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.

Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.

Microglia are highly dynamic immune cells of the CNS and their dynamism is proposed to be regulated by neuronal activities. However, the mechanisms underlying neuronal regulation of microglial dynamism have not been determined. Here, we found an increased number of microglial primary processes in the hippocampus during KA-induced seizure activity. Consistently, global glutamate induced robust microglial process extension toward neurons in both brain slices and in the intact brain in vivo. The mechanism of the glutamate-induced microglial process extension involves the activation of neuronal NMDA receptors, calcium influx, subsequent ATP release, and microglial response through P2Y12 receptors. Seizure-induced increases in microglial process numbers were also dependent on NMDA receptor activation. Finally, we found that P2Y12 KO mice exhibited reduced seizure-induced increases in microglial process numbers and worsened KA-induced seizure behaviors. Our results elucidate the molecular mechanisms underlying microglia-neuron communication that may be potentially neuroprotective in the epileptic brain.

Delayed pharyngeal repolarization promotes abnormal calcium buildup in aging muscle.

In the pharynx of Caenorhabditis elegans, the accessory subunit MPS-4, homolog to human KCNE1, forms a complex with K(+) channel EXP-2 that terminates the action potential. An aspartate residue critical for KCNE1 function, asp76, is conserved in MPS-4 (asp74). Here, we studied the effects of D74N-MPS-4 on the aging pharynx. Electrophysiological studies showed that D74N delays pharyngeal repolarization. Pharynxes of transgenic worms expressing D74N exhibited higher levels of intracellular calcium compared to normal pharynxes. Accordingly, loss of pharyngeal function was accelerated in aging D74N worms. The pharyngeal action potential resembles the action potential that controls the mechanical activity of human left ventricle. Hence, these findings argue that the hearts of patients affected by delayed repolarization, a condition known as long QT syndrome, may experience dysregulated calcium homeostasis.

Biomarkers in Spinal Cord Injury: Prognostic Insights and Future Potentials.

Spinal Cord Injury (SCI) is a major challenge in Neurotrauma research. Complex pathophysiological processes take place immediately after the injury and later on as the chronic injury develops. Moreover, SCI is usually accompanied by traumatic injuries because the most common modality of injury is road traffic accidents and falls. Patients develop significant permanent neurological deficits that depend on the extent and the location of the injury itself and in time they develop further neurological and body changes that may risk their mere survival. In our review, we explored the recent updates with regards to SCI biomarkers. We observed two methods that may lead to the appearance of biomarkers for SCI. First, during the first few weeks following the injury the Blood Spinal Cord Barrier (BSCB) disruption that releases several neurologic structure components from the injured tissue. These components find their way to Cerebrospinal Fluid (CSF) and the systemic circulation. Also, as the injury develops several components of the pathological process are expressed or released such as in neuroinflammation, apoptosis, reactive oxygen species, and excitotoxicity sequences. Therefore, there is a growing interest in examining any correlations between these components and the degrees or the outcomes of the injury. Additionally, some of the candidate biomarkers are theorized to track the progressive changes of SCI which offers an insight on the patients' prognoses, potential-treatments-outcomes assessment, and monitoring the progression of the complications of chronic SCI such as Pressure Ulcers and urinary dysfunction. An extensive literature review was performed covering literature, published in English, until February 2018 using the Medline/PubMed database. Experimental and human studies were included and titles, PMID, publication year, authors, biomarkers studies, the method of validation, relationship to SCI pathophysiology, and concluded correlation were reported. Potential SCI biomarkers need further validation using clinical studies. The selection of the appropriate biomarker group should be made based on the stage of the injuries, the accompanying trauma and with regards to any surgical, or medical interference that might have been done. Additionally, we suggest testing multiple biomarkers related to the several pathological changes coinciding to offer a more precise prediction of the outcome.

A Novel Short Isoform of Cytosolic PSD-95 Interactor (Cypin) Regulates Neuronal Development.

