Expression pattern of the epithelial v-like antigen (Eva) transcript suggests a possible role in placental morphogenesis

According to the amyloid hypothesis for the pathogenesis of Alzheimer's disease (AD), amyloid beta peptide (Abeta) directly affects neurons, leading to neurodegeneration and tau phosphorylation, followed by the production of paired helical filaments (PHF) in neurofibrillary tangles (NFT). To analyze the relationship between the phosphorylation sites of tau and the activation of kinases in response to Abeta, we treated cultured rat hippocampal neurons with a peptide fragment of Abeta, Abeta(25-35). Abeta(25-35) treatment activated tau protein kinase I/glycogen synthase kinase-3beta (TPKI/GSK-3beta) but not glycogen synthase kinase-3alpha (GSK-3alpha) or mitogen activated protein kinase (MAP kinase) in primary culture of hippocampal neurons. Using antibodies that recognize phosphorylated sites of tau, we showed that tau phosphorylation was enhanced in at least five sites (Ser199, Ser202, Ser396, Ser404, and Ser413 numbered according to the human tau isoform containing 441 amino acid residues), to an extent that depended on the level of TPK I/GSK-3beta. Treatment with TPK I/GSK-3beta antisense oligonucleotide inhibited the enhancement of tau phosphorylation induced by Abeta(25-35) exposure. Thus, TPK I/GSK-3beta activation by Abeta(25-35) may lead to extensive tau phosphorylation.     

Examination of phosphorylated tau protein as a PHF-precursor at early stage Alzheimer's disease

Hyperphosphorylated tau protein which can be isolated on the basis of insolubility in 1% sarkosyl (A68-tau fraction) is thought to represent a precursor pool for PHF assembly, associated histologically with neuritic pathology, which feeds into a more resistant tangle-associated PHF pool via cross-linking and proteolysis. We examined these predictions at the earliest detectable stages of neurofibrillary pathology. We report that there is no evidence that neuritic pathology represents an early pathologic stage, no evidence of an association between neuritic pathology and phosphorylated tau, no evidence of selective accumulation of phosphorylated tau at early stages of pathology, and no evidence for a precursor/product relationship between phosphorylated tau and PHFs during progression of pathology. We conclude that altered phosphorylation is a secondary process affecting 5% of PHFs and does not explain PHF assembly in Alzheimer's disease.     

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.     

Prolonged administration of oral etoposide alone or with intravenous carboplatin in stage IV non-small cell lung cancer: a randomized trial

Alzheimer's disease (AD) is characterized by neuronal cell death and two kinds of deposits, neurofibrillary tangles (NFT) and senile plaques. The main component of NFT is paired helical filaments (PHF), which mainly consist of hyperphosphorylated tau protein. Tau protein kinases I and II were found as candidate enzymes responsible for hyperphosphorylation of tau to induce the formation of PHF. Since prior phosphorylation of tau by TPKII strongly enhanced the action of TPKI, it was thought that TPKII was involved in the formation of PHF-tau in concert with TPKI. After cloning, TPKI was found to be identical with glycogen synthase kinase 3 beta (GSK3 beta), while TPKII consists of a novel 23 kDa protein activator and a catalytic subunit that is identical with cyclin-dependent kinase 5 (CDK5). The phosphorylation sites on tau by TPKI and TPKII could account for the most, but not all, of the major phosphorylation sites of fetal tau and PHF-tau. An antibody for a site specifically phosphorylated by TPKI (Ser413) could identify all three neurofibrillary lesions in the AD brain, and double staining for either TPKI or TPKII and NFT in the brain of Down's syndrome patients clearly demonstrated that TPKI and TPKII are both associated with NFT in vivo, suggesting that the level of TPKI or TPKII is elevated in AD brain by some mechanism. On the other hand, the levels of both TPKs change developmentally, being high in the neonatal period when the phosphorylation of fetal tau proceeds actively, suggesting that the TPKI/TPKII cooperative system has an important physiological role in the formation of neural networks. In AD brain, aberrant accumulation of amyloid-beta protein (A beta) occurs ahead of the accumulation of PHF in NFT. When a primary culture of embryonic rat hippocampus was treated with 20 microM A beta, induction of TPKI, extensive phosphorylation of tau and then programmed cell death were observed, indicating that TPKI induced by A beta phosphorylates tau, followed by disruption of axonal transportation and finally cell death. By using a yeast two hybrid system, TPKI was found to interact with pyruvate dehydrogenase (PDH), which is a key enzyme in the glycolytic pathway. PDH was phosphorylated in vitro by TPKI to reduce the activity converting pyruvate into acetyl-CoA, which is required for acetylcholine synthesis. In a primary culture of rat hippocampal cells treated with A beta, PDH was inactivated in inverse relation to the activation of TPKI, resulting in accumulation of pyruvate or lactate, energy failure induced by the disturbance of glucose metabolism, and a shortage of acetylcholine owing to deficiency of acetyl-CoA, all of which are characteristic of AD brain. In cholinergic neurons such as those of the septum, non-aggregated A beta, specifically A beta (1-42), not A beta (1-40), caused a shortage of acetylcholine by activation of TPKI and inactivation of PDH without cell death.     

