Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer disease.

Prostate apoptosis response-4 (Par-4) is a protein containing both a leucine zipper and a death domain that was isolated by differential screening for genes upregulated in prostate cancer cells undergoing apoptosis. Par-4 is expressed in the nervous system, where its function is unknown. In Alzheimer disease (AD), neurons may die by apoptosis, and amyloid beta-protein (A beta) may play a role in this. We report here that Par-4 expression is increased in vulnerable neurons in AD brain and is induced in cultured neurons undergoing apoptosis. Blockade of Par-4 expression or function prevented neuronal apoptosis induced by Ab and trophic factor withdrawal. Par-4 expression was enhanced, and mitochondrial dysfunction and apoptosis exacerbated, in cells expressing presenilin-1 mutations associated with early-onset inherited AD.

Plasticity of hippocampal circuitry in Alzheimer's disease.

Two markers of neuronal plasticity were used to compare the response of the human central nervous system to neuronal loss resulting from Alzheimer's disease with the response of rats to a similar neuronal loss induced by lesions. In rats that had received lesions of the entorhinal cortex, axon sprouting of commissural and associational fibers into the denervated molecular layer of the dentate gyrus was paralleled by a spread in the distribution of tritiated kainic acid-binding sites. A similar expansion of kainic acid receptor distribution was observed in hippocampal samples obtained postmortem from patients with Alzheimer's disease. An enhancement of acetylcholinesterase activity in the dentate gyrus molecular layer, indicative of septal afferent sprouting, was also observed in those patients with a minimal loss of cholinergic neurons. These results are evidence that the central nervous system is capable of a plastic response in Alzheimer's disease. Adaptive growth responses occur along with the degenerative events.

Neuroprotective effects of gelsolin during murine stroke.

Increased Ca2+ influx through activated N-methyl-D-aspartate (NMDA) receptors and voltage-dependent Ca2+ channels (VDCC) is a major determinant of cell injury following brain ischemia. The activity of these channels is modulated by dynamic changes in the actin cytoskeleton, which may occur, in part, through the actions of the actin filament-severing protein gelsolin. We show that gelsolin-null neurons have enhanced cell death and rapid, sustained elevation of Ca2+ levels following glucose/oxygen deprivation, as well as augmented cytosolic Ca2+ levels in nerve terminals following depolarization in vitro. Moreover, major increases in infarct size are seen in gelsolin-null mice after reversible middle cerebral artery occlusion, compared with controls. In addition, treatment with cytochalasin D, a fungal toxin that depolymerizes actin filaments, reduced the infarct size of both gelsolin-null and control mice to the same final volume. Hence, enhancement or mimicry of gelsolin activity may be neuroprotective during stroke.

X-irradiation reduces lesion scarring at the contusion site of adult rat spinal cord.

Spinal cord injury (SCI) results in cell death and tissue destruction, and ultimately cavitation followed by the formation of lesion scars at the injury site. The lesion scars include an astrocytic component (glial scar) and a fibroblastic component (connective tissue scar). The purpose of the present study is to determine if X-irradiation could minimize the formation of lesion scars and reduce the levels of chondroitin sulfate proteoglycans (CSPGs) in the contusion SCI model of the adult rat. Two weeks after SCI, a connective tissue scar formed at the injury site consisting primarily of fibroblasts and exhibits strong CSPG immunoreactivity. The fibroblasts might originate from the connective tissue of pia mater or arachnoid mater. At the same time, reactive astrocytes in the spared tissue accumulate surrounding the lesion cavity to form a thick glial scar with significant enhancement of glial fibrillary acidic protein (GFAP) and CSPG immunoreactivity. After X-irradiation (40 Gy) of the injury site 2 days post-injury, that results in an attenuated dose to the lesion, the connective tissue scar was not observed, and accordingly, almost no CSPG immunoreactivity was detected at this area. Meanwhile, the glial scar and its CSPG immunoreactivity were prominently reduced. X-irradiation did not show significant improvement in locomotor recovery, but resulted in a slight delay of body weight recovery following injury. This preparative treatment could be used to reduce secondary scarring in the lesion resulting in an enriched site for further treatment such as growth related transplantation.

