Post-treatment with cocaine- and amphetamine-regulated transcript enhances infarct resolution, reinnervation, and angiogenesis in stroke rats - an MRI study.

Recent studies have shown that post-treatment with cocaine- and amphetamine-regulated transcript (CART) has neuroregenerative effects in animal models of stroke. The purpose of this study was to characterize CART-mediated neuronal and vascular repairments using non-invasive MRI techniques. Adult male rats were subjected to a 90 min middle cerebral artery occlusion (MCAo). Animals were separated into two groups with similar infarction sizes, measured by T2 -weighted MRI on Day 2 after MCAo, and were treated with CART or vehicle intranasally from Day 3 to Day 12. Diffusion tensor imaging was used to examine changes in plasticity of white matter elements. Susceptibility-weighted imaging (SWI) was used to measure angiogenesis. Post-treatment with CART significantly increased fractional anisotropy (FA) in lesioned cortex on Days 10 and 25 post stroke. A significant correlation between the behavioral recovery in body asymmetry and the change in FA was shown, suggesting that behavioral recovery was associated with reinnervation to the lesioned hemisphere. CART also increased the intensity of SWI and the immunoreactivity of the vascular marker alpha-smooth muscle actin in lesioned cortex. Together, our data support a non-invasive treatment strategy for stroke through angiogenesis and reinnervation by CART. Copyright © 2016 John Wiley & Sons, Ltd.

Post-trauma administration of the pifithrin-α oxygen analog improves histological and functional outcomes after experimental traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Programmed death of neuronal cells plays a crucial role in acute and chronic neurodegeneration following TBI. The tumor suppressor protein p53, a transcription factor, has been recognized as an important regulator of apoptotic neuronal death. The p53 inactivator pifithrin-α (PFT-α) has been shown to be neuroprotective against stroke. A previous cellular study indicated that PFT-α oxygen analog (PFT-α (O)) is more stable and active than PFT-α. We aimed to investigate whether inhibition of p53 using PFT-α or PFT-α (O) would be a potential neuroprotective strategy for TBI. To evaluate whether these drugs protect against excitotoxicity in vitro, primary rat cortical cultures were challenged with glutamate (50mM) in the presence or absence of various concentrations of the p53 inhibitors PFT-α or PFT-α (O). Cell viability was estimated by LDH assay. In vivo, adult Sprague Dawley rats were subjected to controlled cortical impact (CCI, with 4m/s velocity, 2mm deformation). Five hours after injury, PFT-α or PFT-α (O) (2mg/kg, i.v.) was administered to animals. Sensory and motor functions were evaluated by behavioral tests at 24h after TBI. The p53-positive neurons were identified by double staining with cell-specific markers. Levels of mRNA encoding for p53-regulated genes (BAX, PUMA, Bcl-2 and p21) were measured by reverse transcription followed by real time-PCR from TBI animals without or with PFT-α/PFT-α (O) treatment. We found that PFT-α(O) (10 µM) enhanced neuronal survival against glutamate-induced cytotoxicity in vitro more effectively than PFT-α (10 µM). In vivo PFT-α (O) treatment enhanced functional recovery and decreased contusion volume at 24h post-injury. Neuroprotection by PFT-α (O) treatment also reduced p53-positive neurons in the cortical contusion region. In addition, p53-regulated PUMA mRNA levels at 8h were significantly reduced by PFT-α (O) administration after TBI. PFT-α (O) treatment also decreased phospho-p53 positive neurons in the cortical contusion region. Our data suggest that PFT-α (O) provided a significant reduction of cortical cell death and protected neurons from glutamate-induced excitotoxicity in vitro, as well as improved neurological functional outcome and reduced brain injury in vivo via anti-apoptotic mechanisms. The inhibition of p53-induced apoptosis by PFT-α (O) provides a useful tool to evaluate reversible apoptotic mechanisms and may develop into a novel therapeutic strategy for TBI.

Forebrain neuronal specific ablation of p53 gene provides protection in a cortical ischemic stroke model.

