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Biomedical subjects

G Thinakaran

Publications and source records attributed to G Thinakaran.

At least 37 records · Page 2Linked to original sources
PubMed1998

Stable association of presenilin derivatives and absence of presenilin interactions with APP.

Mutations in two related genes, presenilin 1 and 2 presenilin 2 (PS1 and PS2), cosegregate with Alzheimer's disease. PS1 and PS2 are highly homologous polytopic membrane proteins that are subject to endoproteolytic cleavage in vivo. The resulting N- and C-terminal derivatives are the preponderant PS-related species that accumulate in cultured cells and tissue. In earlier studies, we demonstrated that PS1 N- and C-terminal derivatives accumulate to 1:1 stoichiometry and that the absolute levels of fragments are established by a tightly regulated and saturable mechanism. These findings led to the suggestion that the levels of PS1 derivatives might be determined by their association with limiting cellular components. In this study, we use in situ chemical cross-linking and coimmunoprecipitation analyses to document that the N- and C-terminal derivatives of either PS1 or PS2 can be coisolated. Moreover, and in contrast to published reports which documented that PS1 and PS2 form stable heteromeric assemblies with the beta-amyloid precursor protein (APP), we have failed to provide evidence for physiological complexes between PS1 and PS2 holoproteins or their derivatives with APP.

Alzheimer Disease↗
PubMed1998

Axonal transport of mutant superoxide dismutase 1 and focal axonal abnormalities in the proximal axons of transgenic mice.

Superoxide dismutase 1 (SOD1), a ubiquitously expressed enzyme, detoxifies superoxide radicals and participates in copper homeostasis. Mutations in this enzyme have been linked to a subset of autosomal dominant cases of familial amyotrophic lateral sclerosis (FALS), a disorder characterized by selective degeneration of motor neurons. Transgenic mice expressing FALS mutant human (Hu) SOD1 at high levels develop a motor neuron disease, indicating that mutant Hu SOD1 gains properties that are particularly toxic to motor neurons. In this report, we demonstrate that transgenic mice expressing Hu SOD1 with the G37R FALS mutation, but not mice expressing wild-type enzyme, develop focal increases in immunoreactivity in the proximal axons of spinal motor neurons. This SOD1 immunoreactivity and immunoreactivity to hypophosphorylated neurofilament H epitopes are found adjacent to small vacuoles in axons. Using metabolic radiolabeling methods, we show that mutant G37R HuSOD1 as well as endogenous mouse SOD1 are transported anterograde in slow component b in motor and sensory axons of the sciatic nerve. Together, these findings suggest that anterogradely transported mutant SOD1 may act locally to damage motor axons.

Animals↗
PubMed1998

Effects of PS1 deficiency on membrane protein trafficking in neurons.

We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.

Alzheimer Disease↗
PubMed1998

Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides.

Alzheimer's disease (AD) is characterized by the accumulation of cerebral plaques composed of 40- and 42-amino acid beta-amyloid (Abeta) peptides, and autosomal dominant forms of AD appear to cause disease by promoting brain Abeta accumulation. Recent studies indicate that postmenopausal estrogen replacement therapy may prevent or delay the onset of AD. Here we present evidence that physiological levels of 17beta-estradiol reduce the generation of Abeta by neuroblastoma cells and by primary cultures of rat, mouse and human embryonic cerebrocortical neurons. These results suggest a mechanism by which estrogen replacement therapy can delay or prevent AD.

Alzheimer Disease↗
PubMed1998

A role for amyloid precursor-like protein 2 in corneal epithelial wound healing.

PURPOSE: The authors previously showed that the mRNA encoding the amyloid precursor-like protein 2 (APLP2) was one of several genes with upregulated expression in healing corneal epithelium. To elucidate the physiological role of APLP2 in corneal epithelial cells, the expression, distribution, and posttranslational modification of APLP2 were investigated. METHODS: Alternative splicing was assessed by reverse transcription-polymerase chain reaction; translational expression and posttranslational modification were assessed by chondroitinase digestion and Western blotting. The differential distribution of APLP2 in corneal epithelium was examined by in situ hybridization and immunohistochemical analyses. RESULTS: In rat corneal epithelium, the majority of APLP2 mRNA generated from two alternative splicing sites was found to contain the exon encoding a Kunitz protease inhibitor domain in the first site but to lack the exon encoding a 12-amino acid insert in the second site. The absence of the 12-amino acid insert indicated that APLP2 could be modified by the addition of a chondroitin sulfate (CS) glycosaminoglycan chain. The CS proteoglycan nature of APLP2 was verified by chondroitinase digestion. After wounding, APLP2 mRNA and polypeptides were increased markedly in the basal epithelial cells that were actively migrating. Furthermore, APLP2 was observed in the denuded wound bed immediately adjacent to the leading edge of migratory cells and under the epithelial sheet after wound closure. CONCLUSIONS: The wound-induced, basal-cell-specific APLP2 expression correlates with epithelial cell migration. The spatial and temporal expression of Kunitz protease inhibitor-containing, CS-modified APLP2 in healing corneal epithelium is consistent with its hypothesized role(s) in mediating reorganization of the extracellular matrix and dynamic cell-matrix adhesion during reepithelialization.