Cytoskeletal disruption following contusion injury to the rat spinal cord.

Following experimental spinal cord injury (SCI), there is a delayed loss of neurofilament proteins but relatively little is known regarding the status of other cytoskeletal elements. The purpose of the present study was to compare the extent and time course of the MAP2 loss with that of neurofilament proteins, and to examine tau protein levels and distribution following SCI. Within 1 to 6 hours following SCI, there is rapid loss of MAP2, tau, and nonphosphorylated neurofilament proteins at the injury site. In contrast, the loss of phosphorylated neurofilament proteins was not significant until 1 week postinjury. In addition to the loss of MAP2 protein, there was extensive beading of MAP2-immunoreactive dendrites extending into the white matter. This was most pronounced 1 hour after injury and gradually resolved such that beading was no longer evident 2 weeks after SCI. The time course of beading resolution is similar to that of behavioral recovery following SCI, but the functional significance of the beading remains to be determined. Together, these results demonstrate that there are 2 phases of cytoskeletal disruption following SCI; a rapid loss of MAP2, tau, and nonphosphorylated neurofilament proteins, and a delayed loss of phosphorylated neurofilaments.

Neuropil threads are collinear with MAP2 immunostaining in neuronal dendrites of Alzheimer brain.

Alzheimer disease (AD) neuropathology includes neuropil threads (NTs) and neurofibrillary tangles (NFTs). In tangle-bearing neurons, the normal cytoskeleton is severely disrupted and replaced with paired helical filament (PHF) aggregates of aberrantly phosphorylated microtubule-associated protein tau. In this study, double-label immunocytochemistry was used to clarify the relationship between the appearance of neurofibrillary pathology (NTs and NFTs) and the loss of normal cytoskeletal components, such as microtubule-associated protein 2 (MAP2) in 13 AD cases and 6 nondemented elderly control individuals. Brain areas examined included neocortex (cingulate, motor, and inferior parietal cortices), hippocampus, and entorhinal cortex. In mildly affected neurons, PHF-1 immunostained NTs were found in dendrites, frequently at dendritic branch points, and were adjacent to MAP2 immunostaining. In more severely affected neurons, the PHF-1 immunoreactivity occupied distinct dendritic segments and appeared to displace MAP2. Interspersed MAP2 immunopositive dendritic segments were often beaded in appearance. In all instances where dendrites with NTs could be traced back to the soma, the soma also contained PHF-1 immunostained fibrils in various stages of NFT formation. The results suggest that PHFs gradually displace normal microtubules in dendrites, and cause degeneration of dendritic segments between NTs.

N-terminal heterogeneity of parenchymal and cerebrovascular Abeta deposits.

