Expression of reticulon 3 in Alzheimer's disease brain.

AIMS: Reticulon 3 (RTN3), a member of the reticulon family of proteins, interacts with the beta-secretase, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and inhibits its activity to produce beta-amyloid protein. The aim of the present study was to clarify the biological role of RTN3 in the brain and its potential involvement in the neuropathology of Alzheimer's disease (AD). METHODS: We performed immunohistochemical and biochemical analyses using a specific antibody against RTN3 to investigate the expression and subcellular localization of RTN3 in control and AD brain tissue samples. RESULTS: Western blot analysis revealed no significant differences in the RTN3 levels between control and AD brains. Immunohistochemical staining showed that RTN3 immunoreactivity was predominantly localized in pyramidal neurones of the cerebral cortex. The patterns of RTN3 immunostaining were similar in control and AD cerebral cortices, and senile plaques were generally negative for RTN3. Biochemical subcellular fractionation disclosed that RTN3 colocalized with BACE1 in various fractions, including the endoplasmic reticulum and the Golgi apparatus. Double-immunofluorescence staining additionally indicated that RTN3 was localized in both endoplasmic reticulum and Golgi compartments in neurones. CONCLUSIONS: These results show that RTN3 is primarily expressed in pyramidal neurones of the human cerebral cortex and that no clear difference of RTN3 immunoreactivity is observable between control and AD brains. Our data also suggest that there is considerable colocalization of RTN3 with BACE1 at a subcellular level.

Pro-apoptotic effect of presenilin 2 (PS2) overexpression is associated with down-regulation of Bcl-2 in cultured neurons.

Presenilin 2 (PS2) is a polytopic membrane protein that is mutated in some cases of familial Alzheimer's disease (AD). The normal functions of PS2 and its pathogenic role in AD remain unclear. We investigated the biological role of this protein in neurons, using adenovirus-mediated transduction of the PS2 gene into rat primary cortical neurons. Immunocytochemical analyses demonstrated increased PS2 immunoreactivity in most neurons infected with recombinant adenoviruses expressing PS2. Neurons infected with wild-type or mutant (N141I) PS2-expressing adenoviruses showed a significant increase in basal cell death, compared with those infected with control beta-galactosidase-expressing adenovirus. Moreover, PS2 overexpression markedly increased neuronal susceptibility to staurosporine-induced apoptosis. Mutant PS2 was more effective in enhancing apoptosis than its wild-type counterpart. Staurosporine-induced death was significantly inhibited by a specific caspase 3 inhibitor. Western analyses revealed that Bcl-2 protein expression was specifically down-regulated in neurons overexpressing PS2, which temporally corresponded to the accumulation of C- and N-terminal fragments of PS2. Additionally, expression of mutant, but not wild-type PS2, increased the production of beta-amyloid protein (Abeta) 42. These data collectively suggest that the pro-apoptotic effect of PS2 is mediated by down-regulation of Bcl-2. PS2 mutations may increase the susceptibility of neurons to apoptotic stimuli by perturbing the regulation of cell death.

Familial frontotemporal dementia and parkinsonism with a novel mutation at an intron 10+11-splice site in the tau gene.

We report a case of familial frontotemporal dementia and parkinsonism characterized by early onset with mental retardation. The patient died at the age of 54; neuronal loss was severe in the frontal and temporal cortices, globus pallidus, substantia nigra, red nucleus and dentate nucleus. Anti-tau-positive fibrillary changes were observed in neurons and glia in these regions. Although the patient had 2 novel point mutations of the tau gene, P301P (CCG to CCA) and an intron 10+11-splice site (T to C), exon trapping analysis indicated that the latter was pathogenic.

Apoptotic neurons in Alzheimer's disease frequently show intracellular Abeta42 labeling.

It is widely accepted that Abeta plays a pivotal role in the pathogenesis of Alzheimer's disease (AD) [27]. Attention has been focused mainly on how extracellular Abeta exerts its effects on neuronal cells [7,11,16,32]. However, neuronal degeneration from an accumulation of intracellular Abetax-42 (iAbeta42) occurs in presenilin 1 (PS1) mutant mice without extracellular Abeta deposits [5]. In the present study, intracellular deposits of iAbeta42 are correlated with apoptotic cell death in AD and PS-1 familial AD (PS1 FAD) brains by means of triple staining with antibodies to Abeta, TUNEL, and staining with Hoechst 33342. Neurons simultaneously positive for iAbeta42 and the TUNEL assay were significantly more abundant in AD brains than in controls. The number of apoptotic neurons with intracellular neurofibrillary tangles (iNFTs) was insignificant. Our results indicate that intraneuronal deposition of a neurotoxic form of Abeta seems to be an early event in the neurodegeneration of AD.

