Insulin promotes neuronal survival by activating a phosphatidylinositol 3-kinase (PI 3-kinase)/AKT-dependent signaling pathway and reducing caspase activation. We investigated a role for integrin-linked kinase (ILK) in insulin-mediated cell survival in cultured neurons and differentiated R28 cells. We used a serum and depolarization withdrawal model to induce apoptosis in cerebellar granule neurons and a serum withdrawal model to induce apoptosis in differentiated R28 cells. ILK knock-out decreased insulin-mediated protection as did the addition of pharmacological inhibitors of ILK, KP-392 or QLT-0267. Prosurvival effects of insulin were rescued by Boc-Asp (O-methyl)-CH2F (BAF), a pancaspase inhibitor, in the presence of KP-392. Insulin and IGF-1 decreased caspase-3 activation, an effect that was inhibited by KP-392 and QLT-0267. Western blot analysis indicates that insulin-induced stimulation of AKT Ser-473 phosphorylation was decreased after the ILK gene was conditionally knocked-out, following overexpression of AKT-DN or in the presence of QLT-0267. Insulin and IGF-1 stimulated ILK kinase activity in primary neurons and this was inhibited following ILK-DN overexpression. Western blot analysis indicates that insulin exposure upregulated the expression of the cellular inhibitor of apoptosis protein c-IAP2 in an extracellular matrix-dependent manner, an effect blocked by KP-392. These results indicate that ILK is an important effector in insulin-mediated neuroprotection.

After traumatic brain injury (TBI), multiple ongoing processes contribute to worsening and spreading of the primary injury to create a secondary injury. One major process involves disrupted fluid regulation to create vascular and cytotoxic edema in the affected area. Although understanding of factors that influence edema is incomplete, the astrocyte water channel Aquaporin 4 (AQP4) has been identified as an important mediator and therefore attractive drug target for edema prevention. The FDA-approved drug acetazolamide has been administered safely to patients for years in the United States. To test whether acetazolamide altered AQP4 function after TBI, we utilized in vitro and in vivo models of TBI. Our results suggest that AQP4 localization is altered after TBI, similar to previously published reports. Treatment with acetazolamide prevented AQP4 reorganization, both in human astrocyte in vitro and in mice in vivo. Moreover, acetazolamide eliminated cytotoxic edema in our in vivo mouse TBI model. Our results suggest a possible clinical role for acetazolamide in the treatment of TBI.

Tumor hypoxia is associated with poor patient survival and is a characteristic of glioblastoma.Notch signaling is implicated in maintaining glioma stem-like cells (GSCs) within the hypoxic niche, although the molecular mechanisms linking hypoxia to Notch activation have not been clearly delineated.Here we show that Vasorin is a critical link between hypoxia and Notch signaling in GSCs.Vasorin is preferentially induced in GSCs by a HIF1α/STAT3 co-activator complex and stabilizes Notch1 protein at the cell membrane.This interaction prevents Numb from binding Notch1, thereby rescuing it from Numb-mediated lysosomal degradation.Thus, Vasorin

The mechanistic target of rapamycin (mTOR) is an intracellular protein kinase that functions as an energy and nutrient sensor in the cellular microenvironment of neurons. Modulation of mTOR is vital when nutrient and energy sources become limited. Hypoxia, traumatic brain injury, cellular energy states, and growth factors all regulate the phosphorylation and total levels of mTOR in cells. Alterations in the microenvironment induce transduction of signals to downstream proteins by mTOR allowing for cells to make the necessary adjustments to counteract stressors and survive. Progesterone, a hydrophobic steroid hormone, has been shown in studies of non-neural tissue to be a suppressor of mTOR and modulator of mTOR phosphorylation. Our study tested the effects of progesterone on mTOR expression following traumatic brain injury. C57BL/6 mice were treated with progesterone (8 mg/kg) at 1 (intraperitoneal), 6 (subcutaneous), 24 (subcutaneous), and 48 (subcutaneous) hours post closed skull traumatic brain injury. The hippocampus was then harvested 72 hours post injury and prepared for western blot analysis. We found that progesterone significantly decreased total mTOR levels in all groups compared to sham treated with vehicle. This was further confirmed by immunostaining showing decreased cytoplasmic mTOR levels compared to sham. Our study shows progesterone is a significant modulator of mTOR levels in the hippocampus of mice following traumatic brain injury.

