NRG1-Fc improves metabolic health via dual hepatic and central action.

Neuregulins (NRGs) are emerging as an important family of signaling ligands that regulate glucose and lipid homeostasis. NRG1 lowers blood glucose levels in obese mice, whereas the brown fat-enriched secreted factor NRG4 protects mice from high-fat diet-induced insulin resistance and hepatic steatosis. However, the therapeutic potential of NRGs remains elusive, given the poor plasma half-life of the native ligands. Here, we engineered a fusion protein using human NRG1 and the Fc domain of human IgG1 (NRG1-Fc) that exhibited extended half-life in circulation and improved potency in receptor signaling. We evaluated its efficacy in improving metabolic parameters and dissected the mechanisms of action. NRG1-Fc treatment triggered potent AKT activation in the liver, lowered blood glucose, improved insulin sensitivity, and suppressed food intake in obese mice. NRG1-Fc acted as a potent secretagogue for the metabolic hormone FGF21; however, the latter was largely dispensable for its metabolic effects. NRG1-Fc directly targeted the hypothalamic POMC neurons to promote membrane depolarization and increase firing rate. Together, NRG1-Fc exhibits improved pharmacokinetic properties and exerts metabolic benefits through dual inhibition of hepatic gluconeogenesis and caloric intake.

Tracking the in vivo release of bioactive NRG from PLGA and PEG-PLGA microparticles in infarcted hearts.

The growth factor neuregulin (NRG) is one of the most promising candidates in protein therapy as potential treatment for myocardial infarction (MI). In the last few years, biomaterial based delivery systems, such as polymeric microparticles (MPs) made of poly(lactic co glycolic acid) and polyethylene glycol (PLGA and PEG-PLGA MPs), have improved the efficacy of protein therapy in preclinical studies. However, no cardiac treatment based on MPs has yet been commercialized since this is a relatively new field and total characterization of polymeric MPs remains mandatory before they reach the clinical arena. Therefore, the objective of this study was to characterize the in vivo release, bioactivity and biodegradation of PLGA and PEG-PLGA MPs loaded with biotinylated NRG in a rat model of MI. The effect of PEGylation in the clearance of the particles from the cardiac tissue was also evaluated. Interestingly, MPs were detected in the cardiac tissue for up to 12 weeks after administration. In vivo release analysis showed that bNRG was released in a controlled manner throughout the twelve week study. Moreover, the biological cardiomyocyte receptor (ErbB4) for NRG was detected in its activated form only in those animals treated with bNRG loaded MPs. On the other hand, the PEGylation strategy was effective in diminishing phagocytosis of these MPs compared to noncoated MPs in the long term (12 weeks after injection). Taking all this together, we report new evidence in favor of the use of polymeric PLGA and PEG-PLGA MPs as delivery systems for treating MI, which could be soon included in clinical trials.

A corrole nanobiologic elicits tissue-activated MRI contrast enhancement and tumor-targeted toxicity.

Water-soluble corroles with inherent fluorescence can form stable self-assemblies with tumor-targeted cell penetration proteins, and have been explored as agents for optical imaging and photosensitization of tumors in pre-clinical studies. However, the limited tissue-depth of excitation wavelengths limits their clinical applicability. To examine their utility in more clinically-relevant imaging and therapeutic modalities, here we have explored the use of corroles as contrast enhancing agents for magnetic resonance imaging (MRI), and evaluated their potential for tumor-selective delivery when encapsulated by a tumor-targeted polypeptide. We have found that a manganese-metallated corrole exhibits significant T1 relaxation shortening and MRI contrast enhancement that is blocked by particle formation in solution but yields considerable MRI contrast after tissue uptake. Cell entry but not low pH enables this. Additionally, the corrole elicited tumor-toxicity through the loss of mitochondrial membrane potential and cytoskeletal breakdown when delivered by the targeted polypeptide. The protein-corrole particle (which we call HerMn) exhibited improved therapeutic efficacy compared to current targeted therapies used in the clinic. Taken together with its tumor-preferential biodistribution, our findings indicate that HerMn can facilitate tumor-targeted toxicity after systemic delivery and tumor-selective MR imaging activatable by internalization.

