Pharmacological preclinical comparison of tenecteplase and alteplase for the treatment of acute stroke.

Alteplase (rtPA) remains the standard thrombolytic drug for acute ischemic stroke. However, new rtPA-derived molecules, such as tenecteplase (TNK), with prolonged half-lives following a single bolus administration, have been developed. Although TNK is currently under clinical evaluation, the limited preclinical data highlight the need for additional studies to elucidate its benefits. The toxicities of rtPA and TNK were evaluated in endothelial cells, astrocytes, and neuronal cells. In addition, their in vivo efficacy was independently assessed at two research centers using an ischemic thromboembolic mouse model. Both therapies were tested via early (20 and 30 min) and late administration (4 and 4.5 h) after stroke. rtPA, but not TNK, caused cell death only in neuronal cultures. Mice were less sensitive to thrombolytic therapies than humans, requiring doses 10-fold higher than the established clinical dose. A single bolus dose of 2.5 mg/kg TNK led to an infarct reduction similar to perfusion with 10 mg/kg of rtPA. Early administration of TNK decreased the hemorrhagic transformations compared to that by the early administration of rtPA; however, this result was not obtained following late administration. These two independent preclinical studies support the use of TNK as a promising reperfusion alternative to rtPA.

Expanding the genetic and phenotypic spectrum of congenital myasthenic syndrome: new homozygous VAMP1 splicing variants in 2 novel individuals.

We report the cases of two Spanish pediatric patients with hypotonia, muscle weakness and feeding difficulties at birth. Whole-exome sequencing (WES) uncovered two new homozygous VAMP1 (Vesicle Associated Membrane Protein 1) splicing variants, NM_014231.5:c.129+5 G > A in the boy patient (P1) and c.341-24_341-16delinsAGAAAA in the girl patient (P2). This gene encodes the vesicle-associated membrane protein 1 (VAMP1) that is a component of a protein complex involved in the fusion of synaptic vesicles with the presynaptic membrane. VAMP1 has a highly variable C-terminus generated by alternative splicing that gives rise to three main isoforms (A, B and D), being VAMP1A the only isoform expressed in the nervous system. In order to assess the pathogenicity of these variants, expression experiments of RNA for VAMP1 were carried out. The c.129+5 G > A and c.341-24_341-16delinsAGAAAA variants induced aberrant splicing events resulting in the deletion of exon 2 (r.5_131del; p.Ser2TrpfsTer7) in the three isoforms in the first case, and the retention of the last 14 nucleotides of the 3' of intron 4 (r.340_341ins341-14_341-1; p.Ile114AsnfsTer77) in the VAMP1A isoform in the second case. Pathogenic VAMP1 variants have been associated with autosomal dominant spastic ataxia 1 (SPAX1) and with autosomal recessive presynaptic congenital myasthenic syndrome (CMS). Our patients share the clinical manifestations of CMS patients with two important differences: they do not show the typical electrophysiological pattern that suggests pathology of pre-synaptic neuromuscular junction, and their muscular biopsies present hypertrophic fibers type 1. In conclusion, our data expand both genetic and phenotypic spectrum associated with VAMP1 variants.

Intracerebellar injection of monocytic immature myeloid cells prevents the adverse effects caused by stereotactic surgery in a model of cerebellar neurodegeneration.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) constitute a recently discovered bone-marrow-derived cell type useful for dealing with neuroinflammatory disorders. However, these cells are only formed during inflammatory conditions from immature myeloid cells (IMCs) that acquire immunosuppressive activity, thus being commonly gathered from diseased animals. Then, to obtain a more clinically feasible source, we characterized IMCs directly derived from healthy bone marrow and proved their potential immunosuppressive activity under pathological conditions in vitro. We then explored their neuroprotective potential in a model of human cerebellar ataxia, the Purkinje Cell Degeneration (PCD) mouse, as it displays a well-defined neurodegenerative and neuroinflammatory process that can be also aggravated by invasive surgeries. METHODS: IMCs were obtained from healthy bone marrow and co-cultured with activated T cells. The proliferation and apoptotic rate of the later were analyzed with Tag-it Violet. For in vivo studies, IMCs were transplanted by stereotactic surgery into the cerebellum of PCD mice. We also used sham-operated animals as controls of the surgical effects, as well as their untreated counterparts. Motor behavior of mice was assessed by rotarod test. The Purkinje cell density was measured by immunohistochemistry and cell death assessed with the TUNEL technique. We also analyzed the microglial phenotype by immunofluorescence and the expression pattern of inflammation-related genes by qPCR. Parametric tests were applied depending on the specific experiment: one or two way ANOVA and Student's T test. RESULTS: IMCs were proven to effectively acquire immunosuppressive activity under pathological conditions in vitro, thus acting as MDSCs. Concerning in vivo studios, sham-operated PCD mice suffered detrimental effects in motor coordination, Purkinje cell survival and microglial activation. After intracranial administration of IMCs into the cerebellum of PCD mice, no special benefits were detected in the transplanted animals when compared to untreated mice. Nonetheless, this transplant almost completely prevented the impairments caused by the surgery in PCD mice, probably by the modulation of the inflammatory patterns. CONCLUSIONS: Our work comprise two main translational findings: (1) IMCs can be directly used as they behave as MDSCs under pathological conditions, thus avoiding their gathering from diseased subjects; (2) IMCs are promising adjuvants when performing neurosurgery.