Glial cell-derived neurotrophic factor decrease may mediate learning, memory and behavior impairments in rats after neonatal surgery.

Patients who have surgery during the first few years of their lives may have an increased risk of behavioral abnormality. Our previous study has shown a role of glial cell-derived neurotrophic factor (GDNF) in neonatal surgery-induced learning and memory impairment in rats. This study was designed to determine whether neonatal surgery induced hyperactive behavior in addition to learning and memory impairment and whether GDNF played a role in these changes. Postnatal day 7 male and female Sprague-Dawley rats were subjected to right common carotid arterial exposure under sevoflurane anesthesia. Their learning, memory and behavior were tested from 23 days after the surgery. GDNF was injected intracerebroventricularly at the end of surgery. Surgery reduced GDNF expression in the hippocampus. Surgery impaired learning and memory and induced a hyperactive behavior as assessed by Barnes maze, fear conditioning and open field tests. In addition, surgery reduced dendritic arborization and spine density. The effects were attenuated by GDNF injection. These results suggest that surgery induces a hyperactive behavior pattern, impairment of learning and memory, and neuronal microstructural damage later in the lives in rats. GDNF reduction may mediate these surgical effects.

Safety Assessment of AAV2-hGDNF Administered Via Intracerebral Injection in Rats for Treatment of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective biologic in Parkinson's disease models. Adeno-associated viral vector serotype 2 (AAV2)-human GDNF safety was assessed in rats treated with a single intracerebral dose of vehicle, 6.8 × 108, 6.8 × 109, or 5.2 × 1010 vector genomes (vg)/dose followed by interim sacrifices on day 7, 31, 90, and 376. There were no treatment-related effects observed on food consumption, body weight, hematology, clinical chemistry, coagulation parameters, neurobehavioral parameters, organ weights, or serum GDNF and anti-GDNF antibody levels. Increased serum anti-AAV2 neutralizing antibody titers were observed in the 5.2 × 1010 vg/dose group. Histopathological lesions were observed at the injection site in the 6.8 × 109 vg/dose (day 7) and 5.2 × 1010 vg/dose groups (days 7 and 31) and consisted of gliosis, mononuclear perivascular cuffing, intranuclear inclusion bodies, and/or apoptosis on day 7 and mononuclear perivascular cuffing on day 31. GDNF immunostaining was observed in the injection site in all dose groups through day 376 indicating no detectable impacts of anti-AAV2 neutralizing antibody. There was no evidence of increased expression of calcitonin gene-related peptide or Swann cell hyperplasia in the cervical and lumbar spinal cord or medulla oblongata at the 5.2 × 1010 vg/dose level indicating lack of hyperplastic effects. In conclusion, no systemic toxicity was observed, and the local toxicity observed at the injection site appeared to be reversible demonstrating a promising safety profile of intracerebral AAV2-GDNF delivery. Furthermore, an intracerebral dose of 6.8 × 108 AAV2-GDNF vg/dose was considered to be a no observed adverse effect level in rats.

A regulatable adenovector system for GDNF and GFP delivery in the rat hippocampus.

Spatial memory performance declines in both normal aging and Alzheimer's disease. This cognitive deficit is related to hippocampus dysfunction. Gene therapy using neurotrophic factors like Glial cell line-derived neurotrophic factor (GDNF) emerges as a promising approach to ameliorate age-related cognitive deficits. We constructed a two vector regulatable system (2VRS) which consists of a recombinant adenoviral vector (RAd) harboring a Tet-Off bidirectional promoter flanked by GDNF and Green Fluorescent Protein (GFP) genes. A second adenovector, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are cotransduced by the 2VRS, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is silenced. We tested the 2VRS in CHO-K1 cells where we observed a dose-dependent GFP expression that was completely inhibited by DOX (1 mg/ml). The 2VRS injected in the hippocampal CA1 region transduced both neurons and astrocytes and was efficiently inhibited by DOX added to the drinking water. In order to assess GDNF biological activity we injected 2VRS and its Control (CTRL) vector in the hypothalamus and monitored body weight for one month. The results showed that GDNF retards weight recovery 6 days more than CTRL. In conclusion, our 2VRS demonstrated optimal GFP expression and showed a bioactive effect of transgenic GDNF in the brain.

