Yansheng Du

The NF-κB/Rel family of proteins mediates Aβ-induced neurotoxicity and glial activation

The β-amyloid peptide (Aβ) is deposited in neuritic plaques which are characteristic features of Alzheimers disease (AD). Prominent neurodegeneration and glial activation occurs around these plaques leading to the hypothesis that Aβ may play a causative role in the neuronal loss and the inflammatory response associated with AD. Here we show that Aβ-induced toxicity of cultured fetal rat cortical neurons is associated with internucleosomal DNA fragmentation beginning just 6 h after neurons are exposed to Aβ. Additionally, constitutive NF-κB activity readily measured in fetal rat cortical neurons decreases in a concentration- and time-dependent fashion following exposure to Aβ, but there is no corresponding decrease in NF-κB mRNA or protein (p65). An upregulation of both IκBα protein and mRNA which occurs in cortical neurons exposed to Aβ may be responsible for retaining NF-κB in the cytoplasm accounting for the observed decrease in activated NF-κB. The latter is supported by the observation that pretreatment of cortical cultures with an antisense oligonucleotide to IκBα mRNA is neuroprotective. In contrast to cortical neurons, exposure of rat primary astroglial cultures to Aβ results in a concentration- and time-dependent activation of NF-κB with subsequent upregulation of IL-1β and IL-6. Our data suggest that Aβ-induced neurotoxicity as well as astrocyte activation may be medicated by the NF-κB/Rel family of proteins, and thus alterations in NF-κB-directed gene expression may contribute to both the neurodegeneration and inflammatory response which occur in AD.

α2-Macroglobulin as a β-amyloid peptide-binding plasma protein

Abstract: The β‐amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimers disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α2‐macroglobulin (α2M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds 125I‐Aβ(1–42) with high affinity (apparent dissociation constant of 3.8 × 10−10M). Approximately 1 mol of Aβ is bound per mole of α2M. Both native and methylamine‐activated α2M bind 125I‐Aβ(1–42). The binding of 125I‐Aβ(1–42) to α2M is enhanced by micromolar concentrations of Zn2+ (but not Ca2+) and is inhibited by noniodinated Aβ(1–42) and Aβ(1–40) but not by the reverse peptide Aβ(40‐1) or the cytokines interleukin 1β or interleukin 2. α1‐Antichymotrypsin, another plaque‐associated protein, inhibits both the binding of 125I‐Aβ(1–42) to α2M as well as the degradation of 125I‐Aβ(1–42) by proteinase‐activated α2M. Moreover, the binding of 125I‐Aβ(1–42) to α2M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α2M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.

Neuroinflammation and Alzheimer’s disease: critical roles for cytokine/Aβ-induced glial activation, NF-κB, and apolipoprotein E

Neuroscience Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA Departments of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, MO 63110, USA Scios, Inc., Sunnyvale, CA 94086, USA

Peptide inhibitors of caspase-3-like proteases attenuate 1-methyl-4-phenylpyridinum-induced toxicity of cultured fetal rat mesencephalic dopamine neurons.

Abstract Multiple aspartate-specific cysteine proteases have been identified and specific members of this family have been implicated in the apoptotic death of many mammalian cell types. Caspase-3-like proteases seem to play a pivotal role in neuronal apoptosis since mice with germline inactivation of the caspase-3 gene manifest profound alterations in neurogenesis. Moreover, inhibitors of caspase-3-related proteases have been shown to inhibit neuronal apoptosis. Here we extend recent work from our laboratory on the mechanisms mediating the neurotoxic actions of 1-phenyl-4-methylpyridinium using ventral mesencephalon cultures containing dopamine neurons. We demonstrate that low concentrations of 1-phenyl-4-methylpyridinium induce apoptosis in dopamine neurons by morphological and biochemical criteria. Moreover, pretreatment of ventral mesencephalon cultures with the tetrapeptide inhibitors of the caspase-3-like proteases; zVAD-FMK or Ac-DEVD-CHO specifically inhibit death of dopamine neurons neurons induced by low concentrations of 1-phenyl-4-methylpyridinium, whereas the caspase-1-like inhibitor Ac-YVAD-CHO was without effect. Our data indicate that exposure of cultured ventral mesencephalondopamine neurons to low concentrations of 1-phenyl-4-methylpyridinium results in apoptotic death and that caspase-3-like proteases may mediate the neurotoxic apoptotic actions of 1-phenyl-4-methylpyridinium.

Molecular Cloning, Expression, and Chromosomal Localization of a Human Brain-Specific Na+-Dependent Inorganic Phosphate Cotransporter

Abstract: We describe the molecular cloning of a cDNA encoding a human brain Na+‐dependent inorganic phosphate (Pi) cotransporter (hBNPI). The nucleotide and deduced amino acid sequences of hBNPI reveal a protein of 560 amino acids with six to eight putative transmembrane segments. hBNPI shares a high degree of homology with other Na+‐dependent inorganic Pi cotransporters, including those found in rat brain and human and rabbit kidney. Expression of hBNPI in COS‐1 cells results in Na+‐dependent Pi uptake. Northern blot analysis demonstrates that hBNPI mRNA is expressed predominantly in brain and most abundantly in neuron‐enriched regions such as the amygdala and hippocampus. Moderate levels of expression are also observed in glia‐enriched areas such as the corpus callosum, and low levels are observed in the substantia nigra, subthalamic nuclei, and thalamus. In situ hybridization histochemistry reveals relatively high levels of hBNPI mRNA in pyramidal neurons of the cerebral cortex and hippocampus and in granule neurons of dentate gyrus. The level of hBNPI mRNA is quite low in fetal compared with adult human brain, suggesting developmental regulation of hBNPI gene expression. Southern analyses of nine eukaryotic genomic DNAs probed under stringent conditions with hBNPI cDNA revealed that the hBNPI gene is highly conserved during vertebrate evolution and that each gene is most likely present as a single copy. Using fluorescent in situ hybridization, we localized hBNPI to the long arm of chromosome 19 (19q13) in close proximity to the late‐onset familial Alzheimers disease locus.

Rat B2 Sequences Are Induced in the Hippocampal CA1 Region After Transient Global Cerebral Ischemia

Global brain ischemia causes cell death in the CA1 region of the hippocampus 3–5 days after reperfusion. The biological pathway leading to such delayed neuronal damage has not been established. By using differential display analysis, we examined expression levels of poly(A) RNAs isolated from hippocampal extracts prepared from rats exposed to global ischemia and found an up-regulated transcript, clone 17a. Northern blot analysis of clone 17a showed an approximately 35-fold increase in the ischemic brain at 24 h after four-vessel occlusion. Rapid amplification of cDNA ends of clone 17a revealed a family of genes (160–540 base pairs) that had the characteristics of rodent B2 sequences. In situ hybridization demonstrated that the elevated expression of this gene was localized predominantly in the CA1 pyramidal neurons. The level of expression in the CA1 region decreased dramatically between 24 and 72 h after ischemia. The elevated expression of clone 17a was not observed in four-vessel occlusion rats treated with the compound LY231617, an antioxidant known to exert neuroprotection in rats subjected to global ischemia. Since delayed neuronal death has the characteristics of apoptosis, we speculate that clone 17a may be involved in apoptosis. We examined the expression level of clone 17a inin vitro models of apoptosis using cerebellar granule neurons that were subjected to potassium removal, glutamate toxicity, or 6-hydroxydopamine treatment and found that clone 17a transcripts were induced in cerebellar granule neurons by glutamate or 6-hydroxydopamine stimulation but not potassium withdrawal.