Granulocyte-colony stimulating factor (G-CSF) has a multimodal neuroprotective profile and the cumulative preclinical data from numerous translational studies statistically confirmed the efficacy of G-CSF as a treatment option in ischemic stroke. G-CSF activates anti-apoptotic, antioxidative, and anti-inflammatory signaling pathways and stimulates angiogenesis and neurogenesis. In this review, we summarize the role of G-CSF and the corresponding signal transduction pathways regulated by G-CSF in neuroprotection and discuss its potential as a new drug for stroke treatment.

Background context MicroRNAs, a class of small nonprotein-coding RNAs, are thought to control gene translation into proteins. The latter are the ultimate effectors of the biochemical cascade occurring in any physiological and pathological process. MicroRNAs have been shown to change their expression levels during injury of spinal cord in contusion rodent models. Compression is the most frequent mode of damage of neural elements in spinal cord injury. The cellular and molecular changes occurring in the spinal cord during prolonged compression are not very well elucidated. Understanding the underlying molecular events that occur during sustained compression is paramount in building new therapeutic strategies. Purpose The purpose of our study was to probe the relationship between the expression level changes of different miRNAs and the timing of spinal cord decompression in a mouse model. Study design A compression spinal cord injury mouse model was used for the study. Methods A laminectomy was performed in the thoracic spine of C57BL/6 mice. Then, the thecal sac was compressed to create the injury. Decompression was performed early for one group and it was delayed in the second group. The spinal cord at the epicenter of the injury and one level rostral to it were removed at 3, 6, and 24 hours after trauma, and RNA was extracted. Expression levels of six different microRNAs and the relationship to the duration of compression were analyzed. This work was supported in part by the University Research Council Grants Program at the University of Texas Health Science Center San Antonio (Grant 130267). There are no specific conflicts of interest to be disclosed for this work. Results Expression levels of microRNAs in the prolonged compression of spinal cord model were significantly different compared with the expression levels in the short duration of compression spinal cord injury model. Furthermore, microRNAs show a different expression pattern in different regions of the injured spinal cord. Conclusions Our findings demonstrate that spinal cord compression causes alterations in the expression of different miRNAs in the acute phase of injury. Their expression is related to the duration of the compression of the spinal cord. These findings suggest that early decompression of the spinal cord may have an important modulating effect on the molecular cascade triggered during secondary injury through the changes in expression levels of specific microRNAs.

There are currently no federally approved neuroprotective agents to treat traumatic brain injury. Progesterone, a hydrophobic steroid hormone, has been shown in recent studies to exhibit neuroprotective effects in controlled cortical impact rat models. Akt is a protein kinase known to play a role in cell signaling pathways that reduce edema, inflammation, apoptosis, and promote cell growth in the brain. This study aims to determine if progesterone modulates the phosphorylation of Akt via its threonine 308 phosphorylation site. Phosphorylation at the threonine 308 site is one of several sites responsible for activating Akt and enabling the protein kinase to carry out its neuroprotective effects. To assess the effects of progesterone on Akt phosphorylation, C57BL/6 mice were treated with progesterone (8 mg/kg) at 1 (intraperitonally), 6, 24, and 48 hours (subcutaneously) post closed-skull traumatic brain injury The hippocampus was harvested at 72 hours post injury and prepared for western blot analysis. Traumatic brain injury caused a significant decrease in Akt phosphorylation compared to sham operation. However, mice treated with progesterone following traumatic brain injury had an increase in phosphorylation of Akt compared to traumatic brain injury vehicle. Our findings suggest that progesterone is a viable treatment option for activating neuroprotective pathways after traumatic brain injury.