Neurobehavioral Differences Between Mice Receiving Distinct Neuregulin Variants as Neonates; Impact on Sensitivity to MK-801.

Neuregulin-1 (NRG1) is a well-recognized risk gene for schizophrenia and is often implicated in the neurodevelopmental hypothesis of this illness. Alternative splicing and proteolytic processing of the NRG1 gene produce more than 30 structural variants; however, the neuropathological roles of individual variants remain to be characterized. On the basis of the neurodevelopmental hypothesis of schizophrenia, we administered eNRG1 (0.1~1.0 µg/g), a core epidermal growth factor-like (EGF) domain common for all splicing NRG1 variants, to neonatal mice and compared their behavioral performance with mice challenged with a full mature form of type 1 NRG1 variant. During the neonatal stage, recombinant eNRG1 protein administrated from the periphery passed the blood-brain barrier and activated its receptor (ErbB4) in the brain. In adults, the mice receiving the highest dose exhibited lower locomotor activity and deficits in prepulse inhibition and tonedependent fear learning, although the hearing reduction of the eNRG1-treated mice may explain these behavioral deficits. Neonatal eNRG1 treatment also significantly potentiated MK-801-driven locomotor activity in an eNRG1 dose-dependent manner. In parallel eNRG1 treatment enhanced MK-801-driven c-Fos induction and decreased immunoreactivity for NMDA receptor subunits in adult brain. In contrast, mice that had been treated with the same molar dose of a full mature form of type 1 NRG1 as neonates did not exhibit hypersensitivity to MK-801. However, both animal models exhibited similar hypersensitivity to methamphetamine. Collectively, our findings suggest that aberrant peripheral NRG1 signals during neurodevelopment alter later behavioral traits and auditory functions in the NRG1 subtype-dependent manner.

Biodistribution and brain permeability of the extracellular domain of neuregulin-1-ß1.

Neuregulin-1 (NRG1) belongs to a large family of growth and differentiation factors with a key role in the development and maintenance of the brain. Genetic association of NRG1 within brain disorders such as Alzheimer's disease, schizophrenia and neuroprotective properties of certain NRG1 isoforms have led to a variety of studies in corresponding disease models. In the present work, we investigated NRG1 with regard to its peripheral and central biodistribution after systemic application. We first-time radiolabeled the entire biologically active extracellular domain of NRG1 isotype-ß1 (NRG1-ß1 ECD; aa 2-246) with iodine-125 and administered it peripherally to healthy adult C57Bl6 mice. Blood kinetics and relative organ distribution of (125)I-labeled NRG1-ß1 ECD were determined. The blood level of NRG1-ß1 ECD peaked within the first hour after intraperitoneal (i.p.) application. The brain-blood ratios of (125)I-labeled NRG1-ß1 ECD were time-dependently 150-370% higher compared to the brain impermeable control, (131)I-labeled bovine serum albumin. Autoradiographs of brain slices demonstrated that (125)I-labeled NRG1-ß1 ECD accumulated in several regions of the brain e.g. frontal cortex, striatum and ventral midbrain containing the substantia nigra. In addition we found histochemical and biochemical evidence that phosphorylation of the NRG1 prototype receptor ErbB4 was increased in these regions after systemic application of NRG1-ß1 ECD. Our data suggest that NRG1-ß1 ECD passes the blood-brain barrier and activates cerebral ErbB4 receptors.

Expresión y purificación de GDNF para su microencapsulación y aplicación en la enfermedad de Parkinson / Expression and Purification of GDNF for its microencapsulation in drug delivery systems and application in Parkinson’s disease

