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G Thinakaran

Publications and source records attributed to G Thinakaran.

At least 55 records · Page 3Linked to original sources
PubMed1996

Studies on the metabolism and biological function of APLP2.

Amyloid precursor proteins (APP) are a member of a larger family of proteins that include the amyloid precursor-like proteins (APLP) APLPI and APLP2. We have examined the expression and metabolism of APLP2 and document that APLP2 is expressed at high levels in the nervous system and in peripheral tissues. Furthermore, several APLP2 isoforms encoded by alternatively spliced transcripts are posttranslationally modified by a chondroitin sulfate glycosaminoglycan (CSGAG) chain. Furthermore, CSGAG modification is regulated by the insertion of sequences encoded by an alternatively spliced exon. Notably, expression of the CSGAG form of APLP2 appears restricted to embryonic neurons and mature neuronal populations that undergo regeneration, such as olfactory sensory neurons. Thus, differences in posttranslational modifications between the APLP2 isoforms and APP are likely to underlie differences in the regulation and function of these homologues. Our present efforts are directed towards using gene targeting strategies to disrupt the expression of the mouse APP/APLP2 genes to define the normative roles of the encoded molecules in development, plasticity, regeneration, and repair.

Alternative Splicing↗
PubMed1996

Comparative evaluation of synaptophysin-based methods for quantification of synapses.

Development, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the stereological optical dissector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the dissector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereology is the most useful method for estimating synaptic numbers in defined regions of the brain.

Animals↗
PubMed1996

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.

Alzheimer Disease↗
PubMed1996

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.

Amyloid beta-Protein Precursor↗
PubMed1996

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.

Animals↗
PubMed1996

Expression and distribution of amyloid precursor protein-like protein-2 in Alzheimer's disease and in normal brain.

Amyloid precursor-like protein-2 (APLP-2) belongs to a family of homologous amyloid precursor-like proteins. In the present study we report on the expression and distribution of APLP-2 in fetal and adult human brain and in brains of patients with Alzheimer's disease. We demonstrate that APLP-2 mRNAs encoding isoforms predicted to undergo post-translational modification by chondroitin sulfate glycosaminoglycans are elevated in fetal and aging brains relative to the brains of young adults. Immunocytochemical labeling with APLP-2-specific antibodies demonstrates APLP-2 immunoreactivity in cytoplasmic compartments in neurons and astrocytes, in large part overlapping the distribution of the amyloid precursor protein. In Alzheimer's disease brain, APLP-2 antibodies also label a subset of neuritic plaques. APLP-2 immunoreactivity is particularly conspicuous in large dystrophic neurites that also label with antibodies specific for APP and chromogranin A. In view of the age-dependent increase in levels of chondroitin sulfate glycosaminoglycan-modified forms of APLP-2 in aging brain and the accumulation of APLP-2 in dystrophic presynaptic elements, we suggest that APLP-2 may play roles in neuronal sprouting or in the aggregation, deposition, and/or persistence of beta-amyloid deposits.

Adult↗
PubMed1995

A novel protein, amyloid precursor-like protein 2, is present in human brain, cerebrospinal fluid and conditioned media.

A monoclonal antibody, 3B11, was raised to a novel protein, amyloid precursor-like protein 2, which did not recognize amyloid precursor protein. Multiple bands were detected in human brain fractions and cell lysate by Western blotting, indicating the presence of isoforms, 3B11 immunoreactivity was also detected in cerebrospinal fluid and conditioned medium, indicating that the protein is secreted. Immunocytochemistry revealed 3B11 immunoreactivity in sections of human brain.

Amino Acid Sequence↗
PubMed1995

Novel regulation of chondroitin sulfate glycosaminoglycan modification of amyloid precursor protein and its homologue, APLP2.

Alzheimer's disease is characterized by the presence of parenchymal and cerebrovascular deposits of beta-amyloid (A beta). A beta is derived from larger amyloid precursor proteins (APP), a member of a family of related polypeptides that includes amyloid precursor-like proteins, APLP1 and APLP2. APP and APLP2 isoforms are encoded by several alternatively spliced APP and APLP2 transcripts, respectively. We previously reported that the APLP2-751 isoform is modified by the addition of chondroitin sulfate glycosaminoglycan (CS GAG) at Ser-614. In this report, we demonstrate that the APLP2-763 isoform, which contains an insertion of 12 amino acids immediately N-terminal to Ser-614, is not modified by CS GAG. Finally, we demonstrate that like APLP2-751, APP isoforms that lack sequences encoded by exon 15 (L-APP) are also modified by CS GAG, whereas APP forms containing exon 15 are not. We suggest that CS GAG modification of a subset of APP and APLP2 isoforms represents a means of generating functional diversity for these polypeptides.

