Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

Interleukin-15 modulates the response of cortical neurons to ischemia.

OBJECTIVE: Stroke is a major cause of death and disability in the United States. Current acute stroke therapy consists of clot-dissolving drugs, catheter-based interventions and physical rehabilitation. To date, there are no therapies that directly enhance neuronal survival after a stroke. Previous work from our lab demonstrated that Interleukin-15 (IL-15) peptide could rescue cardiomyocytes subjected to hypoxia. We sought to extend these findings to cortical neurons since IL-15 has been implicated to have an important role in neuronal homeostasis. METHODS: We have evaluated the effect of IL-15 peptide on primary cortical neurons derived from embryonic rats in vitro under conditions of anoxia and glucose deprivation, and in vivo following middle cerebral artery occlusion. RESULTS: IL-15 administration rescued neuronal cells subjected to anoxia coupled with glucose deprivation (AGD), as well as with reoxygenation. A hallmark of stroke is the ischemic microenvironment and associated oxidative stress, which results in DNA damage and ER stress, both of which contribute to neuronal cell damage and death. The expression of anoxia, ER stress, and DNA damage factors/markers was evaluated via western blot and correlated with the cellular survival effects of IL-15 in vitro. In addition, IL-15 effect of alleviating ER stress and increasing cell survival was also observed in vivo. INTERPRETATION: Our data indicate, for the first time, that administration of the pleiotropic factor IL-15 reduces neuronal cell death during AGD, which correlates with modulation of multiple cellular stress pathways.

Enhanced Mesenchymal Stromal Cells or Erythropoietin Provide Long-Term Functional Benefit After Neonatal Stroke.

BACKGROUND AND PURPOSE: Perinatal stroke is a common cause of life-long neurobehavioral compromise. Mesenchymal stromal cells (MSCs) and EPO (erythropoietin) have each demonstrated short-term benefit with delayed administration after stroke, and combination therapy may provide the most benefit. The purpose of this study is to determine the long-term histological and functional efficacy of enhanced, intranasal stem cell therapy (MSC preexposed to EPO) compared with standard MSC or multidose systemic EPO. METHODS: Transient middle cerebral artery occlusion or sham surgery was performed in postnatal day (P) 10 Sprague-Dawley rats, who were treated with single-dose intranasal MSC, MSC preexposed to EPO (MSC/EPO), multidose systemic EPO (EPO3; 1000 u/kg per dose×3 every 72 hours), or cell-conditioned media on P13 (day 3 [P13-P19] for EPO), or on P17 (day 7 [P17-P23] for EPO). At 2 months of age, animals underwent novel object recognition, cylinder rearing, and open field testing to assess recognition memory, sensorimotor function, and anxiety in adulthood. RESULTS: MSC, MSC/EPO, and EPO3 improved brain volume when administered at 3 or 7 days after middle cerebral artery occlusion. MSC/EPO also enhanced long-term recognition memory with either day 3 or day 7 treatment, but EPO3 had the most long-term benefit, improving recognition memory and exploratory behavior and reducing anxiety. CONCLUSIONS: These data suggest that single-dose MSC/EPO and multidose systemic EPO improve long-term neurobehavioral outcomes even when administration is delayed, although EPO was the most effective treatment overall. It is possible that EPO represents a final common pathway for improved long-term repair, although the specific mechanisms remain to be determined.

Neuroprotective effects of Sonic hedgehog agonist SAG in a rat model of neonatal stroke.

BACKGROUND: Neonatal stroke affects 1 in 2800 live births and is a major cause of neurological injury. The Sonic hedgehog (Shh) signaling pathway is critical for central nervous system (CNS) development and has neuroprotective and reparative effects in different CNS injury models. Previous studies have demonstrated beneficial effects of small molecule Shh-Smoothened agonist (SAG) against neonatal cerebellar injury and it improves Down syndrome-related brain structural deficits in mice. Here we investigated SAG neuroprotection in rat models of neonatal ischemia-reperfusion (stroke) and adult focal white matter injury. METHODS: We used transient middle cerebral artery occlusion at P10 and ethidium bromide (EB) injection in adult rats to induce damage. Following surgery and SAG or vehicle treatment, we analyzed tissue loss, cell proliferation and fate, and behavioral outcome. RESULTS: We report that a single dose of SAG administered following neonatal stroke preserved brain volume, reduced gliosis, enhanced oligodendrocyte progenitor cell (OPC) and EC proliferation, and resulted in long-term cognitive improvement. Single-dose SAG also promoted proliferation of OPCs following focal demyelination in the adult rat. CONCLUSIONS: These findings indicate benefit of one-time SAG treatment post insult in reducing brain injury and improving behavioral outcome after experimental neonatal stroke. IMPACT: A one-time dose of small molecule Sonic hedgehog agonist protected against neonatal stroke and improved long-term behavioral outcomes in a rat model. This study extends the use of Sonic hedgehog in treating developing brain injury, previously shown in animal models of Down syndrome and cerebellar injury. Sonic hedgehog agonist is one of the most promising therapies in treating neonatal stroke thanks to its safety profile and low dosage.

Delayed erythropoietin therapy improves histological and behavioral outcomes after transient neonatal stroke.

