Encapsulated galanin-producing cells attenuate focal epileptic seizures in the hippocampus.

PURPOSE: Encapsulated cell biodelivery (ECB) is a relatively safe approach, since the devices can be removed in the event of adverse effects. The main objectives of the present study were to evaluate whether ECB could be a viable alternative of cell therapy for epilepsy. We therefore developed a human cell line producing galanin, a neuropeptide that has been shown to exert inhibitory effects on seizures, most likely acting via decreasing glutamate release from excitatory synapses. To explore whether ECB of genetically modified galanin-producing human cell line could provide seizure-suppressant effects, and test possible translational prospect for clinical application, we implanted ECB devices bilaterally into the hippocampus of rats subjected to rapid kindling, a model for recurrent temporal lobe seizures. METHODS: Two clones from a genetically modified human cell line secreting different levels of galanin were tested. Electroencephalography (EEG) recordings and stimulations were performed by electrodes implanted into the hippocampus at the same surgical session as ECB devices. One week after the surgery, rapid kindling stimulations were initiated. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) measurements prior to device implantation showed a release of galanin on average of 8.3 ng/mL/24 h per device for the low-releasing clone and 12.6 ng/mL/24 h per device for the high-releasing clone. High-releasing galanin-producing ECB devices moderately decreased stimulation-induced focal afterdischarge duration, whereas low-releasing ECB devices had no significant effect. SIGNIFICANCE: Our study shows that galanin-releasing ECB devices moderately suppress focal stimulation-induced recurrent seizures. Despite this moderate effect, the study provides conceptual proof that ECB could be a viable alternative approach to cell therapy in humans, with the advantage that the treatment could be terminated by removing these devices from the brain. Thereby, this strategy provides a higher level of safety for future therapeutic applications, in which genetically modified human cell lines that are optimized to produce and release antiepileptic compounds could be clinically evaluated for their seizure-suppressant effects.

Congestive heart failure in rats is associated with increased collecting duct vasopressin sensitivity and vasopressin type 2 receptor reexternalization.

A number of studies have shown that rats with congestive heart failure (CHF) have increased protein levels of the vasopressin (AVP)-regulated water channel aquaporin-2 (AQP2) even during conditions with unchanged circulating levels of AVP, suggesting an increase in the sensitivity of the AVP type 2 (V2) receptor in experimental CHF. The present study was aimed at investigating AVP signaling in rats with moderate CHF (left ventricular end diastolic pressure >10 mmHg; normal plasma AVP levels) induced by ligation of the left anterior descending coronary artery. Sham-operated rats were used as controls. Western blotting analyses revealed an increased abundance of AQP2 in renal cortex (+33 ± 9% of sham; P < 0.05) and in inner medulla (IM) (+54 ± 15% of sham; P < 0.05) in CHF rats compared with sham-operated controls. Dose-response studies on isolated collecting ducts (CDs) showed an increased accumulation of cAMP in response to AVP in CHF rats compared with controls. V2 receptor surface-binding studies in isolated IMCDs showed a marked and comparable AVP-induced V2 receptor internalization in response to AVP in both CHF and control rats. As expected V2 receptor surface binding remained low after AVP challenge in control rats. In contrast to this, V2 receptor surface binding returned to pre-AVP levels within 30 min in the CHF rats, indicating an obtained recycling ability of the V2 receptor in CHF. Together the results indicate the presence of an increased AVP sensitivity in the CDs from CHF rats, associated with an acquired recycling ability of the V2 receptor.

Encapsulated cell-based biodelivery of meteorin is neuroprotective in the quinolinic acid rat model of neurodegenerative disease.

PURPOSE: Encapsulated cell (EC) biodelivery is a promising, clinically relevant technology platform to safely target the delivery of therapeutic proteins to the central nervous system. The purpose of this study was to evaluate EC biodelivery of the novel neurotrophic factor, Meteorin, to the striatum of rats and to investigate its neuroprotective effects against quinolinic acid (QA)-induced excitotoxicity. METHODS: Meteorin-producing ARPE-19 cells were loaded into EC biodelivery devices and implanted into the striatum of rats. Two weeks after implantation, QA was injected into the ipsilateral striatum followed by assessment of neurological performance two and four weeks after QA administration. RESULTS: Implant-delivered Meteorin effectively protected against QA-induced toxicity, as manifested by both near-normal neurological performance and reduction of brain cell death. Morphological analysis of the Meteorin-treated brains showed a markedly reduced striatal lesion size. The EC biodelivery devices produced stable or even increasing levels of Meteorin throughout the study over 6 weeks. CONCLUSIONS: Stereotactically implanted EC biodelivery devices releasing Meteorin could offer a feasible strategy in the treatment of neurological diseases with an excitotoxic component such as Huntington's disease. In a broader sense, the EC biodelivery technology is a promising therapeutic protein delivery platform for the treatment of a wide range of diseases of the central nervous system.

Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo.

Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.5 also in dorsal root ganglions and inner ear but apparently not in the adult nervous system. In vitro, Cometin promotes neurite outgrowth from dorsal root ganglion cells which can be blocked by inhibition of the Janus or MEK kinases. In this assay, additive effects of Cometin and Meteorin are observed indicating separate receptors. Furthermore, Cometin supports migration of neuroblasts from subventricular zone explants to the same extend as stromal cell derived factor 1a. Given the neurotrophic properties in vitro, combined with the restricted inner ear expression during development, we further investigated Cometin in relation to deafness. In neomycin deafened guinea pigs, two weeks intracochlear infusion of recombinant Cometin supports spiral ganglion neuron survival and function. In contrast to the control group receiving artificial perilymph, Cometin treated animals retain normal electrically-evoked brainstem response which is maintained several weeks after treatment cessation. Neuroprotection is also evident from stereological analysis of the spiral ganglion. Altogether, these studies show that Cometin is a potent new neurotrophic factor with therapeutic potential.

Meteorin is a chemokinetic factor in neuroblast migration and promotes stroke-induced striatal neurogenesis.

Ischemic stroke affecting the adult brain causes increased progenitor proliferation in the subventricular zone (SVZ) and generation of neuroblasts, which migrate into the damaged striatum and differentiate to mature neurons. Meteorin (METRN), a newly discovered neurotrophic factor, is highly expressed in neural progenitor cells and immature neurons during development, suggesting that it may be involved in neurogenesis. Here, we show that METRN promotes migration of neuroblasts from SVZ explants of postnatal rats and stroke-subjected adult rats via a chemokinetic mechanism, and reduces N-methyl-D-asparate-induced apoptotic cell death in SVZ cells in vitro. Stroke induced by middle cerebral artery occlusion upregulates the expression of endogenous METRN in cells with neuronal phenotype in striatum. Recombinant METRN infused into the stroke-damaged brain stimulates cell proliferation in SVZ, promotes neuroblast migration, and increases the number of immature and mature neurons in the ischemic striatum. Our findings identify METRN as a new factor promoting neurogenesis both in vitro and in vivo by multiple mechanisms. Further work will be needed to translate METRN's actions on endogenous neurogenesis into improved recovery after stroke.

Lentiviral delivery of meteorin protects striatal neurons against excitotoxicity and reverses motor deficits in the quinolinic acid rat model.

Meteorin is a newly discovered secreted protein involved in both glial and neuronal cell differentiation, as well as in cerebral angiogenesis during development; but effects in the adult nervous system are unknown. The growth factor-like properties and expression of Meteorin during the development of the nervous system raises the possibility that it might possess important neuroprotective or regenerative capabilities. This report is the first demonstration that Meteorin has potent neuroprotective effects in vivo. Lentiviral-mediated striatal delivery of Meteorin to rats two weeks prior to injections of quinolinic acid (QA) dramatically reduced the loss of striatal neurons. The cellular protection afforded by Meteorin was associated with normalization of neurological performance on spontaneous forelimb placing and cylinder behavioral tests and a complete protection against QA-induced weight loss. These benefits were comparable in magnitude to those obtained with lentiviral-mediated delivery of ciliary neurotrophic factor (CNTF), a protein with known neuroprotective properties in the same model system. In naive animals, endogenous levels of both Meteorin and CNTF were increased in glial cells in response to QA lesion indicating that Meteorin may exert its protective effects as part of the reactive gliosis cascade in the injured brain. In summary, these data demonstrate that Meteorin strongly protects striatal neurons and deserves additional evaluation as a novel therapeutic for the treatment of neurological disorders with an excitotoxic component such as Huntington's Disease.

Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the Göttingen minipig basal forebrain.

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Characterization of Meteorin--an evolutionary conserved neurotrophic factor.

Growth factors control cellular growth, proliferation, and differentiation and may have therapeutic applications. In this study, we focus on Meteorin which is a member of a largely uncharacterized evolutionary conserved two-member growth factor family. Our analysis shows that Meteorin is expressed in the central nervous system both during development and in adult mice. Detailed immunohistological analysis of the adult mouse brain reveals that Meteorin is highly expressed in Bergmann glia and in a few discrete neuronal populations residing in the superior colliculus, the ocular motor nucleus, the raphe and pontine nuclei, and in various thalamic nuclei. In addition, low levels of Meteorin is found in astrocytes (S100beta+, OX42-) distributed ubiquitously throughout the brain. Meteorin was cloned and recombinant protein purified allowing N-terminal sequencing and mass spectrometric analysis showing that Meteorin is secreted as an unmodified monomer. This form is bioactive as it induces neurite outgrowth from dorsal root ganglions in vitro. Intrastriatal protein injection and lentiviral studies in vivo showed that Meteorin is a highly diffusible molecule in the brain and cellular uptake is apparent in specific populations which may carry the receptor. In summary, we provide a comprehensive expression analysis and have made and thoroughly validated molecular tools to help investigate the therapeutic potential of Meteorin.

