Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized

We have investigated the synthesis, axonal transport, and processing of the beta-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a t1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]-methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is approximately 110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

Degeneration in vivo of rat hippocampal neurons by wild-type Alzheimer amyloid precursor protein overexpressed by adenovirus-mediated gene transfer

In an attempt to elucidate the pathological implications of intracellular accumulation of the amyloid precursor protein (APP) in postmitotic neurons in vivo, we transferred APP695 cDNA into rat hippocampal neurons by using a replication-defective adenovirus vector. We first improved the efficiency of adenovirus-mediated gene transfer into neurons in vivo by using hypertonic mannitol. When a beta-galactosidase-expressing recombinant adenovirus suspended in 1 M mannitol was injected into a dorsal hippocampal region, a number of neurons in remote areas were positively stained, presumably owing to increased retrograde transport of the virus. When an APP695-expressing adenovirus was injected into the same site, part of the infected neurons in the hippocampal formation underwent severe degeneration in a few days, whereas astrocytes near the injection site showed no apparent degeneration. These degenerating neurons accumulated different epitopes of APP, and beta/A4 protein (Abeta)-immunoreactive materials were undetected in the extracellular space. A small number of degenerating neurons showed nuclear DNA fragmentation. Electron microscopic examinations demonstrated that degenerating neurons had shrunken perikarya along with synaptic abnormalities. Microglial cells/macrophages were often found in close proximity to degenerating neurons, and in some cases they phagocytosed these neurons. These results suggest that intracellular accumulation of wild-type APP695 causes a specific type of neuronal degeneration in vivo in the absence of extracellular Abeta deposition.     

Immunological features of patients with primary biliary cirrhosis (PBC) overlapping systemic sclerosis: a comparison with patients with PBC alone

Mutations within exons 16 and 17 of the beta-amyloid precursor protein (APP) gene were the first known cause of familial Alzheimer's disease. These mutations are rare and have been reported in a handful of families exhibiting autosomal dominant inheritance of Alzheimer's disease with age of onset around 50 years. In vitro and in vivo studies have demonstrated that each of these mutations alters proteolytic processing of APP, resulting in an increase in the production of A beta 42, a highly fibrillogenic peptide, that spontaneously aggregates and deposits in the brain. Transgenic mice carrying a mutant human APP gene also show age-dependent beta-amyloid (A beta) deposition in the brain. The rate of deposition in these mice can be modified by apolipoprotein E expression.     

Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons

Biochemical and molecular mechanisms of neuronal cell death are currently an area of intense research. It is well documented that the lumbar spinal motoneurons of the chick embryo undergo a period of naturally occurring programmed cell death (PCD) requiring new gene expression and activation of caspases. To identify genes that exhibit changed expression levels in dying motoneurons, we used a PCR-based subtractive hybridization protocol to identify messages uniquely expressed in motoneurons deprived of trophic support as compared with their healthy counterparts. We report that one upregulated message in developing motoneurons undergoing cell death is the mRNA for amyloid precursor protein (APP). Increased levels of APP and beta-amyloid protein are also detected within dying motoneurons. The predicted peptide sequence of APP indicates two potential cleavage sites for caspase-3 (CPP-32), a caspase activated in dying motoneurons. When peptide inhibitors of caspase-3 are administered to motoneurons destined to undergo PCD, decreased levels of APP protein and greatly reduced beta-amyloid production are observed. Furthermore, we show that APP is cleaved by caspase-3. Our results suggest that differential gene expression results in increased levels of APP, providing a potential substrate for one of the cell death-activated caspases that may ultimately cause the demise of the cell. These results, combined with information on the toxic role of APP and its proteolytic by-product beta-amyloid, in the neurodegenerative disease Alzheimer's, suggest that events of developmental PCD may be reactivated in early stages of pathological neurodegeneration.     

Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path

Amyloid deposition is a neuropathological hallmark of Alzheimer's disease. The principal component of amyloid deposits is beta amyloid peptide (Abeta), a peptide derived by proteolytic processing of the amyloid precursor protein (APP). APP is axonally transported by the fast anterograde component. Several studies have indicated that Abeta deposits occur in proximity to neuritic and synaptic profiles. Taken together, these latter observations have suggested that APP, axonally transported to nerve terminals, may be processed to Abeta at those sites. To examine the fate of APP in the CNS, we injected [35S]methionine into the rat entorhinal cortex and examined the trafficking and processing of de novo synthesized APP in the perforant pathway and at presynaptic sites in the hippocampal formation. We report that both full-length and processed APP accumulate at presynaptic terminals of entorhinal neurons. Finally, we demonstrate that at these synaptic sites, C-terminal fragments of APP containing the entire Abeta domain accumulate, suggesting that these species may represent the penultimate precursors of synaptic Abeta.     

