Insulin attenuates apoptosis in neuronal cells by an integrin-linked kinase-dependent mechanism.

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.

Acetazolamide Treatment Prevents Redistribution of Astrocyte Aquaporin 4 after Murine Traumatic Brain Injury.

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.

Progesterone modulates mTOR in the hippocampus of mice after traumatic brain injury.

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.

New missions for an old agent: granulocyte-colony stimulating factor in the treatment of stroke patients.

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.

Does progesterone show neuroprotective effects on traumatic brain injury through increasing phosphorylation of Akt in the hippocampus?

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.

Spatial and temporal expression levels of specific microRNAs in a spinal cord injury mouse model and their relationship to the duration of compression.

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.

Spatial distribution of insulin-like growth factor binding protein-2 following hypoxic-ischemic 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.

Rapamycin treatment improves neuron viability in an in vitro model of stroke.

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.

Deciphering the intracellular signaling of erythropoietin in neuronal cells.

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.

Purinergic 2Y1 receptor stimulation decreases cerebral edema and reactive gliosis in a traumatic brain injury model.

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 µM) or MRS2365 (100 µM) 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 IP(3)-signaling pathway. We suggest that enhancing astrocyte mitochondrial metabolism offers a promising neuroprotective strategy for a broad range of brain injuries.

The blood-spinal cord barrier: morphology and clinical implications.

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.

The rostral migratory stream plays a key role in intranasal delivery of drugs into the CNS.

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.

Temporal differences in microRNA expression patterns in astrocytes and neurons after ischemic injury.

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that may lead to novel strategies for therapeutic interventions.

Long-term magnetic resonance imaging of stem cells in neonatal ischemic injury.

OBJECTIVE: Quantitative magnetic resonance imaging (MRI) can serially and noninvasively assess the degree of injury in rat pup models of hypoxic ischemic injury (HII). It can also noninvasively monitor stem cell migration following iron oxide prelabeling. Reports have shown that neural stem cells (NSCs) may help mediate neuroprotection or stimulate neuroreparative responses in adult and neonatal models of ischemic injury. We investigated the ability of high-field MRI to monitor and noninvasively quantify the migration, proliferation, and location of iron oxide-labeled NSCs over very long time periods (58 weeks) in real time while contemporaneously correlating this activity with the evolving severity and extent of neural damage. METHODS: Labeled clonal murine NSCs (mNSCs) were implanted 3 days after unilateral HII in 10-day-old rat pups into the contralateral striatum or ventricle. We developed methods for objectively quantifying key aspects of dynamic NSC behavior (eg, viability; extent, and speed of migration; degree of proliferation; extent of integration into host parenchyma). MRI images were validated with histological and immunohistochemical assessments. RESULTS: mNSCs rapidly migrated (100 µm/day) to the lesion site. Chains of migrating NSCs were observed in the corpus callosum. In pups subjected to HII, though not in intact control animals, we observed a 273% increase in the MR-derived volume of mNSCs 4 weeks after implantation (correlating with the known proliferative behavior of endogenous and exogenous NSCs) that slowly declined over the 58-week time course, with no adverse consequences. Large numbers of now quiescent mNSCs remained at the site of injury, many retaining their iron oxide label. INTERPRETATION: Our studies demonstrate that MRI can simultaneously monitor evolving neonatal cerebral injury as well as NSC migration and location. Most importantly, it can noninvasively monitor proliferation dynamically for prolonged time periods. To be able to pursue clinical trials in newborns using stem cell therapies it is axiomatic that safety be insured through the long-term real time monitoring of cell fate and activity, particularly with regard to observing unanticipated risks to the developing brain. This study supports the feasibility of reliably using MRI for this purpose.

Intranasal erythropoietin therapy in nervous system disorders.

IMPORTANCE OF THE FIELD: Erythropoietin (EPO) is a growth hormone and cytokine that plays an important role in erythropoiesis and neuroprotection. However, EPO treatment for neurological diseases requires repeated injections or high-dose systemic administration, which may cause systemic side effects. The lack of any effective treatment of acute and chronic neurodegenerative diseases and the promising outcome by EPO in animal models in vivo demand a critical evaluation of intranasal EPO delivery to the brain as an alternative administration method. AREAS COVERED IN THIS REVIEW: The current use and intranasal administration of EPO and its derivatives in preclinical studies and recent clinical trials with EPO in neurological diseases. WHAT THE READER WILL GAIN: This paper gives an overview of the therapeutic considerations of intranasal EPO and EPO derivatives for neuroprotection. TAKE HOME MESSAGE: Intranasal delivery (ID) of neuroprotective drugs is an area of great interest. Among the administration strategies used at present, ID of EPO is the most promising. Further preclinical and clinical studies are needed to evaluate the potential significance of this alternative route for increasing EPO bioavailability and decreasing side effects.

Erythropoietin plus insulin-like growth factor-I protects against neuronal damage in a murine model of human immunodeficiency virus-associated neurocognitive disorders.