Cerebral ischemic injury involves death of multiple cell types at the ischemic sites. As a key regulator of cell death, the p53 gene has been implicated in the regulation of cell loss in stroke. Less focal damage is found in stroke animals pre-treated with a p53 inhibitor or in traditional p53 knockout (ko) mice. However, whether the p53 gene plays a direct role in regulating neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 ko mice, we utilized a neuronal specific conditional ko mouse line (CamcreTRP53(loxP/loxP)) to achieve forebrain neuronal specific deletion of p53 and examined the role of the p53 gene in ischemia-induced cell death in neurons. Expression of p53 after stroke is examined using the immunohistochemical method and the outcome of stroke is examined by analysis of infarction size and behavioral deficits caused by stroke. Our data showed that p53 expression is upregulated in the ischemic region in neuronal cells in wildtype (wt) mice but not in CamcreTRP53(loxP/loxP) ko mice. Deletion of the p53 gene in forebrain neurons results in a decreased infarction area in ko mice. Locomotor behavior, measured in automated activity chambers, showed that CamcreTRP53(loxP/loxP) ko mice have less locomotor deficits compared to wt mice after middle cerebral artery occlusion (MCAo). We conclude that manipulation of p53 expression in neurons may lead to unique therapeutic development in stroke.

Enhanced survival of dopaminergic neuronal transplants in hemiparkinsonian rats by the p53 inactivator PFT-α.

A key limiting factor impacting the success of cell transplantation for Parkinson's disease is the survival of the grafted cells, which are often short lived. The focus of this study was to examine a novel strategy to optimize the survival of exogenous fetal ventromesencephalic (VM) grafts by treatment with the p53 inhibitor, pifithrin-α (PFT-α), to improve the biological outcome of parkinsonian animals. Adult male Sprague-Dawley rats were given 6-hydroxydopamine into the left medial forebrain bundle to induce a hemiparkinsonian state. At 7 weeks after lesioning, animals were grafted with fetal VM or cortical tissue into the lesioned striatum and, thereafter, received daily PFT-α or vehicle injections for 5 days. Apomorphine-induced rotational behavior was examined at 2, 6, 9, and 12 weeks after grafting. Analysis of TUNEL or tyrosine hydroxylase (TH) immunostaining was undertaken at 5 days or 4 months after grafting. The transplantation of fetal VM tissue into the lesioned striatum reduced rotational behavior. A further reduction in rotation was apparent in animals receiving PFT-α and VM transplants. By contrast, no significant reduction in rotation was evident in animals receiving cortical grafts or cortical grafts + PFT-α. PFT-α treatment reduced TUNEL labeling and increased TH(+) cell and fiber density in the VM transplants. In conclusion, our data indicate that early postgrafting treatment with PFT-α enhances the survival of dopamine cell transplants and augments behavioral recovery in parkinsonian animals.

Adenoviral gene transfer of bone morphogenetic protein-7 enhances functional recovery after sciatic nerve injury in rats.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß subfamily, function as instructive signals for neuronal lineage commitment and promote neuronal differentiation. However, the mechanism of BMP7 action in vivo after peripheral nerve injury is poorly understood. This study examines the efficacy of gene transfer of adenoviral (Ad) BMP7 on peripheral neuropathy. Transgene expression was found in both Ad-infected sciatic nerves and their respective remote neurons, indicating Ad transduction by a retrograde transport. After AdBMP7 infection to nerves, the sciatic nerves were crushed or transected. Hind limb functional behavior, including rotarod test and sciatic functional index, were conducted in rats weekly after nerve injury. Interestingly, enhanced BMP7 expression significantly improved hind limb functional recovery in AdBMP7-transduced rats when compared with AdGFP-transduced nerve-crushed or transected rats. Furthermore, AdBMP7 transduction reduced injury-induced macrophage activation, nerve demyelination and axonal degeneration. By contrast, AdBMP7 infection did not affect the hyperalgesia paw-withdrawal latency after nerve injury. We further examined the effect of AdBMP7 infection on sciatic nerve explant and Schwann cell cultures. Enhanced cell proliferation was significantly increased by AdBMP7 transduction in both cultures. Taken together, BMP7 overexpression by Ad gene transfer was beneficial in both nerves and Schwann cells on functional recovery after sciatic nerve injury in rats.

MitoPark mice mirror the slow progression of key symptoms and L-DOPA response in Parkinson's disease.

