Biomedical subjects

R Baumeister

Publications and source records attributed to R Baumeister.

At least 19 recordsLinked to original sources

PubMed2004

C. elegans metallothioneins: new insights into the phenotypic effects of cadmium toxicosis.

Metallothioneins are considered to be the primary player in the detoxification of and protection from cadmium, a teratogen, mutagen and potentially lethal heavy metal. The nematode Caenorhabditis elegans has only two metallothioneins, mtl-i and mtIl-2, thus making it an ideal organism to investigate the phenotypic effects of cadmium toxicosis. The functional importance of metallothioneins in cadmium trafficking was highlighted through the generation of viable green fluorescent protein (GFP) expressing transgenes, a metallothionein null allele, as well as RNAi mediated metallothionein knock-downs. A highly sensitive dose and temporal transcriptional response to cadmium, but not copper or zinc, was shown to be equally prevalent in both isoforms. No measurable compensatory up-regulation of mtl-l could be observed in the null allele of mtl-2, suggesting that both isoforms are independent and not synergistic in their mode of action. Exposure to cadmium affected all demographic indices measured, manifested by a reduction in body size, generation time, brood size and lifespan. These effects were magnified in the knock-out or wild-type subjected to a knock down by RNAi, however, only in the presence of cadmium. This substantiates the notion that metallothioneins play a pivotal role in the protection from cadmium toxicosis. Finally, an earthworm metallothionein-GFP construct could be activated in C. elegans upon exposure to cadmium, the results providing further evidence that the transcriptional control of metallothioneins is fundamentally divergent in lower invertebrates and not mediated via MTF-1 as in more complex organisms.

Amino Acid Sequence↗

PubMed2003

Endoscopic-assisted microsurgery: microsurgery in the new millennium? A comparative experimental study.

Endoscopes are already present in almost all plastic surgery departments. The operating microscope is currently an essential piece of equipment for performing microsurgical anastomoses; however, microsurgery could be conducted using other equipment, including the endoscope. By performing 60 vessel and nerve repairs in rats under the operating microscope and the same number using the endoscope as a visual aid, we investigated the technical and clinical differences between the two instruments. We recorded significantly shorter operative, vascular preparation and anastomotic times in the endoscopically assisted group. Based on the data collected during this study, we conclude that microsurgery is possible with the aid of an endoscope. Using the endoscope may make prolonged microvascular procedures shorter and less physically demanding and may increase the comfort level of both the surgeon and the assistant.

Anastomosis, Surgical↗

PubMed2002

Loss of spr-5 bypasses the requirement for the C.elegans presenilin sel-12 by derepressing hop-1.

Presenilins are part of a protease complex that is responsible for the intramembraneous cleavage of the amyloid precursor protein involved in Alzheimer's disease and of Notch receptors. In Caenorhabditis elegans, mutations in the presenilin sel-12 result in a highly penetrant egg-laying defect. spr-5 was identified as an extragenic suppressor of the sel-12 mutant phenotype. The SPR-5 protein has similarity to the human polyamine oxidase-like protein encoded by KIAA0601 that is part of the HDAC-CoREST co-repressor complex. Suppression of sel-12 by spr-5 requires the activity of HOP-1, the second somatic presenilin in C.elegans. spr-5 mutants derepress hop-1 expression 20- to 30-fold in the early larval stages when hop-1 normally is almost undetectable. SPR-1, a C.elegans homologue of CoREST, physically interacts with SPR-5. Moreover, down-regulation of SPR-1 by mutation or RNA interference also bypasses the need for sel-12. These data strongly suggest that SPR-5 and SPR-1 are part of a CoREST-like co-repressor complex in C.elegans. This complex might be recruited to the hop-1 locus controlling its expression during development.

Amino Acid Sequence↗

PubMed2000

A loss of function mutant of the presenilin homologue SEL-12 undergoes aberrant endoproteolysis in Caenorhabditis elegans and increases abeta 42 generation in human cells.

