Kelly J. Conn

Identification of the protein disulfide isomerase family member PDIp in experimental Parkinson's disease and Lewy body pathology.

Parkinsons disease (PD) is a slowly progressing neurodegenerative disorder with no clear etiology. Pathological hallmarks of the disease include the loss of dopaminergic neurons from the substantia nigra (SN) and the presence of Lewy bodies (LBs) (alpha-synuclein and ubiquitin-positive, eosinophilic, cytoplasmic inclusions) in many of the surviving neurons. Experimental modeling of PD neurodegeneration using the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-pyridinium (MPP(+)) has identified changes in gene expression of different endoplasmic reticulum (ER) stress proteins associated with MPTP- and PD-related neurodegeneration. We show that the protein disulfide isomerase (PDI) family member pancreatic protein disulfide isomerase (PDIp), previously considered exclusively expressed in pancreatic tissue, is uniquely upregulated among PDI family members within 24 h following exposure of retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells to either 1 mM MPP(+) or 10 microM of the highly specific proteasome inhibitor lactacystin. RT-PCR confirms PDIp expression in brain of post-mortem human PD subjects and immunohistochemical studies demonstrate PDIp immunoreactivity in LBs. Collectively, these findings suggest that increased PDIp expression in dopaminergic (DA) neurons might contribute to LB formation and neurodegeneration, and that this increased PDIp expression may be the result of proteasome impairment.

Insulin-like Growth Factor-I Regulates Transcription of the Elastin Gene through a Putative Retinoblastoma Control Element A ROLE FOR Sp3 ACTING AS A REPRESSOR OF ELASTIN GENE TRANSCRIPTION

Previous studies have demonstrated that insulin-like growth factor-I (IGF-I) increases elastin gene transcription in aortic smooth muscle cells and that this up-regulation is accompanied by a loss of protein binding to the proximal promoter. Sp1 has been identified as one of the factors whose binding is lost, and in the present study we show that Sp3 binding is also abrogated by IGF-I, but in a selected manner. In functional analyses using Drosophila SL-2 cells, Sp1 expression can drive transcription from the elastin proximal promoter, while co-expression of Sp3 results in a repression of Sp1 activity. Footprint and gel shift analyses position the IGF-I responsive sequences to a putative retinoblastoma control element (RCE). Mutation of the putative RCE sequence as assessed by transient transfection of smooth muscle cells results in an increase in reporter activity equal in magnitude to that conferred by IGF-I on the wild type promoter. Together these results support the hypothesis that IGF-I-mediated increase in elastin transcription occurs via a mechanism of derepression involving the abrogation of a repressor that appears to be Sp3 binding to the RCE.

Insulin degrading enzyme is localized predominantly at the cell surface of polarized and unpolarized human cerebrovascular endothelial cell cultures

Insulin degrading enzyme (IDE) is expressed in the brain and may play an important role there in the degradation of the amyloid beta peptide (Aβ). Our results show that cultured human cerebrovascular endothelial cells (HCECs), a primary component of the blood–brain barrier, express IDE and may respond to exposure to low levels of Aβ by upregulating its expression. When radiolabeled Aβ is introduced to the medium of cultured HCECs, it is rapidly degraded to smaller fragments. We believe that this degradation is largely the result of the action of IDE, as it can be substantially blocked by the presence of insulin in the medium, a competitive substrate of IDE. No inhibition is seen when an inhibitor of neprilysin, another protease that may degrade Aβ, is present in the medium. Our evidence suggests that the action of IDE occurs outside the cell, as inhibitors of internalization fail to affect the rate of the observed degradation. Further, our evidence suggests that degradation by IDE occurs on the plasma membrane, as much of the IDE present in HCECs was biotin‐labeled by a plasma membrane impermeable reagent. This activity seems to be polarity dependent, as measurement of Aβ degradation by each surface of differentiated HCECs shows greater degradation on the basolateral (brain‐facing) surface. Thus, IDE could be an important therapeutic target to decrease the amount of Aβ in the cerebrovasculature.