The MitoPark mouse, in which the mitochondrial transcription factor Tfam is selectively removed in midbrain dopamine (DA) neurons, is a genetic model for Parkinson's disease (PD) that replicates the slow and progressive development of key symptoms. To further validate this model, we have extended both behavioral and biochemical analyses in these animals. We found that vertical movements decline earlier and faster than horizontal movements, possibly modeling the early occurrence of axial, postural instability in PD. L-DOPA induces different locomotor responses depending on the age: in young MitoPark mice the L-DOPA-induced motor activation is small; middle-aged MitoPark mice respond in a dose-dependent manner to L-DOPA, whereas aged MitoPark mice display a double-peaked locomotor response to a high dose of L-DOPA that includes an intermittent period of very low motor activity, similar to the 'on-off' phenomenon in PD. To correlate behavior with biochemical data, we analyzed monoamine levels in three different brain areas that are highly innervated by the DA system: striatum, anterior cortex and olfactory bulb. DA levels declined earlier and faster in striatum than in cortex; only at the latest time-point analyzed, DA levels were found to be significantly lower than control levels in the olfactory bulb. Interestingly, the ratio between homovanillic acid (HVA) and DA differed between regions over time. In striatum and olfactory bulb, the ratio increased steeply indicating increased DA turnover. In contrast, the ratio decreased over time in cortex, revealing important differences between DA cells in substantia nigra and the ventral tegmental area.

Transgenic rodent models of Parkinson's disease.

In the case of Parkinson's disease (PD), classical animal models have utilized dopaminergic neurotoxins such as 6-hydroxydopamine (6OHDA) and 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). More recently, human genetic linkage studies have identified several genes in familial forms of PD. Transgenic models have been made that explore the function of PD-linked genes (e.g. alpha-synuclein, DJ-1, LRRK2, Parkin, UCH-L1, PINK1). Recent evidence suggests mitochondrial dysfunction may play a major role in PD. Manipulation of mitochondrial respiratory genes (e.g. mitochondrial transcription factor A or TFAM) also elicits a PD phenotype in mice. Transgenic mice (MitoPark) were developed that have TFAM selectively knocked out in dopaminergic neurons. The nigral dopamine neurons of MitoPark mice show respiratory chain dysfunction, accompanied by the development of intraneuronal inclusions and eventual cell death. In early adulthood, the MitoPark mice show a slowly progressing loss of motor function that accompanies these cellular changes. The MitoPark mouse enables further study of the role of mitochondrial dysfunction in DA neurons as an important mechanism in the development of PD. Transgenic technology has allowed new insights into mechanisms of neurodegeneration for a number of neurological disorders. This paper will summarize recent studies on several transgenic models of PD.

Nigrostriatal alterations in bone morphogenetic protein receptor II dominant negative mice.

BACKGROUND: We previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson's disease. The purpose of this study is to examine the endogenous neurotrophic properties of BMP Receptor II in dopaminergic neurons of the nigrostriatal pathway. METHODS: Adult male BMPRII dominant negative (BMPRIIDN) mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. A subgroup of BMPRIIDN and WT mice were injected with high doses of methamphetamine (MA) and were sacrificed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry at 4 days after injection. RESULTS: BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantia nigra compacta, TH fiber density in the substantia nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of MA increased TUNEL labeling in the substantia nigra in the BMPRIIDN mice. CONCLUSIONS: Endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA.

Bone morphogenetic protein-7 reduces toxicity induced by high doses of methamphetamine in rodents.

Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. We have previously demonstrated that pretreatment with bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson's disease. In this study, we examined the neuroprotective effects of BMP7 against MA-mediated toxicity in dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase immunoreactivity (THir) while increasing terminal deoxynucleotidyl transferase-mediated dNTP nick end labeling. These toxicities were significantly antagonized by BMP7. Interaction of BMP7 and MA in vivo was first examined in CD1 mice. High doses of MA (10 mg/kgx4 s.c.) significantly reduced locomotor activity and THir in striatum. I.c.v. administration of BMP7 antagonized these changes. In BMP7 +/- mice, MA suppressed locomotor activity and reduced TH immunoreactivity in nigra reticulata to a greater degree than in wild type BMP7 +/+ mice, suggesting that deficiency in BMP7 expression increases vulnerability to MA insults. Since BMP7 +/- mice also carry a LacZ-expressing reporter allele at the BMP7 locus, the expression of BMP7 was indirectly measured through the enzymatic activity of beta-galactosidase (beta-gal) in BMP7 +/- mice. High doses of MA significantly suppressed beta-gal activity in striatum, suggesting that MA may inhibit BMP7 expression at the terminals of the nigrostriatal pathway. A similar effect was also found in CD1 mice in that high doses of MA suppressed BMP7 mRNA expression in nigra. In conclusion, our data indicate that MA can cause lesioning in the nigrostriatal dopaminergic terminals and that BMP7 is protective against MA-mediated neurotoxicity in central dopaminergic neurons.

