Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK

Mutations in ion channels contribute to neurological disorders, but determining the basis of their role in pathophysiology is often unclear. In humans, 2 mutations have been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2 bp frameshift mutation (F139WfsX24) and TRESK-C110R, a missense mutation. Despite the fact that both mutants strongly inhibit TRESK, only TRESK-MT leads to an increase in sensory neuron excitability and is associated with a migraine phenotype. Here, we identify a new mechanism, termed frameshift mutation induced Alternative Translation Initiation (fsATI) that may explain why TRESK-MT but not TRESK-C110R is associated with migraine disorder. fsATI leads, from the same TRESK-MT mRNA, to two proteins: TRESK-MT1 and TRESK-MT2. We show that by co-assembling with and inhibiting TREK1 and TREK2, another subfamily of K2P channels, overexpression of TRESK-MT2 increases trigeminal sensory neuron excitability, a key component of migraine induction, leading to a migraine-like phenotype. This finding identifies TREK as a potential molecular target in migraine pathophysiology and resolves the contradictory lack of effect of TRESK-C110R which targets only TRESK and not TREK. Finally, taking into account the potential for fsATI allowed us to identify a new migraine-related TRESK mutant, Y121LfsX44, which also leads to the production of two TRESK fragments, indicating that this mechanism may be widespread. Together, our results suggest that genetic analysis of disease-related mutations should consider fsATI as a distinct class of mutations

Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor

Retinitis pigmentosa results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. We used a retinal G protein-coupled receptor, mGluR2, which we chemically engineered to respond to light. In retinal ganglion cells (RGCs) of blind rd1 mice, photoswitch-charged mGluR2 ("SNAG-mGluR2") evoked robust OFF responses to light, but not in wild-type retinas, revealing selectivity for RGCs that have lost photoreceptor input. SNAG-mGluR2 enabled animals to discriminate parallel from perpendicular lines and parallel lines at varying spacing. Simultaneous viral delivery of the inhibitory SNAG-mGluR2 and excitatory light-activated ionotropic glutamate receptor LiGluR yielded a distribution of expression ratios, restoration of ON, OFF and ON-OFF light responses and improved visual acuity. Thus, SNAG-mGluR2 restores patterned vision and combinatorial light response diversity provides a new logic for enhanced-acuity retinal prosthetics

Optical Control of Metabotropic Glutamate Receptors

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease

AIDS-related mycoses: the way forward.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries

Use of Anionic Contrast agent Magnetic Resonance Imaging (ACMRI) as a new technique for assessing intervertebral disc degeneration

Glycosaminoglycan (GAG) depletion is a consistent sign of intervertebral disc degeneration, a cause of lower back pain. Anionic contrast agent MRI (ACMRI) has been able to quantify GAG loss in articular cartilage but it has not yet been tested in the intervertebral disc in a controlled setting. We assessed the feasibility of ACMRI to measure GAG depletion in porcine lumbar intervertebral discs. Three studies were undertaken. In study 1, we performed in-vitro dynamic diffusion MR imaging to assess the best method to ensure contrast agent uptake occurred in the disc. Signal intensity of discs bathed in contrast agent was measured at various points over a 10 hour scan. We determined that isolating the disc from the spine and manually exposing the cartilaginous endplates enhanced diffusion into the central nucleus. This result was used in our subsequent studies. Our second study assessed the ability of ACMRI to indirectly assess GAG concentration in the disc. In-vitro contrast agent uptake in healthy and GAG-degenerated discs was measured by calculating T1 times of disc tissue before and after contrast agent exposure. Using Analysis of Variance, we tested the null hypothesis that the magnitude of T1 after contrast uptake and the change in T1 from before to after contrast uptake (z.T1) was the same in healthy and GAG-depleted discs. The nucleus of degenerated specimens had significantly lower post-contrast T1 times and significantly larger AT1 than healthy discs. There were no significant differences found in the annulus of healthy and degenerated discs. In our final study, we designed a research protocol to correlate axial mechanical properties and ACMRI indices of healthy and GAG-degenerated discs. Loading repeatability tests revealed a one degree of rotational freedom rig, combined with facet joint removal will give reproducible results on repeated tests. Six specimens were tested, and compressive stiffness dropped more in GAG-degenerated discs. ACMRI may be useful in creating a new quantifiable scale of disc degeneration. It may also help in assessing the efficacy of disc therapeutic techniques, and to study the effect of GAG health on the in-vivo mechanics of the spine.Applied Science, Faculty ofMechanical Engineering, Department ofGraduat