Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains

OBJECTIVE: Fibroblast growth factor 9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting type factor that exhibits a growth-stimulating effect on primary glial cells. To elucidate the roles of FGF-9 in human brain tumors, the expression and biological activities of FGF-9 were studied using culture cells and surgically obtained tumor specimens. METHODS: Measurement of FGF-9 and basic FGF in conditioned media of cell cultures was performed by using a sandwich enzyme immunoassay. The mitogenic effect of FGF-9 was evaluated by cell growth studies. FGF-9 expression in vivo was demonstrated by immunohistochemistry. RESULTS: One of 4 glioma cell lines and 4 of 16 human meningiomas examined actually secreted detectable amounts of FGF-9 proteins. In comparison, basic FGF production was detected from 3 of 4 glioma cell lines and 11 of 16 human meningiomas. Similarly to basic FGF, recombinant human FGF-9 significantly stimulated the in vitro cell proliferation in three of four glioma cell lines investigated in a dose-dependent manner. A time course growth study using U87 MG cells revealed an accelerated growth stimulation by FGF-9 after Day 4. The growth stimulatory activity was also shown in three of four human meningiomas studied. Moderate to strong immunoreactivity for FGF-9 was observed in 40 (82%) of 49 human brain tumors examined irrespective of origin, tumor type, grade of malignancy, or whether initial or recurrent. In contrast, strong immunostaining was localized in neurons in the normal human cerebral cortex. CONCLUSION: The present findings suggest that FGF-9 may be involved in the biology of human brain tumors with a possible importance in tumor cell growth. Whether the growth factor is more generally involved in oncogenesis of human tumors awaits further investigation.     

An adult case of pneumonia due to Mycoplasma pneumoniae and Chlamydia psittaci

The actin-binding protein drebrin is localized in postsynaptic terminals in adult brain and is considered to be related to synaptic plasticity. Immunocytochemical study demonstrated that widespread drebrin immunoreactivity was observed in hippocampal formations of control human brains, while Alzheimer's disease (AD) brains showed remarkable reductions in this immunoreactivity. Western blot analysis demonstrated that drebrin E (116kD) as well as drebrin A (125 kD) presented in adult human brains, and that these isoforms were decreased in parallel in AD brains. On the other hand, synaptic vesicle-specific 38-kD protein (SVP-38), a presynaptic marker was not so changed in AD brains in comparison with control brains by both techniques. These findings suggest that drebrin E and A in the adult human brain may be co-localized in postsynaptic terminals, and that drebrin may be more sensitive as a marker of synaptic damage than SVP-38, and that the disappearance of drebrin may contribute to the pathogenesis of memory disturbance in AD.     

Human FGF-1 gene delivery protects against quinolinate-induced striatal and hippocampal injury in neonatal rats

Fibroblast growth factors (FGFs) are cell mitogens and differentiating factors with neuroprotective properties in the CNS. We have already shown that endothelial cells genetically engineered to secrete human FGF-1 (RBEZ-FGF) survive implantation to neonatal rat brain (Johnston et al. (1996) J. Neurochem. 67, 1643-1652]. In this study, the effects of cell-based FGF-1 gene delivery on quinolinate-induced neurotoxicity in the developing rat brain were examined. Control endothelial cells (RBE4), and RBEZ-FGF cells were implanted into right striatum at post-natal day (PND) 7. On PND 10, quinolinate (150 nmol), an endogenous N-methyl-d-aspartate (NMDA) receptor agonist, or vehicle alone was injected into striatum ipsilateral to cell implantation. Injury was quantified in coronal sections obtained from PND 17 animals by comparing striatal and hippocampal volumes ipsilateral and contralateral to the site of quinolinate injection. Human FGF-1 specific transgene expression in vivo was shown by Northern blot and RT-PCR up to 14 days after cell implantation in control animals, and up to 4 days after quinolinate exposure. Quinolinate reduced the size of ipsilateral striatum by 37% and hippocampus by 38% in animals preimplanted with control endothelial cells. In contrast, quinolinate reduced the size of striatum by only 14% and had no effect on hippocampal size in animals preimplanted with RBEZ-FGF cells. Thus, FGF-1 gene delivery protected the developing striatum and hippocampus from quinolinate-induced volume loss by 62% and 100%, respectively. Intrastriatal quinolinate resulted in a significant decrease in density of NOS+ CA3 hippocampal neurons (-38%) without affecting the density of NOS+ neurons in hippocampal regions CA1, dentate gyrus or striatum. This response of CA3 NOS+ neurons appeared to be only partially reversed by FGF-1 gene delivery. Our results show that intracerebral FGF-1 gene expression within the developing brain can protect striatum and hippocampus from quinolinate-mediated injury.     

