Patricia B. Eisenhauer

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.

Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin.

ABSTRACT Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic Escherichia coli strains that produce Shiga toxins. Globotriaosylceramide (Gb3) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-α) upregulates Gb3 in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-α treatment enhanced Shiga toxin binding and sensitivity to toxin. This upregulation was specific for Gb3 species containing normal fatty acids (NFA). Central nervous system (CNS) pathology in HUS could involve cytokine-stimulated elevation of endothelial NFA-Gb3 levels. Differential expression of Gb3 species may be a critical determinant of Shiga toxin toxicity and of CNS involvement in HUS.

Toxicity of various amyloid β peptide species in cultured human blood–brain barrier endothelial cells: Increased toxicity of Dutch‐type mutant

The amyloid β peptide (Aβ) is the major component of the neuritic and cerebrovascular amyloid plaques that are one of the characteristic features of Alzheimers disease (AD). This peptide has been shown to be toxic to several relevant cell types, including neurons, cerebrovascular smooth muscle cells, and endothelial cells. We have studied the toxic effects of both soluble and aggregated species of Aβ1–40 and the mutation Aβ1–40Glu→Gln22, which is the major species deposited in the cerebrovascular blood vessels of victims of hereditary cerebral hemorrhage with amyloidosis, Dutch type. We find that aggregates of both peptides, as well as of Aβ1–42 and Aβ25–35, are toxic to cultured human cerebrovascular endothelial cells (hBEC) obtained from the brain of a victim of AD (at doses lower than those that are toxic to CNS neurons or leptomeningeal smooth muscle cells). Soluble Aβ1–40 Gln22 is equally toxic to hBEC, whereas wild‐type Aβ1–40 is toxic only at higher doses. This toxicity is seen at the lowest dose of Aβ1–40 Gln 22 used, 20 nM. The soluble Aβ1–40Gln22 aggregates on the surface of the cells, in contrast to Aβ1–40, and its toxicity can be blocked both by an inhibitor of free radical formation and by Congo red, which inhibits amyloid fibril formation. We discuss the possibility that the enhanced toxicity of Aβ1–40Gln22 is mediated by a Aβ receptor on the endothelial cells. J. Neurosci. Res. 60:804–810, 2000.

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.

p amyloid fragments derived from activated platelets deposit in cerebrovascular endothelium: usage of a novel blood brain barrier endothelial cell model system

Amyloid precursor protein (AβPP) processing results in generation of amyloid β peptide (Aβ) which deposits in the brain parenchyma and cerebrovasculature of patients with Alzheimers disease (AD). Evidence that the vascular deposits derive in part from AβPP fragments originating from activated platelets includes findings that individuals who have had multiple small strokes have a higher prevalence of AD compared to individuals who have taken antiplatelet drugs. Thus, determination ofwhether platelet AβPP fragments are capable of traversing the blood-brain barrier (BBB) is critical. We have established that activated platelets from patients with AD retain more surface trans-membrane-bound AβPP (mAβPP) than control platelets. We report here that this mAβPP can be cleaved to Aβ-containing fragments which pass through a novel BBB model system. This model utilizes human BBB endothelial cells (BEC) isolated from brains of patients with AD. These BEC, after exposure to activated platelets which have been surface-labeled with fluorescein and express surface-retained mAβPP, cleave fluorescein-tagged surface proteins, including mAβPP, resulting in passage to the BEC layer. The data confirm that BEC contribute to processing of platelet-derived mAβPP and show that the processing yields Aβ con-tainingfragments which could potentially contribute to cerebrovascular Aβ deposition.

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.

Blood brain barrier endothelial cells express candidate amyloid precursor protein-cleaving secretases

Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimers disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.

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.