Apoptosis in the nervous system.

Neuronal apoptosis sculpts the developing brain and has a potentially important role in neurodegenerative diseases. The principal molecular components of the apoptosis programme in neurons include Apaf-1 (apoptotic protease-activating factor 1) and proteins of the Bcl-2 and caspase families. Neurotrophins regulate neuronal apoptosis through the action of critical protein kinase cascades, such as the phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase pathways. Similar cell-death-signalling pathways might be activated in neurodegenerative diseases by abnormal protein structures, such as amyloid fibrils in Alzheimer's disease. Elucidation of the cell death machinery in neurons promises to provide multiple points of therapeutic intervention in neurodegenerative diseases.

Amyloid beta interacts with the amyloid precursor protein: a potential toxic mechanism in Alzheimer's disease.

Amyloid beta protein (Abeta) deposition in the brain is a hallmark of Alzheimer's disease (AD). The fibrillar form of Abeta is neurotoxic, although the mechanism of its toxicity is unknown. We showed that conversion of Abeta to the fibrillar form markedly increased binding to specific neuronal membrane proteins, including amyloid precursor protein (APP). Nanomolar concentrations of fibrillar Abeta bound cell-surface holo-APP in cortical neurons. Reduced vulnerability of cultured APP-null neurons to Abeta neurotoxicity suggested that Abeta neurotoxicity involves APP. Thus Abeta toxicity may be mediated by the interaction of fibrillar Abeta with neuronal membrane proteins, notably APP. An Abeta-APP interaction reminiscent of the pathogenic mechanism of prions may thus contribute to neuronal degeneration in AD.

Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta.

Apoptosis, or cellular suicide, is important for normal development and tissue homeostasis, but too much or too little apoptosis can also cause disease. The family of cysteine proteases, the so- called caspases, are critical mediators of programmed cell death, and thus far 14 family members have been identified. Some of these, such as caspase-8, mediate signal transduction downstream of death receptors located on the plasma membrane. Others, such as caspase-9, mediate apoptotic signals after mitochondrial damage. Stress in the endoplasmic reticulum (ER) can also result in apoptosis. Here we show that caspase-12 is localized to the ER and activated by ER stress, including disruption of ER calcium homeostasis and accumulation of excess proteins in ER, but not by membrane- or mitochondrial-targeted apoptotic signals. Mice that are deficient in caspase-12 are resistant to ER stress-induced apoptosis, but their cells undergo apoptosis in response to other death stimuli. Furthermore, we show that caspase-12-deficient cortical neurons are defective in apoptosis induced by amyloid-beta protein but not by staurosporine or trophic factor deprivation. Thus, caspase-12 mediates an ER-specific apoptosis pathway and may contribute to amyloid-beta neurotoxicity.

The pathogenesis of Alzheimer's disease. Is amyloid beta-protein the beginning or the end?

A central issue in Alzheimer's disease research is whether amyloid beta-protein (A beta) is a cause or effect of the pathogenic process. Several independent lines of evidence argue for causality, although human clinical trials may be required to finally settle the issue.

Normal proteolytic processing of the presenilins.

The majority of familial Alzheimer's disease (AD) cases are linked to mutations of the presenilin 1 and 2 (PS1, PS2) genes on chromosomes 14 and 1, respectively (1-3). PS1 and PS2 are about 67% identical in amino acid sequence. Based on hydrophobicity analysis, the presenilins are predicted to have multiple transmembrane domains. Structural analysis (see Chapter 19 }) suggest that presenilins are 6-8 transmembrane proteins which are located in the endoplasmic reticulum (ER) and Golgi. The N- and C-termini and the large hydrophilic loop region are oriented to the cytoplasm (4,5). More than 40 AD-causing mutations have been identified in PS1, whereas only two mutations have been identified in PS2. The disease-causing mutations span most domains of the protein, with clusters of mutations in the second transmembrane domain and the large hydrophilic loop region Fig. 1).

Proteolytic release and nuclear translocation of Notch-1 are induced by presenilin-1 and impaired by pathogenic presenilin-1 mutations.

