Activation of matrix metalloproteinases following anti-Aß immunotherapy; implications for microhemorrhage occurrence.

BACKGROUND: Anti-Aß immunotherapy is a promising approach to the prevention and treatment of Alzheimer's disease (AD) currently in clinical trials. There is extensive evidence, both in mice and humans that a significant adverse event is the occurrence of microhemorrhages. Also, vasogenic edema was reported in phase 2 of a passive immunization clinical trial. In order to overcome these vascular adverse effects it is critical that we understand the mechanism(s) by which they occur. METHODS: We have examined the matrix metalloproteinase (MMP) protein degradation system in two previously published anti-Aß immunotherapy studies. The first was a passive immunization study in which we examined 22 month old APPSw mice that had received anti-Aß antibodies for 1, 2 or 3 months. The second is an active vaccination study in which we examined 16 month old APPSw/NOS2-/- mice treated with Aß vaccination for 4 months. RESULTS: There is a significant activation of the MMP2 and MMP9 proteinase degradation systems by anti-Aß immunotherapy, regardless of whether this is delivered through active vaccination or passive immunization. We have characterized this activation by gene expression, protein expression and zymography assessment of MMP activity. CONCLUSIONS: Since the MMP2 and MMP9 systems are heavily implicated in the pathophysiology of intracerbral hemorrhage, these data may provide a potential mechanism of microhemorrhage due to immunotherapy. Increased activity of the MMP system, therefore, is likely to be a major factor in increased microhemorrhage occurrence.

Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence.

Recently, a small 11-amino acid amidated peptide, dopamine neuron stimulating peptide-11 (DNSP-11), was shown to exert neurotrophic-like actions on primary dopaminergic neurons and in parkinsonian rat models. This suggests smaller neurotrophic-like molecules may be deliverable and modifiable for therapeutic use. Here we evaluate the molecular and cellular protection properties of DNSP-11 and two other amidated-peptides, a 5-mer (DNSP-5) and a 17-mer (DNSP-17), hypothesized to be endoproteolytically processed from the pro- and mature glial cell line-derived neurotrophic factor (GDNF) protein sequence, respectively. Far-UV circular dichroism spectra show that the three DNSPs are soluble and act independently in vitro. Reverse phase HPLC and mass spectrometry analysis show that the three peptides are stable for one month at a variety of storage and experimental conditions. To gain insight into their biodistribution properties in the brain, we used affinity chromatography to show that DNSP-17 binds heparin equally as tight as GDNF, whereas DNSP-5 and DNSP-11 do not bind heparin, which should facilitate their delivery in vivo. Finally, we present data showing that DNSP-11 provides dose-dependent protection of HEK-293 cells from staurosporine and 3-nitropropionate (3-NP) cytotoxicity, thereby supporting its broad mitochondrial-protective properties.

Configuration and rearrangement of the human GAGE gene clusters.

The GAGE protein is detected only in cancer and in testis and is expressed from a cluster of nearly identical gene copies on the X-chromosome. We determined the lengths of these GAGE gene clusters from human families, identical twins, and in clinical samples from cancer patients. The GAGE cluster lengths proved to be highly heterogeneous, ranging from 13 to 39 gene copies, with an average content of 20 GAGE genes per cluster. Low levels of mei-otic rearrangement in families and mitotic rearrangement in adult solid tumors are detectable. Analysis of Rothmund -Thomson syndrome (RTS) kindreds and probands showed GAGE cluster inheritance and stability indistinguishable from that found in non-RTS individuals. These observations support the concept of evolutionarily rapid rearrangement of clustered repetitive sequences in the human genome.