Cypin: A novel target for traumatic brain injury.

Cytosolic PSD-95 interactor (cypin), the primary guanine deaminase in the brain, plays key roles in shaping neuronal circuits and regulating neuronal survival. Despite this pervasive role in neuronal function, the ability for cypin activity to affect recovery from acute brain injury is unknown. A key barrier in identifying the role of cypin in neurological recovery is the absence of pharmacological tools to manipulate cypin activity in vivo. Here, we use a small molecule screen to identify two activators and one inhibitor of cypin's guanine deaminase activity. The primary screen identified compounds that change the initial rate of guanine deamination using a colorimetric assay, and secondary screens included the ability of the compounds to protect neurons from NMDA-induced injury and NMDA-induced decreases in frequency and amplitude of miniature excitatory postsynaptic currents. Hippocampal neurons pretreated with activators preserved electrophysiological function and survival after NMDA-induced injury in vitro, while pretreatment with the inhibitor did not. The effects of the activators were abolished when cypin was knocked down. Administering either cypin activator directly into the brain one hour after traumatic brain injury significantly reduced fear conditioning deficits 5 days after injury, while delivering the cypin inhibitor did not improve outcome after TBI. Together, these data demonstrate that cypin activation is a novel approach for improving outcome after TBI and may provide a new pathway for reducing the deficits associated with TBI in patients.

Time-dependent homeostatic mechanisms underlie brain-derived neurotrophic factor action on neural circuitry.

Plasticity and homeostatic mechanisms allow neural networks to maintain proper function while responding to physiological challenges. Despite previous work investigating morphological and synaptic effects of brain-derived neurotrophic factor (BDNF), the most prevalent growth factor in the central nervous system, how exposure to BDNF manifests at the network level remains unknown. Here we report that BDNF treatment affects rodent hippocampal network dynamics during development and recovery from glutamate-induced excitotoxicity in culture. Importantly, these effects are not obvious when traditional activity metrics are used, so we delve more deeply into network organization, functional analyses, and in silico simulations. We demonstrate that BDNF partially restores homeostasis by promoting recovery of weak and medium connections after injury. Imaging and computational analyses suggest these effects are caused by changes to inhibitory neurons and connections. From our in silico simulations, we find that BDNF remodels the network by indirectly strengthening weak excitatory synapses after injury. Ultimately, our findings may explain the difficulties encountered in preclinical and clinical trials with BDNF and also offer information for future trials to consider.

CCP1, a Tubulin Deglutamylase, Increases Survival of Rodent Spinal Cord Neurons following Glutamate-Induced Excitotoxicity.

Microtubules (MTs) are cytoskeletal elements that provide structural support and act as roadways for intracellular transport in cells. MTs are also needed for neurons to extend and maintain long axons and dendrites that establish connectivity to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Posttranslational modification of MTs is one mechanism by which neurons regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates posttranslational polyglutamylation of MTs. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has also long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Using shRNA-mediated knock-down of CCP1 in embryonic rat spinal cord cultures, we demonstrate that CCP1 protects spinal cord neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system. We previously demonstrated that the CCP1 homolog in Caenorhabditis elegans is important for maintenance of neuronal cilia. Although cilia enhance neuronal survival in some contexts, it is not yet clear whether CCP1 maintains cilia in mammalian spinal cord neurons. We found that knock-down of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons, suggesting that its effect on survival of excitotoxicity is independent of cilia. Our results support the idea that enzyme regulators of MT polyglutamylation might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries.

Inhibitor of Endocannabinoid Deactivation Protects Against In Vitro and In Vivo Neurotoxic Effects of Paraoxon.

The anticholinesterase paraoxon (Pxn) is related to military nerve agents that increase acetylcholine levels, trigger seizures, and cause excitotoxic damage in the brain. In rat hippocampal slice cultures, high-dose Pxn was applied resulting in a presynaptic vulnerability evidenced by a 64% reduction in synapsin IIb (syn IIb) levels, whereas the postsynaptic protein GluR1 was unchanged. Other signs of Pxn-induced cytotoxicity include the oxidative stress-related production of stable 4-hydroxynonenal (4-HNE)-protein adducts. Next, the Pxn toxicity was tested for protective effects by the fatty acid amide hydrolase (FAAH) inhibitor AM5206, a compound linked to enhanced repair signaling through the endocannabinoid pathway. The Pxn-mediated declines in syn IIb and synaptophysin were prevented by AM5206 in the slice cultures. To test if the protective results in the slice model translate to an in vivo model, AM5206 was injected i.p. into rats, followed immediately by subcutaneous Pxn administration. The toxin caused a pathogenic cascade initiated by seizure events, leading to presynaptic marker decline and oxidative changes in the hippocampus and frontal cortex. AM5206 exhibited protective effects including the reduction of seizure severity by 86%, and improving balance and coordination measured 24 h post-insult. As observed in hippocampal slices, the FAAH inhibitor also prevented the Pxn-induced loss of syn IIb in vivo. In addition, the AM5206 compound reduced the 4-HNE modifications of proteins and the ß1 integrin activation events both in vitro and in vivo. These results indicate that Pxn exposure produces oxidative and synaptic toxicity that leads to the behavioral deficits manifested by the neurotoxin. In contrast, the presence of FAAH inhibitor AM5206 offsets the pathogenic cascade elicited by the Pxn anticholinesterase.