect neurons against Abeta toxicity by acting on given receptors and stimulating a cascade of intracellular events, including the cAMP-PKA pathway. We propose that development and testing of specific PGE2 receptor agonists downstream of cyclooxygenase could lead to therapeutic applications.     

Peri-sciatic administration of recombinant rat TNF-alpha induces mechanical allodynia via upregulation of TNF-alpha in dorsal root ganglia and in spinal dorsal horn: the role of NF-kappa B pathway

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) and TNF receptor 1 (TNFR1) in dorsal root ganglia (DRG) and in spinal dorsal horn are upregulated after nerve injury and that many TNF-alpha-containing neurons overexpress TNFR1. In the present study, we found that peri-sciatic administration of rat recombinant TNF-alpha (rrTNF) at the concentrations of 10, 100 and 1000 pg/ml (daily for 2 days) induced mechanical allodynia in bilateral hindpaws, lasting for about 20 days. The immunoreactivity (IR) of TNF-alpha and TNFR1 in the ipsilateral (but not in the contralateral) L4 and L5 DRGs increased significantly on day 1 and day 3 after administration of rrTNF, respectively. Double immunofluorescence staining revealed that in DRGs the increased TNF-alpha-IR was mainly in neuronal cells and with a lesser extent in satellite glial cells, while the upregulation of TNFR1-IR was almost restricted at neuronal cells. TNF-alpha-IR but not TNFR1-IR also increased in bilateral lumbar spinal dorsal horn from day 3 to day 14, which was observed in astrocytes, microglias and neurons. In addition, a progressive infiltration of monocyte/macrophages and T lymphocytes in the ipsilateral L5 DRG and sciatic nerve was observed, starting on day 2 following administration of rrTNF. Intrathecal delivery of PDTC (8.2 ng in 10 microl volume), a nuclear factor-kappa B (NF-kappaB) inhibitor, 30 min before each rrTNF administration blocked mechanical allodynia completely and inhibited the upregulation of TNF-alpha-IR and TNFR1-IR substantially. The results suggest that peri-sciatic administration of rrTNF may induce mechanical allodynia by an autocrine mechanism via activation of the NF-kappaB pathway.     

Transforming growth factor beta2 autocrinally mediates neuronal cell death induced by amyloid-beta

Amyloid beta (Abeta), the major component of the senile plaques of Alzheimer's disease, is implicated in neuronal cell death. We have found that Abeta42, a neurotoxic form of Abeta peptide, induces both neuronal and glial expression of TGFbeta2. We have further demonstrated that the addition into culture media of neutralizing antibody to TGFbeta2 or a large amount of the recombinant soluble amyloid precursor protein alpha, the extracellular domain of amyloid precursor protein (APP) generated by alpha secretase, suppresses death in primary cortical neurons (PCNs) induced by Abeta42 in vitro. Combined with the finding in our recent study indicating that TGFbeta2 is a neuronal cell death-inducing ligand for APP, it is suggested that TGFbeta2 is an autocrinal mediator for Abeta42-induced death in PCNs.     

Interaction of amyloid binding alcohol dehydrogenase/Abeta mediates up-regulation of peroxiredoxin II in the brains of Alzheimer's disease patients and a transgenic Alzheimer's disease mouse model

Alzheimer's patients have increased levels of both the 42 beta amyloid-beta-peptide (Abeta) and amyloid binding alcohol dehydrogenase (ABAD) which is an intracellular binding site for Abeta. The over-expression of Abeta and ABAD in transgenic mice has shown that the binding of Abeta to ABAD results in exaggerating neuronal stress and impairment of learning and memory. From a proteomic analysis of the brains from these animals we identified that peroxiredoxin II levels increase in Alzheimer's diseased brain. This increase in peroxiredoxin II levels protects neurons against Abeta induced toxicity. We also demonstrate, for the first time in living animals, that the expression level of peroxiredoxin II is an indicator for the interaction of ABAD and Abeta as its expression levels return to normal if this interaction is perturbed. Therefore this indicates the possibility of reversing changes observed in Alzheimer's disease and that the Abeta-ABAD interaction is a suitable drug target.     

