Hyperaccumulation of FAD-linked presenilin 1 variants in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.

Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells.

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.

Genetic neurodegenerative diseases: the human illness and transgenic models.

Review The neurodegenerative disorders, a heterogeneous group of chronic progressive diseases, are among the most puzzling and devastating illnesses in medicine. Some of these disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, the prion diseases, and Parkinson's disease, can occur sporadically and, in some instances, are caused by inheritance of gene mutations. Huntington's disease is acquired in an entirely genetic manner. Transgenic mice that express disease-causing genes recapitulate many features of these diseases. This review provides an overview of transgenic mouse models of familial amyotrophic lateral sclerosis, familial Alzheimer's disease, and Huntington's disease and the emerging insights relevant to the underlying molecular mechanisms of these diseases.

Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer's disease.

Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer's disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer's disease.

Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins.

Missense mutations in two related genes, termed presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer's disease (FAD) pedigrees. In a variety of experimental in vitro and in vivo settings, FAD-linked presenilin variants influence the processing of the amyloid precursor protein (APP), leading to elevated levels of the highly fibrillogenic Abeta1-42 peptides that are preferentially deposited in the brains of Alzheimer Disease (AD) patients. In this report, we demonstrate that transgenic animals that coexpress a FAD-linked human PS1 variant (A246E) and a chimeric mouse/human APP harboring mutations linked to Swedish FAD kindreds (APP swe) develop numerous amyloid deposits much earlier than age-matched mice expressing APP swe and wild-type Hu PS1 or APP swe alone. These results provide evidence for the view that one pathogenic mechanism by which FAD-linked mutant PS1 causes AD is to accelerate the rate of beta-amyloid deposition in brain.

An Alzheimer's disease-linked PS1 variant rescues the developmental abnormalities of PS1-deficient embryos.

Mutations in presenilin 1 (PS1) cosegregate with approximately 25% of early onset familial Alzheimer's disease (FAD) pedigrees. A variety of in vitro and in vivo paradigms have established that one mechanism by which PS1 variants cause AD is by elevating the production of highly amyloidogenic Abeta1-42/43 peptides. PS1 is homologous to sel-12, a C. elegans protein that facilitates signaling mediated by the Notch/lin-12 family of receptors. Wild-type human PS1 complements an egg-laying defect in C. elegans lacking sel-12, while FAD-linked PS1 variants exhibit reduced rescue activity. These data suggested that mutant PS1 may cause disease as a result of reduction in PS1 function. To test the function of FAD-linked PS1 in mammals, we examined the ability of the A246E PS1 variant to complement the embryonic lethality and axial skeletal defects in mice lacking PS1. Finally, to examine the influence of reduced PS1 levels on Abeta production, we quantified Abeta1-42/43 peptide levels in PS1 heterozygous null mice (PS1[+/-] mice). We now report that both human wild-type and A246E PS1 efficiently rescue the phenotypes observed in PS1(-/-) embryos, findings consistent with the view that FAD-linked PS1 mutants retain sufficient normal function during mammalian embryonic development. Moreover, the levels of Abeta1-42/43 and Abeta1-40 peptides between PS1(+/-) and control mice are indistinguishable. Collectively, these data lead us to conclude that mutant PS1 causes AD not by loss of normal PS1 function but by influencing amyloid precursor protein (APP) processing in a manner that elevates Abeta1-42/43 production.

Effects of PS1 deficiency on membrane protein trafficking in neurons.

We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.

Age-related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins.

Transgenic FVB/N mice overexpressing human (Hu) or mouse (Mo) Alzheimer amyloid precursor protein (APP695) die early and develop a CNS disorder that includes neophobia and impaired spatial alternation, with diminished glucose utilization and astrogliosis mainly in the cerebrum. Age at onset of neophobia and age at death decrease with increasing levels of brain APP. HuAPP transgenes induce death much earlier than MoAPP transgenes expressed at similar levels. No extracellular amyloid was detected, indicating that some deleterious processes related to APP overexpression are dissociated from formation of amyloid. A similar clinical syndrome occurs spontaneously in approximately 20% of nontransgenic mice when they reach mid- to late-adult life, suggesting that APP overexpression may accelerate a naturally occurring age-related CNS disorder in FVB/N mice.

An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis. Four lines of mice accumulating one of these mutant proteins (G37R) develop severe, progressive motor neuron disease. At lower levels of mutant accumulation, pathology is restricted to lower motor neurons, whereas higher levels cause more severe abnormalities and affect a variety of other neuronal populations. The most obvious cellular abnormality is the presence in axons and dendrites of membrane-bounded vacuoles, which appear to be derived from degenerating mitochondria. Since multiple lines of mice expressing wild-type human SOD1 at similar and higher levels do not show disease, the disease in mice expressing the G37R mutant SOD1 must arise from the acquisition of an adverse property by the mutant enzyme, rather than elevation or loss of SOD1 activity.

Protein topology of presenilin 1.

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.

ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions.