Increase in extracellular calcium in the optic tectum of fish after optic nerve transection

In this study the extracellular distribution of cytochemically generated calcium reaction product in the denervated optic tectum of a cichild fish (Oreochromis mossambicus) was investigated. The left optic nerve had been transected and the fish (5 per experimental condition) maintained for 2, 10 and 21 days. The amount of the calcium-containing precipitates was estimated using energy-filtering transmission electron microscopy (EFTEM) and image analysis. A special degeneration type of the optic terminals (neurofibrillar hypertrophy) was found which seems to be rare in other teleosts and was therefore chosen for quantification of the calcium deposits. These terminals are surrounded by astroglial processes and the calcium reaction product in the extracellular spaces between these glial processes and the terminals was measured and compared to normal optic terminals in nonoperated controls. A distinct and significant increase in the amount of calcium deposits was found 2 and 10 days after surgery which decreased to control levels after 21 days. This rise of deposits around the degenerating terminals was very local as arbitrarily selected extracellular spaces near these terminals showed values which were at the level of the nonoperated controls. Therefore, a transient and local increase in extracellular calcium precipitates was found after optic nerve transection which affected only the degenerating synapses.     

Intracellular calcium parallels motoneuron degeneration in SOD-1 mutant mice

Transgenic mice with Cu,Zn superoxide dismutase (SOD-1) mutations provide a unique model to examine altered Ca homeostasis in selectively vulnerable and resistant motoneurons. In degenerating spinal motoneurons of G93 A SOD-1 mice, developing vacuoles were filled with calcium, while calcium was gradually depleted from the cytoplasm and intact mitochondria. In oculomotor neurons, no degenerative changes, vacuolization, or increased calcium were noted. Motor axon terminals of interosseus muscle gradually degenerated and intracellular calcium was depleted. Oculomotor terminals of mutant SOD-1 mice were smaller and exhibited no degenerative changes, but did exhibit unique membrane-enclosed organelles containing calcium. Spinal motoneurons of SOD-1 mice were shown to have fewer calcium binding proteins, such as parvalbumin, compared with oculomotor neurons. These data suggest that the SOD-1 mutation is associated with impaired calcium homeostasis in motoneurons in vivo, with increased likelihood of degeneration associated with higher levels of intracellular calcium and lower to absent levels of calbindin-D28K and/or parvalbumin, and decreased likelihood of degeneration associated with minimally changed calcium and ample calbindin-D28K and/or parvalbumin.     

Upregulation of actin gene expression in cells expressing exogenous beta-amyloid precursor protein

We investigated the effects of beta-amyloid peptide precursor (APP) overexpression upon the levels of other mRNAs. Using quantitative slot-blot hybridization and immunoblot analysis we observed that enhanced levels of APP elevated the levels of beta-actin and beta-actin mRNA. Our results also suggest that the cytoplasmic domain of APP is crucial for the elevation in beta-actin gene expression.     

Thyroid hormones regulate beta-amyloid gene splicing and protein secretion in neuroblastoma cells

The beta-amyloid protein (Abeta), the major component of the senile plaques found in Alzheimer brains, derives from a larger beta-amyloid precursor protein (APP). Alternative splicing of the APP gene yields three major APP messenger RNAs (mRNAs), which, in turn, give rise to the APP770, APP751, and APP695 protein isoforms. In this study we examined the effects of thyroid hormone on APP expression in N2a-beta neuroblastoma cells. T3 caused a significant increase in the APP770 mRNA band, in detriment of the APP695 mRNA, which was proportionately reduced. In agreement with these results, T3 markedly altered the relative ratio of intracellular APP isoforms, increasing the amount of APP770 and causing an equivalent reduction of the immature APP695 isoform. In accordance with these results, the soluble APP695-derived form was specifically reduced in the culture medium obtained from T3-treated cells. In contrast, the increase in intracellular APP770 was not followed by an enhanced release of soluble derivatives of this isoform. These results suggest that T3 regulates not only APP gene splicing, but also the processing and secretion of the APP peptides. According to our results, thyroid hormone might play a role in the development of Alzheimer's disease by modulating the intracellular and extracellular contents of APP isoforms.     

