Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.

Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration.

DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double-strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aß1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

Non-viral-mediated suppression of AMIGO3 promotes disinhibited NT3-mediated regeneration of spinal cord dorsal column axons.

After injury to the mature central nervous system (CNS), myelin-derived inhibitory ligands bind to the Nogo-66 tripartite receptor complex expressed on axonal growth cones, comprised of LINGO-1 and p75NTR/TROY and induce growth cone collapse through the RhoA pathway. We have also shown that amphoterin-induced gene and open reading frame-3 (AMIGO3) substitutes for LINGO-1 and can signal axon growth cone collapse. Here, we investigated the regeneration of dorsal root ganglion neuron (DRGN) axons/neurites after treatment with a short hairpin RNA (sh) AMIGO3 plasmid delivered with a non-viral in vivo-jetPEI vector, and the pro-survival/axogenic neurotrophin (NT) 3 in vitro and in vivo. A bicistronic plasmid, containing both shAMIGO3 and NT3 knocked down >75% of AMIGO3 mRNA in cultured DRGN and significantly overexpressed NT3 production. In vivo, intra-DRG injection of in vivo-jetPEI plasmids containing shAMIGO3/gfp and shAMIGO3/nt3 both knocked down AMIGO3 expression in DRGN and, in combination with NT3 overexpression, promoted DC axon regeneration, recovery of conduction of compound action potentials across the lesion site and improvements in sensory and locomotor function. These findings demonstrate that in vivo-jetPEI is a potential non-viral, translatable DRGN delivery vehicle in vivo and that suppression of AMIGO3 disinhibits the growth of axotomised DRGN enabling NT3 to stimulate the regeneration of their DC axons and enhances functional recovery.