The Notch family of proteins consists of transmembrane receptors that play a critical role in the determination of cell fate. Genetic studies in Caenorhabditis elegans suggest that the presenilin proteins, which are associated with familial Alzheimer's disease, regulate Notch signaling. Here we show that proteolytic release of the Notch-1 intracellular domain (NICD), an essential step in the activation of Notch signaling, is markedly reduced in presenilin-1 (PS1)-deficient cells and is restored by PS1 expression. Nuclear translocation of the NICD is also markedly reduced in PS1-deficient cells, resulting in reduced transcriptional activation. Mutations in PS1 that are associated with familial Alzheimer's disease impair the ability of PS1 to induce proteolytic release of the NICD and nuclear translocation of the cleaved protein. These results suggest that PS1 plays a central role in the proteolytic activation of the Notch-1-signaling pathway and that this function is impaired by pathogenic PS1 mutations. Thus, dysregulation of proteolytic function may underlie the mechanism by which presenilin mutations cause Alzheimer's disease.

beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation.

Regulation of beta-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of beta-catenin by the Axin-glycogen synthase kinase (GSK)-3beta complex targets beta-catenin for ubiquitination-degradation, and mutations at critical phosphoserine residues stabilize beta-catenin and cause human cancers. How beta-catenin phosphorylation results in its degradation is undefined. Here we show that phosphorylated beta-catenin is specifically recognized by beta-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. beta-catenin harboring mutations at the critical phosphoserine residues escapes recognition by beta-Trcp, thus providing a molecular explanation for why these mutations cause beta-catenin accumulation that leads to cancer. Inhibition of endogenous beta-Trcp function by a dominant negative mutant stabilizes beta-catenin, activates Wnt/beta-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, beta-Trcp plays a central role in recruiting phosphorylated beta-catenin for degradation and in dorsoventral patterning of the Xenopus embryo.

Destabilization of beta-catenin by mutations in presenilin-1 potentiates neuronal apoptosis.

Mutations of the presenilin-1 gene are a major cause of familial early-onset Alzheimer's disease. Presenilin-1 can associate with members of the catenin family of signalling proteins, but the significance of this association is unknown. Here we show that presenilin-1 forms a complex with beta-catenin in vivo that increases beta-catenin stability. Pathogenic mutations in the presenilin-1 gene reduce the ability of presenilin-1 to stabilize beta-catenin, and lead to increased degradation of beta-catenin in the brains of transgenic mice. Moreover, beta-catenin levels are markedly reduced in the brains of Alzheimer's disease patients with presenilin-1 mutations. Loss of beta-catenin signalling increases neuronal vulnerability to apoptosis induced by amyloid-beta protein. Thus, mutations in presenilin-1 may increase neuronal apoptosis by altering the stability of beta-catenin, predisposing individuals to early-onset Alzheimer's disease.

Aging renders the brain vulnerable to amyloid beta-protein neurotoxicity.

The formation of fibrillar deposits of amyloid beta protein (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). A central question is whether Abeta plays a direct role in the neurodegenerative process in AD. The involvement of Abeta in the neurodegenerative process is suggested by the neurotoxicity of the fibrillar form of Abeta in vitro. However, mice transgenic for the Abeta precursor protein that develop amyloid deposits in the brain do not show the degree of neuronal loss or tau phosphorylation found in AD. Here we show that microinjection of plaque-equivalent concentrations of fibrillar, but not soluble, Abeta in the aged rhesus monkey cerebral cortex results in profound neuronal loss, tau phosphorylation and microglial proliferation. Fibrillar Abeta at plaque-equivalent concentrations is not toxic in the young adult rhesus brain. Abeta toxicity in vivo is also highly species-specific; toxicity is greater in aged rhesus monkeys than in aged marmoset monkeys, and is not significant in aged rats. These results suggest that Abeta neurotoxicity in vivo is a pathological response of the aging brain, which is most pronounced in higher order primates. Thus, longevity may contribute to the unique susceptibility of humans to Alzheimer's disease by rendering the brain vulnerable to Abeta neurotoxicity.

Neuronal localization of presenilin-1 and association with amyloid plaques and neurofibrillary tangles in Alzheimer's disease.

Mutations in the presenilin-1 (PS1) gene is a cause of early- onset familial Alzheimer's disease (AD). Endogenous PS1 is associated with the endoplasmic reticulum in the cell body of undifferentiated SH-SY5Y neuroblastoma cells. At early stages of neuronal differentiation in rat hippocampal culture, PS1 appears in all neuritic processes and in growth cones. In mature differentiated neurons, PS1 is concentrated in the somatodendritic compartment but is also present at lower levels in axons. A similar localization of PS1 is observed in vivo in neurons of the adult human cerebral cortex. In sporadic AD, PS1 appears in the dystrophic neurites of mature amyloid plaques and co-localizes with a subset of intraneuronal neurofibrillary tangles (NFTs). About 30% of hippocampal NFTs are labeled with a highly specific antibody to the PS1 C-terminal loop domain but not with an antibody to the PS1 N terminus. This observation is consistent with a potential association of the PS1 C-terminal fragment with NFTs, because PS1 is constitutively cleaved to N- and C-terminal fragments in neurons. These results suggest that PS1 is highly expressed and broadly distributed during early stages of neuronal differentiation, consistent with a role for PS1 in neuronal differentiation. Furthermore, the co-localization of PS1 with NFTs and plaque dystrophic neurites implicates a role for PS1 in the diverse pathological manifestations of AD.

