Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder caused by a CAG repeat expansion. To determine the mechanism of neurotoxicity, we produced transgenic mice and observed a cone-rod dystrophy. Nuclear inclusions were present, suggesting that the disease pathway involves the nucleus. When yeast two-hybrid assays indicated that cone-rod homeobox protein (CRX) interacts with ataxin-7, we performed further studies to assess this interaction. We found that ataxin-7 and CRX colocalize and coimmunoprecipitate. We observed that polyglutamine-expanded ataxin-7 can dramatically suppress CRX transactivation. In SCA7 transgenic mice, electrophoretic mobility shift assays indicated reduced CRX binding activity, while RT-PCR analysis detected reductions in CRX-regulated genes. Our results suggest that CRX transcription interference accounts for the retinal degeneration in SCA7 and thus may provide an explanation for how cell-type specificity is achieved in this polyglutamine repeat disease.

Genomic organization, chromosome location, and expression analysis of mouse beta-synuclein, a candidate for involvement in neurodegeneration.

The synuclein family of proteins is a group of primarily brain-expressed polypeptides that show a high degree of amino acid conservation. alpha-Synuclein is the best known of the synuclein family, as it is a major component of the Lewy body, a cytoplasmic inclusion characteristic of Parkinson's disease as well as a variety of related neurodegenerative disorders. With the discovery that mutations in alpha-synuclein can cause Parkinson's disease, a potential role for the other synuclein family members in neurodegenerative disease is being considered. beta-Synuclein in particular may deserve special attention, as it is co-expressed with alpha-synuclein at presynaptic nerve terminals, is subject to phosphorylation by Ca(2+) calmodulin protein kinase II, appears important for neural plasticity, and forms aggregates in the brains of patients with Parkinson's disease and a related disorder. To facilitate study of beta-synuclein, we have cloned the mouse beta-synuclein gene (Sncb) and determined its genomic organization, size, and intron-exon structure. Using an interspecific backcross mapping panel from The Jackson Laboratory, we were then able to localize Sncb to chromosome 13 at the MGD 35.0 cM position. Like the human beta-synuclein gene, Sncb appears to consist of six exons separated by five introns. Unlike the human beta-synuclein gene, the mouse ortholog possesses a variant GC 5' splice donor sequence at the exon 4 - intron 4 boundary in a highly conserved splice junction consensus. Northern blot analysis and Western blot analysis both indicate that Sncb is highly expressed in the brain. Knowledge of the genomic organization and expression pattern of Sncb will allow functional studies of its potential role in neurodegeneration to commence in the mouse.

Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells.

Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53. The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish. Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas. Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G(1) or G(2) when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein. These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression.

In vivo expansion of trinucleotide repeats yields plasmid and YAC constructs for targeting and transgenesis.

Production of mouse models of inherited neurodegenerative diseases is an important step towards understanding the mechanism of neurotoxicity and for testing potential therapies. We are interested in creating a mouse model for X-linked spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by expansion of a CAG repeat within the androgen receptor (AR) gene. To permit generation of mice that will show a SBMA phenotype within their life span, we decided to obtain a yeast artificial chromosome (YAC) carrying the AR gene and introduce CAG repeat mutations numbering 100 or more triplets. SBMA patients with more than 70 CAGs have never been observed; therefore, we chose to expand a 59 CAG repeat tract in vivo in Escherichia coli. Although we set out to expand this repeat tract using a recombination paradigm involving two plasmid co-propagation, we did not observe large expansions. We were instead able to incrementally generate repeat tracts from 100 to 200 CAGs in a yeast integrating plasmid vector by taking advantage of replication instability. In the course of our experiments that yielded these CAG repeat tracts, we evaluated the role of repeat orientation, vector co-propagation, and recA function on the expansion process. We then used one of the yeast integrating vectors to successfully produce an AR YAC construct carrying 100 CAG repeats. AR YAC CAG100 will serve as a valuable reagent for the production of a SBMA mouse.

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation.

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer's disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer's disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer's disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid beta-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation.

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Overexpression of PS1 mutations in cultured PC12 cells increases their vulnerability to apoptosis-induced trophic factor withdrawal and oxidative insults. We now report that primary hippocampal neurons from PS1 mutant knock-in mice, which express the human PS1M146V mutation at normal levels, exhibit increased vulnerability to amyloid beta-peptide toxicity. The endangering action of mutant PS1 was associated with increased superoxide production, mitochondrial membrane depolarization, and caspase activation. The peroxynitrite-scavenging antioxidant uric acid and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone protected hippocampal neurons expressing mutant PS1 against cell death induced by amyloid beta-peptide. Increased oxidative stress may contribute to the pathogenic action of PS1 mutations, and antioxidants may counteract the adverse property of such AD-linked mutations.

Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice.

