Neurotoxicity from accumulation of misfolded/mutant proteins is thought to drive pathogenesis in neurodegenerative diseases. Since decreasing levels of proteins responsible for such accumulations is likely to ameliorate disease, a therapeutic strategy has been developed to downregulate almost any gene in the CNS. Modified antisense oligonucleotides, continuously infused intraventricularly, have been demonstrated to distribute widely throughout the CNS of rodents and primates, including the regions affected in the major neurodegenerative diseases. Using this route of administration, we found that antisense oligonucleotides to superoxide dismutase 1 (SOD1), one of the most abundant brain proteins, reduced both SOD1 protein and mRNA levels throughout the brain and spinal cord. Treatment initiated near onset significantly slowed disease progression in a model of amyotrophic lateral sclerosis (ALS) caused by a mutation in SOD1. This suggests that direct delivery of antisense oligonucleotides could be an effective, dosage-regulatable means of treating neurodegenerative diseases, including ALS, where appropriate target proteins are known.

Little is known about the mechanisms that account for inhibition of gene expression by antisense oligonucleotides at the level of molecular cell biology. For this purpose, we have selected potent 2-O-(2-methoxy)ethyl antisense oligonucleotides (IC 50 ‫؍‬ 2 and 6 nM) that target the 5 cap region of the human intercellular adhesion molecule 1 (ICAM-1) transcript to determine their effects upon individual processes of mRNA metabolism in HUVECs. Given the functions of the 5 cap structure throughout mRNA metabolism, antisense oligonucleotides that target the 5 cap region of a target transcript have the potential to modulate one or more metabolic stages of the message inside the cell. In this study we found that inhibition of protein expression by these RNase H independent antisense oligonucleotides was not due to effects on splicing or transport of the ICAM-1 transcript, but due instead to selective interference with the formation of the 80 S translation initiation complex. Interestingly, these antisense oligonucleotides also caused an increase in ICAM-1 mRNA abundance in the cytoplasm. These results imply that ICAM-1 mRNA turnover is coupled in part to translation.

Purpose: The L1adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1is released from carcinoma cells in a soluble form. Soluble L1is present in serum and ascites of ovarian carcinoma patients.We investigated the mode of L1cleavage and the function of soluble L1. Experimental Design: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1and L1cleavage byWestern blot analysis and ELISA. Results: We find that in ovarian carcinoma cells the constitutive cleavage of L1proceeds in secretory vesicles. We show that apoptotic stimuli like C 2 -ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. Conclusions: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/ 1 (75%) .

Numerous transmembrane proteins, including the blood pressure regulating angiotensin converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein (APP), are proteolytically shed from the plasma membrane by metalloproteases. We have used an antisense oligonucleotide (ASO) approach to delineate the role of ADAM10 and tumour necrosis factor-a converting enzyme (TACE; ADAM17) in the ectodomain shedding of ACE and APP from human SH-SY5Y cells. Although the ADAM10 ASO and TACE ASO significantly reduced (> 81%) their respective mRNA levels and reduced the a-secretase shedding of APP by 60% and 30%, respectively, neither ASO reduced the shedding of ACE. The mercurial compound 4-aminophenylmercuric acetate (APMA) stimulated the shedding of ACE but not of APP. The APMA-stimulated secretase cleaved ACE at the same Arg-Ser bond in the juxtamembrane stalk as the constitutive secretase but was more sensitive to inhibition by a hydroxamate-based compound. The APMA-activated shedding of ACE was not reduced by the ADAM10 or TACE ASOs. These results indicate that neither ADAM10 nor TACE are involved in the shedding of ACE and that APMA, which activates a distinct ACE secretase, is the first pharmacological agent to distinguish between the shedding of ACE and APP.Keywords: ADAM; antisense oligonucleotide; metalloprotease; secretase; tumour necrosis factor-a converting enzyme.Angiotensin converting enzyme (ACE) is critically involved in blood pressure regulation due to its action in generating angiotensin II and in inactivating bradykinin [1]. The enzyme also has a role in the development of vascular pathology and endothelium remodelling in some disease states [2]. Inhibitors of ACE have emerged as first-line therapy for a range of cardiovascular and renal diseases, including hypertension, congestive heart failure, myocardial infarction and diabetic nephropathy. The transmembrane protein ACE is proteolytically shed from the cell surface by its cognate secretase with the resulting soluble form circulating in the blood and present in other body fluids [3].In addition to ACE, a number of other integral membrane proteins are shed from the cell surface by a post-translational proteolytic cleavage event mediated by zinc metalloproteases [4,5]. Another such shedding process is the nonamyloidogenic processing of the Alzheimer's disease amyloid precursor protein (APP) [6]. Cleavage of APP within the neurotoxic amyloid b region by a-secretase precludes the deposition of intact amyloid b [7] and releases the large soluble ectodomain of APP, sAPPa, which has been shown to have neuroprotective and memory enhancing properties [8]. The APP a-secretase is a membrane-associated metalloprotease [9] that is inhibited by hydroxamic acid-based compounds such as batimastat [10]. Members of the ADAMs (a disintegrin and metalloprotease) family have been put forward as candidate a-secretases, in particular ADAM10 and ADAM17 (tumour necrosis factor-a converting enzyme; TACE) ([11,12] and reviewed in [13]). Although the ACE secretase has no...