The familial Alzheimer's disease-associated presenilins (PSs) occur as a dimeric complex of proteolytically generated fragments, which functionally supports endoproteolysis of Notch and the beta-amyloid precursor protein (betaAPP). A homologous gene, sel-12, has been identified in Caenorhabditis elegans. We now demonstrate that wild-type (wt) SEL-12 undergoes endoproteolytic cleavage in C. elegans similar to the PSs in human tissue. In contrast, SEL-12 C60S protein expressed from the sel-12(ar131) allele is miscleaved in C. elegans, resulting in a larger mutant N-terminal fragment. Neither SEL-12 wt nor C60S undergo endoproteolytic processing upon expression in human cells, suggesting that SEL-12 is cleaved by a C. elegans-specific endoproteolytic activity. The loss of function of sel-12 in C. elegans is not associated with a dominant negative activity in human cells, because SEL-12 C60S and the corresponding PS1 C92S mutation do not interfere with Notch1 cleavage. Moreover, both mutant variants increase the aberrant production of the highly amyloidogenic 42-amino acid version of amyloid beta-peptide similar to familial Alzheimer's disease-associated human PS mutants. Our data therefore demonstrate that the C60S mutation in SEL-12 is associated with aberrant endoproteolysis and a loss of function in C. elegans, whereas a gain of misfunction is observed upon expression in human cells.

Amyloid beta-Peptides↗

PubMed2000

A pathogenic presenilin-1 deletion causes abberrant Abeta 42 production in the absence of congophilic amyloid plaques.

Familial Alzheimer's disease (FAD) is frequently associated with mutations in the presenilin-1 (PS1) gene. Almost all PS1-associated FAD mutations reported so far are exchanges of single conserved amino acids and cause the increased production of the highly amyloidogenic 42-residue amyloid beta-peptide Abeta42. Here we report the identification and pathological function of an unusual FAD-associated PS1 deletion (PS1 DeltaI83/DeltaM84). This FAD mutation is associated with spastic paraparesis clinically and causes accumulation of noncongophilic Abeta-positive "cotton wool" plaques in brain parenchyma. Cerebral amyloid angiopathy due to Abeta deposition was widespread as were neurofibrillary tangles and neuropil threads, although tau-positive neurites were sparse. Although significant deposition of Abeta42 was observed, no neuritic pathology was associated with these unusual lesions. Overexpressing PS1 DeltaI83/DeltaM84 in cultured cells results in a significantly elevated level of the highly amyloidogenic 42-amino acid amyloid beta-peptide Abeta42. Moreover, functional analysis in Caenorhabditis elegans reveals reduced activity of PS1 DeltaI83/DeltaM84 in Notch signaling. Our data therefore demonstrate that a small deletion of PS proteins can pathologically affect PS function in endoproteolysis of beta-amyloid precursor protein and in Notch signaling. Therefore, the PS1 DeltaI83/DeltaM84 deletion shows a very similar biochemical/functional phenotype like all other FAD-associated PS1 or PS2 point mutations. Since increased Abeta42 production is not associated with classical senile plaque formation, these data demonstrate that amyloid plaque formation is not a prerequisite for dementia and neurodegeneration.

Alzheimer Disease↗

PubMed2000

The Caenorhabditis elegans Ldb/NLI/Clim orthologue ldb-1 is required for neuronal function.

LIM homeodomain (LIM-HD) and nuclear LIM-only proteins play important roles in a variety of developmental processes in animals. In some cases their activities are modulated by a nuclear LIM binding protein family called Ldb/NLI/Clim. Here we characterize the Ldb/NLI/Clim orthologue ldb-1 of the nematode Caenorhabditis elegans. Two alternatively spliced variants exist, which differ in their amino-termini. The ldb-1 orthologue of Caenorhabditis briggsae has the same structure as that of C. elegans and is highly conserved throughout the open reading frame, while conservation to fly and vertebrate proteins is restricted to specific domains: the dimerization domain, the nuclear localization sequence, and the LIM interaction domain. C. elegans ldb-1 is expressed in neurogenic tissues in embryos, in all neurons in larval and adult stages, and in vulval cells, gonadal sheath cells, and some body muscle cells. C. elegans LDB-1 is able to specifically bind LIM domains in yeast two-hybrid assays. RNA inactivation studies suggest that C. elegans ldb-1 is not required for the differentiation of neurons that express the respective LIM-HD genes or for LIM-HD gene autoregulation. In contrast, ldb-1 is necessary for several neuronal functions mediated by LIM-HD proteins, including the transcriptional activation of mec-2, the mechanosensory neuron-specific stomatin.

