[Show abstract][Hide abstract] ABSTRACT: Receptor associated protein (RAP) functions in the endoplasmic reticulum (ER) to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP) and lipoprotein receptor 11 (SorLA/LR11). Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP), including processes involved in the generation, catabolism and deposition of beta-amyloid (Abeta) peptides.
Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (p<0.05)]. Changes in the levels of these proteins in the brains of [APPswe/PS1dE9](+/-)/RAP(+/-) mice correlated with 30-40% increases in amyloid deposition by 9 months of age.
Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Abeta catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.
PLoS ONE 09/2008; 3(9):e3159. DOI:10.1371/journal.pone.0003159 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, several studies have demonstrated changes in the levels of small heat shock proteins (sHSP) in the transgenic mouse models of familial amyotrophic lateral sclerosis (fALS) linked to mutations in Cu/Zn superoxide dismutase. Here, we compared the expression of sHSPs in transgenic mouse models of fALS, Parkinson's disease (PD), dentato-rubral pallido-luysian atrophy (DRPLA) and Huntington's disease (HD); where the expression of mutant cDNA genes was under the transcriptional regulation of the mouse prion protein promoter. These models express G37R mutant Cu/Zn superoxide dismutase (SOD1G37R; fALS), A53T mutant alpha-synuclein (alpha-SynA53T; PD), full-length mutant atrophin-1-65Q, and htt-N171-82Q (huntingtin N-terminal fragment; HD). We found that the levels and solubilities of two sHSPs, Hsp25 and alpha B-crystallin, were differentially regulated in these mice. Levels of both Hsp25 and alpha B-crystallin were markedly increased in subgroups of glias at the affected regions of symptomatic SODG37R and alpha-SynA53T transgenic mice; abnormal deposits or cells intensely positive for alpha B-crystallin were observed in SODG37R mice. By contrast, neither sHSP was induced in spinal cords of htt-N171-82Q or atrophin-1-65Q mice, which do not develop astrocytosis or major motor neuron abnormalities. Interestingly, the levels of insoluble alpha B-crystallin in spinal cords gradually increased as a function of age in nontransgenic animals. In vitro, alpha B-crystallin was capable of suppressing the aggregation of alpha-SynA53T, as previously described for a truncated mutant SOD1. The transgenes in these mice are expressed highly in astrocytes and thus our results suggest a role for small heat shock proteins in protecting activated glial cells such as astrocytes in neurodegenerative diseases.
Neurobiology of aging 05/2008; 29(4):586-97. DOI:10.1016/j.neurobiolaging.2006.11.009 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The amino acid sequence of amyloid precursor protein (APP) is highly conserved, and age-related A beta aggregates have been described in a variety of vertebrate animals, with the notable exception of mice and rats. Three amino acid substitutions distinguish mouse and human A beta that might contribute to their differing properties in vivo. To examine the amyloidogenic potential of mouse A beta, we studied several lines of transgenic mice overexpressing wild-type mouse amyloid precursor protein (moAPP) either alone or in conjunction with mutant PS1 (PS1dE9). Neither overexpression of moAPP alone nor co-expression with PS1dE9 caused mice to develop Alzheimer-type amyloid pathology by 24 months of age. We further tested whether mouse A beta could accelerate the deposition of human A beta by crossing the moAPP transgenic mice to a bigenic line expressing human APPswe with PS1dE9. The triple transgenic animals (moAPP x APPswe/PS1dE9) produced 20% more A beta but formed amyloid deposits no faster and to no greater extent than APPswe/PS1dE9 siblings. Instead, the additional mouse A beta increased the detergent solubility of accumulated amyloid and exacerbated amyloid deposition in the vasculature. These findings suggest that, although mouse A beta does not influence the rate of amyloid formation, the incorporation of A beta peptides with differing sequences alters the solubility and localization of the resulting aggregates.