An immortalized rat ventral mesencephalic cell line, RTC4, is protective in a rodent model of stroke.

One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, we have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here we describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100beta, but not the neuronal markers NeuN, MAP2, or beta-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, we examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15-20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, we generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke.

A partial GDNF depletion leads to earlier age-related deterioration of motor function and tyrosine hydroxylase expression in the substantia nigra.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for peripheral organs, spinal cord, and midbrain dopamine (DA) neurons. Levels of GDNF deteriorate in the substantia nigra in Parkinson's disease (PD). A heterozygous mouse model was created to assess whether chronic reductions in this neurotrophic factor impact motor function and the nigrostriatal dopamine system during the aging process. Due to the important role GDNF plays in kidney development, kidney function and histology were assessed and were found to be normal in both wild-type (WT) and GDNF+/- mice up to 22 months of age. Further, the animals of both genotypes had similar weights throughout the experiment. Locomotor activity was assessed for male WT and GDNF+/- mice at 4-month intervals from 4 to 20 months of age. Both GDNF+/- and WT mice exhibited an age-related decline in horizontal activity, although this was found 4 months earlier in GDNF+/- mice, at 12 months of age. Comparison of young (8 month old) and aged (20 month old) GDNF+/- and WT mice on an accelerating rotarod apparatus established a deficiency for aged but not young GDNF+/- mice, while aged WT mice performed as well as young WT mice on this task. Finally, both WT and GDNF+/- mice exhibited an age-related decrease in substantia nigra TH immunostaining, which was accelerated in the GDNF+/- mice. These behavioral and histological alterations suggest that GDNF may be an important factor for maintenance of motor coordination and spontaneous activity as well as DA neuronal function during aging, and further suggest that GDNF+/- mice may serve as a model for neuroprotective or rescue studies.

Neurotrophic effects of bone morphogenetic protein-7 in a rat model of Parkinson's disease.

Previous studies have demonstrated that pretreatment with bone morphogenetic protein-7 (BMP7) reduces ischemic neuronal injury in vivo. Moreover, exogenous application of BMP7 increases both the number of tyrosine hydroxylase (+) cells and dopamine (DA) uptake in rat mesencephalic cell cultures. The purpose of this study was to investigate the in vivo effects of BMP7 on 6-hydroxydopamine (6-OHDA) induced lesioning of midbrain DA neurons. Adult Fischer 344 rats were anesthetized and injected with BMP7 or vehicle into the left substantia nigra, followed by local administration of 9 microg of 6-OHDA into the left medial forebrain bundle. The lesioned animals that received BMP7 pretreatment, as compared to vehicle/6-OHDA controls, had a significant reduction in methamphetamine-induced rotation 1 month after the surgery. BMP7-pretreatment partially preserved KCl-induced dopamine release in the lesioned striatum and significantly increased TH immunoreactivity in the lesioned nigra and striatum. In summary, our data suggest that BMP7 has neuroprotective and/or neuroreparative effects against 6-OHDA lesioning of the nigrostriatal DA pathway in an animal model of Parkinson's disease (PD).

Glial cell line-derived neurotrophic factor is essential for neuronal survival in the locus coeruleus-hippocampal noradrenergic pathway.

It has been shown that the noradrenergic (NE) locus coeruleus (LC)-hippocampal pathway plays an important role in learning and memory processing, and that the development of this transmitter pathway is influenced by neurotrophic factors. Although some of these factors have been discovered, the regulatory mechanisms for this developmental event have not been fully elucidated. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor influencing LC-NE neurons. We have utilized a GDNF knockout animal model to explore its function on the LC-NE transmitter system during development, particularly with respect to target innervation. By transplanting various combinations of brainstem (including LC) and hippocampal tissues from wildtype or GDNF knockout fetuses into the brains of adult wildtype mice, we demonstrate that normal postnatal development of brainstem LC-NE neurons is disrupted as a result of the GDNF null mutation. Tyrosine hydroxylase immunohistochemistry revealed that brainstem grafts had markedly reduced number and size of LC neurons in transplants from knockout fetuses. NE fiber innervation into the hippocampal co-transplant from an adjacent brainstem graft was also influenced by the presence of GDNF, with a significantly more robust innervation observed in transplants from wildtype fetuses. The most successful LC/hippocampal co-grafts were generated from fetuses expressing the wildtype GDNF background, whereas the most severely affected transplants were derived from double transplants from null-mutated fetuses. Our data suggest that development of the NE LC-hippocampal pathway is dependent on the presence of GDNF, most likely through a target-derived neurotrophic function.

