Characterization of behavioral and neuromuscular junction phenotypes in a novel allelic series of SMA mouse models.

A number of mouse models for spinal muscular atrophy (SMA) have been genetically engineered to recapitulate the severity of human SMA by using a targeted null mutation at the mouse Smn1 locus coupled with the transgenic addition of varying copy numbers of human SMN2 genes. Although this approach has been useful in modeling severe SMA and very mild SMA, a mouse model of the intermediate form of the disease would provide an additional research tool amenable for drug discovery. In addition, many of the previously engineered SMA strains are multi-allelic by design, containing a combination of transgenes and targeted mutations in the homozygous state, making further genetic manipulation difficult. A new genetic engineering approach was developed whereby variable numbers of SMN2 sequences were incorporated directly into the murine Smn1 locus. Using combinations of these alleles, we generated an allelic series of SMA mouse strains harboring no, one, two, three, four, five, six or eight copies of SMN2. We report here the characterization of SMA mutants in this series that displayed a range in disease severity from embryonic lethal to viable with mild neuromuscular deficits.

Severe disturbance in the Ca2+ signaling in astrocytes from mouse models of human infantile neuroaxonal dystrophy with mutated Pla2g6.

Infantile neuroaxonal dystrophy (INAD; OMIM #no. 256600) is an inherited degenerative nervous system disorder characterized by nerve abnormalities in brain, spinal cord and peripheral nerves. About 85% of INAD patients carry mutations in the PLA2G6 gene that encodes for a Ca(2+)-independent phospholipase A(2) (VIA iPLA(2)), but how these mutations lead to disease is unknown. Besides regulating phospholipid homeostasis, VIA iPLA(2) is emerging with additional non-canonical functions, such as modulating store-regulated Ca(2+) entry into cells, and mitochondrial functions. In turn, defective Ca(2+) regulation could contribute to the development of INAD. Here, we studied possible changes in ATP-induced Ca(2+) signaling in astrocytes derived from two mutant strains of mice. The first strain carries a hypomorphic allele of the Pla2g6 that reduces transcript levels to 5-10% of that observed in wild-type mice. The second strain carries a point mutation in Pla2g6 that results in inactive VIA iPLA(2) protein with postulated gain in toxicity. Homozygous mice from both strains develop pathology analogous to that observed in INAD patients. The nucleotide ATP is the most important transmitter inducing Ca(2+) signals in astroglial networks. We demonstrate here a severe disturbance in Ca(2+) responses to ATP in astrocytes derived from both mutant mouse strains. The duration of the Ca(2+) responses in mutant astrocytes was significantly reduced when compared with values observed in control cells. We also show that the reduced Ca(2+) responses are probably due to a reduction in capacitative Ca(2+) entry (2.3-fold). Results suggest that altered Ca(2+) signaling could be a central mechanism in the development of INAD pathology.

Aqueous humor concentrations of bimatoprost free acid, bimatoprost and travoprost free acid in cataract surgical patients administered multiple topical ocular doses of LUMIGAN or TRAVATAN.

PURPOSE: To quantify the aqueous humor (AH) concentrations of bimatoprost (amide), travoprost (isopropyl ester), and their hydrolysis products, bimatoprost free acid (BFA) and travoprost free acid (TFA), after multiple topical ocular doses of LUMIGAN and TRAVATAN, respectively, in patients awaiting cataract surgery. METHODS: In 2 separate open-label, sparse-sampling trials, glaucoma patients with cataracts received LUMIGAN (bimatoprost ophthalmic solution, 0.03%) or TRAVATAN (travoprost ophthalmic solution, 0.004%) bilaterally once daily for at least 21 days prior to cataract surgery. Anterior chamber paracentesis was performed at selected times up to 5 h after the last dose and an AH sample was collected. AH samples were assayed by an independent bioanalytical laboratory using a sensitive and validated tandem LC-MS/MS method. The assay lower limits of quantitation were 0.59 nM for bimatoprost, 0.29 nM for BFA, and 0.44 nM for TFA. RESULTS: AH concentrations of BFA (17-phenyl-trinor PGF(2alpha)) were quantifiable in all but one sample at 0.5 h. The maximum concentration achieved (C(max)) of BFA was 30.9 + or - 16.41 nM (n =5), observed at 2 h postdose. AH concentrations of bimatoprost amide were lower than BFA at all time points, with a C(max) of 6.81 + or - 1.36 nM (n = 7) at 1 h postdose. For TFA, measurable AH concentrations were obtained at all time points with a TFA C(max) of 3.91 + or - 2.27 nM (n = 5), which was observed at 3 h after the dose (all data are mean + or - SEM). CONCLUSIONS: Once daily topical ocular administration of LUMIGAN or TRAVATAN for 3 weeks resulted in significant concentrations of BFA and TFA in the AH. Quantifiable levels of bimatoprost amide were also measured. Maximum concentrations of BFA (30.9 nM) and TFA (3.91 nM) in the anterior chamber are sufficient to fully activate the FP prostanoid receptors in the target cells of the ciliary muscle and trabecular meshwork. Both bimatoprost in LUMIGAN and travoprost in TRAVATAN are essentially prodrugs that are rapidly hydrolyzed to their respective free acids that induce the IOP-lowering effect observed with both drugs in vivo.