[Show abstract][Hide abstract] ABSTRACT: The amino acid sequence of amyloid precursor protein (APP) is highly conserved, and age-related Aβ aggregates have been described
in a variety of vertebrate animals, with the notable exception of mice and rats. Three amino acid substitutions distinguish
mouse and human Aβ that might contribute to their differing properties in vivo. To examine the amyloidogenic potential of mouse Aβ, we studied several lines of transgenic mice overexpressing wild-type
mouse amyloid precursor protein (moAPP) either alone or in conjunction with mutant PS1 (PS1dE9). Neither overexpression of
moAPP alone nor co-expression with PS1dE9 caused mice to develop Alzheimer-type amyloid pathology by 24 months of age. We
further tested whether mouse Aβ could accelerate the deposition of human Aβ by crossing the moAPP transgenic mice to a bigenic
line expressing human APPswe with PS1dE9. The triple transgenic animals (moAPP × APPswe/PS1dE9) produced 20% more Aβ but formed
amyloid deposits no faster and to no greater extent than APPswe/PS1dE9 siblings. Instead, the additional mouse Aβ increased
the detergent solubility of accumulated amyloid and exacerbated amyloid deposition in the vasculature. These findings suggest
that, although mouse Aβ does not influence the rate of amyloid formation, the incorporation of Aβ peptides with differing
sequences alters the solubility and localization of the resulting aggregates.
Journal of Biological Chemistry 08/2007; 282(31):22707-22720. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Huntington disease (HD) is caused by the expansion of a glutamine (Q) repeat near the N terminus of huntingtin (htt), resulting in altered conformation of the mutant protein to produce, most prominently in brain neurons, nuclear and cytoplasmic inclusion pathology. The inclusions and associated diffuse accumulation of mutant htt in nuclei are composed of N-terminal fragments of mutant protein. Here, we used a panel of peptide antibodies to characterize the htt protein pathologies in brain tissues from human HD, and a transgenic mouse model created by expressing the first 171 amino acids of human htt with 82Q (htt-N171-82Q). In tissues from both sources, htt pathologic features in nuclei were detected by antibodies to htt peptides 1-17 and 81-90 but not 115-129 (wild-type huntingtin numbering with 23 repeats). Human HEK 293 cells transfected with expression vectors that encode either the N-terminal 233 amino acids of human htt (htt-N233-82Q) or htt-N171-18Q accumulated smaller N-terminal fragments with antibody-binding characteristics identical to those of pathologic peptides. We conclude that the mutant htt peptides that accumulate in pathologic structures of human HD and httN171-82Q in mice are produced by similar, yet to be defined, proteolytic events in a region of the protein near or within amino acids 90-115.
Journal of Neuropathology and Experimental Neurology 05/2007; 66(4):313-20. DOI:10.1097/nen.0b013e318040b2c8 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Familial amyotrophic lateral sclerosis (FALS) has been modeled in transgenic mice by introducing mutated versions of human genomic DNA encompassing the entire gene for Cu,Zn superoxide dismutase (SOD1). In this setting, the transgene is expressed throughout the body and results in mice that faithfully recapitulate many pathological and behavioral aspects of FALS. By contrast, transgenic mice made by introducing recombinant vectors, encoding cDNA genes, that target mutant SOD1 expression to motor neurons, only, or astrocytes, only, do not develop disease. Here, we report that mice transgenic for human SOD1 cDNA with the G37R mutation, driven by the mouse prion promoter, develop motor neuron disease. In this model, expression of the transgene is highest in CNS (both neurons and astrocytes) and muscle. The gene was not expressed in cells of the macrophage lineage. Although the highest expressing hemizygous transgenic mice fail to develop disease by 20 months of age, mice homozygous for the transgene show typical ALS-like phenotypes as early as 7 months of age. Spinal cords and brain stems from homozygous animals with motor neuron disease were found to contain aggregated species of mutant SOD1. The establishment of this SOD1-G37R cDNA transgenic model indicates that expression of mutant SOD1 proteins in the neuromuscular unit is sufficient to cause motor neuron disease. The expression levels required to induce disease coincide with the levels required to induce the formation of SOD1 aggregates.
Neurobiology of Disease 01/2006; 20(3):943-52. DOI:10.1016/j.nbd.2005.06.005 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proteases (secretases) that cleave amyloid-beta (Abeta) peptide from the amyloid precursor protein (APP) have been the focus of considerable investigation in the development of treatments for Alzheimer disease. The prediction has been that reducing Abeta production in the brain, even after the onset of clinical symptoms and the development of associated pathology, will facilitate the repair of damaged tissue and removal of amyloid lesions. However, no long-term studies using animal models of amyloid pathology have yet been performed to test this hypothesis.