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates.

Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and g-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 µm2) compared to autograft (4.62 ± 3.99 µm2) and PCL/Empty (4.52 ± 5.16 µm2) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit.

Drug Delivery for Movement Disorders.

There has been substantial research interest in delivering therapeutic neurotrophic factors directly to the brain for the treatment of Parkinson's Disease (PD) and other movement disorders. Direct infusion of glial cell-line derived neurotrophic factor has been investigated in both pre-clinical models and clinical trials. In this chapter we discuss past and present research investigating the potential of direct drug delivery to the brain for the treatment of PD and other movement disorders.

Research on the repairing effect of polylactic acid-trimethylene carbonate/GNDF slow-release catheter on the injured femoral nerve fiber.

Present research aims to investigate the repairing effect of polylactic acid-trimethylene carbonate/GNDF slow-release catheter on the injured femoral nerve fiber. Adult SD male rats as the subjects were divided into two groups, the GDNF group and the control group, and received the surgery to remove the nerve from the exposed left femoral nerves. Thereafter, rats in the GNDF group and the control group received the GNDF or normal saline, and we evaluated the changes in rats, including the morphological, functional and electrophysiological changes of regenerated nerves. Regenerated axons were found in each group, but enormous regeneration of axons was only identified in GDNF group. Further analysis showed that: At the 4th, 8th and 12th weeks, areas of the regenerated nerves in GDNF group were (0.95±0.06) mm2, (1.14±0.07) mm2 and (1.22±0.06) mm2, respectively; in the control group, these were (0.15±0.01) mm2, (0.25±0.07) mm2 and (0.52±0.05) mm2, respectively. These showed that the outcome of GDNF group was superior to that of control group. In GDNF group, quantities of the myelinated fiber were (0.8119×104±0.0637×104), (1.3371×104±0.0460×104) and (1.7669×104±0.0542×104); while in control group, these were (0.2179×104±0.0097×104), (0.3490×104±0.0329×104) and (0.7737×l04±0.0788×104). Again, these results also indicated that the outcome of GDNF group was superior to that of the control group (p<0.05). In GDNF group, the average diameters of myelinated fibers were (2.25±0.17) µm, (2.42±0.14) µm and (2.80±0.10) µm, which were significantly better than (1.24±0.08) µm, (1.43±0.14) µm and (1.82±0.14) µm in the control group. Degrees of fiber myelination in the GDNF group were (0.71±0.03), (0.64±0.03) and (0.6l±0.0l), respectively, which were also significantly higher than (0.02±0.01), (0.04±0.01) and (0.06±0.02) in the control group (p<0.01). At the 12th week after surgery, HE staining was performed to observe the histological changes in quadriceps femoris for evaluation of atrophy in each group. In the GDNF group, significant amelioration was found in the atrophy of quadriceps femoris with an average area of myofiber of (84.95±3.92) %, while the area of the control group was (57.95±5.78) %, suggesting that the outcome of the GDNF group was better than that of the control group (p<0.05). Electrophysiological examination of nerves was employed to detect the recovery of neurological functions after repair of nerve defect. At the 4th, 8th and 12th weeks after surgery, CMAP amplitudes in the GDNF group were (9.34±0.52) mV, (14.40±0.69) mV and (19.18±0.48) mV, significantly better than (0.39±0.07) mV, (1.44±0.41) mV and (9.27±0.40) in the control group (p<0.01). Polylactic acid-trimethylene carbonate/GNDF slow-release catheter can accelerate the functional recovery of injured nerves, thus promoting the regeneration efficiency of femoral nerves.

Neuroprotective Surgical Strategies in Parkinson's Disease: Role of Preclinical Data.