Background Insulin-like growth factor binding protein-2 (IGFBP-2) regulates the bioavailability, transportation, and localization of insulin-like growth factor-I (IGF-I), an effective neuroprotectant in animal stroke models especially when administered intranasally. Therefore, determining IGFBP-2′s endogenous distribution in the normal and ischemic brain is essential in maximizing the neuroprotective potential of the intranasal IGF-I treatment approach. However, current data on IGFBP-2 is limited to mRNA and in situ hybridization studies. The purpose of this study was to determine if there are any changes in IGFBP-2 protein levels and distribution in ischemic brain and also to determine if IGFBPs play a role in the transportation of intranasally administered IGF-I into the brain. Results Using an in vitro approach, we show that ischemia causes changes in the distribution of IGFBP-2 in primary cortical neurons and astrocytes. In addition, we show using the transient middle cerebral artery occlusion (MCAO) model in mice that there is a significant increase in IGFBP-2 levels in the stroke penumbra and core after 72 h. This correlated with an overall increase in IGF-I after stroke, with the highest levels of IGF-I in the stroke core after 72 h. Brain sections from stroke mice indicate that neurons and astrocytes located in the penumbra both have increased expression of IGFBP-2, however, IGFBP-2 was not detected in microglia. We used binding competition studies to show that intranasally administered exogenous IGF-I uptake into the brain is not receptor mediated and is likely facilitated by IGFBPs. Conclusions The change in protein levels indicates that IGFBP-2 plays an IGF-I-dependent and -independent role in the brain’s acute (neuroprotection) and chronic (tissue remodeling) response to hypoxic-ischemic injury. Competition studies indicate that IGFBPs may have a role in rapid transportation of exogenous IGF-I from the nasal tissue to the site of injury.

Ischemic stroke is the leading cause of serious, long-term adult disability and is associated with sensorimotor and cognitive impairments due to neuronal degeneration. Currently, recombinant tissue plasminogen activator (rTPA) is the only FDA-approved medical therapy for treatment of patients with acute ischemic stroke. However, rTPA can only be given within 3 hours of symptom onset, and only 2% of patients are eligible. Therefore, there is an urgent need for novel neuroprotective treatment options for ischemic stroke. An emerging treatment for a diverse range of neurological disorders associated with neurodegeneration is rapamycin, a key modulator of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway is the primary regulator of the cellular response to nutrient availability, changes in energy status and stress as seen following ischemia and reperfusion. However, rapamycin's effects on mTORC1 and mTORC2 are poorly understood in neurons. In the current study we show that rapamycin can prevent the activation of both mTORC1 and mTORC2 in cortical neurons and improve cell survival following oxygen glucose deprivation (OGD), an in vitro model of ischemic stroke. This work further supports the investigation of rapamycin as a novel neuroprotectant for ischemic stroke.

The search for potential drugs to treat neurodegenerative diseases has been intense in the last two decades. Among many candidates, erythropoietin (EPO) was identified as a potent protectant of neurons suffering from various adverse conditions. A wide array of literature indicates that endogenous or exogenous recombinant human erythropoietin and its variants activate cell signaling that initiates survival-promoting events in neurons and neuronal cells. This chapter gives an overview of the pro-survival signaling induced by endogenous and exogenous erythropoietin in vitro and in vivo and provides methods to further investigate the intracellular signaling. It is important to know that EPO is neuroprotective, but it will greatly enhance our chances to establish EPO as a new drug candidate if we know how EPO protects neurons.The descriptions below summarize our current knowledge in non-neuronal and neuronal signaling pathways induced by EPO. The signaling pathways involved in EPO are multiple; some are well known whereas others are still under intense investigation and few are observed in very specific cell types. It is important to note that neuronal signaling events triggered by EPO are still incomplete and require further research. Therefore, excellent review articles that explore specific EPO-signaling events are referenced.