Constitutive expression of p55TNFR mRNA and mitogen-specific up-regulation of TNF alpha and p75TNFR mRNA in mouse brain

Serum tumor necrosis factor (TNF) functions as a mediator of the immune-to-brain axis. Numerous TNF receptor-mediated effects on the nervous system are described but the knowledge about the regional and cellular expression of TNF receptor p55TNFR and p75TNFR in vivo is far from being complete. It is unclear whether TNF mediates its neuroimmune effects alone or in combination with other factors, e.g., bacterial mitogens. Here, we investigated the distribution of TNFalpha, p55TNFR, and p75TNFR in normal mouse brain and examined the stimulus-specific effects of lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) on the expression of the cerebral TNF system. Both mitogens caused enhanced TNFalpha serum levels and induced c-fos mRNA in the paraventricular nucleus but exhibited different effects on the cerebral gene expression of the TNF system. LPS but not SEB rapidly induced TNFalpha mRNA in circumventricular organs (CVOs) followed by spreading of TNFalpha mRNA into brain parenchyma close to the CVOs. The p55TNFR gene was constitutively expressed in many neurons with high levels in brainstem motor nuclei and in neurons of the sensory mesencephalic trigeminal nucleus. Moderate levels of p75TNFR mRNA were seen in single cells scattered throughout the brain in a pattern resembling microglia. Neither LPS nor SEB modulated the p55TNFR gene expression in any region or cell type of the brain, and LPS but not SEB induced p75TNFR mRNA in the CVOs. Thus, enhanced TNF serum levels able to stimulate c-fos mRNA expression in the paraventricular nucleus did not necessarily result in a modulation of the cerebral TNF system.     

Involvement of microglial receptor for advanced glycation endproducts (RAGE) in Alzheimer's disease: identification of a cellular activation mechanism

Receptor-mediated interactions with amyloid beta-peptide (Abeta) could be important in the evolution of the inflammatory processes and cellular dysfunction that are prominent in Alzheimer's disease (AD) pathology. One candidate receptor is the receptor for advanced glycation endproducts (RAGE), which can bind Abeta and transduce signals leading to cellular activation. Data are presented showing a potential mechanism for Abeta activation of microglia that could be mediated by RAGE and macrophage colony-stimulating factor (M-CSF). Using brain tissue from AD and nondemented (ND) individuals, RAGE expression was shown to be present on microglia and neurons of the hippocampus, entorhinal cortex, and superior frontal gyrus. The presence of increased numbers of RAGE-immunoreactive microglia in AD led us to further analyze RAGE-related properties of these cells cultured from AD and ND brains. Direct addition of Abeta(1-42) to the microglia increased their expression of M-CSF. This effect was significantly greater in microglia derived from AD brains compared to those from ND brains. Increased M-CSF secretion was also demonstrated using a cell culture model of plaques whereby microglia were cultured in wells containing focal deposits of immobilized Abeta(1-42). In each case, the Abeta stimulation of M-CSF secretion was significantly blocked by treatment of cultures with anti-RAGE F(ab')2. Treatment of microglia with anti-RAGE F(ab')2 also inhibited the chemotactic response of microglia toward Abeta(1-42). Finally, incubation of microglia with M-CSF and Abeta increased expression of RAGE mRNA. These microglia also expressed M-CSF receptor mRNA. These data suggest a positive feedback loop in which Abeta-RAGE-mediated microglial activation enhances expression of M-CSF and RAGE, possibly initiating an ascending spiral of cellular activation.     

CCR1 is an early and specific marker of Alzheimer's disease

Chemokines are a diverse group of small proteins that effect cell signaling by binding to G-protein-coupled, seven-trans-membrane receptors. Our group had found previously that the chemokine receptor CCR1 was present in neurons and dystrophic processes in a small sample of Alzheimer's disease cases. This expanded immunohistochemical study shows that the number of CCR1-positive plaque-like structures in the hippocampus and entorhinal cortex is highly correlated to dementia state as measured by the clinical dementia rating score. CCR1 immunoreactivity is found in dystrophic, neurofilament-positive, synaptophysin-negative neurites that are associated with senile plaques containing amyloid beta peptides of the 1-42 species (Abeta42). CCR1 was not, however, associated with diffuse deposits of Abeta42. There was limited expression of CCR1 in neurofibrillary tangle-bearing neuritic processes. Astrocytes and microglia were typically negative for CCR1. Human brains from age-matched, nondemented individuals rarely displayed either CCR1 or Abeta42 immunoreactivity. Seven other types of dementing neurodegenerative diseases were examined, and all failed to demonstrate CCR1 immunopositivity unless Abeta42-positive plaques were also present. Thus, neuronal CCR1 is not a generalized marker of neurodegeneration. Rather, it appears to be part of the neuroimmune response to Abeta42-positive neuritic plaques.     