BACKGROUND AND PURPOSE: Stroke is a major cause of neonatal morbidity, often with delayed diagnosis and with no accepted therapeutic options. The purpose of this study is to investigate the efficacy of delayed initiation of multiple dose erythropoietin (EPO) therapy in improving histological and behavioral outcomes after early transient ischemic stroke. METHODS: 32 postnatal day 10 (P10) Sprague-Dawley rats underwent sham surgery or transient middle cerebral artery occlusion (tMCAO) for 3h, resulting in injury involving the striatum and parieto-temporal cortex. EPO (1000U/kg per dose×3 doses) or vehicle was administered intraperitoneally starting one week after tMCAO (at P17, P20, and P23). At four weeks after tMCAO, sensorimotor function was assessed in these four groups (6 vehicle-sham, 6 EPO-sham, 10 vehicle-tMCAO and 10 EPO-tMCAO) with forepaw preference in cylinder rearing trials. Brains were then harvested for hemispheric volume and Western blot analysis. RESULTS: EPO-tMCAO animals had significant improvement in forepaw symmetry in cylinder rearing trials compared to vehicle-tMCAO animals, and did not differ from sham animals. There was also significant preservation of hemispheric brain volume in EPO-tMCAO compared to vehicle-tMCAO animals. No differences in ongoing cell death at P17 or P24 were noted by spectrin cleavage in either EPO-tMCAO or vehicle-tMCAO groups. CONCLUSIONS: These results suggest that delayed EPO therapy improves both behavioral and histological outcomes at one month following transient neonatal stroke, and may provide a late treatment alternative for early brain injury.

Erythropoietin increases neurogenesis and oligodendrogliosis of subventricular zone precursor cells after neonatal stroke.

BACKGROUND AND PURPOSE: Stroke is a common cause of neonatal brain injury. The subventricular zone is a lifelong source of newly generated cells in rodents, and erythropoietin (EPO) treatment has shown benefit in different animal models of brain injury. The purpose of this study is to investigate the specific role of exogenous EPO on subventricular zone progenitor cell populations in response to neonatal stroke. METHODS: Intraventricular injections of green fluorescent protein (GFP)-expressing lentivirus to label subventricular zone precursor cells were made in postnatal day 1 (P1) Long-Evans rats, which then underwent transient middle cerebral artery occlusion on P7. Middle cerebral artery occlusion and sham rats were treated with either vehicle or EPO (1000 U/kg) at reperfusion, 24 hours, and 7 days later. The density of double-labeled DCx+/GFP+, NeuN+/GFP+, O4+/GFP+, GFAP+/GFP+, as well as single-labeled GFP+ and Ki67+ cells, was calculated to determine cell fate outcome in the striatum at 72 hours and 2 weeks after stroke. RESULTS: There was a significant increase in DCx+/GFP+ and NeuN+/GFP+ neurons and O4+/GFP+ oligodendrocyte precursors, with decreased GFAP+/GFP+ astrocytes at both time points in EPO-middle cerebral artery occlusion animals. There was also a significant increase in GFP+ cells and Ki67+ proliferating cells in EPO compared with vehicle-middle cerebral artery occlusion animals. CONCLUSIONS: These data suggest that subventricular zone neural progenitor cells proliferate and migrate to the site of injury after neonatal stroke and multiple doses of EPO, with a shift in cell fate toward neurogenesis and oligodendrogliosis at both early and late time points. The contribution of local cell proliferation and neurogenesis remains to be determined.

A Photoactivatable Norepinephrine for Probing Adrenergic Neural Circuits.

Norepinephrine (NE) is a critical neuromodulator that mediates a wide range of behavior and neurophysiology, including attention, arousal, plasticity, and memory consolidation. A major source of NE is the brainstem nucleus the locus coeruleus (LC), which sends widespread projections throughout the central nervous system (CNS). Efforts to dissect this complex noradrenergic circuitry have driven the development of many tools that detect endogenous NE or modulate widespread NE release via LC activation and inhibition. While these tools have enabled research that elucidates physiological roles of NE, additional tools to probe these circuits with a higher degree of spatial precision could enable a finer delineation of function. Here, we describe the synthesis and chemical properties of a photo-activatable NE, [Ru(bpy) 2 (PMe 3 )(NE)]PF 6 (RuBi-NE). We validate the one-photon (1P) release of NE using whole-cell patch clamp electrophysiology in acute mouse brain slices containing the LC. We show that a 10 ms pulse of blue light, in the presence of RuBi-NE, briefly modulates the firing rate of LC neurons via α-2 adrenergic receptors. The development of a photo-activatable NE that can be released with light in the visible spectrum provides a new tool for fine-grained mapping of complex noradrenergic circuits, as well as the ability to probe how NE acts on non-neuronal cells in the CNS.

A new genetic strategy for targeting microglia in development and disease.

As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include border-associated meningeal, perivascular and choroid plexus macrophages. Using immunofluorescent labeling, flow cytometry and Cre-dependent ribosomal immunoprecipitations, we describe P2ry12-CreER, a new tool for the genetic targeting of microglia. We use this new tool to track microglia during embryonic development and in the context of ischemic injury and neuroinflammation. Because of the specificity and robustness of microglial recombination with P2ry12-CreER, we believe that this new mouse line will be particularly useful for future studies of microglial function in development and disease.

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Growth factors for the treatment of ischemic brain injury (growth factor treatment).

In recent years, growth factor therapy has emerged as a potential treatment for ischemic brain injury. The efficacy of therapies that either directly introduce or stimulate local production of growth factors and their receptors in damaged brain tissue has been tested in a multitude of models for different Central Nervous System (CNS) diseases. These growth factors include erythropoietin (EPO), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor (IGF-1), among others. Despite the promise shown in animal models, the particular growth factors that should be used to maximize both brain protection and repair, and the therapeutic critical period, are not well defined. We will review current pre-clinical and clinical evidence for growth factor therapies in treating different causes of brain injury, as well as issues to be addressed prior to application in humans.