The Drosophila gene CG9918 codes for a pyrokinin-1 receptor.

The database from the Drosophila Genome Project contains a gene, CG9918, annotated to code for a G protein-coupled receptor. We cloned the cDNA of this gene and functionally expressed it in Chinese hamster ovary cells. We tested a library of about 25 Drosophila and other insect neuropeptides, and seven insect biogenic amines on the expressed receptor and found that it was activated by low concentrations of the Drosophila neuropeptide, pyrokinin-1 (TGPSASSGLWFGPRLamide; EC50, 5 x 10(-8) M). The receptor was also activated by other Drosophila neuropeptides, terminating with the sequence PRLamide (Hug-gamma, ecdysis-triggering-hormone-1, pyrokinin-2), but in these cases about six to eight times higher concentrations were needed. The receptor was not activated by Drosophila neuropeptides, containing a C-terminal PRIamide sequence (such as ecdysis-triggering-hormone-2), or PRVamide (such as capa-1 and -2), or other neuropeptides and biogenic amines not related to the pyrokinins. This paper is the first conclusive report that CG9918 is a Drosophila pyrokinin-1 receptor gene.

Uncoupling of vasopressin signaling in collecting ducts from rats with CBL-induced liver cirrhosis.

Vasopressin (AVP) stimulates collecting duct water reabsorption through cAMP-mediated membrane targeting and increased expression of the aquaporin-2 (AQP2) water channel. Rats with liver cirrhosis induced by common bile duct ligation (CBL) show decreased protein expression of AQP2 despite increased plasma concentrations of AVP. The present study was conducted to investigate possible mechanisms behind this uncoupling of AVP signaling. The rats were examined 4 wk after CBL or sham operation. The CBL rats had increased plasma AVP concentrations (CBL: 3.2 +/- 0.2 vs. sham: 1.4 +/- 0.4 pg/ml, P < 0.05) and reduced AQP2 (0.62 +/- 0.11) and phosphorylated AQP2 (0.50 +/- 0.06) protein expression compared with sham-operated rats. However, examination of subcellular AQP2 localization by immunohistochemistry showed unchanged plasma membrane targeting in CBL rats, indicating a sustained ability of AQP2 short-term regulation. In a separate series of animals, thirsting was found to normalize AQP2 expression, indicating that AVP uncoupling in CBL rats is a physiological compensatory mechanism aimed at avoiding dilutional hyponatremia. Studies on microdissected collecting ducts from CBL rats showed decreased cAMP accumulation in response to AVP stimulation. The presence of the nonspecific phosphodiesterase inhibitor IBMX normalized the cAMP accumulation, indicating that cAMP-phosphodiesterase activity is increased in CBL rats. However, in contrast to this, Western blotting showed a decreased expression of several phosphodiesterase splice variants. We conclude that CBL rats develop an escape from AVP to prevent the formation of dilutional hyponatremia in response to increased plasma AVP concentrations. The mechanism behind AVP escape seems to involve decreased collecting duct sensitivity to AVP as a result of increased cAMP-phosphodiesterase activity.

Effects of renal denervation on tubular sodium handling in rats with CBL-induced liver cirrhosis.

This study was designed to examine the effect of bilateral renal denervation (DNX) on thick ascending limb of Henle's loop (TAL) function in rats with liver cirrhosis induced by common bile duct ligation (CBL). The CBL rats had, as previously shown, sodium retention associated with hypertrophy of the inner stripe of the outer medulla (ISOM) and increased natriuretic effect of furosemide in vivo, and semiquantitative immunoblotting showed increased expression of the furosemide-sensitive Na-K-2Cl cotransporter type 2 (NKCC2) in ISOM from CBL rats. DNX significantly attenuated the sodium retention in the CBL rats, which was associated with normalization of the natriuretic effect of furosemide, as well as a significant reduction in the expression of NKCC2 in the ISOM. However, the marked hypertrophy of the ISOM found in CBL rats was not reversed by DNX. Together, these data indicate that increased renal sympathetic nerve activity known to be present in CBL rats plays a significant role in the formation of sodium retention by stimulating sodium reabsorption in the TAL via increased renal abundance of NKCC2.