Hippocampal volume is associated with memory but not monmemory cognitive performance in patients with mild cognitive impairment

Mild cognitive impairment (MCI) appears to be a transitional stage in the development of Alzheimer's disease (AD). Patients with MCI show impaired memory performance and hippocampal atrophy relative to normal elderly controls. Prior studies indicate that the degree of hippocampal atrophy in MCI patients predicts conversion to AD. In contrast to patients with MCI who have deficits primarily in memory, AD patients have clinically evident impairments in both memory and nonmemory cognitive domains. One explanation for the observation that a smaller hippocampal volume predicts conversion to AD might be that hippocampal atrophy is associated with early impairment in nonmemory cognitive areas as well as memory. A link between hippocampal volume and nonmemory function could occur if hippocampal atrophy was correlated with AD pathology in other brain regions. We therefore sought to determine the relationship of hippocampal volume with performance on memory and nonmemory tasks in patients with MCI. Although we found a significant correlation between hippocampal volume and memory performance, we did not find a significant correlation between hippocampal volume and nonmemory performance. We conclude that the relationship between hippocampal volume and risk of AD is likely tied to reduced memory performance and not associated with impairment in nonmemory cognitive domains.     

MR T2 relaxometry in Alzheimer's disease and age-associated memory impairment

A prolonged MR T2 relaxation time was proposed to mark the presence and severity of Alzheimer's disease (AD). We studied the value of T2 relaxometry in diagnosing early AD. T2 was measured from 54 patients with AD, 25 subjects with age-associated memory impairment (AAMI), 18 elderly and 16 young controls. The AD patients had longer T2 in the right hippocampal head (104 +/- 11 ms) and tail (98 +/- 10 ms) than age-matched controls (95 +/- 5 and 92 +/- 9 ms, respectively). This prolongation was not related to age. In the AD group, the T2 of the left hippocampal head also correlated with the clinical severity. The T2 of the amygdala did not differ across the groups. Increased T2 in the temporal and parietal white matter and the thalamus related to increasing age rather than to the diagnostic category. The AAMI subjects had T2 comparable with those of age-matched controls. Despite the prolongation of T2 in the AD group the possible diagnostic value was compromized by a substantial overlap between the study groups. We, thus, conclude that the T2 relaxometry is not a reliable method for diagnosing early AD.     

Identification and characterization of a novel member of the fibroblast growth factor family

A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.     

18O exchange in suspensions of red blood cells: determination of parameters of mass spectrometer inlet system

A variety of anatomic and functional neuroimaging findings are associated with Alzheimer's disease (AD). One of the strongest imaging associations identified is between AD and hippocampal atrophy. The epsilon4 allele of the apolipoprotein E (ApoE) gene increases the risk of developing AD and lowers the mean age of onset of the disease. The purpose of this study was to assess the association between hippocampal volume and ApoE polymorphisms in elderly control subjects and in patients with probable AD. We performed magnetic resonance imaging-based volume measurements of the hippocampus in 125 cognitively normal elderly controls and 62 patients with probable AD. ApoE genotyping was performed by using standard methods. Hippocampal volumes were significantly smaller in AD cases than in control subjects. Hippocampal volumes did not differ significantly within either clinical group on the basis of ApoE genotype. Both the epsilon4 allele of ApoE and hippocampal atrophy were significantly but independently associated with AD.     

