ABSTRACT
It has been shown that mutations in the Fused in Sarcoma gene (FUS) could explain up to 5% of cases with familial amyotrophic lateral sclerosis (ALS). Our mutation analysis of FUS in a Canadian ALS patient of Chinese origin revealed an unusual novel heterozygous double point mutation (R514S/E516V) confirming that exon 15 is a mutation hot-spot. The substitutions are in cis position to each other and affect highly conserved codons in the RGG-rich region of the FUS protein. The absence of clinical signs of ALS in the relatives of the affected carrier could indicate that this mutation is incompletely penetrant or de novo. The pathologic significance of the R514S/E516V mutation was confirmed by immunohistochemistry. FUS-positive cytoplasmic inclusions were noted in a moderate number in neurons and abundantly in glial cells in the motor cortex and the brainstem. Of interest, a significant number of neuronal and glial FUS-positive inclusions were found in the tegmentum of the brainstem. Importantly, some neurons with inclusions showed retention of the normal nuclear FUS immunostaining.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Point Mutation/genetics , RNA-Binding Protein FUS/genetics , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/physiopathology , Asian People/genetics , Brain Stem/metabolism , Brain Stem/pathology , DNA Mutational Analysis , Humans , Male , Middle Aged , Motor Cortex/metabolism , Motor Cortex/pathologyABSTRACT
We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.
Subject(s)
Amyloid beta-Protein Precursor/genetics , Amyloidosis/genetics , Brain/pathology , Cognition Disorders/genetics , Aging , Amino Acid Substitution , Amyloid/analysis , Amyloid/genetics , Amyloid beta-Protein Precursor/analysis , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Amyloidosis/pathology , Amyloidosis/psychology , Animals , Brain/growth & development , Cognition Disorders/pathology , Crosses, Genetic , Female , Humans , Male , Maze Learning/physiology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Restriction MappingABSTRACT
Dominant mutations in the Presenilin 1 gene are linked to an aggressive, early-onset form of familial Alzheimer's Disease (FAD). Spatial memory of transgenic (Tg) mice expressing either mutant (lines Tg(M146L)1, Tg(M146L)76, Tg(L286V)198) or wild type (line Tg(PS1wt)195) human PS1 transgenes was investigated in the Morris water maze (WM) test at 6 and 9 months of age. The results showed that the mutated Tg mice had increased swim speed when compared to non-Tg littermates or Tg PS1 wild type mice. The swim speed difference did not, however, significantly affect the spatial learning in the WM test and all groups showed comparable search paths during training and similar spatial bias during probe trials. When re-tested at 9 months, all mice showed significantly improved learning acquisition of spatial information. The lack of progressive spatial learning impairment in mice expressing the mutated human PS1 transgene in the WM does not preclude impairments in other cognitive tasks but suggests that full phenotypic expression of mutant PS1 alleles may require co-expression of human versions of other AD-associated genes.
Subject(s)
Alzheimer Disease/physiopathology , Maze Learning/physiology , Membrane Proteins/genetics , Space Perception/physiology , Alzheimer Disease/genetics , Animals , Cognition , Discrimination Learning/physiology , Disease Models, Animal , Exploratory Behavior/physiology , Gene Expression/physiology , Genotype , Humans , Longitudinal Studies , Mice , Mice, Inbred C57BL , Mice, Transgenic , Presenilin-1 , Swimming , Transgenes/geneticsABSTRACT
The novel locus Prnd is 16 kb downstream of the mouse prion protein (PrP) gene Prnp and encodes a 179 residue PrP-like protein designated doppel (Dpl). Prnd generates major transcripts of 1.7 and 2.7 kb as well as some unusual chimeric transcripts generated by intergenic splicing with Prnp. Like PrP, Dpl mRNA is expressed during embryogenesis but, in contrast to PrP, it is expressed minimally in the CNS. Unexpectedly, Dpl is upregulated in the CNS of two PrP-deficient (Prnp(0/0)) lines of mice, both of which develop late-onset ataxia, suggesting that Dpl may provoke neurodegeneration. Dpl is the first PrP-like protein to be described in mammals, and since Dpl seems to cause neurodegeneration similar to PrP, the linked expression of the Prnp and Prnd genes may play a previously unrecognized role in the pathogenesis of prion diseases or other illnesses.
