Inosine augments the effects of a Nogo receptor blocker and of environmental enrichment to restore skilled forelimb use after stroke.

Stroke is the leading cause of disability in much of the world, with few treatment options available. Following unilateral stroke in rats, inosine, a naturally occurring purine nucleoside, stimulates the growth of projections from the undamaged hemisphere into denervated areas of the spinal cord and improves skilled use of the impaired forelimb. Inosine augments neurons' intrinsic growth potential by activating Mst3b, a component of the signal transduction pathway through which trophic factors regulate axon outgrowth. The present study investigated whether inosine would complement the effects of treatments that promote plasticity through other mechanisms. Following unilateral stroke in the rat forelimb motor area, inosine combined with NEP1-40, a Nogo receptor antagonist, doubled the number of axon branches extending from neurons in the intact hemisphere into the denervated side of the spinal cord compared with either treatment alone, and restored rats' level of skilled reaching using the impaired forepaw to preoperative levels. Similar functional improvements were seen when inosine was combined with environmental enrichment (EE). The latter effect was associated with changes in gene expression in layer 5 pyramidal neurons of the undamaged cortex well beyond those seen with inosine or EE alone. Inosine is now in clinical trials for other indications, making it an attractive candidate for the treatment of stroke patients.

Inosine alters gene expression and axonal projections in neurons contralateral to a cortical infarct and improves skilled use of the impaired limb.

Recovery after stroke and other types of brain injury is restricted in part by the limited ability of undamaged neurons to form compensatory connections. Inosine, a naturally occurring purine nucleoside, stimulates neurons to extend axons in culture and, in vivo, enhances the ability of undamaged neurons to form axon collaterals after brain damage. The molecular changes induced by inosine are unknown, as is the ability of inosine to restore complex functions associated with a specific cortical area. Using a unilateral injury model limited to the sensorimotor cortex, we show that inosine triples the number of corticospinal tract axons that project from the unaffected hemisphere and form synaptic bouton-like structures in the denervated half of the spinal cord. These changes correlate with improved recovery in animals' ability to grasp and consume food pellets with the affected forepaw. Studies using laser-capture microdissection and microarray analysis show that inosine profoundly affects gene expression in corticospinal neurons contralateral to the injury. Inosine attenuates transcriptional changes caused by the stroke, while upregulating the expression of genes associated with axon growth and the complement cascade. Thus, inosine alters gene expression in neurons contralateral to a stroke, enhances the ability of these neurons to form connections on the denervated side of the spinal cord, and improves performance with the impaired limb.

Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1.

Synaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts with the PDZ domain protein PICK1, which plays a critical role in regulating the expression of glutamate receptors at excitatory synapses. A yeast two-hybrid screen of a neuronal library using the carboxyl tail of GLT1b yielded clones expressing PICK1. The GLT1b C-terminal peptide bound to PICK1 with high affinity (K(i) = 6.5 +/- 0.4 microM) in an in vitro fluorescence polarization assay. We also tested peptides based on other variants of GLT1 and other glutamate transporters. GLT1b co-immunoprecipitated with PICK1 from rat brain lysates and COS7 cell lysates derived from cells transfected with plasmids expressing PICK1 and GLT1b. In addition, expression of GLT1b in COS7 cells changed the distribution of PICK1, bringing it to the surface. GLT1b and PICK1 co-localized with each other and with synaptic markers in hippocampal neurons in culture. Phorbol ester, an activator of protein kinase C (PKC), a known PICK1 interactor, had no effect on glutamate transport in rat forebrain neurons in culture. However, we found that exposure of neurons to a myristolated decoy peptide with sequence identical to the C-terminal sequence of GLT1b designed to block the PICK1-GLT1b interaction rendered glutamate transport into neurons responsive to phorbol ester. These results suggest that the PICK1-GLT1b interaction regulates the modulation of GLT1 function by PKC.

The glutamate transporter EAAT2 is transiently expressed in developing human cerebral white matter.