Developmental regulation of presenilin-1 processing in the brain suggests a role in neuronal differentiation.

Most cases of early-onset familial Alzheimer's disease are caused by mutations in the presenilin genes. Presenilin-1 (PS1) is subject to proteolytic cleavage resulting in the accumulation of N- and C-terminal fragments. In this report, we show that the proteolytic cleavage of PS1 is developmentally regulated in the brain. Low levels of full-length PS1 and higher levels of 30-kDa N-terminal and 20-kDa C-terminal fragments are identified at all developmental stages in the rat brain. However, in the adult brain, additional 36-kDa N-terminal and 14-kDa C-terminal fragments appear and become major PS1 species. Alternative N-terminal PS1 fragments also appear in the adult human brain, but are more heterogenous than in the rat brain. The alternative PS1 fragments are not detected at significant levels in rat or human peripheral tissues that express PS1. The alternative cleavage of PS1 is also detected in primary cultures of rat hippocampal neurons, but not in astrocytes, and is induced by neuronal differentiation. Furthermore, alternative PS1 cleavage is detected in rat PC12 cells and human neuroblastoma SH-SY5Y cells following induction of neuronal differentiation. These results suggest that an alternative pathway of PS1 proteolytic processing is induced in the brain by neuronal differentiation. PS1 may therefore play an important role in brain development and neuronal function, which may relate to the brain-specific pathological effects of PS1 mutations.

Distinct processing of endogenous and overexpressed recombinant presenilin 1.

The presenilin 1 (PS1) gene has been identified by positional cloning. More than 30 mutations were detected in this gene which cosegregate with Alzheimer's disease (AD). Understanding their role in disease pathogenesis requires a characterization of the PS1 protein. We have generated a set of antibodies against the three major hydrophilic domains of the deduced amino acid sequence. Analyzing cultured cells and brain samples, we identified the endogenous PS1 polypeptide as well as amino- and carboxy-terminal fragments. These metabolites were much more abundant than the full-length molecule, indicating substantial processing. Overexpression of human PS1 markedly increased the full-length polypeptide but hardly altered the amount of the metabolites. Instead, additional proteolytic fragments appeared suggesting a different metabolism of the excess PS1, which may impede studies in transfected cells. Our results indicate a tight regulation of the endogenous PS1 metabolites. PS1 and its fragments are shown to be integral membrane proteins of the endoplasmic reticulum. The mechanisms regulating the generation of the metabolites, their potential function, and role in AD remain to be studied.

Amyloid fibril toxicity in Alzheimer's disease and diabetes.

Several lines of evidence suggest that amyloid deposition in the brain contributes to neuronal degeneration in Alzheimer's disease (AD). In the AD brain, diffuse plaques composed mostly of amorphous beta amyloid (Am-beta A) are inert, whereas compact plaques composed of beta amyloid fibrils (Fib-beta A) are associated with neurodegenerative changes. The effects of these two types of amyloid were tested on primary rat hippocampal neurons. Fib-beta A induced the formation of dystrophic neurites and caused neuronal cell death, whereas Am-beta A was not toxic. In addition, Fib-beta A caused synapse loss in the remaining viable neurons, whereas Am-beta A did not significantly affect synapse number. We also examined the effects of amylin, the primary constituent of the amyloid fibrils that form in the pancreas in adult-onset diabetes. Amylin was toxic to rat and human insulin-producing islet cells in the concentration range of fibril formation. The relative toxic potencies of amylin peptides of different species correlated with their fibril-forming capacity. Soluble amylin was not toxic. The amyloid fibril-binding dye Congo red inhibited the toxicity of both beta A and amylin. Congo red afforded protection against toxicity by a dual mechanism. When present during the phase of fibril polymerization, Congo red could inhibit fibril formation from some peptides. When added to preformed fibrils, Congo red bound to fibrils rendering them nontoxic. These results suggest that fibril formation is necessary for both beta A and amylin toxicity. Congo red appears to be a general inhibitor of amyloid fibril toxicity and may therefore be a useful prototype for drugs targeted to the amyloid pathology of AD and adult-onset diabetes.