Alternative Splicing↗
PubMed1997

Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors.

Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for "replacement" we expressed familial Alzheimer's disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).

Animals↗
PubMed1997

Endoproteolytic processing and stabilization of wild-type and mutant presenilin.

Presenilin 1 (PS1), mutated in pedigrees of early-onset familial Alzheimer's disease, is a polytopic integral membrane protein that is endoproteolytically cleaved into 27-kDa N-terminal and 17-kDa C-terminal fragments. Although these fragments are the principal PS1 species found in normal mammalian brain, the role of endoproteolysis in the maturation of PS1 has been unclear. The present study, which uses stably transfected mouse neuroblastoma N2a cells, demonstrates that full-length polypeptides, derived from either wild-type or A246E FAD-mutant human (hu) PS1, are relatively short-lived (t1/2 1.5 h) proteins that give rise to the N- and C-terminal PS1 fragments, which are more stable (t1/2 approximately 24 h). N-terminal fragments, generated artificially by engineering a stop codon at amino acid 306 (PS1-306) of wild-type huPS1, were short-lived, whereas an FAD-linked variant that lacked exon 9 (DeltaE9) and was not endoproteolytically cleaved exhibited a long half-life. These observations suggest that endoproteolytic cleavage and stability are not linked, leading us to propose a model in which wild-type full-length huPS1 molecules are first stabilized then subsequently endoproteolytically cleaved to generate the N- and C-terminal fragments. These fragments appear to represent the mature and functional forms of wild-type huPS1.

Animals↗
PubMed1997

Evidence for phosphorylation and oligomeric assembly of presenilin 1.

Pathogenic mutations in presenilin 1 (PS1) are associated with approximately 50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12, 13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20-23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage lambda protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.

Animals↗
PubMed1997

Processing of presenilin 1 in brains of patients with Alzheimer's disease and controls.

Mutations in the presenilin genes are involved in the aetiology of the majority of familial early-onset Alzheimer disease (AD) cases. Presenilin 1 (PS1) is proteolytically processed in vivo, resulting in the accumulation of N-terminal approximately 27-28 kDa and C-terminal approximately 18 kDa derivatives. To examine the metabolism of PS1 in brains of patients with AD harbouring PS1 mutations I143T and G384A, we performed immunoblot analyses of brain homogenates using well characterized antibodies. We document that approximately 27-28 kDa N-terminal and approximately 18 kDa C-terminal PS1 proteolytic fragments accumulate in brain of these individuals, and that in large part the accumulation pattern is indistinguishable from that observed in brains from individuals with sporadic AD or controls. Notably, little, if any, full-length PS1 was detected in brain tissue of patients carrying PS1 mutations or in those with sporadic AD, indicating that failed proteolysis of PS1 is not a central feature of pathogenesis in these patients.

Adult↗
PubMed1997

Generation of Alzheimer beta-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation.

beta-amyloid protein (A beta) formation was reconstituted in permeabilized neuroblastoma cells expressing human Alzheimer beta-amyloid precursor protein (beta APP) harboring the Swedish double mutation associated with familial early-onset Alzheimer disease. Permeabilized cells were prepared following metabolic labeling and incubation at 20 degrees C, a temperature that allows beta APP to accumulate in the trans-Golgi network (TGN) without concomitant A beta formation. Subsequent incubation at 37 degrees C led to the generation of A beta. A beta production in the TGN persisted even under conditions in which formation of nascent post-TGN vesicles was inhibited by addition of guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable GTP analogue, or by omission of cytosol. These and other results indicate that vesicle budding and trafficking may not be required for proteolytic metabolism of beta APP to A beta, a process that includes "gamma-secretase" cleavage within the beta APP transmembrane domain.