HSV amplicon delivery of glial cell line-derived neurotrophic factor is neuroprotective against ischemic injury.

Direct intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) is neuroprotective against ischemia-induced cerebral injury. Utilizing viral vectors to deliver and express therapeutic genes presents an opportunity to produce GDNF within localized regions of an evolving infarct. We investigated whether a herpes simplex virus (HSV) amplicon-based vector encoding GDNF (HSVgdnf) would protect neurons against ischemic injury. In primary cortical cultures HSVgdnf reduced oxidant-induced injury compared to the control vector HSVlac. To test protective effects in vivo, HSVgdnf or HSVlac was injected into the cerebral cortex 4 days prior to, or 3 days, after a 60-min unilateral occlusion of the middle cerebral artery. Control stroke animals developed bradykinesia and motor asymmetry; pretreatment with HSVgdnf significantly reduced such motor deficits. Animals receiving HSVlac or HSVgdnf after the ischemic insult did not exhibit any behavioral improvement. Histological analyses performed 1 month after stroke revealed a reduction in ischemic tissue loss in rats pretreated with HSVgdnf. Similarly, these animals exhibited less immunostaining for glial fibrillary acidic protein and the apoptotic marker caspase-3. Taken together, our data indicate that HSVgdnf pretreatment provides protection against cerebral ischemia and supports the utilization of the HSV amplicon for therapeutic delivery of trophic factors to the CNS.

The noradrenergic system of aged GDNF heterozygous mice.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for noradrenergic (NE) neurons of the pontine nucleus locus coeruleus (LC). Decreased function of the LC-NE neurons has been found during normal aging and in neurodegenerative disorders. We have previously shown that GDNF participates in the differentiation of LC-NE neurons during development. However, the continued role of GDNF for LC-NE neurons during maturation and aging has not been addressed. We examined alterations in aged mice that were heterozygous for the GDNF gene (Gdnf+/-). Wild-type (Gdnf+/+) and Gdnf+/- mice (18 months old) were tested for locomotor activity and brain tissues were collected for measuring norepinephrine levels and uptake, as well as for morphological analysis. Spontaneous locomotion was reduced in Gdnf+/- mice in comparison with Gdnf+/+ mice. The reduced locomotor activity of Gdnf+/- mice was accompanied by reductions in NE transporter activity in the cerebellum and brain stem as well as decreased norepinephrine tissue levels in the LC. Tyrosine hydroxylase (TH) immunostaining demonstrated morphological alterations of LC-NE cell bodies and abnormal TH-positive fibers in the hippocampus, cerebellum, and frontal cortex of Gdnf+/- mice. These findings suggest that the LC-NE system of Gdnf+/- mice is impaired and suggest that GDNF plays an important role in continued maintenance of this neuronal system throughout life.

Differential expression of the cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in heterozygous Gfralpha1 null-mutant mice after stroke.