Hematopoietic growth factors (HGFs) are a family of cytokines that play important roles in the survival, proliferation, and differentiation of hematopoietic progenitors, as well as in the functional activation of mature cells. However, there is a growing body evidences have suggested that HGFs have important non-hematopoietic functions in the central nervous system. Preclinical studies have demonstrated significant benefits from the use of these cytokines in the acute and chronic treatment of various neurological disorders. Unfortunately, the efficacy of these candidate neuroprotectants in animal experiments does not reliably predict efficacy in stroke patients. This chapter presents a broad overview of data from preclinical neuroprotection and clinical trial studies.

Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults. Neuroprotective agents that may promote repair or counteract damage after injury do not currently exist. We recently reported that stimulation of the purinergic receptor subtype P2Y(1)R using 2-methylthioladenosine 5' diphosphate (2MeSADP) significantly reduced cytotoxic edema induced by photothrombosis. Here, we tested whether P2Y(1)R stimulation was neuroprotective after TBI. A controlled closed head injury model was established for mice using a pneumatic impact device. Brains were harvested at 1, 3, or 7 days post-injury and assayed for morphological changes by immunocytochemistry, Western blot analysis, and wet/dry weight. Cerebral edema and expression of both aquaporin type 4 and glial fibrillary acidic protein were increased at all time points examined. Immunocytochemical measurements in both cortical and hippocampal slices also revealed significant neuronal swelling and reactive gliosis. Treatment of mice with 2MeSADP (100 mu M) or MRS2365 (100 mu M) 30 min after trauma significantly reduced all post-injury symptoms of TBI including edema, neuronal swelling, reactive gliosis, and AQ4 expression. The neuroprotective effect was lost in IP(3)R2-/- mice treated with 2MeSADP. Immunocytochemical labeling of brain slices confirmed that P2Y(1)R expression was defined to cortical and hippocampal astrocytes, but not neurons. Taken together, the data show that stimulation of astrocytic P2Y(1)Rs significantly reduces brain injury after acute trauma and is mediated by the IP3-signaling pathway. We suggest that enhancing astrocyte mitochondrial metabolism offers a promising neuroprotective strategy for a broad range of brain injuries.

BACKGROUND:The blood brain barrier (BBB) is impermeable to most drugs, impeding the establishment of novel neuroprotective therapies and strategies for many neurological diseases. Intranasal administration offers an alternative path for efficient drug delivery into the CNS. So far, the anatomical structures discussed to be involved in the transport of intranasally administered drugs into the CNS include the trigeminal nerve, olfactory nerve and the rostral migratory stream (RMS), but the relative contributions are debated.METHODS AND FINDINGS:In the present study we demonstrate that surgical transection, and the resulting structural disruption of the RMS, in mice effectively obstructs the uptake of intranasally administered radioligands into the CNS. Furthermore, using a fluorescent cell tracer, we demonstrate that intranasal administration in mice allows agents to be distributed throughout the entire brain, including olfactory bulb, hippocampus, cortex and cerebellum.CONCLUSIONS:This study provides evidence of the vital role the RMS has in the CNS delivery of intranasally administered agents. The identification of the RMS as the major access path for intranasally administered drugs into the CNS may contribute to the development of treatments that are tailored for efficient transport within this structure. Research into the RMS needs to continue to elucidate its limitations, capabilities, mechanisms of transport and potential hazards before we are able to advance this technique into human research.

The blood–spinal cord barrier (BSCB) is the functional equivalent of the blood–brain barrier (BBB) in the sense of providing a specialized microenvironment for the cellular constituents of the spinal cord. Even if intuitively the BSCB could be considered as the morphological extension of the BBB into the spinal cord, evidence suggests that this is not so. The BSCB shares the same principal building blocks with the BBB; nevertheless, it seems that morphological and functional differences may exist between them. Dysfunction of the BSCB plays a fundamental role in the etiology or progression of several pathological conditions of the spinal cord, such as spinal cord injury, amyotrophic lateral sclerosis, and radiation‐induced myelopathy. This review summarizes current knowledge of the morphology of the BSCB, the methodology of studying the BSCB, and the potential role of BSCB dysfunction in selected disorders of the spinal cord, and finally summarizes therapeutic approaches to the BSCB. Ann Neurol 2011;