The kinetic parameters of sodium currents in maturing acutely isolated rat hippocampal CA1 neurones

Whole-cell voltage clamp techniques were used to characterize the kinetics of INa in immature (P3-5) and older (P > 25) acutely isolated rat CA1 hippocampal neurones. Fast-rising and fast-inactivating currents were recorded at all stages of maturation, evocable from Vm values of -55 to -50 mV. Currents were sensitive to TTX (1 microM) and to sodium removal from the perfusate. Current density and maximum slope conductance increased with maturation. Current decay was described by two exponentials, the faster component dominating at -35 mV or more depolarized Vm values; the ratio fast/slow inactivating component decreased with maturation. The voltage-dependence of conductance was taken as an approximation of m infinity. In younger cells, V1/2 values of the steady-state inactivation (h infinity) and activation curves (m infinity) were depolarized. Shifts of h infinity and m infinity curves were accompanied by shifts in the corresponding tau h and tau m voltage-dependence curves. In younger cells, activation curves had comparatively higher slope factors (Vs), which is an indication of a lower voltage sensitivity of activation. m infinity, tau m, h infinity, and tau h parameters were used to calculate the forward and backward activation and inactivation rate constants (alpha m, beta m, alpha h and beta h). P3-5 cells had relatively higher beta m values accounting for the lower voltage sensitivity of activation. The findings are an indication of a dominant channel variety in the younger cells with a closed state higher probability. The results are consistent with lower depolarization rates previously reported in CA1 cells at early stages of maturation. Faster inactivation due to poor expression of the slower inactivating component may compensate for poorer repolarization mechanisms due to the immaturity of outward currents previously reported at early stages of maturation.     

Secreted beta-amyloid precursor protein stimulates mitogen-activated protein kinase and enhances tau phosphorylation

Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. We report that the secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulated the 43-kDa form of MAPK by > 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevented the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer disease, we sought and found a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation. In the ras dominant inhibitory cell line, beta-APP failed to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau.     

Study of proline-directed protein kinases involved in phosphorylation of the heavy neurofilament subunit

The high-molecular-mass neurofilament subunit (NFH) is normally hypophosphorylated in the neuronal perikaryon and undergoes extensive phosphorylation after entering the initial axon segment. Aberrant hyperphosphorylation of perikaryal NFH is a common feature of many neurological diseases. In a previous study (), we demonstrated a correlation between phosphorylation of perikaryal NFH and induction of stress-activated protein kinase (SAPK)-gamma. In this report, we present direct evidence showing that the in vivo activation of SAPKs by an upstream activator (MEKK-1) caused extensive NFH phosphorylation. We also show that stress-activated p38 kinases were not involved in the phosphorylation of perikaryal NFH in cultured dorsal root ganglion neurons and that this process was reversible. SAPKgamma was shown to be located in both the cell body and the neurites of the cultured neurons, suggesting that it is likely to be involved in the phosphorylation of cytoplasmic substrates. These could include neuritic NFH, which is highly phosphorylated despite the demonstrated lack of cyclin-dependent kinase-5 activity in these neurons. Neuritic NFH was also highly phosphorylated in neuronal cultures devoid of Schwann cells, indicating that this form of post-translational modification does not require cues stemming from Schwann cell-axon contacts. Collectively, these findings provide significant new insights into mechanisms involved in NFH phosphorylation in normal neurons and in disease states characterized by aberrant phosphorylation of neurofilaments.     