We have generated a transgenic mouse model that genetically mimics the arrest of Abeta production expected from treatment with secretase inhibitors. These mice overexpress mutant APP from a vector that can be regulated by doxycycline. Under normal conditions, high-level expression of APP quickly induces fulminant amyloid pathology. We show that doxycycline administration inhibits transgenic APP expression by greater than 95% and reduces Abeta production to levels found in nontransgenic mice. Suppression of transgenic Abeta synthesis in this model abruptly halts the progression of amyloid pathology. However, formation and disaggregation of amyloid deposits appear to be in disequilibrium as the plaques require far longer to disperse than to assemble. Mice in which APP synthesis was suppressed for as long as 6 mo after the formation of Abeta deposits retain a considerable amyloid load, with little sign of active clearance.
This study demonstrates that amyloid lesions in transgenic mice are highly stable structures in vivo that are slow to disaggregate. Our findings suggest that arresting Abeta production in patients with Alzheimer disease should halt the progression of pathology, but that early treatment may be imperative, as it appears that amyloid deposits, once formed, will require additional intervention to clear.
PLoS Medicine 01/2006; 2(12):e355. DOI:10.1371/journal.pmed.0020355 · 14.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mice expressing variants of superoxide dismutase-1 (SOD1) encoding C-terminal truncation mutations linked to familial amyotrophic lateral sclerosis (FALS) have begun to define the role of misfolding and aggregation in the pathogenesis of disease. Here, we examine transgenic mice expressing SOD1-L126Z (Z = stop-truncation of last 28 amino acids), finding that detergent-insoluble mutant protein specifically accumulates in somatodendritic compartments. Soluble forms of the SOD1-L126Z were virtually undetectable in spinal cord at any age and the levels of accumulated protein directly correlated with disease symptoms. Neither soluble nor insoluble forms of SOD1-L126Z were transported to distal axons. In vitro, small heat shock protein (Hsp) alphaB-crystallin suppressed the in vitro aggregation of SOD1-L126Z. In vivo, alphaB-crystallin immunoreactivity was most abundant in oligodendrocytes and up-regulated in astrocytes of symptomatic mice; neither of these cell-types accumulated mutant SOD1 immunoreactivity. These results suggest that damage to motor neuron cell bodies and dendrites within the spinal cord can be sufficient to induce motor neuron disease and that the activities of chaperones may modulate the cellular specificity of mutant SOD1 accumulation.
Human Molecular Genetics 09/2005; 14(16):2335-47. DOI:10.1093/hmg/ddi236 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiological studies suggest that individuals with greater education or more cognitively demanding occupations have diminished risk of developing dementia. We wanted to test whether this effect could be recapitulated in rodents using environmental enrichment, a paradigm well documented to attenuate behavioral deficits induced by various pathological insults. Here, we demonstrate that learning and memory deficits observed in a transgenic mouse model of Alzheimer's disease can be ameliorated by enrichment. Female transgenic mice overexpressing amyloid precursor protein and/or presenilin-1 and nontransgenic controls were placed into enriched or standard cages at 2 months of age and tested for cognitive behavior after 6 months of differential housing. Enrichment significantly improved performance of all genotypes in the radial water maze and in the classic and repeated-reversal versions of the Morris water maze. However, enrichment did not benefit all genotypes equally. Mice overproducing amyloid-beta (Abeta), particularly those with amyloid deposits, showed weaker memory for the platform location in the classic Morris water maze and learned new platform positions in the repeated-reversals task less quickly than their nontransgenic cagemates. Nonetheless, enrichment normalized the performance of Abeta-overproducing mice to the level of standard-housed nontransgenic mice. Moreover, this functional preservation occurred despite increased neuritic plaque burden in the hippocampus of double-transgenic animals and elevated steady-state Abeta levels, because both endogenous and transgene-derived Abeta are increased in enriched animals. These results demonstrate that the generation of Abeta in vivo and its impact on the function of the nervous system can be strongly modulated by environmental factors.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 06/2005; 25(21):5217-24. DOI:10.1523/JNEUROSCI.5080-04.2005 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transgenic mice made by crossing animals expressing mutant amyloid precursor protein (APPswe) to mutant presenilin 1 (PS1dE9) allow for incremental increases in Abeta42 production and provide a model of Alzheimer-type amyloidosis. Here, we examine cognition in 6- and 18-month old transgenic mice expressing APPswe and PS1dE9, alone and in combination. Spatial reference memory was assessed in a standard Morris Water Maze task followed by assessment of episodic-like memory in Repeated Reversal and Radial Water maze tasks. We then used factor analysis to relate changes in performance in these tasks with cholinergic markers, somatostatin levels, and amyloid burden. At 6 months of age, APPswe/PS1dE9 double-transgenic mice showed visible plaque deposition; however, all genotypes, including double-transgenic mice, were indistinguishable from nontransgenic animals in all cognitive measures. In the 18-month-old cohorts, amyloid burdens were much higher in APPswe/PS1dE9 mice with statistically significant but mild decreases in cholinergic markers (cortex and hippocampus) and somatostatin levels (cortex). APPswe/PS1dE9 mice performed all cognitive tasks less well than mice from all other genotypes. Factor and correlation analyses defined the strongest correlation as between deficits in episodic-like memory tasks and total Abeta loads in the brain. Collectively, we find that, in the APPswe/PS1dE9 mouse model, some form of Abeta associated with amyloid deposition can disrupt cognitive circuits when the cholinergic and somatostatinergic systems remain relatively intact; and that episodic-like memory seems to be more sensitive to the toxic effects of Abeta.