Excitotoxicity, a form of neuronal injury in which excessive activation of glutamate receptors results in cellular calcium overload, has been implicated in the pathogenesis of Alzheimer disease (AD), although direct evidence is lacking. Mutations in the presenilin-1 (PS1) gene on chromosome 14 are causally linked to many cases of early-onset inherited AD (refs. 5,6). We generated PS1 mutant mice (PS1M146VKI) that express the PS1 M146V targeted allele at normal physiological levels. Although PS1M146VKI mice have no overt mutant phenotype, they are hypersensitive to seizure-induced synaptic degeneration and necrotic neuronal death in the hippocampus. Cultured hippocampal neurons from PS1M146VKI mice have increased vulnerability to death induced by glutamate, which is correlated with perturbed calcium homeostasis, increased oxidative stress and mitochondrial dysfunction. Agents that suppress calcium influx or release and antioxidants protect neurons against the excitotoxic action of the PS1 mutation. These findings establish a direct link between a genetic defect that causes AD and excitotoxic neuronal degeneration, and indicate new avenues for therapeutic intervention in AD patients.

The Werner syndrome protein is a DNA helicase.

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

Alzheimer's presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid beta-peptide: involvement of calcium and oxyradicals.

Most autosomal dominant inherited forms of early onset Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS-1) gene on chromosome 14. PS-1 is an integral membrane protein with six to nine membrane-spanning domains and is expressed in neurons throughout the brain wherein it is localized mainly in endoplasmic reticulum (ER). The mechanism or mechanisms whereby PS-1 mutations promote neuron degeneration in AD are unknown. Recent findings suggest links among deposition of amyloid beta-peptide (Abeta), oxidative stress, disruption of ion homeostasis, and an apoptotic form of neuron death in AD. We now report that expression of the human PS-1 L286V mutation in PC12 cells increases their susceptibility to apoptosis induced by trophic factor withdrawal and Abeta. Increases in oxidative stress and intracellular calcium levels induced by the apoptotic stimuli were exacerbated greatly in cells expressing the PS-1 mutation, as compared with control cell lines and lines overexpressing wild-type PS-1. The antiapoptotic gene product Bcl-2 prevented apoptosis after NGF withdrawal from differentiated PC12 cells expressing mutant PS-1. Elevations of [Ca2+]i in response to thapsigargin, an inhibitor of the ER Ca2+-ATPase, were increased in cells expressing mutant PS-1, and this adverse effect was abolished in cells expressing Bcl-2. Antioxidants and blockers of calcium influx and release from ER protected cells against the adverse consequences of the PS-1 mutation. By perturbing cellular calcium regulation and promoting oxidative stress, PS-1 mutations may sensitize neurons to apoptotic death in AD.

Alzheimer's PS-1 mutation perturbs calcium homeostasis and sensitizes PC12 cells to death induced by amyloid beta-peptide.

Mutations in the presenilin-1 (PS-1) gene on chromosome 14 are linked to autosomal dominant early-onset Alzheimer's disease. The amino acid sequence of PS-1 predicts an integral membrane protein and immunocytochemical studies indicate that PS-1 is localized to endoplasmic reticulum (ER). We report that expression of PS-1 mutation L286V in cultured PC12 cells exaggerates Ca2+ responses to agonists (carbachol and bradykinin) that induce Ca2+ release from ER. Cells expressing L286V exhibit enhanced elevations of [Ca2+]i following exposure to amyloid beta-peptide (A beta) and increased vulnerability to A beta toxicity. An antagonist of voltage-dependent calcium channels (nifedipine), and a blocker of Ca2+ release from ER (dantrolene), counteract the adverse consequences of the PS-1 mutation. By perturbing Ca2+ homeostasis, PS-1 mutations may sensitize neurons to A beta-induced apoptosis.

Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain.

Proteolytic cleavage of beta-amyloid precursor protein (beta APP) by alpha-secretase results in release of one secreted form (sAPP) of APP (sAPP alpha), whereas cleavage by beta-secretase releases a C-terminally truncated sAPP (sAPP beta) plus amyloid beta-peptide (A beta). beta APP mutations linked to some inherited forms of Alzheimer's disease may alter its processing such that levels of sAPP alpha are reduced and levels of sAPP beta increased. sAPP alpha s may play important roles in neuronal plasticity and survival, whereas A beta can be neurotoxic. sAPP alpha was approximately 100-fold more potent than sAPP beta in protecting hippocampal neurons against excitotoxicity, A beta toxicity, and glucose deprivation. Whole-cell patch clamp and calcium imaging analyses showed that sAPP beta was less effective than sAPP alpha in suppressing synaptic activity, activating K+ channels, and attenuating calcium responses to glutamate. Using various truncated sAPP alpha and sAPP beta APP695 products generated by eukaryotic and prokaryotic expression systems, and synthetic sAPP peptides, the activity of sAPP alpha was localized to amino acids 591-612 at the C-terminus. Heparinases greatly reduced the actions of sAPP alpha s, indicating a role for a heparin-binding domain at the C-terminus of sAPP alpha in receptor activation. These findings indicate that alternative processing of beta APP has profound effects on the bioactivity of the resultant sAPP products and suggest that reduced levels of sAPP alpha could contribute to neuronal degeneration in Alzheimer's disease.