Fibroblast growth factor-2 protects entorhinal layer II glutamatergic neurons from axotomy-induced death

The entorhinal cortex is a major relay between the hippocampus and other cortical and subcortical regions. Glutamatergic axons from layer II neurons form the entorhinal cortical projection to the hippocampus via the perforant pathway. We have demonstrated previously that lesion of the perforant pathway causes the death of approximately 30% of entorhinal layer II (ECL2) neurons. To elucidate mechanisms contributing to neuronal death and to investigate strategies preventing it, we identified the phenotype of the vulnerable neuronal population. Sections were immunolabeled with antibodies to the neuronal markers NeuN, glutamate, and calbindin-D28k, and to receptors for fibroblast growth factor-2 (FGFR1) and NMDA (NMDAR1) and were examined using confocal microscopy. Calbindin immunoreactivity was strikingly lamina-specific to ECL2, where one-third of all ECL2 neurons were calbindin-positive. Localization of glutamate revealed that half of the glutamatergic ECL2 neurons coexpressed calbindin. Quantification using unbiased stereology at 9 weeks after lesion of the perforant pathway revealed that the only ECL2 neuronal population that experienced a significant (70%) loss (20% of the total) was the population of glutamatergic ECL2 neurons that did not coexpress calbindin. All ECL2 neurons expressed FGFR1; therefore, we tested the role of FGF-2 in the survival of glutamatergic ECL2 neurons. We grafted fibroblasts genetically engineered to express nerve growth factor or FGF-2 and found that only FGF-2 grafts prevented loss of the vulnerable glutamatergic/calbindin-negative neurons. We present a hypothesis for the selective vulnerability of these glutamatergic/calbindin-negative ECL2 neurons and address the role of FGF-2 in neuronal rescue.     

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.     

Kinetics of conformational changes associated with potassium binding to and release from Na+/K(+)-ATPase

Previous studies have demonstrated that embryonal carcinoma (EC) cells express both fibroblast growth factor-4 (FGF-4) and FGF receptors. It has also been established that differentiation of EC cells represses the expression of the FGF-4 gene. Currently, the role of FGF-4 in the growth and differentiation of EC cells is unclear. In this study, we examined whether the differentiation of EC cells requires the repression of FGF-4 expression. To address this and related questions, F9 EC cells were transfected with an expression vector that uses the human beta-actin promoter to drive the constitutive expression of recombinant FGF-4. Unlike their untransfected counterparts, F9 EC cells transfected with this plasmid continue to produce recombinant FGF-4 after they differentiate. However, constitutive expression of this growth factor does not block morphological differentiation of the cells, nor does it alter the expression of six genes regulated by the differentiation of EC cells. Constitutive expression of recombinant FGF-4 also did not noticeably alter the growth of the transfected F9 EC cells before or after differentiation. Furthermore, unlike immortalized fibroblasts, which are known to grow in soft agar after transfection with FGF-4 expression plasmids, continued expression of recombinant FGF-4 activity did not enhance the ability of the EC-derived differentiated cells to form colonies in soft agar. These findings argue that continuous expression of recombinant FGF-4 activity does not block the differentiation of EC cells and that repression of the FGF-4 gene after EC cells differentiate does not appear, on its own, to be responsible for the loss of tumorigenicity that accompanies the differentiation of EC cells.     

Synaptic density in the inner molecular layer of the hippocampal dentate gyrus in Alzheimer disease

We examined the inner molecular layer (IML) of the hippocampal dentate gyrus for possible changes in synaptic density. Material was obtained from 9 individuals with Alzheimer disease (AD) and compared to samples obtained from 10 age-matched, postmortem-matched neurologically normal controls, employing standard ultrastructural techniques. Statistical analyses demonstrated a significant decline in synaptic numbers between controls and AD subjects. This decline was accompanied by a significant increase in apposition length and resulted in a significant correlation with the synaptic density. As the number of synapses declined, the apposition length increased. Assessment was also made of the granule cells density and the analyses showed a significant decline in the synapse to granule cell ratio in the AD group. This decline in the density of synaptic contacts in the IML reflects a more widespread decline in plasticity in AD and may be related to the memory problems associated with the disease.     

