Cypin Inhibition as a Therapeutic Approach to Treat Spinal Cord Injury-Induced Mechanical Pain.

Cypin (cytosolic postsynaptic density protein 95 interactor) is the primary guanine deaminase in the central nervous system (CNS), promoting the metabolism of guanine to xanthine, an important reaction in the purine salvage pathway. Activation of the purine salvage pathway leads to the production of uric acid (UA). UA has paradoxical effects, specifically in the context of CNS injury as it confers neuroprotection, but it also promotes pain. Since neuropathic pain is a comorbidity associated with spinal cord injury (SCI), we postulated that small molecule cypin inhibitor B9 treatment could attenuate SCI-induced neuropathic pain, potentially by interfering with UA production. However, we also considered that this treatment could hinder the neuroprotective effects of UA and, in doing so, exacerbate SCI outcomes. To address our hypothesis, we induced a moderate midthoracic contusion SCI in female mice and assessed whether transient intrathecal administration of B9, starting at 1 d postinjury (dpi) until 7 dpi, attenuates mechanical pain in hindlimbs at 3 weeks pi. We also evaluated the effects of B9 on the spontaneous recovery of locomotor function. We found that B9 alleviates mechanical pain but does not affect locomotor function. Importantly, B9 does not exacerbate lesion volume at the epicenter. In accordance with these findings, B9 does not aggravate glutamate-induced excitotoxic death of SC neurons in vitro. Moreover, SCI-induced increased astrocyte reactivity at the glial scar is not altered by B9 treatment. Our data suggest that B9 treatment reduces mechanical pain without exerting major detrimental effects following SCI.

Carboxypeptidase E Independently Changes Microtubule Glutamylation, Dendritic Branching, and Neuronal Migration.

Neuronal migration and dendritogenesis are dependent on dynamic changes to the microtubule (MT) network. Among various factors that regulate MT dynamics and stability, post-translational modifications (PTMs) of MTs play a critical role in conferring specificity of regulatory protein binding to MTs. Thus, it is important to understand the regulation of PTMs during brain development as multiple developmental processes are dependent on MTs. In this study, we identified that carboxypeptidase E (CPE) changes tubulin polyglutamylation, a major PTM in the brain, and we examine the impact of CPE-mediated changes to polyglutamylation on cortical neuron migration and dendrite morphology. We show, for the first time, that overexpression of CPE increases the level of polyglutamylated α-tubulin while knockdown decreases the level of polyglutamylation. We also demonstrate that CPE-mediated changes to polyglutamylation are dependent on the CPE zinc-binding motif and that this motif is necessary for CPE action on p150Glued localization. However, overexpression of a CPE mutant that does not increase MT glutamylation mimics the effects of overexpression of wild type CPE on dendrite branching. Furthermore, although overexpression of wild type CPE does not alter cortical neuron migration, overexpression of the mutant may act in a dominant-negative manner as it decreases the number of neurons that reach the cortical plate (CP), as we previously reported for CPE knockdown. Overall, our data suggest that CPE changes MT glutamylation and redistribution of p150Glued and that this function of CPE is independent of its role in shaping dendrite development but plays a partial role in regulating cortical neuron migration.

Uric acid released from poly(ε-caprolactone) fibers as a treatment platform for spinal cord injury.

Spinal cord injury (SCI) is characterized by a primary mechanical phase of injury, resulting in physical tissue damage, and a secondary pathological phase, characterized by biochemical processes contributing to inflammation, neuronal death, and axonal demyelination. Glutamate-induced excitotoxicity (GIE), in which excess glutamate is released into synapses and overstimulates glutamate receptors, is a major event in secondary SCI. GIE leads to mitochondrial damage and dysfunction, release of reactive oxygen species (ROS), DNA damage, and cell death. There is no clinical treatment that targets GIE after SCI, and there is a need for therapeutic targets for secondary damage in patients. Uric acid (UA) acts as an antioxidant and scavenges free radicals, upregulates glutamate transporters on astrocytes, and preserves neuronal viability in in vitro and in vivo SCI models, making it a promising therapeutic candidate. However, development of a drug release platform that delivers UA locally to the injured region in a controlled manner is crucial, as high systemic UA concentrations can be detrimental. Here, we used the electrospinning technique to synthesize UA-containing poly(ɛ-caprolactone) fiber mats that are biodegradable, biocompatible, and have a tunable degradation rate. We optimized delivery of UA as a burst within 20 min from uncoated fibers and sustained release over 2 h with poly(ethylene glycol) diacrylate coating. We found that both of these fibers protected neurons and decreased ROS generation from GIE in organotypic spinal cord slice culture. Thus, fiber mats represent a promising therapeutic for UA release to treat patients who have suffered a SCI.

Current advances in in vitro models of central nervous system trauma.

CNS trauma is a prominent cause of mortality and morbidity, and although much effort has focused on developing treatments for CNS trauma-related pathologies, little progress has been made. Pre-clinical models of TBI and SCI suffer from significant drawbacks, which result in substantial failures during clinical translation of promising pre-clinical therapies. Here, we review recent advances made in the development of in vitro models of CNS trauma, the promises and drawbacks of current in vitro CNS injury models, and the attributes necessary for future models to accurately mimic the trauma microenvironment and facilitate CNS trauma drug discovery. The goal is to provide insight for the development of future CNS injury models and to aid researchers in selecting effective models for pre-clinical research of trauma therapeutics.

The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis.

Lipid metabolism plays a critical role in female reproduction. During oogenesis, maturing oocytes accumulate high levels of neutral lipids that are essential for both energy production and the synthesis of other lipid molecules. Metabolic pathways within the ovary are partially regulated by protein kinases that link metabolic status to oocyte development. Although the functions of several kinases in this process are well established, the roles that many other kinases play in coordinating metabolic state with female germ cell development are unknown. Here, we demonstrate that the catalytic activity of casein kinase 2 (CK2) is essential for Drosophila oogenesis. Using an unbiased biochemical screen that leveraged an unusual catalytic property of the kinase, we identified a novel CK2 substrate in the Drosophila ovary, the lipid droplet-associated protein Jabba. We show that Jabba is essential for modulating ovarian lipid metabolism and for regulating female fertility in the fly. Our findings shed light on a CK2-dependent signaling pathway governing lipid metabolism in the ovary and provide insight into the long-recognized but poorly understood association between energy metabolism and female reproduction.