The major brain abnormality underlying cerebral palsy in premature infants is periventricular leukomalacia (PVL), a lesion of the immature cerebral white matter. Oligodendrocyte precursors (pre-OLs; O4(+)O1(-)) predominate in human cerebral white matter during the peak time frame for PVL (24-32 gestational weeks) and are vulnerable to excitotoxicity. We hypothesize that PVL reflects, in part, excitotoxicity to pre-OLs resulting from cerebral ischemia/reperfusion. Reversal of glutamate transport in the setting of energy failure is a major source of pathologic accumulation of extracellular glutamate. Here, we identify and localize the glutamate transporters in human cerebral white matter during the age range of PVL. In situ hybridization was performed with digoxigenin-labeled probes directed against the full-length coding regions of EAAT1, EAAT2, and EAAT3. EAAT2 mRNA was abundant in human fetal white matter during the period of peak incidence of PVL and virtually disappeared by 2 postnatal months. Its developmental profile differed significantly from that of both EAAT1 and EAAT3 mRNA. Immunoblotting demonstrated that EAAT2 protein was highly expressed in early development relative to adult values. Double-label immunocytochemistry detected EAAT2 in OLs but not astrocytes or axons in the human fetal white matter. We conclude that transient expression of EAAT2 occurs during the window of peak vulnerability for PVL, suggesting that this developmentally up-regulated transporter may be a major source of extracellular glutamate in ischemic injury to the cerebral white matter of the preterm infant.

Axon regeneration in goldfish and rat retinal ganglion cells: differential responsiveness to carbohydrates and cAMP.

Mammalian retinal ganglion cells (RGCs) do not normally regenerate their axons through an injured optic nerve, but can be stimulated to do so by activating macrophages intraocularly. In a cell culture model of this phenomenon, we found that a small molecule that is constitutively present in the vitreous, acting in concert with macrophage-derived proteins, stimulates mature rat RGCs to regenerate their axons if intracellular cAMP is elevated. In lower vertebrates, RGCs regenerate their axons spontaneously in vivo, and in culture, the most potent axon-promoting factor for these cells is a molecule that resembles the small vitreous-derived growth factor from the rat. This molecule was isolated chromatographically and was shown by mass spectrometry to be a carbohydrate. In agreement with this finding, D-mannose proved to be a potent axon-promoting factor for rat RGCs (ED50 approximately 10 microm); this response was cAMP-dependent and was augmented further by macrophage-derived proteins. Goldfish RGCs showed far less selectivity, responding strongly to either D-mannose or D-glucose in a cAMP-independent manner. These findings accord well with the success or failure of optic nerves to regenerate in higher and lower vertebrates in vivo. The axon-promoting effects of mannose are highly specific and are unrelated to energy metabolism or glycoprotein synthesis.

Macrophage-derived factors stimulate optic nerve regeneration.

After optic nerve injury in mature mammals, retinal ganglion cells (RGCs) are normally unable to regenerate their axons and undergo delayed apoptosis. However, if the lens is damaged at the time of nerve injury, many RGCs survive axotomy and regenerate their axons into the distal optic nerve. Lens injury induces macrophage activation, and we show here that factors secreted by macrophages stimulate RGCs to regenerate their axons. When macrophages were activated by intravitreal injections of Zymosan, a yeast cell wall preparation, the number of RGC axons regenerating into the distal optic nerve was even greater than after lens injury. These effects were further enhanced if Zymosan was injected 3 d after nerve crush. In a grafting paradigm, intravitreal Zymosan increased the number of RGCs that regenerated their axons through a 1.5 cm peripheral nerve graft twofold relative to uninjected controls and threefold if injections were delayed 3 d. In cell culture, media conditioned by activated macrophages stimulated adult rat RGCs to regenerate their axons; this effect was potentiated by a low molecular weight factor that is constitutively present in the vitreous humor. After gel-filtration chromatography, macrophage-derived proteins > or =30 kDa were found to be toxic to RGCs, whereas proteins <30 kDa reversed this toxicity and promoted axon regeneration. The protein(s) that stimulated axon growth is distinct from identified polypeptide trophic factors that were tested. Thus, macrophages produce proteins with both positive and negative effects on RGCs, and the effects of macrophages can be optimized by the timing of their activation.

Expression of a variant form of the glutamate transporter GLT1 in neuronal cultures and in neurons and astrocytes in the rat brain.

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of approximately 3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons.

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3'-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14-29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.

Inosine stimulates axon growth in vitro and in the adult CNS.