Amyloid beta-Peptides↗
PubMed1997

Ectodomain phosphorylation of beta-amyloid precursor protein at two distinct cellular locations.

The beta-amyloid precursor protein (betaAPP) is a transmembrane protein that is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of betaAPP phosphorylation, we took advantage of an antibody that specifically detects the free C terminus of beta-secretase-cleaved betaAPP containing the Swedish missense mutation (APPssw-beta). This antibody previously established the cellular location of the beta-secretase cleavage of Swedish betaAPP as a post-Golgi secretory compartment (Haass, C., Lemere, C., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nature Med. 1, 1291-1296). We have now localized the selective ectodomain phosphorylation of betaAPP to the same compartment. Moreover, the phosphorylation sites of betaAPP were identified at Ser198 and Ser206 of betaAPP695 by tryptic peptide mapping, mass spectrometry, and site-directed mutagenesis. Intracellular phosphorylation of betaAPP was inhibited by Brefeldin A and by incubating cells at 20 degrees C, thus excluding phosphorylation in the endoplasmic reticulum or trans-Golgi network. Ectodomain phosphorylation within a post-Golgi compartment occurred not only with mutant Swedish betaAPP, but also with wild type betaAPP. In addition to phosphorylation within a post-Golgi compartment, betaAPP was also found to undergo phosphorylation at the cell surface by an ectoprotein kinase. Therefore, this study revealed two distinct cellular locations for betaAPP phosphorylation.

Amyloid beta-Protein Precursor↗
PubMed1997

Identification of candidate proteins binding to prion protein.

Prion diseases are disorders of protein conformation that produce neurodegeneration in humans and animals. Studies of transgenic (Tg) mice indicate that a factor designated protein X is involved in the conversion of the normal cellular prion protein (PrPC) into the scrapie isoform (PrPSc); protein X appears to interact with PrPC but not with PrPSc. To search for PrPC binding proteins, we fused PrP with alkaline phosphatase (AP) to produce a soluble, secreted probe. PrP-AP was used to screen a lambdagt11 mouse brain cDNA library, and six clones were isolated. Four cDNAs are novel while two clones are fragments of Nrf2 (NF-E2 related factor 2) transcription factor and Aplp1 (amyloid precursor-like protein 1). The observation that PrP binds to a member of the APP (amyloid precursor protein) gene family is intriguing, in light of possible relevance to Alzheimer's disease. Four of the isolated clones are expressed preferentially in the mouse brain and encode a similar motif.

Amino Acid Sequence↗
PubMed1997

Hyperaccumulation of FAD-linked presenilin 1 variants in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.

Actins↗
PubMed1997

Amyotrophic lateral sclerosis and Alzheimer disease. Lessons from model systems.

The human neurodegenerative diseases, including motor neuron disease and Alzheimer's disease (AD), are characterized by a selective involvement of certain regions of the brain/spinal cord and selected populations of neurons. Sporadic amyotrophic lateral sclerosis (ALS) is an age-associated disease with cytoskeletal abnormalities and death of motor neurons; familial ALS (FALS), an autosomal dominant disease linked to mutations in superoxide dismutase 1 (SOD1), is manifested by inclusions and degeneration of motor neurons. Autosomal dominant familial AD (FAD), linked to mutations in presenilin (PS1 and PS2) genes or the amyloid precursor protein (APP) gene, shows brain abnormalities (e.g., neurofibrillary tangles, deposits of .-amyloid A., and death of subsets of neurons) similar to those that occur in sporadic AD, the risk of which is enhanced by the presence of one or two copies of apolipoprotein E4 (apoE4) alleles. To examine the mechanisms of these diseases, investigators have used a variety of animal models, including experimentally produced, spontaneously occurring, or genetically engineered models of disease. Studies of models of degeneration of motor neurons (axotomy) and cytoskeletal abnormalities seen in motor neuron disease (i.e., axonopathy induced by .,.'-iminodipropionitrile (IDPN), hereditary canine spinal muscular atrophy (HCSMA), and neurofilament NF transgenic Tg mice) have demonstrated that NF-filled swellings of axons are related to alterations in the biology of NF transport. Tg mice with SOD1 mutations, which develop the clinical features of FALS, show selective degeneration of motor neurons, which is attributed to the acquisition of toxic properties by mutant SOD1. Models of AD include: aged monkeys that show both cognitive/memory deficits and cellular abnormalities (amyloid deposition/cytoskeletal abnormalities of neurons) in cortex and hippocampus; and Tg mice that express mutant human FAD-linked genes (i.e., APP and PS1) and show increased levels of A.42, amyloid deposits, dystrophic neurites, and local responses of astrocytes and microglia. This review discusses the behavioral/neuropathological features of AD, the results of investigations of mechanisms of disease in model systems, and potential utility of some of these models for testing new therapies.