The guanine deaminase cypin (cytosolic PSD-95 interactor) binds to PSD-95 (postsynaptic density protein 95) and regulates dendrite branching by promoting microtubule polymerization. Here, we identify a novel short isoform of cypin, termed cypinS, which is expressed in mouse and human, but not rat, tissues. Cypin and cypinS mRNA and protein levels peak at P7 and P14 in the mouse brain, suggesting a role for these isoforms during development. Interestingly, although cypinS lacks guanine deaminase activity, overexpression of cypinS increases dendrite branching. This increase occurs further away from soma than do increases resulting from overexpression of cypin. In contrast, overexpression of cypin, but not cypinS, decreases dendritic spine density and maturity. This suggests that changes to spines, but not to dendrites, may be dependent on guanine deaminase activity. Furthermore, overexpression of either cypin or cypinS increases miniature excitatory postsynaptic current (mEPSC) frequency, pointing to a presynaptic role for both isoforms. Interestingly, overexpression of cypinS results in a significantly greater increase in frequency than does overexpression of cypin. Thus, cypin and cypinS play distinct roles in neuronal development.

Overexpression of cypin alters dendrite morphology, single neuron activity, and network properties via distinct mechanisms.

OBJECTIVE: This study investigates the effect that overexpression of cytosolic PSD-95 interactor (cypin), a regulator of synaptic PSD-95 protein localization and a core regulator of dendrite branching, exerts on the electrical activity of rat hippocampal neurons and networks. APPROACH: We cultured rat hippocampal neurons and used lipid-mediated transfection and lentiviral gene transfer to achieve high levels of cypin or cypin mutant (cypinΔPDZ; PSD-95 non-binding) expression cellularly and network-wide, respectively. MAIN RESULTS: Our analysis revealed that although overexpression of cypin and cypinΔPDZ increase dendrite numbers and decrease spine density, cypin and cypinΔPDZ distinctly regulate neuronal activity. At the single cell level, cypin promotes decreases in bursting activity while cypinΔPDZ reduces sEPSC frequency and further decreases bursting compared to cypin. At the network level, by using the Fano factor as a measure of spike count variability, cypin overexpression results in an increase in variability of spike count, and this effect is abolished when cypin cannot bind PSD-95. This variability is also dependent on baseline activity levels and on mean spike rate over time. Finally, our spike sorting data show that overexpression of cypin results in a more complex distribution of spike waveforms and that binding to PSD-95 is essential for this complexity. SIGNIFICANCE: Our data suggest that dendrite morphology does not play a major role in cypin action on electrical activity.

Role of Akt-independent mTORC1 and GSK3ß signaling in sublethal NMDA-induced injury and the recovery of neuronal electrophysiology and survival.

Glutamate-induced excitotoxicity, mediated by overstimulation of N-methyl-D-aspartate (NMDA) receptors, is a mechanism that causes secondary damage to neurons. The early phase of injury causes loss of dendritic spines and changes to synaptic activity. The phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt/ mammalian target of rapamycin (PI3K/Akt/mTOR) pathway has been implicated in the modulation and regulation of synaptic strength, activity, maturation, and axonal regeneration. The present study focuses on the physiology and survival of neurons following manipulation of Akt and several downstream targets, such as GSK3ß, FOXO1, and mTORC1, prior to NMDA-induced injury. Our analysis reveals that exposure to sublethal levels of NMDA does not alter phosphorylation of Akt, S6, and GSK3ß at two and twenty four hours following injury. Electrophysiological recordings show that NMDA-induced injury causes a significant decrease in spontaneous excitatory postsynaptic currents at both two and twenty four hours, and this phenotype can be prevented by inhibiting mTORC1 or GSK3ß, but not Akt. Additionally, inhibition of mTORC1 or GSK3ß promotes neuronal survival following NMDA-induced injury. Thus, NMDA-induced excitotoxicity involves a mechanism that requires the permissive activity of mTORC1 and GSK3ß, demonstrating the importance of these kinases in the neuronal response to injury.

Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function.

Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function.