Exogenous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ischemia-induced cerebral infarction. Cerebral ischemia induces gene expression of GDNF, GDNF-receptor alpha-1 (GFRalpha-1) and c-Ret, suggesting that a GDNF signaling cascade mechanism may be involved in endogenous neuroprotection during ischemia. In the present study, we examined if this endogenous neuroprotective pathway was altered in Gfralpha-1 deficient mice. Since mice homozygous for the Gfralpha-1 deletion (-/-) die within 24 h of birth, stroke-induced changes in the levels of Gfralpha-1 mRNA were studied in Gfralpha-1 heterozygous (+/-) mice and their wild-type (+/+) littermates. The right middle cerebral artery was transiently ligated for 45 min in anesthetized mice. Animals were killed at 0, 6, 12 and 24 h after the onset of reperfusion and levels of Gfralpha-1 mRNA were measured by in situ hybridization histochemistry. Previously, we showed that Gfralpha-1 (+/-) mice are more vulnerable to focal cerebral ischemia. In the present study, we found that basal levels of GFRalpha-1 mRNA were at similar low levels in cortex and striatum in adult Gfralpha-1 (+/+) and Gfralpha-1 (+/-) mice and that ischemia/reperfusion induced up-regulation of Gfralpha-1 mRNA in the lesioned and contralateral sides of cortex and striatum in both Gfralpha-1 (+/+) and GFRalpha-1 (+/-) mice. However, the ischemia/reperfusion induction of Gfralpha-1 mRNA was significantly higher in the cortex of wild type mice, as compared to Gfralpha-1 (+/-) mice. Moreover, the increased expression of Gfralpha-1 in striatum after reperfusion occurred earlier in the GFRalpha-1 (+/+) than in the Gfralpha-1 (+/-) mice. These results indicate that after ischemia, there is a differential up-regulation of Gfralpha-1 expression in Gfralpha-1 (+/+) and Gfralpha-1 (+/-) mice. Since GDNF has neuroprotective effects, the reduced up-regulation of Gfralpha-1 in Gfralpha-1 (+/-) mice at early time points after ischemia suggests that the responsiveness to GDNF and GDNF receptor mediated neuroprotection is attenuated in these genetically modified animals and may underlie their greater vulnerability.

Glial cell line neurotrophic factor-family receptor alpha-1 is present in central neurons with distinct phenotypes.

Glial cell line neurotrophic factor(GDNF) is a potent survival factor for several types of neurons. GDNF binds with high affinity to the GDNF-family receptor alpha-1 (GFRalpha-1) which is expressed in different brain areas. In the present study, by using anatomical techniques, we document the phenotypic diversity among GFRalpha-1 expressing neurons in the CNS. GFRalpha-1 expression was found in GABA (gamma-aminobutyric acid)-containing neurons distributed in the cortex, reticular thalamic nucleus and septum. While high expression of GFRalpha-1 was often observed in cholinergic motoneurons in the spinal cord, very few septal cholinergic neurons were found to express GFRalpha-1. GFRalpha-1 transcripts were also detected in catecholaminergic neurons in the periventricular hypothalamic nucleus, dorsal raphe nucleus and locus ceruleus. Within the raphe nucleus, GFRalpha-1 expression was prominent in many serotonergic neurons and in few neurons containing the enzyme nitric oxide synthase. As GFRalpha-1 is activated by GDNF and GDNF-related neurotrophic factors, the widespread distribution of GFRalpha-1 in neurons with different phenotypes indicates that the neuronal activity of these neurons is likely to be affected by GDNF and GDNF-related neurotrophic factors. This would result in the regulation of diverse neuronal pathways in the adult brain. Published by Elsevier Science Ltd on behalf of IBRO.

The Noradrenergic System of Aged GDNF Heterozygous Mice.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for noradrenergic (NE) neurons of the pontine nucleus locus coeruleus (LC). Decreased function of the LC-NE neurons has been found during normal aging and in neurodegenerative disorders. We have previously shown that GDNF participates in the differentiation of LC-NE neurons during development. However, the continued role of GDNF for LC-NE neurons during maturation and aging has not been addressed. We examined alterations in aged mice that were heterozygous for the GDNF gene (Gdnf+/-). Wild-type (Gdnf+/+) and Gdnf+/- mice (18 months old) were tested for locomotor activity and brain tissues were collected for measuring norepinephrine levels and uptake, as well as for morphological analysis. Spontaneous locomotion was reduced in Gdnf+/- mice in comparison with Gdnf+/+ mice. The reduced locomotor activity of Gdnf +/- mice was accompanied by reductions in NE transporter activity in the cerebellum and brain stem as well as decreased norepinephrine tissue levels in the LC. Tyrosine hydroxylase (TH) immunostaining demonstrated morphological alterations of LC-NE cell bodies and abnormal TH-positive fibers in the hippocampus, cerebellum, and frontal cortex of Gdnf+/- mice. These findings suggest that the LC-NE system of Gdnf+/- mice is impaired and suggest that GDNF plays an important role in continued maintenance of this neuronal system throughout life.

Bone morphogenetic proteins are involved in fetal kidney tissue transplantation-induced neuroprotection in stroke rats.