The goals of this study were twofold: to determine whether species differences in Abeta N-terminal heterogeneity explain the absence of neuritic plaques in the aged dog and aged bear in contrast to the human; and to compare Abeta N-terminal isoforms in parenchymal vs cerebrovascular Abeta (CVA) deposits in each of the species, and in individuals with Alzheimer disease (AD) vs nondemented individuals. N-terminal heterogeneity can affect the aggregation, toxicity, and stability of Abeta. The human, polar bear, and dog brain share an identical Abeta amino acid sequence. Tissues were immunostained using affinity-purified polyclonal antibodies specific for the L-aspartate residue of Abeta at position one (AbetaN1[D]), D-aspartate at N1 (AbetaN1[rD]), and pyroglutamate at N3 (AbetaN3[pE]) and p3, a peptide beginning with leucine at N17 (AbetaN17[L]). The results demonstrate that each Abeta N-terminal isoform can be present in diffuse plaques and CVA deposits in AD brain, nondemented human, and the examined aged animal models. Though each Abeta N-terminal isoform was present in diffuse plaques, the average amyloid burden of each isoform was highest in AD vs polar bear and dog (beagle) brain. Moreover, the ratio of AbetaN3(pE) (an isoform that is resistant to degradation by most aminopeptidases) vs AbetaN17(L)-x (the potentially nonamyloidogenic p3 fragment) was greatest in the human brain when compared with aged dog or polar bear. Neuritic plaques in AD brain typically immunostained with antibodies against AbetaN1(D) and AbetaN3(pE), but not AbetaN17(L) or AbetaN1(rD). Neuritic deposits in nondemented individuals with atherosclerotic and vascular hypertensive changes could be identified with AbetaN1(D), AbetaN3(pE), and AbetaN1(rD). The presence of AbetaN1(rD) in neuritic plaques in nondemented individuals with atherosclerosis or hypertension, but not in AD, suggests a different evolution of the plaques in the two conditions. AbetaN1(rD) was usually absent in human CVA, except in AD cases with atherosclerotic and vascular hypertensive changes. Together, the results demonstrate that diffuse plaques, neuritic plaques, and CVA deposits are each associated with distinct profiles of Abeta N-terminal isoforms.

Alterations in tau immunostaining in the rat hippocampus following transient cerebral ischemia.

Previous studies in gerbils have shown that cytoskeletal disruption and a loss of the dendritic microtubule-associated protein, MAP2, may occur after short periods of transient global ischemia. tau, a predominantly axonal microtubule-associated protein, has not been examined following ischemia. We compared neuronal damage with alterations in MAP2, tau, and 72-kD heat shock protein (HSP72) immunostaining at various reperfusion times following 20 min of ischemia in the rat four-vessel occlusion model. tau accumulated in neuronal cell bodies throughout the hippocampal formation 30 min to 2 h after the ischemic insult. Perikaryal tau immunostaining was transient in most regions, but persisted in polymorphic hilar neurons. This was accompanied by a loss of immunostaining in the target of many hilar neurons, the inner molecular layer of the dentate gyrus. The same neuronal populations that exhibited increased tau immunostaining of perikarya later displayed an induction of HSP72 immunoreactivity. In contrast, loss of MAP2 immunostaining was not consistently observed before neuronal death and did not correspond to HSP72 induction. The altered tau immunostaining is not the direct result of excitotoxic insult, as intrahippocampal injection of kainic acid did not cause the somal accumulation of tau, but did cause disruption of MAP2 immunostaining. Taken together, the results suggest that the somal accumulation of tau is an early, sensitive, and selective marker of ischemic insult.

Microtubular proteolysis in focal cerebral ischemia.

Calpain, a neutral protease activated by calcium, may promote microtubular proteolysis in ischemic brain. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. The earliest sign of tissue injury was observed after no more than 15 min of ischemia, with coiling of apical dendrites immunolabeled to show microtubule-associated protein 2 (MAP2). After 6 h of ischemia, MAP2 immunoreactivity was markedly diminished in the infarct zone. Quantitative Western analysis demonstrated that MAP2 was almost unmeasurable after 24 h of ischemia. An increase in calpain activity, shown by an antibody recognizing calpain-cleaved spectrin fragments, paralleled the loss of MAP2 immunostaining. Double-labeled immunofluorescent studies showed that intraneuronal calpain activity preceded evidence of MAP2 proteolysis. Perikaryal immunolabeling of tau protein became increasingly prominent between 1 and 6 h in neurons located within the transition zone between ischemic and unaffected tissue. Western blot experiments confirmed that dephosphorylation of tau protein occurred during 24 h of ischemia, but was not associated with significant loss of tau antigen. We conclude that focal cerebral ischemia is associated with early microtubular proteolysis caused by calpain.

Plasticity in Alzheimer's disease: too much or not enough?