Alternative Splicing↗
PubMed1995

Metabolism of the amyloid precursor-like protein 2 in MDCK cells. Polarized trafficking occurs independent of the chondroitin sulfate glycosaminoglycan chain.

Deposition of beta-amyloid peptide in senile plaques is a principal neuropathological hallmark of Alzheimer's disease. beta-Amyloid peptide is derived from larger amyloid precursor proteins. Amyloid precursor protein is a member of a family of integral membrane glycoproteins that includes amyloid precursor-like protein (APLP) 1 and 2. Alternatively spliced pre-mRNAs encode several APLP2 isoforms; the APLP2-751 isoform is a substrate for modifications by a chondroitin sulfate glycosaminoglycan (CS GAG) chain, whereas the APLP2-763 isoform does not undergo CS GAG modification. In this report, we have examined the sorting and metabolism of APLP2-751 and APLP2-763 in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that, despite differences in post-translational modifications, both the APLP2-751 proteoglycan and APLP2-763 isoform were targeted and secreted to the basolateral compartment of MDCK cells. We document that the kinetics of intracellular maturation of full-length forms and secretion of soluble derivatives generated from each isoform were indistinguishable. Our results are consistent with the view that, in MDCK cells, the CS GAG chain of APLP2 has little influence on intracellular trafficking and that the principal basolateral targeting determinants are likely to reside in the APLP2 core protein.

Alternative Splicing↗
PubMed1995

Distribution of an APP homolog, APLP2, in the mouse olfactory system: a potential role for APLP2 in axogenesis.

Deposition of beta-amyloid (A beta) in senile plaques is a major pathological characteristic of Alzheimer's disease. A beta is generated by proteolytic processing of amyloid precursor proteins (APP). APP is a member of a family of related polypeptides that includes amyloid precursor-like proteins APLP1 and APLP2. To examine the distribution of APLP2 in the nervous system, we generated antibodies specific for APLP2 and used these reagents in immunocytochemical and biochemical studies of the rodent nervous system. In this report, we document that in cortex and hippocampus, APLP2 is enriched in postsynaptic compartments. In the olfactory system, however, APLP2 is abundant in olfactory sensory axons, and axon terminals in glomeruli. Confocal microscopy revealed that APLP2 is present in both pre- and postsynaptic compartments in the olfactory bulb. Notably, mRNA encoding chondroitin sulfate glycosaminoglycan (CS GAG)-modified forms of APLP2 are enriched in the olfactory epithelium, relative to alternatively-spliced mRNA, encoding CS GAG-free forms of APLP2. In addition, we demonstrate that CS-modified APLP2 forms accumulate in the olfactory bulb. CS proteoglycans are known to play an important role in regulating cell migration and neuronal outgrowth. Since sensory neurons in the olfactory epithelium are in a state of continual turnover, axons of newly generated cells must establish synaptic connections with neurons in the olfactory bulb in adult life. The presence of APLP2 in olfactory sensory axons and glomeruli is consistent with the view that this protein may play an important role in axonal pathfinding and/or synaptogenesis.

Amyloid beta-Protein Precursor↗
PubMed1994

Amyloid precursor-like protein 2 (APLP2) is modified by the addition of chondroitin sulfate glycosaminoglycan at a single site.

beta-Amyloid, the principal component of senile plaques in individuals with Alzheimer's disease, is derived from larger integral membrane glycoproteins, termed amyloid precursor proteins (APP). APP is a member of a family of proteins that includes the amyloid precursor-like proteins APLP1 and APLP2. The present study examines the metabolism of mouse APLP2 in cultured mammalian cells. We report that in stably transfected Chinese hamster ovary and transiently transfected African green monkey kidney (COS-1) cells, APLP2 is modified by glycosaminoglycan (GAG) addition. The sensitivity of GAG-modified APLP2 to digestion with chondroitinase AC indicates that chondroitin sulfate (CS) chains are the preponderant GAG on APLP2. CS GAG modification of APLP2 occurs in a region with little homology to APP. Contained within this heterologous region is a predicted CS modification site, ENEGSGMAEQ (APLP2 residues 610-619); APLP2 polypeptides harboring a serine-to-alanine substitution at position 614 fail to undergo CS GAG modification. Our observation that APLP2 is modified by a pathway distinct from APP suggests that the two molecules may be functionally divergent.