Although there have been many pharmacological agents considered to be neuroprotective therapy in Parkinson's disease (PD) patients, neurosurgical approaches aimed to neuroprotect or restore the degenerative nigrostriatal system have rarely been the focus of in depth reviews. Here, we explore the neuroprotective strategies involving invasive surgical approaches (NSI) using neurotoxic models 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA), which have led to clinical trials. We focus on several NSI approaches, namely deep brain stimulation of the subthalamic nucleus, glial neurotrophic derived factor (GDNF) administration and cell grafting methods. Although most of these interventions have produced positive results in preclinical animal models, either from behavioral or histological studies, they have generally failed to pass randomized clinical trials to validate each approach. We argue that NSI are promising approaches for neurorestoration in PD, but preclinical studies should be planned carefully in order not only to detect benefits but also to detect potential adverse effects. Further, clinical trials should be designed to be able to detect and disentangle neuroprotection from symptomatic effects. In summary, our review study evaluates the pertinence of preclinical models to study NSI for PD and how this affects their efficacy when translated into clinical trials.

Temporally controlled growth factor delivery from a self-assembling peptide hydrogel and electrospun nanofibre composite scaffold.

Tissue-specific self-assembling peptide (SAP) hydrogels designed based on biologically relevant peptide sequences have great potential in regenerative medicine. These materials spontaneously form 3D networks of physically assembled nanofibres utilising non-covalent interactions. The nanofibrous structure of SAPs is often compared to that of electrospun scaffolds. These electrospun nanofibers are produced as sheets that can be engineered from a variety of polymers that can be chemically modified to incorporate many molecules including drugs and growth factors. However, their macroscale morphology limits them to wrapping and bandaging applications. Here, for the first time, we combine the benefits of these systems to describe a two-component composite scaffold from these biomaterials, with the design goal of providing a hydrogel scaffold that presents 3D structures, and also has temporal control over drug delivery. Short fibres, cut from electrospun scaffolds, were mixed with our tissue-specific SAP hydrogel to provide a range of nanofibre sizes found in the extracellular matrix (10-300 nm in diameter). The composite material maintained the shear-thinning and void-filling properties of SAP hydrogels that have previously been shown to be effective for minimally invasive material injection, cell delivery and subsequent in vivo integration. Both scaffold components were separately loaded with growth factors, important signaling molecules in tissue regeneration whose rapid degradation limits their clinical efficacy. The two biomaterials provided sequential growth factor delivery profiles: the SAP hydrogel provided a burst release, with the release rate decreasing over 12 hours, while the electrospun nanofibres provided a more constant, sustained delivery. Importantly, this second release commenced 6 days later. The design rules established here to provide temporally distinct release profiles can enable researchers to target specific stages in regeneration, such as the acute immune response versus sustained protection and survival of cells following injury. In summary, this novel composite material combines the physical advantages of SAP hydrogels and electrospun nanofibres, while additionally providing a superior vehicle for the stabilisation and controlled delivery of growth factors necessary for optimal tissue repair.

Glucose-6-phosphatase-α participates in dopaminergic differentiation.

OBJECTIVE:  Induction of dopaminergic (DA) differentiation is a cell-based therapy for Parkinson's disease (PD). Here, we explore the key factors of DA differentiation with a focus on glucose-6-phosphatase (G6Pase), a marker enzyme for the endoplasmic reticulum (ER) associated with cell differentiation. METHODS:  We cultured SH-SY5Y human neuroblastoma cells, a model system for PD research, and added glial cell-derived neurotrophic factor (GDNF; 25, 50, or 100 ng/ml) to stimulate differentiation. Subsequently, several methods, such as microRNA/mRNA microarrays, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to detect target genes and proteins respectively. RESULTS:  Light microscopy revealed that 50 ng/ml GDNF most effectively induced DA differentiation. MicroRNA/mRNA microarrays identified that G6PC mRNA was significantly upregulated, which might be influenced by three downregulated microRNAs. Follow-up qRT-PCR results were consistent with the microarray findings, and western blots also supported the results. DISCUSSION: Taken together, our results demonstrate that G6PC, a subunit of G6Pase, participates in DA differentiation. Our findings may contribute to provide a foundation for the research on the mechanism of DA differentiation as well as cell-based therapy for PD.