High levels of familial Amyotrophic Lateral Sclerosis (ALS)-linked SOD1 mutants G93A and G37R were previously shown to mediate disease in mice through an acquired toxic property. We report here that even low levels of another mutant, G85R, cause motor neuron disease characterized by an extremely rapid clinical progression, without changes in SOD1 activity. Initial indicators of disease are astrocytic inclusions that stain intensely with SOD1 antibodies and ubiquitin and SOD1-containing aggregates in motor neurons, features common with some cases of SOD1 mutant-mediated ALS. Astrocytic inclusions escalate markedly as disease progresses, concomitant with a decrease in the glial glutamate transporter (GLT-1). Thus, the G85R SOD1 mutant mediates direct damage to astrocytes, which may promote the nearly synchronous degeneration of motor neurons.

Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA.

Dentatorubral and pallidoluysian atrophy (DRPLA) is a member of a family of progressive neurodegenerative diseases caused by polyglutamine repeat expansion. Transgenic mice expressing full-length human atrophin-1 with 65 consecutive glutamines exhibit ataxia, tremors, abnormal movements, seizures, and premature death. These mice accumulate atrophin-1 immunoreactivity and inclusion bodies in the nuclei of multiple populations of neurons. Subcellular fractionation revealed 120 kDa nuclear fragments of mutant atrophin-1, whose abundance increased with age and phenotypic severity. Brains of DRPLA patients contained apparently identical 120 kDa nuclear fragments. By contrast, mice overexpressing atrophin-1 with 26 glutamines were phenotypically normal and did not accumulate the 120 kDa fragments. We conclude that the evolution of neuropathology in DRPLA involves proteolytic processing of mutant atrophin-1 and nuclear accumulation of truncated fragments.

Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo.

The majority of early-onset cases of familial Alzheimer's disease (FAD) are linked to mutations in two related genes, PS1 and PS2, located on chromosome 14 and 1, respectively. Using two highly specific antibodies against nonoverlapping epitopes of the PS1-encoded polypeptide, termed presenilin 1 (PS1), we document that the preponderant PS1-related species that accumulate in cultured mammalian cells, and in the brains of rodents, primates, and humans are approximately 27-28 kDa N-terminal and approximately 16-17 kDa C-terminal derivatives. Notably, a FAD-linked PS1 variant that lacks exon 9 is not subject to endoproteolytic cleavage. In brains of transgenic mice expressing human PS1, approximately 17 kDa and approximately 27 kDa PS1 derivatives accumulate to saturable levels, and at approximately 1:1 stoichiometry, independent of transgene-derived mRNA. We conclude that PS1 is subject to endoproteolytic processing in vivo.

Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 genes cosegregate with the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. We now document that the Abeta1-42(43)/Abeta1-40 ratio in the conditioned media of independent N2a cell lines expressing three FAD-linked PS1 variants is uniformly elevated relative to cells expressing similar levels of wild-type PS1. Similarly, the Abeta1-42(43)/Abeta1-40 ratio is elevated in the brains of young transgenic animals coexpressing a chimeric amyloid precursor protein (APP) and an FAD-linked PS1 variant compared with brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP. These studies provide compelling support for the view that one mechanism by which these mutant PS1 cause AD is by increasing the extracellular concentration of Abeta peptides terminating at 42(43), species that foster Abeta deposition.

SOD1 rescues cerebral endothelial dysfunction in mice overexpressing amyloid precursor protein.

Peptides derived from proteolytic processing of the beta-amyloid precursor protein (APP), including the amyloid-beta peptide, are important for the pathogenesis of Alzheimer's dementia. We found that transgenic mice overexpressing APP have a profound and selective impairment in endothelium-dependent regulation of the neocortical microcirculation. Such endothelial dysfunction was not found in transgenic mice expressing both APP and superoxide dismutase-1 (SOD1) or in APP transgenics in which SOD was topically applied to the cerebral cortex. These cerebrovascular effects of peptides derived from APP processing may contribute to the alterations in cerebral blood flow and to neuronal dysfunction in Alzheimer's dementia.

Synthesis and trafficking of prion proteins in cultured cells.

Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrPSc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells synthesize PrPSc from the normal PrP isoform (PrPC) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrPSc. Removal of BFA after the chase permitted synthesis of PrPSc to resume. BFA also blocked the export of nascent PrPC to the cell surface but did not alter the distribution of intracellular deposits of PrPSc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrPSc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrPSc and that the synthesis of PrPSc occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrPSc.

Transgenic models of neurodegenerative diseases.

Identification of genetic mutations linked to familial neurodegenerative diseases have made it possible to generate useful transgenic animal models. Studies using these transgenic animals indicate that many familial neurodegenerative diseases, such as motor neuron disease, Alzheimer's disease, prion diseases and trinucleotide repeat diseases, result from a gain of deleterious properties. The disease-specific pathology in transgenic mice demonstrates the utility of these models in elucidating pathogenic mechanisms of the disease and in developing therapeutic strategies.

Loss of functional prion protein: a role in prion disorders?

To understand the normal function of the prion protein (PrP) and its role in prion disorders, several groups have generated mice lacking PrP. Some of these mice develop symptoms associated with prion diseases, but other experimental evidence suggests that the loss of functional PrP is not the instigating factor in these disorders.

Transgenic mouse models of Alzheimer's disease and amyotrophic lateral sclerosis.

Over the past several years, there has been enormous progress in generating transgenic mice that model aspects of human neurodegenerative diseases. These studies build upon the efforts of molecular geneticists who have identified a number of genes that, when mutated, cause familial forms of these diseases. In this review, we focus on the mutations that cause familial forms of Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), and transgenic mouse models that develop clinical and pathological abnormalities resembling those occurring in the human diseases.