Neurodegenerative mechanisms in Alzheimer disease. A role for oxidative damage in amyloid beta protein precursor-mediated cell death.

We have established a stably transformed human neuroblastoma cell line (MC65) that conditionally expresses a C-terminal derivative of the amyloid beta protein precursor (beta PP) termed S beta C (a fusion protein composed of the amino-17 and carboxyl-99 residues of beta PP). Conditional expression of S beta C (mediated by the withdrawal of tetracycline from the culture medium) induces pronounced nuclear DNA fragmentation and cytotoxicity in this cell line. These effects are enhanced by hyperoxygen and suppressed by hypooxygen and antioxidants. This cell line is relatively insensitive to the extracellular application of amyloid beta 25-35, and coculture experiments suggest that this cytotoxicity is mediated by an intracellular process. These findings suggest that the overexpression of the C-terminal domain of beta PP can disrupt normal cellular processes in these cells in such a way as to induce a directed (deoxyribonuclease-mediated) mechanism of cell death. This process appears to be modulated and/or mediated by a reactive oxygen specie(s) (ROS). Consistent with a role for ROS in the process of S beta C-mediated toxicity, we have found that the MC65 cell line is hypersensitive to oxidative stress and that it is this sensitivity that appears (at least in part) to underlie its susceptibility to S beta C.

cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein.

Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP). A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region. The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein. This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2. These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP. Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID). Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins. RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology. The human Fe65 mRNA was cloned from a fetal brain cDNA library. The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein. The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization.

Molecular cloning and characterization of bovine beta-mannosidase.

Deficiency of lysosomal beta-mannosidase activity results in a severe neurodegenerative disease in goats and cattle and a relatively milder phenotype in humans. A cDNA coding for the entire beta-mannosidase protein is described. Mixed oligonucleotides derived from bovine beta-mannosidase peptide sequences were used to screen a bovine thyroid cDNA library. Clones covering about 80% of the C-terminal region were recovered. The missing 5'-region was obtained using the technique of 5'-rapid amplification of cDNA ends. The composite cDNA contains 3852 nucleotides, encoding 879 amino acids. The N-terminal methionine is followed by 16 amino acids displaying the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrates a single 4.2-kilobase transcript in various tissues from both normal and affected goats and calves. The mRNA level is decreased in tissues of affected beta-mannosidosis animals. The gene encoding beta-mannosidase is localized to human chromosome 4 as shown by Southern analysis of rodent/human somatic cell hybrids. This is the first report of cloning of lysosomal beta-mannosidase.

Cytotoxicity mediated by conditional expression of a carboxyl-terminal derivative of the beta-amyloid precursor protein.

The beta amyloid peptide which accumulates within the brains of patients with Alzheimer's disease (AD) is proteolytically derived from a precursor protein (beta PP). We established and characterized four stably transformed human neuroblastoma cell lines which conditionally expressed a partial beta PP fusion protein (amino-17 residues+carboxyl-99 residues; S beta C). Conditional expression of S beta C was achieved using a tetracycline-responsive promoter system. Expression of this fusion protein in one of the cell lines resulted in pronounced cytotoxicity. Addition of n6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate and/or fetal bovine serum to the culture medium of this cell line further elevated the level of S beta C expression and enhanced the associated cytotoxicity. Conditioned medium, acquired from cells expressing S beta C, was not cytotoxic. These findings suggest that modulation of beta PP expression and/or metabolism can have cytotoxic consequences. This is the first report of cytotoxic effects mediated by conditional expression of a beta PP derivative. This immortal cell line provides a unique opportunity to screen for complementary DNAs which suppress this toxicity. Such cDNAs could help elucidate the processes underlying S beta C mediated cytotoxicity which in turn could further our understanding of the pathogenesis of AD and could also provide additional candidate genes for various forms of familial AD.

Bovine kidney beta-mannosidase: purification and characterization.

Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity.

Purification and characterization of goat lysosomal beta-mannosidase using monoclonal and polyclonal antibodies.

Goat beta-mannosidase was purified 120,000-fold in 26% yield from kidney using concanavalin A-Sepharose chromatography followed by immunoaffinity and cation-exchange chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie Blue staining, the purified enzyme preparation consists of 90- and 100-kDa peptides. Both these peptides react with anti-beta-mannosidase monoclonal antibodies and produce similar electrophoretic peptide patterns when subjected to limited proteolysis. Deglycosylation reduces the size of the 90- and 100-kDa peptides to 86 and 91 kDa, respectively. Goat kidney tissues lacking beta-mannosidase activity, acquired from animals affected with beta-mannosidosis, do not contain detectable quantities of the 90- and 100-kDa peptides as judged by monoclonal antibody reactivity. We postulate that the 90- and 100-kDa peptides represent two related forms of beta-mannosidase.