Thermosensitive heparin-poloxamer hydrogels enhance the effects of GDNF on neuronal circuit remodeling and neuroprotection after spinal cord injury.

Traumatic spinal cord injury (SCI) results in paraplegia or quadriplegia, and currently, therapeutic interventions for axonal regeneration after SCI are not clinically available. Animal studies have revealed that glial cell-derived neurotrophic factor (GDNF) plays multiple beneficial roles in neuroprotection, glial scarring remodeling, axon regeneration and remyelination in SCI. However, the poor physicochemical stability of GDNF, as well as its limited ability to cross the blood-spinal cord barrier, hampers the development of GDNF as an effective therapeutic intervention in clinical practice. In this study, a novel temperature-sensitive heparin-poloxamer (HP) hydrogel with high GDNF-binding affinity was developed. HP hydrogels showed a supporting scaffold for GDNF when it was injected into the lesion epicenter after SCI. GDNF-HP by orthotopic injection on lesioned spinal cord promoted the beneficial effects of GDNF on neural stem cell proliferation, reactive astrogliosis inhibition, axonal regeneration or plasticity, neuroprotection against cell apoptosis, and body functional recovery. Most interestingly, GDNF demonstrated a bidirectional regulation of autophagy, which inhibited cell apoptosis at different stages of SCI. Furthermore, the HP hydrogel promoted the inhibition of autophagy-induced apoptosis by GDNF in SCI. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2816-2829, 2017.

Intranasal Administration of GDNF Protects Against Neural Apoptosis in a Rat Model of Parkinson's Disease Through PI3K/Akt/GSK3ß Pathway.

Glial cell line-derived neurotrophic factor (GDNF) plays important roles in protecting the damaged or dying dopamine neurons in the animal models of Parkinson's disease (PD). This study was to determine the effect and mechanisms of GDNF on the apoptosis of neurons in 6-hydroxydopamine (6-OHDA) induced Parkinson's disease model of rats. Healthy male Sprague-Dawley rats (220-240 g) were randomly divided into six groups (n = 10). 6-OHDA was used to establish the PD rat model. Tyrosine hydroxylase (TH) immunohistochemistry was used to assess the neuron loss in 6-OHDA-lesioned rats. TUNEL and western blot were used to identify the effects and mechanisms of GDNF in the rat model of PD. The numbers of TH-positive neurons in the 6-OHDA-injected lesioned substantia nigra (SN) decreased significantly compared with the Sham group. GDNF treatment effectively ameliorated the apoptosis of neuronal cells in SN induced by 6-OHDA. In addition, GDNF significantly increased serine protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3ß) phosphorylation induced by 6-OHDA. In contrast, application of LY294002 or triciribine reversed the roles of GDNF in PD models. The results implicated that the anti-apoptosis effects of GDNF in neurons might be mediated through PI3K/Akt/GSK3ß pathway. Therefore, GDNF may be a promising agent for PD treatment.

Morphological Changes in a Severe Model of Parkinson's Disease and Its Suitability to Test the Therapeutic Effects of Microencapsulated Neurotrophic Factors.