Specific up-regulation of GADD153/CHOP in 1-methyl-4-phenyl-pyridinium-treated SH-SY5Y cells

Growth arrest DNA damage‐inducible 153 (GADD153) expression was increased in 1‐methyl‐4‐phenyl‐pyridinium (MPP+)‐treated human SH‐SY5Y neuroblastoma cells as determined by gene microarray analysis. GADD153 expression increased after 24 hr of MPP+ (1 mM) exposure and preceded activation of caspase 3. Comparison of GADD153 expression among cultures treated with other toxins whose primary mode of action is either via mitochondrial impairment (rotenone) or via oxidative stress (6‐hydroxydopamine or hydrogen peroxide) showed that GADD153 was uniquely up‐regulated by MPP+. Together these data suggest that a cellular mechanism distinct from mitochondrial impairment or oxidative stress contributes significantly to the up‐regulation of GADD153 by MPP+ and that GADD153 may function as an inducer of apoptosis following MPP+ exposure. Published 2002 Wiley‐Liss, Inc.

Insulin degrading enzyme is expressed in the human cerebrovascular endothelium and in cultured human cerebrovascular endothelial cells

Insulin degrading enzyme (IDE) is found in the cytosol, peroxisomes and plasma membrane of many cells. Although it preferentially cleaves insulin it can also cleave many other small proteins with diverse sequences including the monomeric form of the amyloid beta peptide (A beta). In the brain, IDE has been reported to be expressed predominantly in neurons. In this study, IDE expression was detected in cultured human cerebrovascular endothelial cells. Using laser capture microdissection followed by PCR analysis, it was found that IDE mRNA is expressed in human brain blood vessels. Using immunofluorescence and multiphoton microscopy IDE was localized to the endothelium of the cerebrovascular blood vessels in human.

Decreased expression of the NADH:ubiquinone oxidoreductase (complex I) subunit 4 in 1-methyl-4-phenylpyridinium -treated human neuroblastoma SH-SY5Y cells

Oxidative stress and mitochondrial dysfunction have been implicated in Parkinsons disease (PD) pathology. NADH:ubiquinone oxidoreductase (complex I) (EC 1.6.99.3) enzyme activity is aberrant in both PD and 1-methyl-4-phenylpyridinium (MPP(+)) models of PD. Reverse transcription polymerase chain reaction of RNA isolated from MPP(+)-treated human neuroblastoma SH-SY5Y cells identified changes in steady-state mRNA levels of the mitochondrial transcript for subunit 4 of complex I (ND4). Expression of ND4 decreased to nearly 50% after 72 h of MPP(+) (1 mM) exposure. The expression of other mitochondrial transcripts did not change significantly under the same conditions. Pre-incubation of cells with the free-radical spin-trap, N-tert-butyl-alpha-(2-sulfophenyl)-nitrone prior to MPP(+) exposure, prevented decreases in cell viability and ND4 expression. This suggests that functional defects in complex I enzyme activity in PD and MPP(+) toxicity may result from changes in steady-state mRNA levels and that free radicals may be important in this process.

cDNA Microarray Analysis of Changes in Gene Expression Associated with MPP+ Toxicity in SH-SY5Y Cells

AbstractcDNA microarray analysis of 1-methyl-4-phenyl-pyridinium (MPP+) toxicity (1 mM, 72 h) in undifferentiated SH-SY5Y cells identified 48 genes that displayed a signal intensity greater than the mean of all differentially expressed genes and a two-fold or greater difference in normalized expression. RT-PCR analysis of a subset of genes showed that c-Myc and RNA-binding protein 3 (RMB3) expression decreased by ∼50% after 72 h of exposure to MPP+ (1 mM) but did not change after 72 h of exposure to 6-hydroxydopamine (25 μM), rotenone (50 nM), and hydrogen peroxide (600 μM). Exposure of retinoic acid (RA)-differentiated SH-SY5Y cells to MPP+ (1 mM, 72 h) also resulted in a decrease in RMB3 expression and an increase in GADD153 expression. In contrast, c-Myc expression was slightly increased in RA-differentiated cells. Collectively, these data provide new insights into the molecular mechanisms of MPP+ toxicity and show that MPP+ can elicit distinct patterns of gene expression in undifferentiated and RA-differentiated SH-SY5Y cells.