Mutational analysis of intrinsic regions of presenilin 2 that determine its endoproteolytic cleavage and pathological function.

To investigate the significance of endoproteolytic processing of presenilin 2 (PS2) on its pathological function, we constructed PS2 cDNAs causing amino acid substitutions or deletions around the cleavage site. We found that a PS2 mutant (Del3) with a 20-amino acid deletion was not endoproteolytically processed, while other PS2s with amino acid substitutions and short deletions were cleaved. Overproduction of all the mutant proteins led to a compensatory decrease of endogenous PS1 fragments, but did not affect the amyloid beta peptide X-42/Abeta X-40 ratio without the familial Alzheimer's disease mutation. The Del3 mutant did not exhibit significant deficits in gamma-secretase activity. The turnover rate of the Del3 holoprotein was the same as that of full-length PS2. These data suggest that the determinants of the PS2 cleavage site reside within a large region and that the pathological function of PS2 is exerted by familial Alzheimer's disease mutations not related to the cleavage of holoproteins. We also found that PS2 with an 18-amino acid deletion at the C-terminal end was not processed. Its overexpression led neither to diminished accumulation of endogenous PS1 fragments nor to increased production of amyloid beta peptide X-42. The C-terminal end of PS2 seems to possess the signal for entry into the processing pathway.

Overexpression of presenilin-2 enhances apoptotic death of cultured cortical neurons.

Presenilin-2 (PS2) is a gene of unknown function linked with some forms of familial Alzheimer's disease. To investigate the biological role of PS2 in neurons, we overexpressed PS2 in primary cortical neurons using recombinant adenoviral vectors. Western blot and immunohistochemical analyses showed enhanced expression of PS2 proteins in infected neurons after infection of recombinant adenoviruses containing the human wild-type or mutant PS2 gene. Neuronal survival was decreased by approximately 30% in cultures infected with adenovirus expressing either wild-type or mutant PS2, as compared with those infected with adenovirus expressing the LacZ gene. Fragmented nuclei were frequently observed in dying neurons. These data suggest that apoptotic death of cultured cortical neurons is enhanced by PS2 overexpression.

Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of A beta1-42, indicating that PS-1 is involved in amyloidogenesis. However, PS-1 transgenic mice have failed to show amyloid plaques in their brains. Because PS-1 mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited A beta42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.

Increased expression of amyloid precursor protein and amyloid precursor-like protein 2 during trophic factor withdrawal-induced death of neuronal PC12 cells.

Programmed cell death (PCD) (apoptosis) is implicated in the neuronal cell death of Alzheimer's disease (AD). We investigated expression of amyloid precursor protein (APP) and amyloid precursor-like protein 2 (APLP2) during trophic factor deprivation-induced PCD of neuronally differentiated PC12 cells. Neuronal PC12 cells underwent PCD within two days following withdrawal of nerve growth factor (NGF) from the culture medium. Total APP mRNA levels increased gradually after 24 h, reaching levels 250% higher than those in control cells at 48 h after NGF withdrawal, and total APLP2 mRNA levels also increased similarly at 48 h. Analysis of the three major APP mRNA isoforms APP695, APP751, and APP770 by reverse transcription polymerase chain reaction showed a substantial increase in the proportion of APP770 at 48 h after NGF withdrawal. Basic fibroblast growth factor, which prevented the appearance of PCD after NGF withdrawal, inhibited the increases in APP and APLP2 mRNA levels as well as the increase in the proportion of APP770. Cellular holoprotein levels of total APP, APP containing the Kunitz protease inhibitor domain, and APLP2 also increased by approximately 60%, 100%, and 30%, respectively, at 48 h after NGF withdrawal. These data indicate that in neuronal PC12 cells undergoing PCD following trophic factor withdrawal, the syntheses of both APP and APLP2 are upregulated, and the alternative splicing of the APP gene is modified. This implies a linkage between APP and APLP2 expression and neuronal PCD.

How is membrane phospholipid biosynthesis controlled in neural tissues?

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.

Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth.