Unlike mammals, lower vertebrates can regenerate their optic nerves and certain other CNS pathways throughout life. To identify the molecular bases of this phenomenon, we developed a cell culture model and found that goldfish retinal ganglion cells will regenerate their axons in response to the purine nucleoside inosine. Inosine acts through a direct intracellular mechanism and induces many of the changes in gene expression that underlie regenerative growth in vivo, e.g., upregulation of GAP-43, T alpha-1 tubulin, and the cell-adhesion molecule, L1. N-kinase, a 47-49-kDa serine-threonine kinase, may mediate the effects of inosine and serve as part of the modular signal transduction pathway that controls axon growth. In vivo, inosine stimulates extensive axon growth in the mature rat corticospinal tract. Following unilateral transection of the corticospinal tract, inosine applied to the intact sensorimotor cortex stimulated layer 5 pyramidal cells to upregulate GAP-43 expression and to sprout axon collaterals. These collaterals crossed the midline at the level of the cervical enlargement and reinnervated regions whose normal connections had been served. Further understanding of the molecular changes that lie upstream and downstream of N-kinase may lead to new insights into the control of axon growth and to novel methods to improve functional outcome in patients with CNS injury.

VEGF, which is elevated in the CSF of patients with hydrocephalus, causes ventriculomegaly and ependymal changes in rats.

Hydrocephalus is a condition characterized primarily by excessive accumulation of fluid in the ventricles of the brain for which there is currently no effective pharmacological treatment. Surgery, often accompanied by complications, is the only current treatment. Extensive research in our laboratory along with work from others has suggested a link between hydrocephalus and vascular function. We hypothesized that vascular endothelial growth factor (VEGF), the major angiogenic factor, could play a role in the pathogenesis of hydrocephalus. We tested this hypothesis by examining two predictions of such a link: first, that VEGF is present in many cases of clinical hydrocephalus; and second, that exogenous VEGF in an animal model could cause ventricular enlargement and tissue changes associated with hydrocephalus. Our results support the idea that VEGF elevation can potentiate hydrocephalus. The clinical relevance of this work is that anti-angiogenic drugs may be useful in patients with hydrocephalus, either alone or in combination with the currently available surgical treatments.

Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways.

Mammalian sterile 20-like kinase-3b (Mst3b, encoded by Stk24), regulates axon outgrowth in embryonic cortical neurons in culture, but its role in vivo and in neural repair is unknown. Here we show that Mst3b mediates the axon-promoting effects of trophic factors in mature rat retinal ganglion cells (RGCs) and dorsal root ganglion (DRG) neurons, and is essential for axon regeneration in vivo. Reducing Mst3b levels using short hairpin RNA prevented RGCs and DRG neurons from regenerating axons in response to growth factors in culture, as did expression of a kinase-dead Mst3b mutant. Conversely, expression of constitutively active Mst3b enabled both types of neurons to extend axons without growth factors. In vivo, RGCs lacking Mst3b failed to regenerate injured axons when stimulated by intraocular inflammation. DRG neurons regenerating axons in vivo showed elevated Mst3b activity, and reducing Mst3b expression attenuated regeneration and p42/44 MAPK activation. Thus, Mst3b regulates axon regeneration in both CNS and PNS neurons.

Nerve growth factor controls GAP-43 mRNA stability via the phosphoprotein ARPP-19.

The membrane phosphoprotein GAP-43 is involved in axon growth and synaptic plasticity. In PC12 pheochromocytoma cells, induction of a neuronal phenotype by nerve growth factor (NGF) is accompanied by a marked increase in GAP-43 levels. NGF regulates GAP-43 expression by altering the half-life of its mRNA. We report here that the phosphoprotein ARPP-19 mediates this regulation. In an NGF-dependent manner, ARPP-19 bound to a region in the 3' end of GAP-43 mRNA previously found to be important for regulating the half-life of the mRNA. Overexpression of wild-type ARPP-19 in PC12 cells increased the NGF-dependent expression of a reporter construct linked to the critical 3' region of GAP-43 mRNA. Mutation of serine 104, the site of phosphorylation by protein kinase A in ARPP-19, to either alanine or aspartate abolished this regulation in PC12 cells. These findings demonstrate that ARPP-19 is an important link between NGF signaling and post-transcriptional control of neuronal gene expression.