Subject(s)
Ataxia/genetics , Prions/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Central Nervous System/cytology , Central Nervous System/metabolism , Central Nervous System/pathology , Cloning, Molecular , Embryo, Mammalian/metabolism , GPI-Linked Proteins , Gene Deletion , Glycosylation , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Molecular Sequence Data , Prions/chemistry , Prions/metabolism , Prions/physiology , Purkinje Cells/metabolism , Purkinje Cells/pathology , RNA, Messenger/analysis , RNA, Messenger/genetics , Sequence Alignment , Trans-Splicing/genetics , Up-RegulationABSTRACT
The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. Here we show that one of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala resulted in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Drosophila Proteins , Homeodomain Proteins/metabolism , Threonine/metabolism , Alanine/genetics , Alanine/metabolism , Animals , Animals, Genetically Modified , Aspartic Acid/genetics , Aspartic Acid/metabolism , Binding Sites , Cell Line , DNA/metabolism , Drosophila/embryology , Drosophila/metabolism , Fushi Tarazu Transcription Factors , Homeodomain Proteins/genetics , Homeostasis , Mutagenesis, Site-Directed , Phosphorylation , Proto-Oncogene Proteins/genetics , Structure-Activity Relationship , Threonine/genetics , Transcription Factors/genetics , Transcription, Genetic , Wnt1 ProteinABSTRACT
The normal cellular form of prion protein (PrPC) is a precursor to the pathogenic protease-resistant forms (PrPSc) believed to cause scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease. Its amino terminus contains the octapeptide PHGGGWGQ, which is repeated four times and is among the best-preserved regions of mammalian PrPC. Here we show that the amino-terminal domain of PrPC exhibits five to six sites that bind copper (Cu(II)) presented as a glycine chelate. At neutral pH, binding occurs with positive cooperativity, with binding affinity compatible with estimates for extracellular, labile copper. Two lines of independently derived PrPC gene-ablated (Prnp0/0) mice exhibit severe reductions in the copper content of membrane-enriched brain extracts and similar reductions in synaptosomal and endosome-enriched subcellular fractions. Prnp0/0 mice also have altered cellular phenotypes, including a reduction in the activity of copper/zinc superoxide dismutase and altered electrophysiological responses in the presence of excess copper. These findings indicate that PrPC can exist in a Cu-metalloprotein form in vivo.
Subject(s)
Copper/metabolism , PrPC Proteins/metabolism , Animals , Brain/metabolism , Cells, Cultured , Cerebellum/cytology , Cerebellum/metabolism , Copper/blood , Electrophysiology , Humans , In Vitro Techniques , Kidney/metabolism , Liver/metabolism , Mice , Mice, Inbred C57BL , Muscles/metabolism , Protein Binding , Purkinje Cells/metabolism , SynapsesABSTRACT
PrP(C), the cellular isoform of the prion protein (PrP) serves as a precursor to abnormal PrP isoforms which accumulate in diseases such as scrapie in sheep, and Creutzfeldt-Jakob disease in humans. Since prions can replicate in photoreceptors we surmised that PrP(C) must be expressed in these cells. Accordingly, monoclonal antisera directed against two epitopes of hamster PrP(C) produced retinal immunostaining in hamsters, and in mice bearing a hamster PrP transgene. Immunostaining was most prominent in the inner and outer segments of rod photoreceptors, coinciding with the earliest site of pathologic changes in scrapie-infected hamsters. These data define PrP(C) expression in an experimentally-accessible population of neurons and suggest that the retina may comprise a useful system for studying the biology of wild-type and mutant prion proteins.
Subject(s)
Photoreceptor Cells/metabolism , Prions/biosynthesis , Animals , Cricetinae , Mesocricetus , Mice , Mice, TransgenicABSTRACT
The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.
Subject(s)
Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Membrane Proteins/genetics , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/immunology , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Brain/metabolism , Brain/pathology , Cell Line , Enzyme-Linked Immunosorbent Assay , Genetic Vectors , Hippocampus/metabolism , Hippocampus/pathology , Humans , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Mutation , Peptide Fragments/genetics , Peptide Fragments/metabolism , Presenilin-1 , Presenilin-2 , TransfectionABSTRACT
Thirty-nine eyes of 20 premature infants, mean birthweight 922 g, mean gestational age 27 weeks, with active retinopathy of prematurity (ROP), were treated. Thirty-one eyes with stage 3+ ROP and 180 degrees to 360 degrees of preretinal neovascularization received cryotherapy to ablate the zone of peripheral avascular retina. All underwent complete regression of active disease. Eight of these eyes subsequently developed retinal detachments due to ongoing vitreous traction. One detachment was inoperable. Six eyes were successfully reattached following scleral buckling surgery. One of these six redetached and became inoperable after 20 months. Pars plicata vitrectomy was not successful in reattaching the eighth case. Twenty-eight of these 31 eyes retain useful visual acuity with follow-up of 15 to 70 months (mean 41 months). There have been no complications resulting from cryotherapy. Eight other eyes with stage 4 ROP (traction retinal detachment) were treated with cryotherapy and scleral buckling surgery. All were initially reattached, but ongoing vitreoretinal traction caused redetachment in five. One was inoperable. "Open sky" vitrectomy was successful in reattaching three of the other four. Of the six cases that remained reattached with follow-up of 6 to 51 months, only two retain useful vision.