Butyrylcholinesterase in the life cycle of amyloid plaques

Deposits of diffuse beta-amyloid (Abeta) may exist in the brain for many years before leading to neuritic degeneration and dementia. The factors that contribute to the putative transformation of the Abeta amyloid from a relatively inert to a pathogenic state remain unknown and may involve interactions with additional plaque constituents. Matching brain sections from 2 demented and 4 nondemented subjects were processed for the demonstration of Abeta immunoreactivity, butyrylcholinesterase (BChE) enzyme activity, and thioflavine S binding. Additional sections were processed for the concurrent demonstration of two or three of these markers. A comparative analysis of multiple cytoarchitectonic areas processed with each of these markers indicated that Abeta plaque deposits are likely to undergo three stages of maturation, ie, a "diffuse" thioflavine S-negative stage, a thioflavine S-positive (ie, compact) but nonneuritic stage, and a compact neuritic stage. A multiregional analysis showed that BChE-positive plaques were not found in cytoarchitectonic areas or cortical layers that contained only the thioflavine S-negative, diffuse type of Abeta plaques. The BChE-positive plaques were found only in areas containing thioflavine S-positive compact plaques, both neuritic and nonneuritic. Within such areas, almost all (>98%) BChE-containing plaques bound thioflavine S, and almost all (93%) thioflavine S plaques contained BChE. These results suggest that BChE becomes associated with amyloid plaques at approximately the same time that the Abeta deposit assumes a compact beta-pleated conformation. BChE may therefore participate in the transformation of Abeta from an initially benign form to an eventually malignant form associated with neuritic tissue degeneration and clinical dementia.     

Developmental regulation and PKC dependence of Alzheimer's-type tau phosphorylations in cultured fetal rat hippocampal neurons

Attempts to describe a mechanism of neurofibrillary tangle formation often focus on site specific phosphorylations of tau protein. These have typically been described in both Alzheimer's disease and developing brains. Therefore, study of the developmental regulation of Alzheimer epitope tau phosphorylations may help explain their persistence or recurrence during Alzheimer's disease. Using fetal rat hippocampal cultures, we report a spatial and temporal expression of tau phosphorylation during neuronal differentiation. We have examined phosphorylation at the epitopes recognized by monoclonal antibodies, PHF-1 and Tau 1. Tau was highly phosphorylated at the PHF-1 epitope at all culture ages examined using both immunohistochemical staining and Western blots. Tau was heavily phosphorylated at the Tau 1 epitope only in older cultures. The populations of tau recognized by the two antibodies also exhibited different solubilities, suggesting different microtubule binding behaviors: tau phosphorylated at PHF-1 was retained in axons following solubilization whereas Tau 1 immunoreactive tau was not retained in any cell compartment. Finally, in this culture system, maintenance of phosphorylation at the PHF-1 epitope, but not the Tau 1 epitope, required protein kinase C activity. These results indicate unique regulatory mechanisms and roles for each of these phosphorylated tau epitopes.     

Insulin and insulin-like growth factor-1 regulate tau phosphorylation in cultured human neurons

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary lesions associated with Alzheimer's disease. Hyperphosphorylation reduces the affinity of tau for microtubules and is thought to be a critical event in the pathogenesis of this disease. Recently, glycogen-synthase kinase-3 has been shown to phosphorylate tau in vitro and in non-neuronal cells transfected with tau. The activity of glycogen-synthase kinase-3 can be down-regulated in response to insulin or insulin-like growth factor-1 through the activation of the phosphatidylinositol 3-kinase pathway. We therefore hypothesize that insulin or insulin-like growth factor-1 may affect tau phosphorylation through the inhibition of glycogen-synthase kinase-3 in neurons. Using cultured human neuronal NT2N cells, we demonstrate that glycogen-synthase kinase-3 phosphorylates tau and reduces its affinity for microtubules and that insulin and insulin-like growth factor-1 stimulation reduces tau phosphorylation and promotes tau binding to microtubules. We further demonstrate that these effects of insulin and insulin-like growth factor-1 are mediated through the inhibition of glycogen-synthase kinase-3 via the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.     

Activation of the Xenopus oocyte mitogen-activated protein kinase pathway by Mos is independent of Raf

Transgenic mice expressing the oncogenic protein-serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimer's disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.     

The place of laparoscopy in pediatric surgery]

Histopathological features of Alzheimer's disease (AD) include extracellular deposits of amyloid beta (A beta) fibrils in the cores of senile plaques, intracellular neurofibrillary tangles (NFT) which are composed of paired helical filaments (PHF), and neuronal cell loss. The main component of PHF is highly phosphorylated tau protein. We identified a protein kinase converting normal tau into a PHF-like state. The kinase is tau protein kinase (TPK) I/glycogen synthase kinase (GSK)-3 beta. Using a neuronal cell culture system as an AD model, it was recognized that TPK I/GSK-3 beta plays a central role in AD pathology. We hypothesize that A beta-induced neuronal cell death occurs by the following mechanism. A beta inactivates PI3-kinase and activates TPK I/GSK-3 beta, which in turn phosphorylates and inactivates both tau and pyruvate dehydrogenase (PDH). After the ability of tau to promote microtubule assembly is diminished by phosphorylation, soluble tau molecules aggregate into PHF by an unknown mechanism. Destabilization of microtubule arrays causes inhibition of axonal transport and accumulation of amyloid precursor protein (APP). Phosphorylation of PDH inhibits the reaction converting pyruvate to acetyl-CoA, resulting in inhibition of energy metabolism and a decrease in acetylcholine, both of which are also characteristics of AD. These changes may lead to neuronal cell death.     

Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons

Whereas GABA is a major inhibitory neurotransmitter in the adult central nervous system, recent experiments performed in our laboratory have shown that the activation of GABAA receptors in the hippocampus leads to excitatory effects during the early post-natal period. The possible consequence of a depolarizing effect of GABA was assessed on the neuritic outgrowth of embryonic hippocampal neurons in culture. No morphological alterations were observed when hippocampal neurons were cultured for three days in the presence of muscimol, a GABAA receptor agonist. In contrast, the neuritic outgrowth of cultured hippocampal neurons was profoundly affected by the presence of bicuculline in the culture medium. In the presence of this GABAA receptor antagonist neurons displayed a reduction in the number of primary neurites and branching points, resulting in a concomitant decrease of the total neuritic length. Thus, this study suggests that GABA, acting on GABAA subtype of receptors, is able to affect the development of the hippocampus.     

Proteolytic degradation of microtubule associated protein tau by thrombin

Deposits in the brain of beta-amyloid and tau proteins constitute the two major characteristics of Alzheimer's disease. It is unknown how the deposits are formed, but several studies have suggested that proteases might play a crucial role. Consequently, the search for proteases responsible for processing tau and amyloid precursor protein has become relevant. Here, the ability of thrombin to process tau in vitro is examined. Thrombin, which is found in blood but presumably also in the nervous system, cleaves tau and generates a stable 25 kDa fragment. Immunoblot and amino acid sequencing reveals that the fragment is derived from the C-terminal of tau, and a microtubule assembly assay shows that it has a reduced capacity to promote microtubule assembly compared with full length tau.     

Lithium reduces tau phosphorylation by inhibition of glycogen synthase kinase-3

Lithium is one of the most widely used drugs for treating bipolar (manic-depressive) disorder. Despite its efficacy, the molecular mechanism underlying its action has not been elucidated. One recent study has proposed that lithium inhibits glycogen synthase kinase-3 and thereby affects multiple cellular functions. Because glycogen synthase kinase-3 regulates the phosphorylation of tau (microtubule-binding protein that forms paired helical filaments in neurons of the Alzheimer's disease brain), we hypothesized that lithium could affect tau phosphorylation by inhibiting glycogen synthase kinase-3. Using cultured human NT2N neurons, we demonstrate that lithium reduces the phosphorylation of tau, enhances the binding of tau to microtubules, and promotes microtubule assembly through direct and reversible inhibition of glycogen synthase kinase-3. These results provide new insights into how lithium mediates its effects in the central nervous system, and these findings could be exploited to develop a novel intervention for Alzheimer's disease.     

Oligodendroglial precursor cell susceptibility to hypoxia is related to poor ability to cope with reactive oxygen species

Paired helical filaments, one of the major hallmarks of Alzheimer's disease brains at autopsy, consist mainly of aberrantly phosphorylated tau. This aberrant tau phosphorylation can be induced in the human neuroblastoma cell line TR14 by a hyperstimulating mixture, consisting of nerve growth factor (NGF), db-cAMP, gangliosides and sodium butyrate (NaBut) [20,23]. Evidence is presented that exposing these cells to increasing concentrations of NaBut alone in the 0.5-2 mM dose-range is sufficient to induce aberrant tau phosphorylation within 24 h, measured by AT-8 immunocytochemistry and Western blotting. This process is associated with increased morphological differentiation. Furthermore, the aberrant tau phosphorylation is followed by neurotoxicity. This neurotoxicity has features of programmed cell death, such as fragmentation on a DNA agarose gel, fragmented nuclei and chromatin condensation and inhibition by the protein synthesis inhibitor cycloheximide. The mechanism by which NaBut induces these modified tau proteins and neurotoxicity are largely unknown but the data suggest an involvement of cytoskeletal proteins.     

