Not all of the effects of telomerase can be easily explained by its nuclear actions. Growing evidence suggests that TERT, the catalytic subunit of telomerase, can reversibly translocate from the nucleus to organelles (including the mitochondria), in a dose-and time-dependent manner, in response to stressors, Objective: We examined the hypothesis that telomerase activity modulates microvascular flow-mediated dilation, and loss of telomerase activity contributes to the change of mediator from nitric oxide to mitochondrial hydrogen peroxide in patients with coronary artery disease (CAD). Clinical Track Methods and Results:Human coronary and adipose arterioles were isolated for videomicroscopy. Flow-mediated dilation was measured in vessels pretreated with the telomerase inhibitor BIBR-1532 or vehicle. Statistical differences between groups were determined using a 2-way analysis of variance repeated measure (n≥4; P<0.05). L-NAME (N ω -nitro-L-arginine methyl ester; nitric oxide synthase inhibitor) abolished flow-mediated dilation in arterioles from subjects without CAD, whereas polyethylene glycol-catalase (PEG-catalase; hydrogen peroxide scavenger) had no effect. After exposure to BIBR-1532, arterioles from non-CAD subjects maintained the magnitude of dilation but changed the mediator from nitric oxide to mitochondrial hydrogen peroxide (% max diameter at 100 cm H 2 O: vehicle 74.6±4.1, L-NAME 37.0±2.0*, PEG-catalase 82.1±2.8; BIBR-1532 69.9±4.0, L-NAME 84.7±2.2, PEG-catalase 36.5±6.9*). Conversely, treatment of microvessels from CAD patients with the telomerase activator AGS 499 converted the PEG-catalase-inhibitable dilation to one mediated by nitric oxide (% max diameter at 100 cm H 2 O: adipose, AGS 499 78.5±3.9; L-NAME 10.9±17.5*; PEG-catalase 79.2±4.9). Endothelial-independent dilation was not altered with either treatment. Conclusions:

Agrin is a synapse-organizing protein likely to mediate nerve-induced aggregation of acetylcholine receptors and other postsynaptic components at the neuromuscular junction. We used in situ hybridization and polymerase chain reaction (PCR) to define the localization of agrin mRNA and its alternatively spliced forms in the chick embryo nervous system. Agrin cRNA probes intensely labeled motor neurons, dorsal root ganglia, cerebellar Purkinje neurons, and retinal ganglion cells. Neuronal layers in optic tectum and ventricular regions were also labeled. Analysis by PCR showed that all parts of the nervous system at embryonic day 10 contained three major forms of agrin mRNA. Our results raise the possibility that agrin isoforms play a role in synapse formation or other aspects of neuronal development in the central nervous system.

Agrin is a synapse-organizing protein synthesized and externalized by motor neurons in the spinal cord, which organizes the postsynaptic apparatus of the developing neuromuscular junction. Agrin mRNA in the nervous system consists of several alternatively spliced variants. Splicing of agrin gene transcripts at the major site of variability results in four variants: encoding 8 (B8) or 11 (B11) amino acid inserts, both (B19), or predominant variant (B0) without inserts. The insert-containing variants are neuron specific and encode agrin proteins with greater synapse-organizing activity than the B0 variant. Here, we report the localization and developmental regulation of agrin mRNA variants in chick embryo spinal cord and dorsal root ganglia. In situ hybridization using antisense oligodeoxynucleotide (ODN) probes specific for the B8 and B11 sequences shows that the neuron-specific variants are concentrated in ventrolateral cells of the chick embryo spinal cord, presumably motor neurons, beginning at embryonic day 4 (E4). By E14, the insert-containing mRNAs are found almost exclusively in presumptive motor neurons. These variants are also found in dorsal root ganglia and sympathetic ganglia, but not in non-neural tissues. Analysis by polymerase chain reaction showed that the B11 and B19 mRNA variants appeared in spinal cord at E4, whereas the B8 variant was first seen at E14. During development, B11 decreased and disappeared by E20, whereas B8 increased from E14 to E20. A similar time course was seen in dorsal root ganglia. The greatest acetylcholine receptor-aggregating activity in the spinal cord was seen from E6 to E10, coincident with the highest proportion of B11-containing transcripts and with the peak of synaptogenesis in limb muscles. These data provide the first evidence linking appearance of the neuron-specific agrin mRNA variants with expression of the functional protein. The B11 and B19 variants appeared in E2 (stage 15) neural tubes cultured for 2 days with or without notochord and trunk tissues, indicating that there is no peripheral signal required to induce these agrin mRNA variants in developing motor neurons.