Alba Galan

In vivo recruitment by painful stimuli of AMPA receptor subunits to the plasma membrane of spinal cord neurons

&NA; The persistent increase in pain sensitivity observed after injury, known as hyperalgesia, depends on synaptic plasticity in the pain pathway, particularly in the spinal cord. Several potential mechanisms have been proposed, including post‐synaptic exocytosis of the AMPA subclass of glutamate receptors (AMPA‐R), which is known to play a critical role in synaptic plasticity in the hippocampus. AMPA‐R trafficking has been described in spinal neurons in culture but it is unknown if it can also occur in spinal neurons in vivo, or if it can be induced by natural painful stimulation. Here we have induced referred mechanical hyperalgesia in vivo by intracolonic instillation of capsaicin in mice and have observed a recruitment of GluR1 AMPA‐R subunits to neuronal plasma membranes in the lumbar spinal cord. Intracolonic capsaicin induced a rapid (10 min) increase in GluR1, but not GluR2/3 in the synaptosomal membrane fraction which lasted at least 3 h and a decrease in GluR1 subunit in the cytosolic fraction. Capsaicin treatment also provoked CaMKII activation and pre‐treatment with a specific CaMKII inhibitor prevented the GluR1 trafficking. Brefeldin‐A, an antibiotic that inhibits exocytosis of proteins, not only prevented GluR1 trafficking to the membrane but also inhibited referred hyperalgesia in capsaicin‐treated mice. Our results show that delivery of GluR1 AMPA receptor subunits to the cell membrane through a CaMKII activity‐dependent exocytotic regulated pathway contributes to the development of hyperalgesia after a painful stimulus. We conclude that AMPA‐R trafficking contributes to the synaptic strengthening induced in the pain pathway by natural stimulation.

Painful stimuli induce in vivo phosphorylation and membrane mobilization of mouse spinal cord NKCC1 co-transporter.

The Na+ --Cl- --K+ isoform 1 (NKCC1) is a co-transporter that increases the intracellular concentration of chloride. NKCC1 plays a critical role in neuronal excitability and it has been recently suggested that it can contribute to hyperalgesic states by modulating the chloride concentration inside nociceptive neurons. In the spinal cord, trafficking of neurotransmitter receptors from the cytosol to the plasma membrane has been demonstrated to contribute to the development of hyperalgesia. However, it is unknown if trafficking of co-transporters can also occur in the nervous system or if it can be induced by painful stimulation. In this study, we have induced referred mechanical hyperalgesia in vivo by intracolonic instillation of capsaicin in mice. Using subcellular fractionation of proteins and cross-linking of membrane proteins we have observed that intracolonic capsaicin induced a 50% increase in NKCC1 in the plasma membrane of lumbosacral spinal cord 90 and 180 min after instillation, in parallel with a similar decrease in the cytosolic fraction. These effects returned to basal levels 6 h after capsaicin treatment. Intracolonic capsaicin also evoked a rapid (10 min) and transient phosphorylation of NKCC1, however, intracolonic saline did not produce significant changes in either NKCC1 trafficking or phosphorylation and none of the treatments induced any alterations of NKCC1 in the thoracic spinal cord. These results suggest that phosphorylation and recruitment of NKCC1 might play a role in referred mechanical hyperalgesia evoked by a painful visceral stimulus. The time course of the effects observed suggests that phosphorylation could contribute to the initial generation of hyperalgesia whereas trafficking could participate in the maintenance of hyperalgesic states observed at longer time points.

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75NTR. During retinal injury, endogenous NGF, TrkA, and p75NTR are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75NTR affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75NTR agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75NTR action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α2-macroglobulin. Antagonists of p75NTR inhibit TNF-α and α2-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.

Light triggers expression of philanthotoxin-insensitive Ca2+-permeable AMPA receptors in the developing rat retina.