Monoclonal antibodies against the human metalloprotease EC 3.4.24.15 label neurofibrillary tangles in Alzheimer's disease brain

Abstract: Alzheimers disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β‐precursor protein. In our search for proteases that can generate the N‐terminus of amyloid β protein (β‐secretases), we discovered a thiol‐dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine‐aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N‐terminus of amyloid β protein, and generates amyloidogenic fragments from full‐length recombinant amyloid β‐precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not “ghost” tangles or amyloid in sections taken from Alzheimers disease brain. This finding provides further evidence for the metalloproteases relevance to Alzheimers disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.

Upregulation of clusterin/apolipoprotein J in lactacystin‐treated SH‐SY5Y cells

Clusterin (apolipoprotein J) is a highly conserved, multifunctional, vertebrate glycoprotein. Several isoforms of clusterin have been described including the predominant secreted isoform (sCLU) and several nuclear isoforms (nCLU) associated with cell death. sCLU has been shown to bind a variety of partly unfolded, stressed proteins including those associated with Lewy bodies (LBs) in patients with Parkinsons disease (PD). The development of familial and sporadic PD has been associated with the ubiquitin‐proteasome system (UPS) dysfunction and aberrant protein degradation. This suggests that failure of the UPS to degrade abnormal proteins may underlie nigral degeneration and LB formation in PD. The effects of toxin‐mediated proteasomal impairment on changes in gene expression and cell viability were studied in differentiated SH‐SY5Y cells. Clusterin expression was increased in cells exposed for 24 hr to the proteasomal inhibitor lactacystin (10 μM) as determined by gene microarray analysis. RT‐PCR showed that sCLU, not nCLU, was the major clusterin isoform expressed in both control and lactacystin‐treated cells. Western blot analysis identified statistically significant increases in sCLU in total cell lysates after 24 hr of lactacystin exposure and showed that sCLU fractionates with the endoplasmic reticulum. Time‐course studies demonstrated that maximal decreases in proteasome activity (4 hr) preceded maximal increases in clusterin expression (24 hr). Together these data suggest that proteasome impairment results in the upregulation of sCLU in SH‐SY5Y cells, supporting the hypothesis that the association of clusterin with LBs in PD may be related to UPS failure.

Identification of full length β-amyloid precursor protein in human neuronal and non-neuronal cell culture supernatant: a possible extracellular source for the generation of Aβ

β protein (Aβ) which is deposited in the amyloid plaques and vasculature in brains of Alzheimers Disease (AD) patients is a 39 to 43 amino acid peptide proteolytically derived from the amyloid precursor protein (AβPP). Three major isoforms are expressed in the brain: AβPP751 and AβPP770 which contain a Kunitz-like protease inhibitor domain (KPI), and AβPP695 To date it is still unknown which AβPP isoforms are the precursors of Aβ, which proteolytic pathways are involved in its production, and if the processing occurs intracellularly and/or extracellularly. We now report the identification, by Western blot analysis, of an Aβ-containing AβPP protein which co-migrates with full length recombinant AβPP751 in the culture supernatant of two human neuroblastoma cell lines and in one human kidney cell line. This protein is recognized with six different antibodies towards AβPP targeting intracellular, extracel lular, and the Aβ region of AβPP. The immunodetection of this Aβ precursor is shown to be specific by ab...