Amino Acid Sequence↗

PubMed2000

Presenilin is required for proper morphology and function of neurons in C. elegans.

Mutations in the human presenilin genes cause the most frequent and aggressive forms of familial Alzheimer's disease (FAD). Here we show that in addition to its role in cell fate decisions in non-neuronal tissues, presenilin activity is required in terminally differentiated neurons in vivo. Mutations in the Caenorhabditis elegans presenilin genes sel-12 and hop-1 result in a defect in the temperature memory of the animals. This defect is caused by the loss of presenilin function in two cholinergic interneurons that display neurite morphology defects in presenilin mutants. The morphology and function of the affected neurons in sel-12 mutant animals can be restored by expressing sel-12 only in these cells. The wild-type human presenilin PS1, but not the FAD mutant PS1 A246E, can also rescue these morphological defects. As lin-12 mutant animals display similar morphological and functional defects to presenilin mutants, we suggest that presenilins mediate their activity in postmitotic neurons by facilitating Notch signalling. These data indicate cell-autonomous and evolutionarily conserved control of neural morphology and function by presenilins.

Animals↗

PubMed2000

Protein interaction surface of the POU transcription factor UNC-86 selectively used in touch neurons.

The Caenorhabditis elegans POU protein UNC-86 specifies the HSN motor neurons, which are required for egg-laying, and six mechanosensory neurons. To investigate how UNC-86 controls neuronal specification, we characterized two unc-86 mutants that do not respond to touch but show wild-type egg-laying behavior. Residues P145 and L195, which are altered by these mutations, are located in the POU-specific domain and abolish the physical interaction of UNC-86 with the LIM homeodomain protein, MEC-3. This results in a failure to maintain mec-3 expression and in loss of expression of the mechanosensory neuron-specific gene, mec-2. unc-86-dependent expression of genes in other neurons is not impaired. We conclude that distinct residues in the POU domain of UNC-86 are involved in modulating UNC-86 activity during its specification of different neurons. A structural model of the UNC-86 POU domain, including base pairs and amino acid residues required for MEC-3 interaction, revealed that P145 and L195 are part of a hydrophobic pocket which is similar to the OCA-B-binding domain of the mammalian POU protein, Oct-1.

Amino Acid Sequence↗

PubMed2000

Separation of presenilin function in amyloid beta-peptide generation and endoproteolysis of Notch.

Most of the genetically inherited Alzheimer's disease cases are caused by mutations in the presenilin genes, PS1 and PS2. PS mutations result in the enhanced production of the highly amyloidogenic 42/43 amino acid variant of amyloid beta-peptide (Abeta). We have introduced arbitrary mutations at position 286 of PS1, where a naturally occurring PS1 mutation has been described (L286V). Introduction of charged amino acids (L286E or L286R) resulted in an increase of Abeta42/43 production, which reached almost twice the level of the naturally occurring PS1 mutation. Although pathological Abeta production was increased, endoproteolysis of Notch and nuclear transport of its cytoplasmic domain was significantly inhibited. These results demonstrate that the biological function of PS proteins in the endoproteolysis of beta-amyloid precursor protein and Notch can be separated.

Alzheimer Disease↗

PubMed2000

Presenilin-1 differentially facilitates endoproteolysis of the beta-amyloid precursor protein and Notch.