The goal of optimizing restorative sprouting in Alzheimer's disease is based on the premise that sprouting is beneficial and is deficient in AD. The beneficial aspects of neuronal plasticity have been questioned, however, and other studies suggest that some aspects of sprouting may be exaggerated in AD and contribute to the formation of plaques, tangles, and other neuropathological hallmarks of this disorder. Manipulation of the sprouting response may represent a promising treatment strategy in AD, but whether the goal is to augment or impede sprouting may depend upon the extent of the damage and the severity of the disease state.

Plasticity, pathology, and Alzheimer's disease.

Much basic data support the notion that growth factors are important in the maintenance and repair of the CNS. Although local increases in growth factors might contribute to aberrant growth and senile plaque formation in Alzheimer's disease (AD), the administration of growth factor inhibitors as a therapeutic intervention would be predicted to decrease the vulnerability of remaining healthy neurons. The administration of neurotrophic agents in AD may help to protect and maintain neurons, and thereby minimize neuronal death and the resultant sprouting response.

Elevated phosphocholine and phosphatidylcholine following rat entorhinal cortex lesions.

At early stages of Alzheimer's disease, phosphomonoesters (PMEs) including phosphocholine (P-choline) are present at elevated levels. PMEs also are elevated in the developing brain during the period of neurite extension. To determine if the elevation of PMEs in AD could reflect neuritic sprouting, 31P-NMR was used to examine phospholipid metabolites and membrane phospholipids at various times following unilateral lesions of the entorhinal cortex, a well-defined model of neuritic sprouting. Two to 7 days postlesion, P-choline levels were elevated 48% in the hippocampus ipsilateral to the entorhinal cortex lesion, but not in the contralateral hippocampus or cerebral cortex. P-choline levels declined by day 15, and reached control levels 45 days following the lesion. The lesion-induced elevation in P-choline could result from increased P-choline synthesis via choline kinase, decreased activity of CTP:phosphocholine cytidylyltransferase, or breakdown of phosphatidylcholine (PC). To distinguish between these possibilities, the membrane phospholipids PC and phosphatidylethanolamine (PE) were measured. Both phospholipids were maintained at or above control levels at each of the postlesion time points, arguing against membrane breakdown or decreased PC synthesis contributing to the elevation of P-choline levels. Other alterations included a widespread elevation in inositol phosphate 2 days postlesion, but not at later time points. The alterations in phospholipid metabolites observed in the rat hippocampus following entorhinal cortex lesions closely resemble those observed in the human brain in the early stages of AD.

Perikaryal accumulation and proteolysis of neurofilament proteins in the post-mortem rat brain.

Investigations of neurofilament alterations in neurodegenerative disorders utilize postmortem human tissues obtained at autopsy. To determine if alterations in the levels or distribution of neurofilament proteins might occur during the interval between death and autopsy, the postmortem cooling curve of the human brain was modeled in Sprague-Dawley rats and neurofilament proteins were examined by immunocytochemistry and immunoblots. One hour after death, enhanced perikaryal immunostaining of NF-M and both phosphorylated and nonphosphorylated NF-H epitopes was observed throughout the hippocampal formation. A greater number of neurons exhibited increased somatic immunostaining 4-h postmortem. In addition, loss of neurofilament protein immunostaining was observed in the neuropil, particularly in the molecular layer of the dentate gyrus. This corresponded with, but lagged behind, the pattern of calpain activation determined using an antibody against calpain-cleaved alpha-spectrin. Immunoblots confirmed the postmortem loss of neurofilament proteins in both triton-soluble and insoluble fractions. These results demonstrate that the levels and localization of neurofilament proteins observed in tissues obtained at autopsy even with short postmortem intervals may not accurately reflect the premortem condition.

Age-specific onset of beta-amyloid in beagle brains.