Alzheimer Disease↗
PubMed1994

Expression of a ubiquitous, cross-reactive homologue of the mouse beta-amyloid precursor protein (APP).

Alzheimer's disease is characterized by the presence of senile plaques comprised primarily of deposits of the beta-amyloid protein (A beta) derived from larger amyloid precursor proteins (APP). We have identified a cDNA that encodes a 751-amino acid APP-like protein (designated APLP2) from the mouse that, with exception of the A beta region, is highly homologous to APP. In situ hybridization and quantitative polymerase chain reaction reveal that APLP2 and APP mRNA are expressed in similar, if not identical, neuronal populations and at similar levels. APLP2 appears to mature through the same unusual secretory/cleavage pathway as APP. Furthermore, widely utilized antibodies generated against non-overlapping epitopes of APP do not discriminate between APP and APLP2. Although APLP2 cannot give rise to A beta, its near identity to APP outside the A beta domain confounds the interpretation of previous immunocytochemical and biochemical characterizations of APP biosynthesis and metabolism.

Alzheimer Disease↗
PubMed1993

Expression of c-jun/AP-1 during myogenic differentiation in mouse C2C12 myoblasts.

Mitogen withdrawal triggers myogenic differentiation in skeletal myoblasts in culture. We have examined the expression of the proto-oncogene c-jun during this process in mouse C2C12 myoblasts. c-jun belongs to a family of immediate early genes whose expression is activated in cultured cells in response to the addition of serum growth factors. Interestingly, expression of c-jun was maintained in mouse C2C12 and rat L6 myoblasts undergoing myogenic differentiation under low-serum conditions. Previously it has been reported that expression of c-jun is downregulated during differentiation of C2 cells. However, our results using C2C12 cells, a subclone of the C2 line, show that c-jun mRNA, protein and the activator-protein 1 (AP-1) DNA-binding activity were easily detected in proliferating myoblasts and differentiated myotubes. Although overexpression of c-jun has been shown to block myogenic differentiation in C2 cells, results presented here suggest that expression of c-jun at physiological levels may not interfere with skeletal myogenesis.

Animals↗
PubMed1991

Alterations in the expression of muscle-specific genes mediated by troponin C antisense oligodeoxynucleotide.

The effectiveness of an antisense oligodeoxynucleotide to troponin C (TnC) mRNA in blocking expression of TnC in differentiated chicken myotubes was examined. An 18-nucleotide-long sequence common to both fast and slow isoforms of TnC mRNAs was chosen as the target sequence. The oligomer was found to be efficiently taken up by myotubes. However, the intracellular half-life of the oligomer was found to be only 3 h. Results of studies using different concentrations of oligomer for 3 h in the culture medium showed that compared to the untreated control culture, myotubes incubated with 20 microns antisense oligomer showed a 30% reduction in the steady-state level of TnC mRNAs. An increase of incubation period to 12 h with additions of fresh culture medium containing 20 microns antisense oligomer every 3 h failed to produce any further reduction of TnC mRNA level. Concomitant to the decrease of TnC mRNAs in antisense oligomer-treated cells, the steady-state levels of alpha-actin and alpha-tropomyosin mRNAs were also reduced by approximately 20 to 40%. Analysis of the homology of the sense sequence of this oligomer with that of alpha-actin and alpha-tropomyosin mRNAs suggested that reduction in the level of alpha-actin and alpha-tropomyosin mRNAs was not due to direct hybridization of the antisense oligomer to these mRNAs. Comparison of TnC polypeptide synthesis in untreated and oligomer-treated myotubes showed approximately 70% reduction of fast TnC polypeptide synthesis in antisense oligomer-treated cells. In contrast, slow TnC polypeptide synthesis was not significantly reduced in treated cells. Similarly, alpha-actin and alpha-tropomyosin polypeptide synthesis remained close to the level of untreated cells. Furthermore, analysis of transcription of various muscle-specific mRNAs showed increased synthesis of both TnC and alpha-tropomyosin mRNAs in antisense oligomer-treated myotubes. On the other hand, synthesis of actin mRNAs was not altered by this treatment. These results showed that antisense oligomer was effective in significantly reducing TnC polypeptide synthesis in chicken myotubes. Furthermore, these results suggest that treatment of myotubes with antisense oligomer to TnC mRNA may have triggered a complex array of compensatory processes.