Ca2+‐permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca2+ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca2+‐permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca2+‐permeable AMPARs. Before eye‐opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca2+‐permeable AMPARs. After eye‐opening, however, a subpopulation of Ca2+‐permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM‐1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX‐insensitive AMPARs failed to express in retinae from dark‐reared animals demonstrating that light entering the eye triggers their expression. Eye‐opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca2+‐permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina.

Neuronal Injury External to the Retina Rapidly Activates Retinal Glia, Followed by Elevation of Markers for Cell Cycle Re-Entry and Death in Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.

The Paradoxical Signals of Two TrkC Receptor Isoforms Supports a Rationale for Novel Therapeutic Strategies in ALS

Full length TrkC (TrkC-FL) is a receptor tyrosine kinase whose mRNA can be spliced to a truncated TrkC.T1 isoform lacking the kinase domain. Neurotrophin-3 (NT-3) activates TrkC-FL to maintain motor neuron health and function and TrkC.T1 to produce neurotoxic TNF-α; hence resulting in opposing pathways. In mouse and human ALS spinal cord, the reduction of miR-128 that destabilizes TrkC.T1 mRNA results in up-regulated TrkC.T1 and TNF-α in astrocytes. We exploited conformational differences to develop an agonistic mAb 2B7 that selectively activates TrkC-FL, to circumvent TrkC.T1 activation. In mouse ALS, 2B7 activates spinal cord TrkC-FL signals, improves spinal cord motor neuron phenotype and function, and significantly prolongs life-span. Our results elucidate biological paradoxes of receptor isoforms and their role in disease progression, validate the concept of selectively targeting conformational epitopes in naturally occurring isoforms, and may guide the development of pro-neuroprotective (TrkC-FL) and anti-neurotoxic (TrkC.T1) therapeutic strategies.

The route of administration influences the therapeutic index of an anti-proNGF neutralizing mAb for experimental treatment of Diabetic Retinopathy

Many neurodegenerative retinal diseases are treated with monoclonal antibodies (mAb) delivered by invasive intravitreal injection (IVT). In Diabetic Retinopathy there is a scarcity of effective agents that can be delivered using non-invasive methods, and there are significant challenges in the validation of novel therapeutic targets. ProNGF represents a potential novel target, and IVT administration of a function-blocking anti-proNGF mAb is therapeutic in a mouse model of DR. We therefore compared invasive IVT to less invasive systemic intravenous (IV) and local subconjunctival (SCJ) administration, for therapy of Diabetic Retinopathy. The IV and SCJ routes are safe, afford sustained pharmacokinetics and tissue penetration of anti-proNGF mAb, and result in long–term therapeutic efficacy that blocks retinal inflammation, edema, and neuronal death. SCJ may be a more convenient and less-invasive approach for ophthalmic use and may enable reduced frequency of intervention for the treatment of retinal pathologies.

In retinitis pigmentosa TrkC.T1-dependent vectorial Erk activity upregulates glial TNF-α, causing selective neuronal death

In some diseases the TrkC.T1 isoform is upregulated in glia, associated with glial TNF-α production and neuronal death. What remains unknown are the activating signals in glia, and how paracrine signals may be selective for a targeted neuron while sparing other proximate neurons. We studied these questions in the retina, where Müller glia contacts photoreceptors on one side and retinal ganglion cells on the other. In a mutant Rhodopsin mouse model of retinitis pigmentosa (RP) causing progressive photoreceptor death—but sparing retinal ganglion cells—TrkC.T1 and NT-3 ligand are upregulated in Müller glia. TrkC.T1 activity generates p-Erk, which causes increased TNF-α. These sequential events take place predominantly in Müller fibers contacting stressed photoreceptors, and culminate in selective death. Each event and photoreceptor death can be prevented by reduction of TrkC.T1 expression, by pharmacological antagonism of TrkC or by pharmacological inhibition Erk. Unmasking the sequence of non-cell autologous events and mechanisms causing selective neuronal death may help rationalize therapies.