Alzheimer Disease↗
PubMed1996

Assessment of normal and mutant human presenilin function in Caenorhabditis elegans.

We provide evidence that normal human presenilins can substitute for Caenorhabditis elegans SEL-12 protein in functional assays in vivo. In addition, six familial Alzheimer disease-linked mutant human presenilins were tested and found to have reduced ability to rescue the sel-12 mutant phenotype, suggesting that they have lower than normal presenilin activity. A human presenilin 1 deletion variant that fails to be proteolytically processed and a mutant SEL-12 protein that lacks the C terminus display considerable activity in this assay, suggesting that neither presenilin proteolysis nor the C terminus is absolutely required for normal presenilin function. We also show that sel-12 is expressed in most neural and nonneural cell types in all developmental stages. The reduced activity of mutant presenilins and as yet unknown gain-of-function properties may be a contributing factor in the development of Alzheimer disease.

Animals↗
PubMed1996

Expression of presenilin 1 and 2 (PS1 and PS2) in human and murine tissues.

Mutations in genes encoding related proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2), are linked to the majority of cases with early-onset familial Alzheimer's disease (FAD). To clarify potential function(s) of presenilins and relationships of presenilin expression to pathogenesis of AD, we examined the expression of PS1 and PS2 mRNA and PS1 protein in human and mouse. Semi-quantitative PCR of reverse-transcribed RNA (RT-PCR) analysis revealed that PS1 and PS2 mRNA are expressed ubiquitously and at comparable levels in most human and mouse tissues, including adult brain. However, PS1 mRNA is expressed at significantly higher levels in developing brain. In situ hybridization studies of mouse embryos revealed widespread expression of PS1 mRNA with a neural expression pattern that, in part, overlaps that reported for mRNA encoding specific Notch homologs. In situ hybridization analysis in adult mouse brain revealed that PS1 and PS2 mRNAs are enriched in neurons of the hippocampal formation and entorhinal cortex. Although PS1 and PS2 mRNA are expressed most prominently in neurons, lower but significant levels of PS1 and PS2 transcripts are also detected in white matter glial cells. Moreover, cultured neurons and astrocytes express PS1 and PS2 mRNAs. Using PS1-specific antibodies in immunoblot analysis, we demonstrate that PS1 accumulates as approximately 28 kDa N-terminal and approximately 18 kDa C-terminal fragments in brain. Immunocytochemical studies of mouse brain reveal that PS1 protein accumulates in a variety of neuronal populations with enrichment in somatodendritic and neuropil compartments.

Adult↗
PubMed1996

Metabolism of the "Swedish" amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the "beta-secretase" site occurs in the golgi apparatus.

The 4-kDa beta-amyloid peptide (Abeta), a principal component of parenchymal amyloid deposits in Alzheimer's disease, is derived from amyloid precursor proteins (APP). To identify potential intracellular compartments involved in Abeta production, we expressed human APP-695 (APPwt) and APP-695 harboring the Swedish double mutation (APPswe) associated with familial early-onset Alzheimer's disease, in mouse N2a cells. We demonstrate that cells expressing APPswe secrete high levels of Abeta peptides and beta-secretase-generated soluble APP derivatives (APP s beta) relative to cells expressing APPwt. In addition, we observed a concomitant diminution in the levels of alpha-secretase-generated soluble APP derivatives (APP s alpha). Our interpretation of these findings is that beta-secretase cleavage occurs in an intracellular compartment and disables those substrates which would normally be cleaved by alpha-secretase. As anticipated, the levels of APPswe are diminished relative to the steady-state levels of surface-bound APPwt; moreover, surface-bound APPswe and APPwt molecules are released from the plasma membrane after cleavage by alpha-secretase, but not by beta-secretase. Finally, by examining the rate of appearance of specific APP metabolites generated by beta-secretase, we now unequivocally demonstrate that beta-secretase cleavage of APPswe occurs within the Golgi apparatus, as early as the medial compartment.

Alzheimer Disease↗