Automatic weighing of individual laying hens in aviary housing systems

Administration of a goitrogen (methimazole) and a low iodine diet to rats over a two-week period resulted in hypothyroidism and thyroid hyperplasia compared with controls (control: total serum thyroxine (T4) 66 +/- 4 nmol/l, thyroid weight 5 +/- 1 mg/100 g body weight; experimental: T4 undetectable, thyroid weight 27 +/- 4 mg/100 g body weight after 2 weeks of treatment; mean +/- S.D., n = 10). Immunohistochemistry carried out using a specific endothelial cell marker, CD31, and morphometric analysis (point counting of immunopositive cells) revealed that the progression of goitre in the rat thyroid is accompanied by an increase in capillary endothelial cell growth (neovascularisation). Fibroblast growth factor-2 (FGF-2) immunohistochemistry revealed widespread staining for the protein in the follicular cells of control glands. Less intense staining was found in the stroma and follicular cell nuclei. During hyperplasia and subsequent neovascularisation there was a progressive increase in the FGF-2 immunoreactivity at all locations during the two-week treatment period. Thrombospondin-1 (TSP1) immunoreactivity in the control rat thyroid was found in the stroma and in the endothelial cells, while weak follicular cell staining was also present. In the goitrous rat thyroid the TSP immunoreactivity was present after 1 week of treatment in the endothelial cells and most follicular cells, whilst stroma localisation was weak. After week 2 of treatment the endothelial cell and stromal localisation was no longer apparent, although a follicular localisation was still present. Transforming growth factor-beta 1 (TGF beta 1) immunoreactivity was present in the cytoplasm of a minority of the follicular cells in control rat thyroids, while their nuclei were unstained. In the goitrous rat thyroid an increase intensity of staining for TGF beta 1 was seen in all follicular cells, many of which now also demonstrated immuno-positive nuclei, within one week of goitrogen administration. These results show that in the hyperplastic thyroid increases in FGF-2 and TGF beta 1, and decreases in TSP1 accompany angiogenesis. These factors may interact in an autocrine/paracrine relationship to stimulate the neovascularisation that occurs during goitre formation.     

Membrane potential of hepatic mitochondria after acute cocaine administration in rats--the role of mitochondrial reduced glutathione

Occurrence of the classical pathway complement proteins C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9 was studied in human hippocampus and temporal cortex by immunohistochemistry and Western blotting. In Alzheimer disease (AD) cases, positive staining for all of these proteins was observed in pyramidal neurons and senile plaques. In control cases, weaker pyramidal neuron staining was observed except for C1q and C1s which were not detected. On Western blots of AD hippocampal extracts, bands corresponding to those detected in normal serum were found for each of the complement proteins. Comparable bands were also detected in normal hippocampal extracts with the exception of C1s which was not observed. The intensity of the bands was generally stronger in AD than in normal extracts, but, in the latter, there was considerable variability between cases and between bands in a single case. These data suggest that pyramidal neurons may be a source of the complement components known to be associated with Alzheimer lesions.     

Distribution of fibroblast growth factor (FGF)-2 and FGF receptor-1 messenger RNA expression and protein presence in the mid-trimester human fetus

Fibroblast growth factors (FGF) are known to have key roles in embryonic growth and morphogenesis, but their presence and contributions to fetal development are unclear. In particular, little information exists as to the relevance of FGF and their specific receptors to human fetal development. We studied the anatomical distribution of messenger RNA encoding FGF-2 and one of its high affinity receptors, FGFR1, using in situ hybridization in a variety of human fetal tissues in early second trimester. Corresponding protein distributions were determined by immunohistochemistry. Both FGF-2 and FGFR1 mRNA and proteins were found to be present in every organ and tissue examined, but with defined cellular localizations. In skeletal muscle, both FGF-2 and FGFR1 mRNA and peptides were present in differentiated fibers, and both co-localized to proliferating chondrocytes of the epiphyseal growth plate. FGF-2 and FGFR1 mRNA and peptides were also present within cardiac or gastrointestinal smooth muscle. Within the gastrointestinal tract FGF-2 mRNA and peptide were located in the submucosal tissue, whereas FGFR1 was expressed within the overlying mucosa. Similarly, in skin, FGF-2 was expressed within the dermis whereas FGFR1 mRNA and peptide were most apparent in the stratum germinativum of the epidermis. In kidney and lung, FGFR1 mRNA was located in the tubular and alveolar epithelia respectively, whereas FGF-2 was expressed in both epithelial and mesenchymal cell populations. Both growth factor and receptor were widespread in both neuroblasts and glioblasts in the cerebral cortex of the brain. Immunoreactivity for FGF-2 and FGFR1 was seen in all vascular endothelial cells of major vessels and capillaries. Within the skin, kidney, lung, and intestine FGF-2 immunoreactivity was found in basement membranes underlying epithelia, and was associated with the extracellular matrix and plasma membranes of many cell types. The results show that FGF-2 and one of its receptors are widely expressed anatomically in the mid-trimester human fetus.     