Presenilin 1 regulates the processing of beta-amyloid precursor protein C-terminal fragments and the generation of amyloid beta-protein in endoplasmic reticulum and Golgi

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.     

Axonal contact regulates expression of alpha2 and beta2 isoforms of Na+, K+-ATPase in Schwann cells: adhesion molecules and nerve regeneration

Three isoforms of catalytic alpha subunits and two isoforms of beta subunits of Na+,K+-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na+,K+-ATPase was highly resistant to ouabain. The ouabain-resistant alpha1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na+,K+-ATPase activity. After sciatic nerve injury, the alpha3 and beta1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, alpha2 and beta2 isoform expression and Na+,K+-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the alpha2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that alpha3 and beta1 isoforms are exclusive for the axon and alpha2 and beta2 isoforms are exclusive for the Schwann cell, although axonal contact regulates alpha2 and beta2 isoform expressions. Because the beta2 isoform of Na+,K+-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/beta2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/beta2 may act as an adhesion molecule in peripheral nerve regeneration.     

Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called beta-amyloid (Abeta) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Abeta that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-beta-cyclodextrin, we show that the formation of Abeta is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Abeta formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Abeta formation to occur and imply a link between cholesterol, Abeta, and Alzheimer's disease.     

Rapid increase in amyloid precursor protein immunoreactivity in the supraoptic and paraventricular nuclei of the rat hypothalamus after osmotic stress

The effects of water deprivation or i.p. injection of hypertonic salt solution on the expression of the amyloid precursor polypeptide (APP) were studied immunohistochemically in the rat brain, in particular in the supraoptic and paraventricular nuclei, both known to be involved in electrolytic and water homeostasis and to contain mRNAs coding for the various forms of APP. In parallel, the expression of the immediate early gene c-fos was also studied by immunohistochemistry. Both hypertonic saline injection and water deprivation resulted in a rapid and dramatic increase in the levels of amyloid precursor protein-like immunoreactivity in neurones of the supraoptic and paraventricular nuclei. These increases paralleled those seen using c-fos immunohistochemistry. In contrast, no changes were observed in other brain areas, including the subfornical organ, which also contained mRNA and APP-like immunoreactivity. The results indicate that levels of the beta-amyloid precursor protein can be rapidly increased by stressors affecting the activity of well characterized cell populations in the rat hypothalamus. These results suggest the involvement of the beta-amyloid precursor protein in the secretory activities of these cells, or in the initiation of morphological changes which are known to occur after osmotic stress in the supraoptic and paraventricular neurones. Interestingly, the changes were limited to neurones and no modification of beta-amyloid precursor protein levels was observed in glial cells, which are also known to be modified by osmotic stress.     

The beta-amyloid epitope masking activity in human brain is identified as albumin

Human brain homogenate proteins were analyzed for binding and processing activity in relation to brain beta-amyloid precursor protein (APP). The homogenate was purified by arginine-Sepharose 4B affinity chromatography, which traps proteins with affinity to certain groups of arginine residue, such as serine proteases and zymogens. A 69 kDa protein that masks epitope(s) of brain APP was found in a weakly bound fraction. The nature of the 69 kDa brain protein was identified as albumin by N-terminal amino acid sequencing and Western blot analysis using anti-human albumin antibody. Western blot analysis with domain-specific anti-APP antibodies revealed that the masking activity is complete for beta-amyloid epitope(s), but incomplete for cytoplasmic and extracellular domain epitopes, suggesting that the interaction site of the albumin is beta-amyloid itself. Therefore, it seems that brain albumin is not merely a carrier protein for beta-amyloid in cerebrospinal fluid, but also a modulator which interferes with processing of beta-amyloid precursor protein and its peptides.     