Phospholipids are the major components of cell membranes and are required for cellular growth. We studied membrane phosphatidylcholine (PtdCho) biosynthesis in neuronal cells undergoing neurite outgrowth, by using PC12 cells as a model system. When neurite outgrowth was induced by exposing PC12 cells to nerve growth factor for 2 and 4 days, the amounts of [14C]choline incorporated into [14C]phosphatidylcholine per cell (i.e., per DNA) increased approximately 5- and 10-fold, respectively, as compared with control cells, reflecting increases in the rate of PtdCho biosynthesis. [14C]choline uptake was not affected. Analysis of the three major PtdCho biosynthetic enzymes showed that the activity of CDPcholine:1,2-diacylglycerol cholinephosphotransferase was increased by approximately 50% after nerve growth factor treatment, but the activities of choline kinase or choline-phosphate cytidylyltransferase were unaltered; the cholinephosphotransferase displayed a high Km value ( approximately 1,200 microM) for diacylglycerol. Moreover, free cellular diacylglycerol levels increased by approximately 1.5- and 4-fold on the second and fourth days, respectively. These data indicate that PtdCho biosynthesis is enhanced when PC12 cells sprout neurites, and the enhancement is mediated primarily by changes in cholinephosphotransferase activity and its saturation with diacylglycerol. This suggests a novel regulatory role for diacylglycerol in membrane phospholipid biosynthesis.

Stimulation of amyloid precursor protein synthesis by adrenergic receptors coupled to cAMP formation.

Amyloid plaques in Alzheimer disease are primarily aggregates of Abeta peptides that are derived from the amyloid precursor protein (APP). Neurotransmitter agonists that activate phosphatidylinositol hydrolysis and protein kinase C stimulate APP processing and generate soluble, non-amyloidogenic APP (APPs). Elevations in cAMP oppose this stimulatory effect and lead to the accumulation of cell-associated APP holoprotein containing amyloidogenic Abeta peptides. We now report that cAMP signaling can also increase cellular levels of APP holoprotein by stimulating APP gene expression in astrocytes. Treatment of astrocytes with norepinephrine or isoproterenol for 24 h increased both APP mRNA and holoprotein levels, and these increases were blocked by the beta-adrenergic antagonist propranolol. Treatment with 8-bromo-adenosine 3',5'-cyclic monophosphate or forskolin for 24 h similarly increased APP holoprotein levels; astrocytes were also transformed into process-bearing cells expressing increased amounts of glial fibrillary acidic protein, suggesting that these cells resemble reactive astrocytes. The increases in APP mRNA and holoprotein in astrocytes caused by cAMP stimulation were inhibited by the immunosuppressant cyclosporin A. Our study suggests that APP overexpression by reactive astrocytes during neuronal injury may contribute to Alzheimer disease neuropathology, and that immunosuppressants can inhibit cAMP activation of APP gene transcription.

Secretion kinetics of Alzheimer's amyloid beta-protein differs from secreted beta-amyloid precursor protein.

Amyloid beta-protein (A beta) and secreted beta-amyloid precursor protein (sAPP), derived from beta-amyloid precursor protein (APP), are normally released by cultured mammalian cells. We investigated by pulse-chase analysis the secretion kinetics of these two APP derivatives using mouse cholinergic SN49 cell lines stably transfected with mouse APP695 cDNA. After both A beta and sAPP peaked at about the second hour, sAPP decreased with a half-life of approximately 5 hours, but A beta remained almost unchanged for at least 14 hours. These results indicate that A beta is more stable than sAPP in the SN49 conditioned medium.

Characterization of neurotrophic factors produced by immortalized mouse brain glial cells (VR-2g).

We analyzed the neurotrophic factors secreted by immortalized fetal mouse brain glial cells (VR-2g). Concentrated conditioned medium of VR-2g cells were applied to a gel filtration column and the trophic activities of the fractions were determined by the bioassay method with primary fetal rat striatal neurons. Several peaks of neurotrophic activity were detected, the most prominent of which was found in a fraction of molecular weight 5-7 kDa. In peak fractions of molecular weight 13-26 kDa, molecules reactive to anti-NGF antibodies were detected by Western blotting only in non-reduced condition of SDS-PAGE. Anti-NGF antibodies absorbed 40% of the neurotrophic activities in the conditioned medium of VR-2g cells.

The decrement of muscarinic receptor-mediated calcium influx by overexpression of APP in a mouse cholinergic cell line.