Both bone morphogenetic proteins (BMPs) and glial cell line-derived neurotrophic factor (GDNF) reduce ischemia-induced cerebral injury in rats. Intracerebral transplantation of fetal kidney tissue, which normally expresses BMPs and GDNF during development, reduces ischemic injury in cerebral cortex. In this study, we tested the hypothesis that BMP is involved in this neuroprotective response. Fetal kidney tissue was cut into small pieces and transplanted into cortical areas adjacent to the right middle cerebral artery (MCA) in adult rats. In situ hybridization of brain indicated that these fetal kidney transplants contained high levels of BMP-7 mRNA three days after grafting. Immunohistochemical analysis of grafted brain showed co-localization of BMP-7 and PAX-2 immunoreactivity in the graft, suggesting that these transplants contained BMP protein. Some animals were grafted with fetal kidney tissue after intraventricular administration (ICV) of the BMP antagonist noggin (1 micro g) or after vehicle, followed by MCA ligation for 60 min. Animals receiving fetal kidney tissue transplantation developed significantly less body asymmetry, as compared to stroke animals that either did not receive transplantation or received fetal kidney grafts and noggin pretreatment. Analysis of these brains after triphenyltetrazolium chloride staining showed that fetal kidney tissue transplantation reduced the volume of infarction in the cerebral cortex. Noggin pretreatment reduced the protection induced by fetal kidney grafting, although noggin itself did not cause increase in cerebral infarction. Eight hours after ischemia, brain homogenates were obtained from grafted and control animals to assay caspase-3 enzymatic activity. This analysis demonstrated that fetal kidney grafts significantly reduced ischemia-induced caspase-3 activity. Reduction of caspase-3 activity could also be antagonized by noggin pretreatment. In conclusion, our data suggest that fetal kidney transplantation reduces ischemia/reperfusion-induced cortical infarction and behavioral deficits in adult rats, which are, at least partially, mediated through the effect of BMPs from the transplants.

GFRalpha-1 mRNA in dopaminergic and nondopaminergic neurons in the substantia nigra and ventral tegmental area.

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for several types of neurons, including dopaminergic (DAergic) neurons. GDNF binds with high affinity to the GDNF family receptor alpha-1 (GFRalpha-1), which is highly expressed in the midbrain. Using anatomical and lesion techniques, we demonstrated that GFRalpha-1 was expressed in DAergic and non-DAergic neurons in the rat midbrain. Immunohistochemical characterization of GFRalpha-1-expressing neurons indicated that most of the neurons that were immunopositive for the DAergic marker tyrosine hydroxylase (TH) expressed GFRalpha-1 in the substantia nigra pars compacta (SNC). In contrast, fewer TH-containing neurons expressed GFRalpha-1 in the substantia nigra pars reticulata (SNR) and the ventral tegmental area (VTA). Depletion of GFRalpha-1/TH neurons was observed in the SNC following treatment with the neurotoxin 6-hydroxydopamine (6-OHDA); however, GFRalpha-1 expression remained in some neurons located in the SNR. The gamma-aminobutyric acid (GABA)ergic nature of GFRalpha-1-expressing neurons located in the SNR, which were resistant to (6-hydroxydopamine) 6-OHDA, was established by their expression of glutamic acid decarboxylase (GAD; the synthesizing enzyme for GABA). Further analysis indicated that coexpression of GFRalpha-1 and GAD varied in a rostrocaudal gradient in the SNR, substantia nigra pars lateralis (SNL), and VTA. Midbrain DAergic and GABAergic neurons have been previously classified according to their Ca(2+) binding protein (CaBP) content; thus, we also sought to investigate the proportion of midbrain GFRalpha-1-expressing neurons containing parvalbumin (PV), calbindin (CB), and calretinin (CR) in the midbrain. Although GFRalpha-1 expression was found mainly in CB- and CR-immunoreactive neurons, it was rarely observed in PV-immunolabeled neurons. Analysis of the proportion of GFRalpha-1-expressing neurons for each CaBP subpopulation indicated the coexistence of GFRalpha-1 with CR in the VTA and all subdivisions of the SN; double-labeled GFRalpha-1/CR neurons were distributed in the SNC, SNR, SNL, and VTA. GFRalpha-1/CB neurons were also detected in the SNC, SNL, and VTA. Expression of GFRalpha-1 in DAergic and non-DAergic neurons in the rat SN and VTA suggests that GDNF, via GFRalpha-1, might modulate DAergic and GABAergic functions in the nigrostriatal, mesolimbic, and nigrothalamic circuits of the adult rat.