Neurobiology of Disease 05/2005; 18(3):602-17. DOI:10.1016/j.nbd.2004.10.022 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: More than 70 different mutations in presenilin 1 (PS1) have been associated with inherited early onset Alzheimer's disease (AD). How all these different mutations cause disease has not been clearly delineated. Our laboratory has previously shown that co-expression of mutant PS1 in mice transgenic for amyloid precursor protein (APPswe) dramatically accelerates the rate of amyloid deposition in the brain. In our original animals mutant PS1 was substantially over-expressed, and the stabilized pool of mouse PS1 fragments was largely replaced by the human protein. In this setting the accelerated amyloid pathology in the double transgenic mice could have been due, in part, to decreased endogenous PS1 activity. To investigate this possibility, we generated APP transgenic mice with reduced levels of endogenous PS1. We find that mice harboring only one functional PS1 allele and co-expressing Mo/HuAPPswe do not develop amyloid deposits at ages comparable to mice expressing mutant PS1. We next tested whether hypo-expression of mutant PS1 could accelerate the rate of amyloid deposition using an unusual line of transgenic mice expressing PS1dE9 at low levels, finding no significant acceleration. Our findings demonstrate that the accelerated amyloid pathology, caused by so many different mutations in PS1, is clearly not a result of haplo-insufficiency that might result from inactivating mutations. Instead, our data are consistent with a gain of property mechanism.
Neurobiology of Aging 09/2004; 25(7):885-92. DOI:10.1016/j.neurobiolaging.2003.09.008 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amyloid precursor protein (APP) is endoproteolytically processed by BACE1 and gamma-secretase to release amyloid peptides (Abeta40 and 42) that aggregate to form senile plaques in the brains of patients with Alzheimer's disease (AD). The C-terminus of Abeta40/42 is generated by gamma-secretase, whose activity is dependent upon presenilin (PS 1 or 2). Missense mutations in PS1 (and PS2) occur in patients with early-onset familial AD (FAD), and previous studies in transgenic mice and cultured cell models demonstrated that FAD-PS1 variants shift the ratio of Abeta40 : 42 to favor Abeta42. One hypothesis to explain this outcome is that mutant PS alters the specificity of gamma-secretase to favor production of Abeta42 at the expense of Abeta40. To test this hypothesis in vivo, we studied Abeta40 and 42 levels in a series of transgenic mice that co-express the Swedish mutation of APP (APPswe) with two FAD-PS1 variants that differentially accelerate amyloid pathology in the brain. We demonstrate a direct correlation between the concentration of Abeta42 and the rate of amyloid deposition. We further show that the shift in Abeta42 : 40 ratios associated with the expression of FAD-PS1 variants is due to a specific elevation in the steady-state levels of Abeta42, while maintaining a constant level of Abeta40. These data suggest that PS1 variants do not simply alter the preferred cleavage site for gamma-secretase, but rather that they have more complex effects on the regulation of gamma-secretase and its access to substrates.