Association of memory and cognition in Alzheimer's disease with volumetric estimates of temporal lobe structures.

The hypothesis that explicit memory impairment in Alzheimer's disease (AD) depends in part on hippocampal formation atrophy was tested in 47 persons with AD. Volumes of the hippocampal formation, parahippocampal gyrus, and temporal neocortex (excluding the hippocampal formation, amygdala, and parahippocampal gyrus) were estimated by reconstruction of magnetic resonance images. Tests of explicit memory, language, and constructional praxis were administered. Psychometric-volumetric associations were evaluated in regression analyses controlling for age, gender, education, and intracranial volume. Hippocampal formation volume was associated with a delayed-recall measure but not with immediate recall: temporal neocortical volume was correlated with performance on measures of language and constructional praxis. The results suggest that patterns of mnemonic and cognitive impairment in AD are due in part to differences in the distribution of pathology in the temporal lobe. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

FGF-3 and FGF-4 elicit distinct oncogenic properties in mouse mammary myoepithelial cells

Fibroblast Growth Factors 3 (FGF-3) and 4 (FGF-4) were compared for the effects they each exert on EF43 mouse cells. This non-transformed mammary cell line appears to be myoepithelial mainly because it expresses alpha-smooth muscle actin. The EF43 cells were infected with similar vectors that carry either the short fgf-3 sequence (the product of which goes into the secretory pathway), fgf-4 or the selection gene only as control. In syngeneic animals, EF43.fgf-3 cells were tumorigenic only when orthotopically implanted whereas EF43.fgf-4 cells invariably gave rise to aggressive tumors. However, both tumor types were metastatic as evidenced by the blue micrometastases observed when the implanted cells expressed lacZ. In vitro, the FGF-3 producing cells were strongly invasive in matrigel coated chambers whereas the EF43.fgf-4 cells only were invasive in type I-collagen gels. Interestingly, FGF-3 production greatly stimulated the synthesis of pro-MMP-9 (Matrix Metalloprotease-9) and, to a lesser extent, that of pro-MMP-2. FGF-3 also up-regulated the production of plasminogen activators. In contrast, FGF-4 had no effect on these secretions and the medium conditioned by the EF43.fgf-4 cells displayed the largest plasminogen activator-inhibitor activity. These results show that FGF-3 and FGF-4 have distinct mechanisms of action on myoepithelial cells.     

Effect of chronic hypoxia on adrenoceptor responses of ovine foetal umbilical vessels

OBJECTIVE: To evaluate the relevance of hypometabolism in the hippocampal head to the pathophysiology of memory impairment. BACKGROUND: Neurofunctional imaging studies with an image reslicing technique provided by using software suggest that dysfunction of the amygdalohippocampal system causes memory impairment. However, metabolic and morphologic profiles of the whole hippocampal formation have not been evaluated in detail. METHODS: By tilting the gantry of a high-resolution PET scanner in a plane parallel to the hippocampal longitudinal axis determined beforehand by MRI, we performed quantitative measurement of glucose metabolism in the subdivisions of the hippocampal formation (head, body, tail) in 10 patients of normal intelligence with pure amnesia, in eight patients with AD, and in eight normal subjects. RESULTS: Although the volumes of the amygdala and hippocampal formation in pure amnesics were not different significantly from those of normal subjects, glucose metabolism in the head of the hippocampus was significantly lower in pure amnesics. In patients with AD, marked hypometabolism was found extending to the amygdala, the hippocampal head, and the parietotemporal cortex, along with amygdalohippocampal atrophy. CONCLUSION: Hippocampal head dysfunction plays an important role in memory impairment in amnesic patients. Further metabolic impairment over the amygdalohippocampal system and the surrounding association cortex reflects the pathophysiology of AD.     