As part of an effort to characterize Alzheimer's disease-like neuropathy in the canine brain we have determined the age of onset of spontaneous beta-amyloid deposition in 103 laboratory-raised beagles. Tissue samples for each subject were obtained from hippocampal and cortical regions and examined for the incidence and density of beta-amyloid deposition after staining with modified Bielschowsky silver stain and immunohistochemistry. Amyloid deposition was characterized as diffuse plaque or cloud-like formation. The diffuse type of beta-amyloid plaque formation predominated in all brain regions examined. A threshold effect of plaque development was observed; no plaques were apparent in dogs before the age of 10 years, while 36% of dogs aged 11.1-12.9, 60% of dogs aged 13.0-15.0, and 73% of dogs aged 15.1-17.8 developed beta-amyloid deposits. Additionally, a significant increase in plaque density was observed with increasing age.

N-methyl-D-aspartate receptors and Alzheimer's disease.

The results of several studies now suggest that the density of N-methyl-D-aspartate (NMDA) receptors is maintained in many Alzheimer's disease (AD) cases, although loss of these receptors can occur in specific regions as a consequence of severe neuronal loss. Recent findings demonstrate that there are at least two subtypes of the NMDA receptor which are allosterically regulated. To determine the status of the NMDA receptor in AD, studies are required which will examine the activation state of the NMDA receptor and the properties of subtypes in relation to neuronal density and structure.

Protein L-isoaspartyl methyltransferase in postmortem brains of aged humans.

The specific activity of protein L-isoaspartyl methyltransferase, an enzyme implicated in the metabolism of damaged, isoaspartate-containing proteins, has been measured in postmortem samples of parietal cortex from 30 individuals (19 with Alzheimer's disease and 11 controls). Methyltransferase specific activity was positively correlated with age at death, increasing by 2.9 pmol/min/mg of protein for every ten years of age (r = .51, p less than 0.005). This correlation was significant in the control and Alzheimer's disease groups alike. Specific activity also appeared to be about 15% higher in females than in age- and diagnosis-matched males (p less than 0.05). No significant differences were observed between age- and sex-matched Alzheimer patients and controls, suggesting that a deficiency in this enzyme is not responsible for the accumulation of abnormal proteins in Alzheimer's disease.

Comparison of neuropathologic criteria for the diagnosis of Alzheimer's disease.

The National Institute on Aging and Reagan Institute (NIA-RI) criteria, and other neuropathologic criteria for Alzheimer's disease (AD), were compared with the clinical diagnosis of dementia in a well defined population of Catholic sisters. The 47-participant subset examined in this study were college educated and lacked complicating conditions such as brain infarcts or diffuse Lewy body disease. Sixteen participants had a clinical diagnosis of dementia. The NIA-RI criteria imply a perfect correlation between neuritic plaque (NP) density and neurofibrillary tangle distribution. However, NP density often did not coincide with tangle distribution. As a result, it was not possible to categorize many of the participants using the NIA-RI guidelines. The 'high likelihood' category of the NIA-RI criteria for AD research settings (neocortical Braak stage and frequent neocortical NP) had relatively high specificity (90% of nondemented participants did not meet this criteria). However, only half of the demented participants were in this category. Neuropathologic criteria requiring the presence of neocortical tangles (rather than neocortical Braak stage) had relatively high sensitivity, accounting for 87-94% of participants with dementia, but also included 32-35% of nondemented participants. Criteria based on neocortical NP or senile plaques had 100% sensitivity, but a majority of nondemented participants also met these criteria. The results support consideration of both tangles and NP for the neuropathologic diagnosis of AD, but indicate that refinement of the NIA-RI criteria is necessary. A possible refinement is suggested for further consideration.

Carboxy terminal of beta-amyloid deposits in aged human, canine, and polar bear brains.