Maturation and differentiation of cultured fetal stomach. Effects of corticosteroids, pentagastrin, and cytochalasin B

Gastric mucosa of fetal rats undergoes striking developmental changes during the last few days of gestation in utero. To investigate some aspects of this process, gastric explants from 18-day fetuses (4 days before birth) were maintained in organ culture for 3 days, then assessed by light and electron microscopy. The epithelia from base line uncultured stomachs were stratified and morphologically undifferentiated. During culture in basic medium (Leibovitz L 15), modest maturation of antral and fundic architecture occurred, characterized by epithelial invagination to produce small pit-glands. Secretory granules appeared in occasional epithelial cells, and cytochemistry indicated that most were mucous granules. Addition of pentagastrin (10(-9) to 2 X 10(-7) M) did not induce further morphological maturation in this system. However, addition of cortisol (10(-6) to 10(-5) M) resulted in a marked, dose-related increase of pit-gland formation and of cytological differentiation (appearance of secretory granules). This cortisol-induced architectural maturation was completely inhibited by the mold metabolite cytochalasin B (10(-5) M). The results indicate that fetal gastric maturation can be partially reproduced by culture in chemically defined media, and also suggest that corticosteroids may plan an important role in gastric organogenesis.     

Casein kinase II preferentially phosphorylates human tau isoforms containing an amino-terminal insert. Identification of threonine 39 as the primary phosphate acceptor

The in vitro phosphorylation of the microtubule-associated protein tau by casein kinase II was studied. Purified human brain tau was phosphorylated by casein kinase II to a stoichiometry of 0.7 mol of 32P/mol of tau. Individual recombinant human tau isoforms were phosphorylated to stoichiometries ranging from 0.2 to 0.8 mol of 32P/mol of tau. Casein kinase II catalyzed a 4-fold greater incorporation of phosphate into the tau isoform containing a 58-amino acid insert near its amino terminus (T4L) than the isoforms without the 58-amino acid insert (T3 and T4). Phosphopeptide mapping of casein kinase II phosphorylated human tau and recombinant tau isoforms suggested that the isoforms containing an amino-terminal insert constitute the major substrates for casein kinase II within the tau family. The sites of phosphorylation on T4L were identified by digesting phosphorylated T4L with the protease Asp-N, separating the peptides by reversed phase high performance liquid chromatography, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase amino-terminal sequencing. Thr39 was identified as the predominant phosphorylation site, which is located 5 residues from the amino-terminal insert in T4L. Phosphopeptide mapping of tau isolated from LA-N-5 neuroblastoma cells indicates that Thr39 is phosphorylated in situ. To our knowledge, this is the first demonstration of a differential phosphorylation of the human tau isoforms, with the isoforms containing the acidic amino-terminal insert being the preferred substrates of casein kinase II.     

Transgenic mice expressing Alzheimer amyloid precursor proteins

Nearly a decade after the identification of the Alzheimer amyloid precursor protein (APP) gene several groups of investigators have created transgenic mice expressing APP that simulate some of the prominent behavioral and pathological features of Alzheimer's disease (Quon et al., 1991; Games et al., 1995; Hsiao et al., 1995, 1996; Moechars et al., 1996; Sturchler-Pierrat et al., 1997). These features, which are present to various degrees in different lines of mice, include age-related impairment in learning and memory, neuronal loss, gliosis, neuritic changes, amyloid deposition, and abnormal tau phosphorylation. No mouse model exhibiting every neuropathological feature of Alzheimer's disease exists. Whether an exact simulation of Alzheimer neuropathology is required to understand neural dysfunction in Alzheimer's disease is unclear. Various mouse models of Alzheimer's disease are summarized in this article.     

Beta-amyloid accumulation in aged canine brain: a model of early plaque formation in Alzheimer's disease

We characterized eight aged beagles (maintained from birth in a laboratory colony) and one black Labrador using Bielschowsky's, thioflavine S, and Congo red staining, and antibodies to the beta-amyloid peptide, dystrophic neurites, and other plaque components. All plaques within these canine brains were of the diffuse subtype and were neither thioflavine S- nor Congo red-positive. The majority of plaques in the entorhinal cortex contained numerous neurons within them while plaques in the dentate gyrus did not. beta-Amyloid immunoreactivity was also present within select neurons and neuronal processes and was detected as a diffuse linear zone corresponding to the terminal fields of the perforant path. There was no significant correlation between extent of beta-amyloid accumulation and neuron number in entorhinal cortex. Neither tau-1, PHF-1, nor SMI-31-immunostaining revealed dystrophic fibers, confirming the classification of these plaques as diffuse. Canine plaques did not appear to contain bFGF- or HS-positive immunostaining. This may explain why neuritic involvement was not detected within these canine plaques. It is possible that the beta-amyloid within the canine brain has a unique primary structure or may not be in an assembly state that adversely affects neurons.