Alzheimer's-specific effects of soluble beta-amyloid on protein kinase C-alpha and -gamma degradation in human fibroblasts

Alzheimer's disease (AD) is a multifactorial disease in which beta-amyloid peptide (betaAP) plays a critical role. We report here that the soluble fraction 1-40 of betaAP differentially degrades protein kinase C-alpha and -gamma (PKCalpha and PKCgamma) isoenzymes in normal (age-matched controls, AC) and AD fibroblasts most likely through proteolytic cascades. Treatment with nanomolar concentrations of betaAP(1-40) induced a 75% decrease in PKCalpha, but not PKCgamma, immunoreactivity in AC fibroblasts. In the AD fibroblasts, a 70% reduction of the PKCgamma, but not PKCalpha, immunoreactivity was observed after betaAP treatment. Preincubation of AC or AD fibroblasts with 50 microM lactacystine, a selective proteasome inhibitor, prevented beta-AP(1-40)-mediated degradation of PKCalpha in the AC cells, and PKCgamma in the AD fibroblasts. The effects of betaAP(1-40) on PKCalpha in AC fibroblasts were prevented by inhibition of protein synthesis and reversed by PKC activation. A 3-hr treatment with 100 nM phorbol 12-myristate 13-acetate restored the PKCalpha signal in treated AC cells but it did not reverse the effects of betaAP(1-40) on PKCgamma in the AD fibroblasts. Pretreatment with the protein synthesis inhibitor, cycloheximide (CHX, 100 microM), inhibited the effects of betaAP(1-40) on PKCalpha and blocked the rescue effect of phorbol 12-myristate 13-acetate in AC fibroblasts but did not modify PKCgamma immunoreactivity in AD cells. These results suggest that betaAP(1-40) differentially affects PKC regulation in AC and AD cells via proteolytic degradation and that PKC activation exerts a protective role via de novo protein synthesis in normal but not AD cells.     

Abeta associated neuropil changes: correlation with neuronal loss and dementia

Although genetic studies clearly implicate beta-amyloid peptide (Abeta) as a pathogenic agent in Alzheimer disease (AD), it is puzzling that the total amount of Abeta immunoreactivity does not correlate closely with neuronal loss or degree of dementia. We hypothesized that Abeta deposits could vary in the extent to which they disrupt the neuropil, and that the degree to which this occurs might then correlate with the degree of dementia. We used 3 dimensional triple immunofluorescent confocal microscopy to examine the fine structural relationships between Abeta deposits and neurites in their vicinity. In non-demented elderly, Abeta deposits were porous structures with numerous normal appearing processes coursing through them. In AD, dendrites within Abeta deposits, compared with dendrites in the surrounding neuropil, were likely to have decreased SMI32 immunoreactivity and increased Alz-50 immunoreactivity. We found that the degree to which Abeta deposits disrupt the neuropil, as assessed by local loss of SMI32 immunoreactivity, correlates closely with the amount of neuronal loss and with duration of dementia. These observations support the hypothesis that a subset of Abeta deposits contribute directly to neural system failure in AD.     

In situ hybridization analysis of presenilin 1 mRNA in Alzheimer disease and in lesioned rat brain

Presenilin-1 (PS-1) gene mutations are responsible for the majority of the early onset familial forms of Alzheimer disease (AD). Neither PS-1's anatomic distribution in brain nor expression in AD have been reported. Using in situ hybridization in the rat forebrain, we show that PS-1 mRNA expression is primarily in cortical and hippocampal neurons, with less expression in subcortical structures, in a regional pattern similar to APP695. Excitotoxic lesions lead to loss of PS-1 signal. A neuronal pattern of expression of PS-1 mRNA was also observed in the human hippocampal formation. AD and control levels did not differ. PS-1 is expressed in brain areas vulnerable to AD changes more so than in areas spared in AD; however, PS-1 expression is not sufficient to mark vulnerable regions. Collectively, these data suggest that the neuropathogenic process consequent to PS-1 mutations begins in neuronal cell populations.