Immunocytochemistry, using antibodies specific for different carboxy termini of beta-amyloid. A beta 40 and A beta 42(43), was used to compare beta-amyloid deposits in aged animal models to nondemented and demented Alzheimer's disease human cases. Aged beagle dogs exhibit diffuse plaques in the absence of neurofibrillary pathology and the aged polar bear brains contain diffuse plaques and PHF-1-positive neurofibrillary tangles. The brains of nondemented human subjects displayed abundant diffuse plaques, whereas the AD cases had both diffuse and mature (cored) neuritic plaques. Diffuse plaques were positively immunostained with an antibody against A beta 42(43) in all examined species, whereas A beta 40 immunopositive mature plaques were observed only in the human brain. Anti-A beta 40 strongly immunolabeled cerebrovascular beta-amyloid deposits in each of the species examined, although some deposits in the polar bear brain were preferentially labeled with anti-A beta 42(43). beta-amyloid deposition was evident in the outer molecular layer of the dentate gyrus in the aged dog, polar bear, and human. Within this layer, A beta 42 was present as diffuse deposits, although these deposits were morphologically distinct in each of the examined animal models. In dogs, A beta 42 was cloud-like in nature; the polar bear demonstrated a more aggregated type of deposition, and the nondemented human displayed well-defined deposits. Alzheimer's disease cases were most frequently marked by neuritic plaques in this region. Taken together, the data indicate that beta-amyloid deposition in aged mammals is similar to the earliest stages observed in human brain. In each species, A beta 42(43) is the initially deposited isoform in diffuse plaques.

Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD.

The cytokine tumor necrosis factor (TNF) is toxic to some mitotic cells, but protects cultured neurons from a variety of insults by mechanisms that are unclear. Pretreatment of neurons or astrocytes with TNF caused significant increases in MnSOD activity, and also significantly attenuated 3-nitropropionic acid (3-NP) induced superoxide accumulation and loss of mitochondrial transmembrane potential. In oligodendrocytes, however, MnSOD activity was not increased, and 3-NP toxicity was unaffected by TNF. Genetically engineered PC6 cells that overexpress MnSOD also were resistant to 3-NP-induced damage. TNF pretreatment and MnSOD overexpression prevented 3-NP induced apoptosis, and shifted the mode of death from necrosis to apoptosis in response to high levels of 3-NP. Mitochondria isolated from either MnSOD overexpressing PC6 cells or TNF-treated neurons maintained resistance to 3-NP-induced loss of transmembrane potential and calcium homeostasis, and showed attenuated release of caspase activators. Overall, these results indicate that MnSOD activity directly stabilizes mitochondrial transmembrane potential and calcium buffering ability, thereby increasing the threshold for lethal injury. Additional studies showed that levels of oxidative stress and striatal lesion size following 3-NP administration in vivo are increased in mice lacking TNF receptors.

N-methyl-D-aspartate receptors in the cortex and hippocampus of baboon (Papio anubis and Papio papio).

In vitro autoradiography was used to examine the N-methyl-D-aspartate receptor in the brain of a baboon species, Papio anubis, and compared to that of Papio papio which exhibits a photosensitive epilepsy. The epilepsy originates in the frontal cortex and is accompanied by an enhanced sensitivity to N-methyl-D-aspartate. In both Papio anubis and Papio papio, the density of N-methyl-D-aspartate receptors was greatest in the hippocampus, followed by associational areas including frontal cortex, and low in primary sensory areas such as the visual cortex. The receptors were concentrated in the outer cortical layers I-III, very low in layer IV except in primary visual cortex, and of intermediate density in layer V. The density of binding sites was approximately two-fold lower than previously observed in the rodent brain, whereas the affinity of the receptor for [3H]L-glutamate was greater in the primate versus the rodent brain. Glycine potentiated the binding of [3H]L-glutamate in both cortex and hippocampus. No significant differences in the properties of N-methyl-D-aspartate receptors were observed between the two baboon species, suggesting that the photosensitivity of Papio papio is not due to alterations in the binding of L-glutamate to the N-methyl-D-aspartate receptor complex.