Immunohistochemical studies on neurofilamentous hypertrophy in degenerating retinal terminals of the olivary pretectal nucleus in the rat

Following section of the optic nerve, degenerating retinal terminals reveal an accumulation of neurofilaments (neurofilamentous hypertrophy) as demonstrated by silver impregnation techniques or electron microscopy. The present study examined degenerating retinal terminals by means of immunohistochemistry and antibodies specific for the triplet of neurofilament proteins of low (NF-L), medium (NF-M), and high (NF-H) molecular weight class. Following unilateral optic nerve section in the rat and survival of 1, 2, 4, 8, and 21 days, brains were perfused with aldehyde fixative, sliced on a vibratome and stained for neurofilaments by using the peroxidase-antiperoxidase technique. Other brains were frozen, cut in the native state, and slide-mounted sections were fixed by acetone. Side comparisons in visual pathways were made in frontal sections, taking advantage of the near complete crossing of retinal fibers in the rat. Anterograde degeneration of axons occurred in the optic tract and branchium colliculi. Changes of terminals were investigated in the olivary pretectal nucleus, which contains a dense aggregation of retinal terminals in the core region. The optic tract and branchium colliculi showed a reduction in immunostaining for neurofilament proteins following axotomy. Within the core region of the olivary pretectal nucleus, strong increases of immunoreactivity of NF-L and NF-M were detected beginning at 2 days postlesion and persisting at 8 days. No changes in NF-H proteins were found in the terminal regions with three different antibody probes. The increase in immunostaining reflects the accumulation of neurofilament proteins in the degenerating retinal terminals, i.e., neurofilamentous hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)     

A strategy for designing inhibitors of beta-amyloid toxicity

beta-Amyloid peptide is the major protein component of Alzheimer's plaques. When aggregated into amyloid fibrils, the peptide is toxic to neuronal cells. Here, an approach to the design of inhibitors of beta-amyloid toxicity is described; in this strategy, a recognition element, which interacts specifically with beta-amyloid, is combined with a disrupting element, which alters beta-amyloid aggregation pathways. The synthesis, biophysical characterization, and biological activity of such an inhibitor is reported. This prototype inhibitor is composed of residues 15-25 of beta-amyloid peptide, designed to function as the recognition element, linked to an oligolysine disrupting element. The inhibitor does not alter the apparent secondary structure of beta-amyloid nor prevent its aggregation; rather, it causes changes in aggregation kinetics and higher order structural characteristics of the aggregate. Evidence for these effects includes changes in fibril morphology and a reduction in thioflavin T fluorescence. In addition to its influence on the physical properties of beta-amyloid aggregates, the inhibitor completely blocks beta-amyloid toxicity to PC-12 cells. Together, these data suggest that this general strategy for design of beta-amyloid toxicity inhibitors is effective. Significantly, these results demonstrate that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity.     

A comprehensive study of the spatiotemporal pattern of beta-amyloid precursor protein mRNA and protein in the rat brain: lack of modulation by exogenously applied nerve growth factor

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons, a population that degenerates and dies in Alzheimer's disease (AD). It has been suggested that NGF be used to treat AD patients. However, in vivo administration of NGF to the developing hamster brain was shown to induce the expression of the beta-amyloid precursor protein (beta APP) gene. The association of alterations in beta APP gene expression with AD-like neuropathological changes and cognitive impairment in animals, and with AD-like neurodegeneration in Down syndrome patients suggests that NGF-mediated increases in beta APP expression could negate or attenuate NGF's neurotrophic activity in AD treatment trials. The present study was undertaken to explore further the influence of NGF on beta APP expression, and to determine which, if any, of the beta APP mRNAs is altered in response to NGF treatment. We first examined the spatiotemporal pattern of beta APP-695 and Kunitz protease inhibitor (KPI)-containing beta APP mRNA expression in the rat brain. Specific oligonucleotide probes were used to show that these mRNAs are present during embryonic development. In addition, we evaluated postnatal expression in nine brain regions and showed that beta APP mRNAs were readily detected in all regions at postnatal day 2. In human brain, the relative levels of beta APP-695 and beta APP-KPI mRNA and their protein are discordant, in that the level of beta APP-695 mRNA is slightly higher than that of beta APP-KPI, but beta APP-KPI protein predominates. In contrast, the several-fold excess of beta APP-695 mRNA relative to beta APP-KPI mRNA in the rat brain was also reflected at the protein level. Surprisingly, administration of exogenous NGF failed to affect rat beta APP mRNA levels either in vitro or during postnatal development in vivo.