The unilateral 6-hydroxydopamine (6-OHDA) lesion of medial forebrain bundle (MFB) in rats affords us to study the advanced stages of Parkinson's disease (PD). Numerous evidences suggest synergic effects when various neurotrophic factors are administered in experimental models of PD. The aim of the present work was to assess the morphological changes along the rostro-caudal axis of caudo-putamen complex and substantia nigra (SN) in the referred model in order to test the suitability of a severe model to evaluate new neurorestorative therapies. Administration of 6-OHDA into MFB in addition to a remarkable depletion of dopamine in the nigrostriatal system induced an increase of glial fibrillary acidic protein (GFAP)-positive cells in SN and an intense immunoreactivity for OX-42, vascular endothelial growth factor (VEGF), and Lycopersycum esculentum agglutinin (LEA) in striatum and SN. Tyrosine hydroxylase (TH) immunostaining revealed a significant decrease of the TH-immunopositive striatal volume in 6-OHDA group from rostral to caudal one. The loss of TH-immunoreactive (TH-ir) neurons and axodendritic network (ADN) was higher in caudal sections. Morphological recovery after the implantation of microspheres loaded with VEGF and glial cell line-derived neurotrophic factor (GDNF) in parkinsonized rats was related to the preservation of the TH-ir cell number and ADN in the caudal region of the SN. In addition, these findings support the neurorestorative role of VEGF+GDNF in the dopaminergic system and the synergistic effect between both factors. On the other hand, a topological distribution of the dopaminergic system was noticeable in the severe model, showing a selective vulnerability to 6-OHDA and recovering after treatment.

In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay.

In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. The bead-derived GDNF signals were able to induce the stratified aggregate formation of GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted Asingle and marginal Apaired-Aaligned GFRα1-positive spermatogonia and was surrounded by Aaligned GFRα1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFRα1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis.

Gelatin-methacrylamide gel loaded with microspheres to deliver GDNF in bilayer collagen conduit promoting sciatic nerve growth.

In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide(®)). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 µm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable artificial nerve guide for nerve tissue engineering.

Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

Transplantation of Neural Stem Cells Cotreated with Thyroid Hormone and GDNF Gene Induces Neuroprotection in Rats of Chronic Experimental Allergic Encephalomyelitis.

The present study investigates whether transplantation of NSCs treated with T3 alone (T3/NSCs), or in conjunction with GDNF gene (GDNF-T3/NSCs), provides a better therapeutic effect than NSCs for chronic EAE. EAE rats were, respectively, injected with NSCs, T3/NSCs, GDNF-T3/NSCs, and saline at 10 days and sacrificed at 60 days after EAE immunization. The three cell grafted groups showed a significant reduction in clinical scores, inflammatory infiltration, and demyelination compared with the saline-injected group, and among the cell grafted groups, the reduction in GDNF-T3/NSCs group was the most notable, followed by T3/NSCs group. Grafted T3/NSCs and GDNF-T3/NSCs acquired more MAP2, GalC, and less GFAP in brain compared with grafted NSCs, and grafted GDNF-T3/NSCs acquired most MAP2 and least GalC among the cell grafted groups. Furthermore, T3/NSCs and GDNF-T3/NSCs grafting increased the expression of mRNA for PDGFαR, GalC, and MBP in lesion areas of brain compared with NSCs grafting, and the expression of mRNA for GalC and MBP in GDNF-T3/NSCs group was higher than that in T3/NSCs group. In conclusion, T3/NSCs grafting, especially GDNF-T3/NSCs grafting, provides a better neuroprotective effect for EAE than NSCs transplantation.

Macrophage-mediated inflammation and glial response in the skeletal muscle of a rat model of familial amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and loss of large motor neurons in the spinal cord and brain stem. While much research has focused on mechanisms of motor neuron cell death in the spinal cord, degenerative processes in skeletal muscle and neuromuscular junctions (NMJs) are also observed early in disease development. Although recent studies support the potential therapeutic benefits of targeting the skeletal muscle in ALS, relatively little is known about inflammation and glial responses in skeletal muscle and near NMJs, or how these responses contribute to motor neuron survival, neuromuscular innervation, or motor dysfunction in ALS. We recently showed that human mesenchymal stem cells modified to release glial cell line-derived neurotrophic factor (hMSC-GDNF) extend survival and protect NMJs and motor neurons in SOD1(G93A) rats when delivered to limb muscles. In this study, we evaluate inflammatory and glial responses near NMJs in the limb muscle collected from a rat model of familial ALS (SOD1(G93A) transgenic rats) during disease progression and following hMSC-GDNF transplantation. Muscle samples were collected from pre-symptomatic, symptomatic, and end-stage animals. A significant increase in the expression of microglial inflammatory markers (CD11b and CD68) occurred in the skeletal muscle of symptomatic and end-stage SOD1(G93A) rats. Inflammation was confirmed by ELISA for inflammatory cytokines interleukin-1 ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in muscle homogenates of SOD1(G93A) rats. Next, we observed active glial responses in the muscle of SOD1(G93A) rats, specifically near intramuscular axons and NMJs. Interestingly, strong expression of activated glial markers, glial fibrillary acidic protein (GFAP) and nestin, was observed in the areas adjacent to NMJs. Finally, we determined whether ex vivo trophic factor delivery influences inflammation and terminal Schwann cell (TSC) response during ALS. We found that intramuscular transplantation of hMSC-GDNF tended to exhibit less inflammation and significantly maintained TSC association with NMJs. Understanding cellular responses near NMJs is important to identify suitable cellular and molecular targets for novel treatment of ALS and other neuromuscular diseases.

Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression.

Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.

Functionalized composite scaffolds improve the engraftment of transplanted dopaminergic progenitors in a mouse model of Parkinson's disease.

With the brain's limited capacity for repair there is a need for new and innovative therapies to promote regeneration. Stem/progenitor cell transplantation has received increasing attention, and whilst clinical trials demonstrating functional integration exist, inherent variability between patients has hindered development of this therapy. Variable outcomes have largely been attributed to poor survival and insufficient reinnervation of target tissues due in part to the suboptimal host environment. Here we examined whether improving the physical properties of the host milieu, by way of bioengineered scaffolds, may enhance engraftment. We developed a composite scaffold, incorporating electrospun poly(l-lactic acid) short nanofibers embedded within a thermo-responsive xyloglucan hydrogel, which could be easily injected into the injured brain. Furthermore, to improve the trophic properties of the host brain, glial derived neurotrophic factor (GDNF), a protein known to promote cell survival and axonal growth, was blended into and/or covalently attached onto the composite scaffolds to provide controlled delivery. In vitro we confirmed the ability of the scaffolds to support ventral midbrain (VM) dopamine progenitors, and provide sustained delivery of GDNF - capable of eliciting effects on cell survival and dopaminergic axon growth. In Parkinsonian mice, we show that these composite scaffolds, whilst having no deleterious impact on the host immune response, enhanced the survival of VM grafts and reinnervation of the striatum, an effect that was augmented through the scaffold delivery of GDNF. Taken together, these functionalized composite scaffolds provide a means to significantly improve the milieu of the injured brain, enabling enhanced survival and integration of grafted neurons.

Intranasal Administration of chitosan-Coated Nanostructured Lipid Carriers Loaded with GDNF Improves Behavioral and Histological Recovery in a Partial Lesion Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder, but current therapies are only symptomatic. A promising alternative to address the neurodegenerative process is the use of neurotrophic factors, such as the glial cell-derived neurotrophic factor (GDNF). However, its clinical use has been limited due to its short half-life and rapid degradation after in vivo administration, in addition to difficulties in crossing the blood-brain barrier (BBB). This barrier is a limiting factor in brain drug development, making the future progression of neurotherapeutics difficult. In the past few years, intranasal drug delivery has appeared as an alternative non-invasive administration route to bypass the BBB and target drugs directly to the CNS. Thus, the aim of this work was to study the in vivo neuroprotective effect of intranasally administered GDNF, encapsulated in chitosan-coated nanostructured lipid carrier (CS-NLC-GDNF), in a 6-OHDA partially lesioned rat model. The developed CS-NLC-GDNF showed a particle size of approximately 130 nm and high encapsulation efficiency. The in vitro study in PC-12 cells demonstrated the ability of the encapsulated GDNF to protect these cells against 6-OHDA toxin. After two weeks of daily intranasal administration of treatments, the administration of CS-NLC-GDNF achieved a behavioral improvement in rats, as well as a significant improvement in both the density of TH+ fibres in the striatum and the TH+ neuronal density in the SN. Thus, it can be concluded that the nose-to-brain delivery of CS-NLC-GDNF could be a promising therapy for the treatment of PD.