Gamma-glutamylcysteine ethyl ester-induced up-regulation of glutathione protects neurons against Abeta(1-42)-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease

Glutathione (GSH) is an important endogenous antioxidant found in millimolar concentrations in the brain. GSH levels have been shown to decrease with aging. Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging and oxidative stress. Abeta(1-42) has been shown to induce oxidative stress and has been proposed to play a central role in the oxidative damage detected in AD brain. It has been shown that administration of gamma-glutamylcysteine ethyl ester (GCEE) increases cellular levels of GSH, circumventing the regulation of GSH biosynthesis by providing the limiting substrate. In this study, we evaluated the protective role of up-regulation of GSH by GCEE against the oxidative and neurotoxic effects of Abeta(1-42) in primary neuronal culture. Addition of GCEE to neurons led to an elevated mean cellular GSH level compared with untreated control. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine (BSO) led to a 98% decrease in total cellular GSH compared with control, which was returned to control levels by addition of GCEE. Taken together, these results suggest that GCEE up-regulates cellular GSH levels which, in turn, protects neurons against protein oxidation, loss of mitochondrial function, and DNA fragmentation induced by Abeta(1-42). These results are consistent with the notion that up-regulation of GSH by GCEE may play a viable protective role in the oxidative and neurotoxicity induced by Abeta(1-42) in AD brain.     

Sublethal concentrations of prion peptide PrP106-126 or the amyloid beta peptide of Alzheimer's disease activates expression of proapoptotic markers in primary cortical neurons

Neurodegenerative disorders such as prion diseases and Alzheimer's disease (AD) are characterized by neuronal dysfunction and accumulation of amyloidogenic protein. In vitro studies have demonstrated that these amyloidogenic proteins can induce cellular oxidative stress and therefore may contribute to the neuronal dysfunction observed in these illnesses. Although the neurotoxic pathways are not fully elucidated, recent studies in AD have demonstrated up-regulation of caspases in neurons treated with amyloid beta (Abeta) peptide, suggesting involvement of apoptotic processes. To examine the role of proapoptotic pathways in prion diseases we treated primary mouse cortical neurons with the toxic prion protein peptide PrP106-126 and measured caspase activation and annexin V binding. We found that PrP106-126 induced a rapid and marked elevation in caspase 3, 6, and 8-like activity in neuronal cultures. Increased annexin V binding was observed predominantly on cortical cell neurites in peptide-treated cultures. Interestingly, these effects were induced by sublethal (5-50 microM) or lethal (100-200 microM) concentrations of PrP106-126. Sublethal concentrations of PrP106-126 maintained elevated caspase activation for at least 10 days with no loss of cell viability. Abeta1-40 also up-regulated caspase 3 activity and annexin V binding at both sublethal (5 microM) and lethal (25 microM) concentrations. There were no changes to proapoptotic marker expression in cultures treated with scrambled PrP106-126 (200 microM) or Abeta1-28 (25 microM) peptides. These studies demonstrate that amyloidogenic peptides can induce prolonged activation of proapoptotic marker expression in cultured neurons even at sublethal concentrations. These effects could contribute to chronic neuronal dysfunction and increase susceptibility to additional metabolic insults in neurodegenerative disorders. If so, targeting of therapeutic strategies against neuronal caspase activation early in the disease course could be beneficial in AD and prion diseases.     

Alzheimer's pathology in human temporal cortex surgically excised after severe brain injury

Traumatic brain injury (TBI) is a risk factor for the development of Alzheimer's disease (AD). This immunohistochemical study determined the extent of AD-related changes in temporal cortex resected from individuals treated surgically for severe TBI. Antisera generated against Abeta species (total Abeta, Abeta(1-42), and Abeta(1-40)), the C-terminal of the Abeta precursor protein (APP), apolipoprotein E (apoE), and markers of neuron structure and degeneration (tau, ubiquitin, alpha-, beta-, and gamma-synuclein) were used to examine the extent of Abeta plaque deposition and neurodegenerative changes in 18 TBI subjects (ages 18-64 years). Diffuse cortical Abeta deposits were observed in one third of subjects (aged 35-62 years) as early as 2 h after injury, with only one (35-year old) individual exhibiting "mature", dense-cored plaques. Plaque-like deposits, neurons, glia, and axonal changes were also immunostained with APP and apoE antibodies. In plaque-positive cases, the only statistically significant change in cellular immunostaining was increased neuronal APP (P = 0.013). There was no significant correlation between the distribution of Abeta plaques and markers of neuronal degeneration. Diffuse tau immunostaining was localized to neuronal cell soma, axons or glial cells in a larger subset of individuals. Tau-positive, neurofibrillary tangle (NFT)-like changes were detected in only two subjects, both of more advanced age and who were without Abeta deposits. Other neurodegenerative changes, evidenced by ubiquitin- and synuclein-immunoreactive neurons, were abundant in the majority of cases. Our results demonstrate a differential distribution and course of intra- and extra-cellular AD-like changes during the acute phase following severe TBI in humans. Abeta plaques and early evidence of neuronal degenerative changes can develop rapidly after TBI, while fully developed NFTs most likely result from more chronic disease- or injury-related processes. These observations lend further support to the hypothesis that head trauma significantly increases the risk of developing pathological and clinical symptoms of AD, and provide insight into the molecular mechanisms that initiate these pathological cascades very early during severe brain injury.