Human Molecular Genetics 02/2004; 13(2):159-70. DOI:10.1093/hmg/ddh019 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiological studies of Alzheimer patients from a wide variety of ethnic and socioeconomic backgrounds have identified education and occupation as environmental factors that can affect the risk of developing disease. A model of environmental manipulation in rodents uses enriched housing to provide cognitive and social stimulation. Previous studies have established elevations in synaptic number and function in rodents housed under enriched conditions. Recent experiments in hippocampal cultures have demonstrated that synaptic activity can influence the processing of amyloid precursor protein (APP). Here we examined whether changes in synaptic activity brought about by enriched housing might also influence the deposition of amyloid plaques in vivo using a transgenic mouse model of Alzheimer disease (AD). Mice co-expressing mutant APP and presenilin 1 (PS1) were housed in either enriched or standard cages from 2 months of age and then killed for pathological evaluation several months later. We find that, as compared to littermates housed in standard cages, the enriched APP/PS1 transgenic mice develop a higher amyloid burden with commensurate increases in aggregated and total A beta. These results suggest that A beta deposition can be exacerbated by the neuronal changes associated with enrichment, and demonstrate a substantial, albeit paradoxical, environmental influence on the progression of pathology in a mouse model of AD.
Journal of Neuropathology and Experimental Neurology 01/2004; 62(12):1220-7. · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cu/Zn superoxide dismutase (SOD1), a crucial cellular antioxidant, can in certain settings mediate toxic chemistry through its Cu cofactor. Whether this latter property explains why mutations in SOD1 cause FALS has been debated. Here, we demonstrate motor neuron disease in transgenic mice expressing a SOD1 variant that mutates the four histidine residues that coordinately bind Cu. In-depth analyses of this new mouse model, previously characterized models and FALS human tissues revealed that the accumulation of detergent-insoluble forms of SOD1 is a common feature of the disease. These insoluble species include full-length SOD1 proteins, peptide fragments, stable oligomers and ubiquitinated entities. Moreover, chaperones Hsp25 and alphaB-crystallin specifically co-fractionated with insoluble SOD1. In cultured cells, all 11 of the FALS variants tested produced insoluble forms of mutant SOD1. Importantly, expression of recombinant peptide fragments of wild-type SOD1 in cultured cells also produced insoluble species, suggesting that SOD1 possesses elements with an intrinsic propensity to aggregate. Thus, modifications to the protein, such as FALS mutations, fragmentation and possibly covalent modification, may simply act to augment a natural, but potentially toxic, propensity to aggregate.
Human Molecular Genetics 12/2003; 12(21):2753-64. DOI:10.1093/hmg/ddg312 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in Cu/Zn superoxide dismutase 1 (SOD1) have been linked to dominantly inherited forms of amyotrophic lateral sclerosis (FALS). To test the hypothesis that the toxicity of mutant SOD1 originates in Cu(2+)-mediated formation of toxic radicals, we generated transgenic mice that express human SOD1 that encodes disease-linked mutations at two of the four histidine residues that are crucial for the coordinated binding of copper (H46R/H48Q). We demonstrate that mice expressing this mutant, which possesses little or no superoxide scavenging activity, develop motor neuron disease. Hence, mutations in SOD1 that disrupt the copper-binding site do not eliminate toxicity. We note that the pathology of the H46R/H48Q mice is dominated by fibrillar (Thioflavin-S-positive) inclusions and that similar inclusions were evident in mouse models that express the G37R, G85R, and G93A variants of human SOD1. Overall, our data are consistent with the hypothesis that the aberrant folding/aggregation of mutant SOD1 is a prominent feature in the pathogenesis of motor neuron disease.
Neurobiology of Disease 08/2002; 10(2):128-38. DOI:10.1006/nbdi.2002.0498 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extracellular and/or intracellular aggregates are pathological features of many, if not all, neurodegenerative diseases. In Alzheimer disease (AD), extracellular aggregates of beta-amyloid (Abeta) and intracellular aggregates of tau or a-synuclein are key diagnostic markers of the disease. We report here a method to rapidly detect these protein aggregates that relies on size exclusion filtration and immunostaining of trapped material, a method termed filter trapping. We demonstrate that aggregated forms of Abeta and tau are readily trapped in 0.22 microm cellulose acetate filter membranes, which can then be immunostained with specific antibodies in a manner similar to the standard immunoblot. Coupling this method with serial dilution permits a rapid assessment of relative aggregate burden.