La enfermedad de Parkinosn (EP) es un proceso neurodegenerativodel sistema nervioso central que afecta a las neuronasde dopamina de la sustancia negra, núcleo mesoencefálico delcontrol motor. La perdida en el cerebro de este neurotransmisorvital causa los síntomas de la enfermedad. La EP afectaactualmente a 200 de cada 100.000 personas y a 2 de cada100 entre los mayores de 60 años. En España hay unos110.000 enfermos. Además, hoy por hoy no se conoce nadaque pueda prevenir o curar la enfermedad, ni existe ningunaprueba de laboratorio que permita diagnosticarla. Recentiementese ha demostrado que el GDNF, factor neurotrófico derivadode las células gliales, es capaz de proteger las neuronasdopaminérgicas e incluso inducir la regeneración del tejidodopaminérgico dañado in vivo.El objetivo del trabajo fue diseñar y desarrolar un método deexpresión y purificación de GDNF bioactivo para su posteriormicroencapsulación y aplicación en la enfermedad de Parkison.El sistema escogido para expresar el GDNF fue el sistema decélulas eucariotas de mamífero. El vector utilizado para la produccióndel GDNF en células eucariotas fue el pDEST26 (TecnologíaGateway de Invitrogen). Como sistema de expresión deGDNF se utilizaron las líneas celulares eucariota BHK, 293 yCOS 7. Estas células fueron cultivadas en medio D-MEM (Invitrogen)complementado con un 10% de suero fetal bovino(FBS) y Penicilina/Streptomicina (100u/ml) (Invitrogen). Latransfección se realizó con Lipofectamine Plus (Invitrogen). Seanalizó la expresión de GDNF a nivel de mRNA mediante PCR ya nivel de proteína mediante Western Blot del medio condicionado. Los clones positivos se crecieron en botellas de cultivode 850 cm2 (Corning) y se realizaron ciclos de recolección delmedio. Cada ciclo fue analizado por SDS-PAGE y Western Blot.Para evaluar la actividad de la proteína se ha desarrollado unensayo de actividad en el que se demuestra la diferenciaciónmorfológica de células PC-12 inducida por GDNF. La presenciade los receptores GFRa1 y RET, necesarios para que el GDNFejerza su acción, fue determinada por PCR.Las conclusiones obtenidas de este estudio son la obtenciónde GDNF recombinante a partir de un sistema de expresión encélulas eucariotas, el desarrollo de un protocolo para su posteriorpurificación y la obtención de GDNF recombinante biológicamenteactivo

Parkinson’s disease (PD) is a slowly progressive neurodegenerativedisorder caused by decreased levels of dopamine in thesubstantia nigra, brain region responsible for movement. Thelack of dopamine is believe to be responsible of the symptomsof PD. Parkinson’s affects 200 in 100000 people and 2 inevery 100 persons over 60 years old. In Spain, there areabout 110000 people with PD. There is no cure at this time,and there are no prevention techniques or therapies. Recently,it has been demonstrated that GDNF, a glial-derived neurotrophicfactor is able to protect the dopaminergic neurons of thesubstancia nigra and it can also induce regeneration of injuredneurons in the central nervous system in vivo.The aim of this work was to develop a procedure for theexpression and purification of bioactive GDNF in view of itsmicroencapsulation for the treatment of PD.The cDNA of the GDNF gene was cloned in the expressionvector pDEST26 (Invitrogen) using the Gateway® Technology.Several eukaryotic mammalian cell lines (BHK, COS-7, and293) were stably transfected with the construction with LipofectaminePlus (Invitrogen). In those clones in which the presenceof the mRNA of the GDNF was confirmed by PCR studies,the expression of the recombinant protein in the serumfree medium was analyzed by Western Blot studies. The highestGDNF-producing clone, obtained from the BHK cell line,was cultured in 850 cm2 roller bottles (Corning) alternatingthe presence or the absence of FBS in the medium every 24hours, being collected only the serum free medium for theproduction of the recombinant GDNF. Each cycle protein expression was analyzed by SDS-PAGE and by Western Blot.The secreted protein was purified by several chromatographysteps. After each step of the purification procedure the fractionsobtained were analyzed by SDS-PAGE, Western Blotand silver staining analysis. A neuronal differentiation PC-12cell-based bioassay was also developed to confirm the biologicalactivity of the purified recombinant protein. Previously,the presence of GFRa1 and RET, receptors required forGDNF activity were confirmed by PCR.In conclusion, recombinant GDNF was produced in an eukaryoticmammalian cell line-based system. The protein purificationprocedure developed, allowed to obtain a highly purifiedrecombinant GDNF. Furthermore, the recombinant protein isbioactive