Growth/differentiation factor-15 deficiency compromises dopaminergic neuron survival and microglial response in the 6-hydroxydopamine mouse model of Parkinson's disease.

Growth/differentiation factor-15 (Gdf-15) is a member of the TGF-ß superfamily and a pleiotropic, widely distributed cytokine, which has been shown to play roles in various pathologies, including inflammation. Analysis of Gdf-15(-/-) mice has revealed that it serves the postnatal maintenance of spinal cord motor neurons and sensory neurons. In a previous study, exogenous Gdf-15 rescued 6-hydroxydopamine (6-OHDA) lesioned Gdf-15(+/+) nigrostriatal dopaminergic (DAergic) neurons in vitro and in vivo. Whether endogenous Gdf-15 serves the physiological maintenance of nigrostriatal DAergic neurons in health and disease is not known and was addressed in the present study. Stereotactic injection of 6-OHDA into the medial forebrain bundle (MFB) led to a significant decline in the numbers of DAergic neurons in both Gdf-15(+/+) and Gdf-15(-/-) mice over a time-period of 14days. However, this decrease was exacerbated in the Gdf-15(-/-) mice, with only 5.5% surviving neurons as compared to 24% in the Gdf-15(+/+) mice. Furthermore, the microglial response to the 6-OHDA lesion was reduced in Gdf-15(-/-) mice, with significantly lower numbers of total and activated microglia and a differential cytokine expression as compared to the Gdf-15(+/+) mice. Using in vitro models, we could demonstrate the importance of endogenous Gdf-15 in promoting DAergic neuron survival thus highlighting its relevance in a direct neurotrophic supportive role. Taken together, these results indicate the importance of Gdf-15 in promoting survival of DAergic neurons and regulating the inflammatory response post 6-OHDA lesion.

Differential proteome of the striatum from hemiparkinsonian rats displays vivid structural remodeling processes.

Parkinson's disease is a multifactorial, neurodegenerative disease where etiopathogenetic mechanisms are not fully understood. Animal models like the neurotoxic 6-OHDA-hemiparkinsonian rat model are used for standardized experiments. Here, we analyzed proteome changes of the striatum three months after 6-OHDA lesions of the nigral dopaminergic cell population. Striata were removed and proteins were separated by 2DE followed by differential spot analysis. Proteins in spots were identified by MALDI-TOF-MS. Most up-regulations of proteins were concerning energy metabolism in mitochondria. Proteins of calcium homeostasis like annexin A3, annexin A7, calbindin, calmodulin, calreticulin, and reticulocalbin 1 also were differentially regulated. Moreover, proteins involved in antioxidative mechanisms like superoxide dismutase, protein disulfide isomerase 1 and 3, N(G),N(G)-dimethylarginindimethyl-aminotransferase 2, and thioredoxin-dependent peroxide reductase were up-regulated. Interestingly, most cytoskeletal proteins belonging to the axon cytoskeleton and synapse were up-regulated pointing to long-distance axon remodeling. In addition, transcription factors, proteins of nucleic acid metabolism, chaperones, and degrading proteins (UCHL1) were up-regulated as well. In conclusion, the neurotoxin-induced proteome alterations indicate vivid long-distance remodeling processes of dendrites, axons, and synapses that are still ongoing even three months after perturbation, indicating a high plasticity and regeneration potential in the adult rat brain.

Selective targeting of adenoviral vectors to neural precursor cells in the hippocampus of adult mice: new prospects for in situ gene therapy.

The adult brain contains neural precursor cells (NPC) that are attracted to brain lesions, such as areas of neurodegeneration, ischemia, and cancer. This suggests that NPC engineered to promote lineage-specific differentiation or to express therapeutic genes might become a valuable tool for restorative cell therapy and for targeting therapeutic genes to diseased brain regions. Here we report the identification of NPC-specific ligands from phage display peptide libraries and show their potential to selectively direct adenovirus-mediated gene transfer to NPC in adult mice. Identified peptides mediated specific virus binding and internalization to cultured neurospheres. Importantly, peptide-mediated adenoviral vector infection was restricted to precursor cells in the hippocampal dentate gyrus of pNestin-green fluorescent protein transgenic or C57BL/6 mice. Our approach represents a novel method for specific manipulation of NPC in the adult brain and may have major implications for the use of precursor cells as therapeutic delivery vehicles in the central nervous system.