Mutations in the presenilin-1 (PS1) gene are associated with Alzheimer's disease and cause increased secretion of the neurotoxic amyloid-beta peptide (Abeta). Critical intramembraneous aspartates at residues 257 and 385 are required for the function of PS1 protein. Here we investigate the biological function of a naturally occurring PS1 splice variant (PS1 Deltaexon 8), which lacks the critical aspartate 257. Cell lines that stably express PS1 Deltaexon 8 or a PS1 protein in which aspartate residue 257 is mutated secrete significant levels of Abeta, whereas Abeta generation is severely reduced in cells transfected with PS1 containing a mutation of aspartate 385. In contrast, endoproteolytic processing of Notch is almost completely inhibited in cell lines expressing any of the PS1 variants that lack one of the critical aspartates. These data indicate that PS1 may differentially facilitate gamma-secretase-mediated generation of Abeta and endoproteolysis of Notch.

Alternative Splicing↗

PubMed2000

Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases.

Endoproteolysis of beta-amyloid precursor protein (betaAPP) and Notch requires conserved aspartate residues in presenilins 1 and 2 (PS1 and PS2). Although PS1 and PS2 have therefore been proposed to be aspartyl proteases, no homology to other aspartyl proteases has been found. Here we identify homology between the presenilin active site and polytopic aspartyl proteases of bacterial origin, thus supporting the hypothesis that presenilins are novel aspartyl proteases.

Amyloid beta-Peptides↗

PubMed2000

Identification of amino acid residues in the Caenorhabditis elegans POU protein UNC-86 that mediate UNC-86-MEC-3-DNA ternary complex formation.

The POU homeodomain protein UNC-86 and the LIM homeodomain protein MEC-3 are essential for the differentiation of the six mechanoreceptor neurons in the nematode Caenorhabditis elegans. Previous studies have indicated that UNC-86 and MEC-3 bind cooperatively to at least three sites in the mec-3 promoter and synergistically activate transcription. However, the molecular details of the interactions of UNC-86 with MEC-3 and DNA have not been investigated so far. Here we used a yeast system to identify the functional domains in UNC-86 required for transcriptional activation and to characterize the interaction of UNC-86 with MEC-3 in vivo. Our results suggest that transcriptional activation is mediated by the amino terminus of UNC-86, whereas amino acids in the POU domain mediate DNA binding and interaction with MEC-3. By random mutagenesis, we identified mutations that only affect the DNA binding properties of UNC-86, as well as mutations that prevent coactivation by MEC-3. We demonstrated that both the POU-specific domain and the homeodomain of UNC-86, as well as DNA bases adjacent to the proposed UNC-86 binding site, are involved in the formation of a transcriptionally active complex with MEC-3. These data suggest that some residues involved in the contact of UNC-86 with MEC-3 also contribute to the interaction of the functionally nonrelated POU protein Oct-1 with Oca-B, whereas other positions have different roles.

Amino Acid Sequence↗

PubMed1999

The influence of endoproteolytic processing of familial Alzheimer's disease presenilin 2 on abeta42 amyloid peptide formation.

Mutant presenilins (PS) contribute to the pathogenesis of familial Alzheimer's disease (FAD) by enhancing the production of Abeta42 from beta-amyloid precursor protein. Presenilins are endoproteolytically processed to N-terminal and C-terminal fragments, which together form a stable 1:1 complex. We have mapped the cleavage site in the PS2 protein by direct sequencing of its C-terminal fragment isolated from mouse liver. Three different N-terminal residues were identified starting at Val-299, Thr-301, and Leu-307 that correspond closely to the previously described N termini of the C-terminal fragment of human PS1. Mutational analysis of the PS2 cleavage site indicates that the principal endoproteolytic cleavage occurs at residues Met-298/Val-299 and that the N terminus is subsequently modified by secondary proteolytic cleavages. We have generated cleavage defective PS2 constructs, which accumulate exclusively as full-length polypeptides in transfected Neuro2a cells. Functional analysis of such cleavage defective PS2 carrying the FAD mutation Asn-141 --> Ile showed that its Abeta42 producing activity was strongly reduced compared with cleavage-competent FAD PS2. In contrast, cleavage defective PS2 was active in rescuing the egg-laying defect of a sel-12 mutant in Caenorhabditis elegans. We conclude that PS2 endoproteolytic cleavage is not an absolute requirement for its activities but may rather selectively enhance or stabilize its functions.

