Identification of alternative splicing of spinocerebellar ataxia type 2 gene.

Spinocerebellar ataxia 2 (SCA-2) is a neurodegenerative disorder caused by the expansion of an unstable CAG/polyglutamine repeat located at the NH(2)-terminus of ataxin-2 protein. Ataxin-2 is composed by 1312 aminoacids and it is expressed ubiquitously in human tissues. To date, the function of ataxin-2 is not known. In this study, we report the characterization of an alternative splice variant of human ataxin-2. The splice transcript lacks the exon 21 and connects exon 20 to exon 22 with the same reading frame of the full length mRNA. This novel isoform of ataxin-2 is conserved in the mouse. It is named type IV to differentiate it from type II splice variant lacking exon 10 (present in human and mouse cDNAs) and from type III, lacking exon 10 and exon 11 seen in mouse. Type IV of human ataxin-2 cDNA is predicted to encode a protein of 1294 residues. Both the full length and the type IV transcript of ataxin-2 are present in several human tissues, including brain, spinal cord, cerebellum, heart and placenta. These findings allow the hypothesis that type I, II and IV of human ataxin-2 might perform different functions.

Intravenous prostaglandin E1 reduces soluble vascular cell adhesion molecule-1 in peripheral arterial obstructive disease.

OBJECTIVES: Elevated levels of soluble (s) vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1, pointing to activation of cells involved in vascular inflammation, have been previously reported in peripheral arterial obstructive disease (PAOD). We tested the hypothesis that intravenous prostaglandin E(1) (PGE(1)) treatment, which produces clinical benefits in this condition, might decrease such levels. METHODS: Ten subjects (age range 58 +/- 10 years, 6 male, 4 female) with characterized Fontaine stage IIa to IV PAOD (ankle/arm pressure index <0.96) were entered into a treatment protocol with twice daily intravenous infusions of PGE(1) (alprostadil) at 120 microg per day, repeated for 10 consecutive days. Preinfusion and postinfusion plasma samples were stored for blind enzyme immunoassays of soluble adhesion molecules and the fibrinolytic marker tissue plasminogen activator, type-1 plasminogen-activator inhibitor, and D -dimer. RESULTS: Estimates of severity of pain at rest, consumption of analgesics, magnitude of trophic lesions, remission to lower Fontaine stages, and favorable changes in the venoarteriolar reflex documented significant beneficial effects of the treatment. Significant (P <.01) pretreatment and posttreatment reductions of in all soluble markers explored were found. Particularly, sVCAM-1 exhibited a significant decrease after each infusion, which was sustained at the last day of treatment (from 854 +/- 214 ng/mL to 775 +/- 215 ng/mL across the first infusion, from 773 +/- 146 ng/mL to 680 +/- 110 ng/mL across the last infusion). CONCLUSION: Thus a global decrease of vascular cell activation appears to occur as a result of PGE(1) administration and may contribute to the observed clinical benefits in PAOD.

High levels of adhesion molecules are associated with impaired endothelium-dependent vasodilation in patients with peripheral arterial disease.

Soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cellular adhesion molecule-1 (sVCAM-1) were measured alongside flow-mediated vasodilation (FMD) in 34 patients with intermittent claudication and 14 control subjects. Patients with plasma sICAM-1 >253 ng/mL (median value) showed lower FMD than those with sICAM-1 < 253 ng/mL (5.6 +/- 1.8% vs 9.6 +/- 4.2%, p < 0.01). Similarly, in the 17 patients with plasma sVCAM-1 > 414 ng/mL, FMD was lower than in the remaining 17 patients (6.1 +/- 1.9% vs 9.2 +/- 4.5%, p < 0.05). Additionally, when endothelial dysfunction was defined as FMD < or = 5.5%, patients with FMD below this value had higher plasma concentrations of sICAM-1 and sVCAM-1 than those with FMD > 5.5%. Therefore, our findings indicate a close association between elevated plasma levels of adhesion molecules and endothelial dysfunction. As impaired endothelial function is one of the first steps in atherogenesis, our findings have clinical relevance since they serve as the basis for further evaluation of sICAM-1 and sVCAM-1 as potential plasma markers for progression of atherosclerosis in a population at high risk.

Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication.

Adhesion molecules play a relevant role in the pathogenesis of vascular diseases. In 21 patients with intermittent claudication and 18 sex- and age-matched control subjects, we measured plasma levels of the circulating form of the adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) alongside von Willebrand factor (vWF), at rest, at maximally tolerated exercise and 5, 15 and 30 min after exercise. In controls, plasma sICAM-1 levels did not change with exercise, while in claudicants they increased from 285+/-15 to 317+/-16 ng/ml (p<0.01). Also for sVCAM-1 exercise did not modify plasma levels of sVCAM-1 in controls but increased it in claudicants from 671+/-45 to 751+/-47 ng/ml (p<0.05). Similarly, vWF did not change with exercise in controls, but increased in claudicants from 100+/-9% to 111+/-8% of value for pooled normal plasma (p<0.05). Exercise-induced changes in sICAM-1 negatively correlated with the maximal tolerated walking time, which is an index of disease severity. These findings indicate that, in claudicants, exercise is associated with increase in plasma levels of sICAM-1 and sVCAM-1.

The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates.

A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.

Polyglutamine-mediated aggregation and cell death.

The expansion of CAG repeats is the genetic defect underlying eight neurodegenerative diseases. A common feature of these disorders is the presence of intracellular aggregates in neuronal cells. It is still unclear the significance of these cellular inclusions in the neurodegenerative process, since cell death without aggregate formation has been reported. We have constructed a synthetic fusion protein containing 17 or 43 CAG repeats and the green fluorescent protein that recapitulates the features of CAG-expanded alleles. Expression of 43, but not 17 CAG repeats results in formation of nuclear aggregates in human neuroblastoma cells. Moreover, the normal allele (17 CAG) is sequestered in the inclusion bodies. The presence of nuclear inclusions tightly correlates with apoptosis in cells expressing the protein encoding 43 CAG repeats. Cells harboring nuclear aggregates stop proliferation and undergo apoptosis. Moreover, the inhibition of protein degradation pathway increases intracellular aggregates and cell death. These data indicate that intranuclear aggregates induce apoptosis and suggest that the degradation of unfolded proteins improves cell survival.

Transglutaminase activity is related to CAG repeat length in patients with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder associated with CAG repeat expansion. We measured transglutaminase (TGase) activity in lymphocytes from 35 HD patients and from healthy individuals to ascertain whether it was altered in this condition. TGase activity was above maximum control levels in 25% of HD patients; it was correlated with the age of the patient and inversely correlated with the CAG repeat length. These results suggest that: (1) HD could be biochemically heterogeneous, and (2) the length of the CAG repeat expansion/TGase ratio could be important in the manifestation of HD.