Actins↗
PubMedDate not supplied

Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice.

Amyloid precursor protein (APP) is a member of a larger gene family including amyloid precursor-like proteins (APLP), APLP2 and APLP1. To examine the function of APLP2 in vivo, we generated APLP2 knockout (KO) mice. They are of normal size, fertile, and appear healthy up to 22 months of age. We observed no impaired axonal outgrowth of olfactory sensory neurons following bulbectomy, suggesting against an important role for APLP2 alone in this process. Because APLP2 and APP are highly homologous and may serve similar functions in vivo, we generated mice with targeted APLP2 and APP alleles. Approximately 80% of double KO mice die within the first week after birth, suggesting that APLP2 and APP are required for early postnatal development. The surviving approximately 20% of double KO mice are 20-30% reduced in weight and show difficulty in righting, ataxia, spinning behavior, and a head tilt, suggesting a deficit in balance and/or strength. Adult double KO mice mate poorly, despite apparent normal ovarian and testicular development. Otherwise, double KO mice appear healthy up to 13 months of age. We conclude, that APLP2 and APP can substitute for each other functionally.

Amyloid beta-Protein Precursor↗
PubMedDate not supplied

Lack of requirement for presenilin1 in Notch1 signaling.

Studies in invertebrates have indicated a functional requirement for presenilin (PS) genes in the Notch pathway [1-5]. One model of Notch signal transduction suggests that proteolysis releases an activated Notch fragment that migrates to the nucleus and regulates gene transcription in concert with CBF1/Su(H)/lag1 (CSL) proteins [6-9]. Recent studies suggest that PS genes control the proteolysis and nuclear access of the Notch intracellular domain [3,4,10,11], offering a basis for the functional interaction of PS and Notch genes [12]. Here, we report that Notch1 signaling elicited by the ligand Delta1 was quantitatively unchanged in PS1-deficient primary embryonic fibroblasts (PEFs). Notch1 signals were measured by both the activation of the hairy/enhancer of split (HES1) promoter and by the antagonism of MyoD-induced muscle creatine kinase (MCK) promoter activity. A membrane-tethered ligand-independent Notch1 construct also showed full efficacy in both assays, despite its presumed requirement for cleavage. Although signaling through Notch1 persisted in PS1-deficient cells, we found a marked reduction in the appearance of a complex of a cleaved, intracellular Notch fragment (NICD) and a CSL protein, as previously reported [6] [10]. These studies reveal that PS1 is not required for ligand-dependent Notch signaling, and that PS1 and PS2 may be redundant. Our data also suggest that the identified NICD fragment may not be necessary for Notch signal transduction [9].

Animals↗
PubMedDate not supplied

Regulation of c-jun/AP-1 expression in rat L6 myoblasts.

Myogenic differentiation of skeletal myoblasts in culture is triggered by withdrawal of serum mitogens. Expression of one of the serum-inducible, immediate early genes, the protooncogene c-jun, is maintained under low-serum conditions during myogenic differentiation of L6 myoblasts. In this report we have used agents that modulate protein kinases and Ca2+ levels to investigate how the expression of c-jun and myogenin mRNA and also the activator protein 1 (AP-1) DNA-binding activity are regulated in differentiating L6 cells. Our results show that expression of c-jun and myogenin are regulated independent of each other. Furthermore, down regulation of c-jun expression does not cause an increase in myogenin expression, suggesting that c-jun does not suppress myogenin expression in these cells. Electrophoretic mobility shift and ultraviolet cross-linking analyses revealed that the AP-1 complexes of proliferating myoblasts and differentiating myotubes are formed of similar set of polypeptides, and the AP-1 binding activity is probably modulated by posttranslational modifications in differentiating L6 cells.

Animals↗