Alzheimer Disease↗

PubMed1999

A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling.

Presenilin-1 (PS1) facilitates gamma-secretase cleavage of the beta-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid beta-peptide (Abeta42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of beta-amyloid precursor protein indicating a defect in gamma-secretase activity. The reduced gamma-secretase activity results in the almost complete inhibition of Abeta and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Abeta and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks gamma-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Abeta production and Notch signaling by facilitating similar proteolytic cleavages.

Amyloid beta-Peptides↗

PubMed1999

Thermal avoidance in Caenorhabditis elegans: an approach to the study of nociception.

Upon perception of a noxious stimulus, an organism executes defense mechanisms, such as escape responses. The molecular basis of these mechanisms is poorly understood. In this paper we show that upon exposure to noxious temperature, Caenorhabditis elegans reacts by a withdrawal reflex. To analyze this thermal avoidance behavior, we developed a laser-based assay to quantify the response. The escape reflex can be observed in 98% of the adult animals, but is not executed in animals in diapause. The thermal avoidance response differs significantly from the thermotaxis behavior that is based on the perception of physiological temperature. It involves different neurons and is influenced by mutations in distinct genes. As in mammals, the strength of the thermal avoidance response is increased by application of capsaicin, the pungent ingredient in chili peppers. We find that thermal avoidance is strongly reduced in mutants affecting the neural transmission modulated by glutamate and neuropeptides as well as in mutants affecting the structure and function of sensory neurons. We suggest that the study of this nociceptive behavior in C. elegans can be used to understand the genetic and molecular basis of thermal nociception.

Animals↗

PubMed1999

The biological and pathological function of the presenilin-1 Deltaexon 9 mutation is independent of its defect to undergo proteolytic processing.

The two homologous presenilins are key factors for the generation of amyloid beta-peptide (Abeta), since Alzheimer's disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid Abeta (Abeta42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total Abeta production. Presenilins undergo proteolytic processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1Deltaexon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1Deltaexon9 is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of Abeta42. Like PS1Deltaexon9, the resulting presenilin variant (PS1Deltaexon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1Deltaexon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the AD-associated mutation. Therefore not only the increased Abeta42 production but also the decreased biological function of PS1Deltaexon9 is due to a point mutation and independent of the lack of proteolytic processing.

Alzheimer Disease↗

PubMed1999

The biological and pathological function of presenilin proteins--simple cell systems and a worm in Alzheimer's disease research.

Alzheimer's disease (AD) is the most common form of dementia. In a small number of cases AD is genetically inherited. Mutations are associated with so far three genes. These genes encode the beta-amyloid precursor protein (beta APP), as well as presenilin (PS) 1 and -2. Mutations in all three genes affect the generation of amyloid beta-peptide (A beta), which is the major component of senile plaques. Mutations in the PS genes occur much more frequently as those associated with the beta APP gene and can cause the earliest onset of AD ever recorded. PS genes are not only involved in familial AD but also play a functional role in the general production of A beta. Therefore PS proteins are key molecules, which will allow us to understand fundamental aspects of the molecular mechanisms involved in AD. Here we will summarize the pathological as well as biological function of PS and demonstrate that simple systems, such as cultured cells and the worm Caenorhabditis elegans can be used for modern AD research.

Aged↗

PubMed1999

The physiological role of presenilins in cellular differentiation: lessons from model organisms.

Mutations in the human presenilin genes cause the most frequent and aggressive forms of Alzheimer's Disease. They results in an increase of the 42 amino acid variant of amyloid beta peptide that rapidly aggregates into neurotoxic plaques. In addition, lack of presenilin activity prevents the proteolytic cleavage of the Notch receptor of intercellular signaling. The biological role of presenilins is evolutionary conserved in animals. This review summarizes recent results obtained from animal models to understand presenilin activity and malfunction.

Alzheimer Disease↗