OBJECTIVE: Prolonged human immunodeficiency virus-1 (HIV-1) infection leads to neurological debilitation, including motor dysfunction and frank dementia. Although pharmacological control of HIV infection is now possible, HIV-associated neurocognitive disorders (HAND) remain intractable. Here, we report that chronic treatment with erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) protects against HIV/gp120-mediated neuronal damage in culture and in vivo. METHODS: Initially, we tested the neuroprotective effects of various concentrations of EPO, IGF-I, or EPO+IGF-I from gp120-induced damage in vitro. To assess the chronic effects of EPO+IGF-I administration in vivo, we treated HIV/gp120-transgenic or wild-type mice transnasally once a week for 4 months and subsequently conducted immunohistochemical analyses. RESULTS: Low concentrations of EPO+IGF-I provided neuroprotection from gp120 in vitro in a synergistic fashion. In vivo, EPO+IGF-I treatment prevented gp120-mediated neuronal loss, but did not alter microgliosis or astrocytosis. Strikingly, in the brains of both humans with HAND and gp120-transgenic mice, we found evidence for hyperphosphorylated tau protein (paired helical filament-I tau), which has been associated with neuronal damage and loss. In the mouse brain following transnasal treatment with EPO+IGF-I, in addition to neuroprotection we observed increased phosphorylation/activation of Akt (protein kinase B) and increased phosphorylation/inhibition of glycogen synthase kinase (GSK)-3beta, dramatically decreasing downstream hyperphosphorylation of tau. These results indicate that the peptides affected their cognate signaling pathways within the brain parenchyma. INTERPRETATION: Our findings suggest that chronic combination therapy with EPO+IGF-I provides neuroprotection in a mouse model of HAND, in part, through cooperative activation of phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling. This combination peptide therapy should therefore be tested in humans with HAND.

Erythropoietin in stroke: quo vadis.

IMPORTANCE OF THE FIELD: Recombinant erythropoietin (rEPO) failed in a recent clinical study to protect from damages induced by ischemic stroke. The lack of acute treatments in ischemic stroke and the promising outcome in numerous preclinical studies in vivo demands a more critical evaluation of the future use of EPO as an acute treatment. AREAS COVERED IN THIS REVIEW: The current use and administration of rhEPO and its analogs in animal models and the future use of this cytokine in the treatment of ischemic stroke. WHAT THE READER WILL GAIN: In this review the potential reasons for the failure of EPO in the clinical trial are analysed and whether the preclinical trials sufficiently evaluated the true potential of recombinant EPO and its analogs is assessed. Alternative methods for administration of EPO to enhance its potential as a neuroprotective drug in ischemic stroke are discussed. TAKE HOME MESSAGE: Failure in clinical trial does not necessarily indicate the lack of therapeutic potential of EPO. This review encourages further investigation of the true potential of EPO as a candidate drug for the treatment of ischemic stroke by improved preclinical experimental design and utilization of alternative administration methods.

Intranasal delivery of erythropoietin plus insulin-like growth factor-I for acute neuroprotection in stroke. Laboratory investigation.

OBJECT: Individually, the cytokines erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) have both been shown to reduce neuronal damage significantly in rodent models of cerebral ischemia. The authors have previously shown that EPO and IGF-I, when administered together, provide acute and prolonged neuroprotection in cerebrocortical cultures against N-methyl-D-aspartate-induced apoptosis. The aim of this study was to determine whether intranasally applied EPO plus IGF-I can provide acute neuroprotection in an animal stroke model and to show that intranasal administration is more efficient at delivering EPO plus IGF-I to the brain when compared with intravenous, subcutaneous, or intraperitoneal administration. METHODS: The EPO and IGF-I were administered intranasally to mice that underwent transient middle cerebral artery occlusion (MCAO). Stroke volumes were measured after 1 hour of MCAO and 24 hours of reperfusion. To evaluate the long-term effects of this treatment, behavioral outcomes were assessed at 3, 30, 60, and 90 days following MCAO. Radiography and liquid scintillation were used to visualize and quantify the uptake of radiolabeled 125I-EPO and 125I-IGF-I into the mouse brain after intranasal, intravenous, subcutaneous, or intraperitoneal administration. RESULTS: Intranasal administration of EPO plus IGF-I reduced stroke volumes within 24 hours and improved neurological function in mice up to 90 days after MCAO. The 125I-EPO and 125I-IGF-I were found in the brain within 20 minutes after intranasal administration and accumulated within the injured areas of the brain. In addition, intranasal administration delivered significantly higher levels of the applied 125I-EPO and 125I-IGF-I to the brain compared with intravenous, subcutaneous, or intraperitoneal administration. CONCLUSIONS: The data demonstrate that intranasal EPO plus IGF-I penetrates into the brain more efficiently than other drug delivery methods and could potentially provide a fast and efficient treatment to prevent chronic effects of stroke.

IGF-I regulated phosphorylation and translocation of PDK-1 in neurons.

3'-phosphoinositide-dependent protein kinase-1 (PDK-1) is a crucial serine/threonine kinase in the insulin-like growth factor-I (IGF-I)/AKT signaling pathway, but its function and localization in the nervous system has not been fully characterized. In this study, we compared the localization of PDK-1 in adult neurons and non-neuronal PC-3 cells. We showed that PC-3 cells expressed phosphorylated and nonphosphorylated PDK-1 in the cytoplasm and nucleoplasm. In contrast, neuronal PDK-1 was located in the nucleoplasm and the phosphorylated form was located along the perinuclear region. Furthermore, we found that IGF-I transiently increased phosphorylation of neuronal PDK-1, resulting in its translocation to other cellular compartments. Our findings suggest that IGF-I may regulate neuronal PDK-1 differently than in non-neuronal cells, which may indicate a novel role for PDK-1 in IGF-I-mediated neuroprotective signaling.