The effects of beta-amyloid precursor protein (APP) overexpressing on cell metabolisms of cholinergic neuronal hybridoma cell line (SN49) were examined. The cells stably overexpressing APP contained higher amount of GTP binding protein Go and cytosolic inactive protein kinase C epsilon, and showed less Ca2+ influx through muscarinic acetylcholine receptor ml compared to original and mock cells which had been transfected with a vector alone. The contents of sn-1, 2-diacylglycerol and cyclic AMP were also reduced in the APP transfectants, although the similar changes were observed in the mock cells. These findings strongly suggest that the overexpression of APP affect the transient receptor-mediated ion channel and calcium-related cell metabolisms in neuronal cells.

Demonstration of amyloid beta-protein secretion in a mouse neuronal cell line.

We investigated the secretion of amyloid beta-protein (beta AP) in a mouse neuronal cell line SN49. SN49 cells stably transfected with mouse beta-amyloid precursor protein (APP) 695 cDNA released approximately three times greater amounts of a 4 kDa protein immunoreactive with anti-beta AP antibodies than untransfected and mock-transfected cells. This 4 kDa protein was further identified as mouse beta AP by direct amino acid sequence analysis. These results strongly suggest that the beta AP secretion occurs in mouse neuronal cells as in human cells.

Immortalization of fetal mouse brain glial cells by human papillomavirus type 16 E7 genes.

Fetal mouse brain glial cells in primary cultures were immortalized by recombinant retroviruses containing human papillomavirus type 16 E7 genes, and named VR-2g cells. The presence and expression of E7 genes in VR-2g cells were demonstrated by the Southern and the Northern blot analyses. VR-2g cells did not form colonies in soft agar culture, indicating that VR-2g cells have no transforming phenotypes. By the karyotype analysis, VR-2g cells consisted of two cell populations, the pseudo-diploid and the pseudo-tetraploid. VR-2g cells were positive in immunostaining with mono- and polyclonal antibodies against glial fibrillary acidic proteins (glial-specific intermediate filaments). In addition, VR-2g cells secreted neurotrophic factors as assayed with primary cultures of fetal rat striatum neurons, although molecular characterization of the factor(s) was not yet determined. These results indicate that the present method for cell immortalization will be useful for establishing untransformed cell lines from primary cultures of fetal brain cells.

Trophic effect of beta-amyloid precursor protein on cerebral cortical neurons in culture.

We investigated the effect of human beta-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons.

Aberrant accumulation of phospholipase C-delta in Alzheimer brains.

Since phosphoinositide-specific phospholipase C (PLC) is one of the key molecules in signal transduction, the authors assessed its involvement in Alzheimer's disease (AD). Immunostaining of a specific antibody against the PLC isozyme, PLC-delta, demonstrated that this enzyme was abnormally accumulated in neurofibrillary tangles (NFT), the neurites surrounding senile plaque (SP) cores, and neuropil threads in AD brains. Western blot analysis confirmed that PLC-delta was concentrated in the paired helical filament (PHF)-rich fraction of AD brains. Antibodies to other PLC isozymes did not produce positive immunostaining of these pathologic structures. Moreover, diffuse and amorphous deposits of PLC-delta were found to precede the accumulation of fibrillary deposits. These results suggest that PLC-delta accumulation is a crucial event that ultimately may contribute to the formation of PHF.

Presence of calpain II immunoreactivity in senile plaques in Alzheimer's disease.

Calpains are calcium-dependent neutral cysteine proteinases. We utilized a specific anti-calpain II antibody to examine immunohistochemically whether calpain II is associated with pathological changes in Alzheimer's disease (AD). Calpain II was mainly expressed in the neurons in control human brains. In AD brains, intense immunoreactivity was present in the dystrophic neurites of senile plaques. These results suggest that calpain II is involved in the pathogenesis of AD.

Familial hypobetalipoproteinaemia complicated by cerebellar ataxia and steatocystoma multiplex.

A 55-year-old man with cerebellar ataxia and steatocystoma multiplex was found to have reduced serum concentrations of total cholesterol, betalipoprotein and apolipoprotein B. Computed tomography revealed atrophy of the cerebellum and brain stem. Of the six family members examined, four had hypobetalipoproteinaemia, and one had mild ataxia. Similar skin lesions were noted in five male relatives. This case represents a rare combination of familial hypobetalipoproteinaemia, cerebellar ataxia and steatocystoma multiplex.