Effects of systemic PSI administration on catecholaminergic cells in the brain, adrenal medulla and carotid body in Wistar rats.

Traditional Parkinson's disease models in rats have several disadvantages. A promising alternative in terms of a more physiological model was proposed by McNaught et al. [McNaught, K.S., Perl, D.P., Brownell, A.L., Olanow, C.W., 2004. Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease. Ann. Neurol. 56, 149-162.] inhibiting the proteasomal protein degradation in vivo where they observed in Sprague-Dawley rats distinct symptoms of Parkinson's disease, a typical slow progredient loss of dopaminergic neurons in the substantia nigra and a lack of dopaminergic afferences in the striatum. We administered to Wistar rats a synthetic proteasome inhibitor (PSI) analogous to the published method. Locomotor changes were analysed by a footprint test. Brain slices containing the substantia nigra and the striatum were stained immunohistochemically against tyrosine hydroxylase, neuronal nuclei antigen, glial fibrillary acidic protein, alpha-synuclein and microglia. Standard histological stainings (haematoxylin eosin or Nissl) were also performed. The proteasome inhibitor effect on the glomerular layer of the olfactory bulb, the adrenal medulla and the carotid body was examined. We observed no PSI-induced motor deficits and loss of tyrosine hydroxylase immunoreactivity in the substantia nigra or the striatum. However, we detected a distinct increase of tyrosine hydroxylase immunoreactivity in the glomerular layer of the olfactory bulb and in the adrenal medulla. Our results fall in line with reports of other research groups which failed to reproduce the original report, but here for the first time McNaughts model could not be reproduced in Wistar rats. The observed effects on the olfactory bulb and peripheral catecholaminergic organs speak for an impermeability of the blood brain barrier for PSI.

Targeting of neural stem cells in the hippocampus of adult rats by custom-made Ad vectors.

Adult hippocampal neural stem cells (NSC) are an intriguing source for cell replacement or could serve as delivery vehicles for therapeutic genes. We recently reported selective transduction of adult mouse NSC in the DG by in vivo injection of GFP encoding adenoviral (Ad) vectors engineered to bind NSC-specific peptides. Here, we investigated the specificity of these peptide-tagged vectors in the adult rat DG, and whether they can be used to follow differentiation of infected cells over time. The virus-containing solution was injected into the DG by stereotaxic surgery. Specific transduction of NSC was demonstrated by the radial glia-like morphology of GFP-expressing type-1 cells and co-labeling with nestin or glial fibrillary acidic protein. Three days post-injection more than 82% of GFP-containing cells were nestin-immunoreactive, as revealed by unbiased stereology and no GFP-expressing neurons were observed. However, 30 days after injection, the amount of GFP and nestin-containing cells declined (56%), whereas now neurons that contained NeuN or possessed the typical granular nerve cell morphology expressed GFP, indicating that they were derived from initially transduced NSC. Importantly, still more than 20% of nestin-immunoreactive NSC was found to be GFP-positive 90 days after infection, but unfortunately at this time point no GFP-containing neurons were detectable. Our results demonstrate that Ad vectors tagged with NSC-specific ligands can be used to target type-1 NSC, the low-proliferating cell population, in the rat hippocampus. They are a valuable tool to monitor the differentiation of their descendants, at least over short time periods.

Ciliary neurotrophic factor overexpression in neural progenitor cells (ST14A) increases proliferation, metabolic activity, and resistance to stress during differentiation.

Neurotrophic factors exert considerable neuroprotective and neurorestorative effects in neurodegenerative diseases. Because neuronal progenitor cells have, at least in part, the potency to restore degenerated neuronal networks, transgenic high-dosage expression of neurotrophins by these cells in neurotransplantation may be advantageous. In the present study, a retroviral vector containing the gene of rat ciliary neurotrophic factor (rCNTF) was permanently transfected into a striatal neuronal progenitor cell line. Qualitative and quantitative analyses demonstrated a sustained expression of the transgene; i.e., rCNTF was present at the mRNA level and protein level. Moreover, cocultivation in separate chambers of transgenic CNTF-ST14A cells and CNTF-dependent TF1 cells exerted typical biological effects, such as increased proliferation and differentiation of the TF1 cells, indicating the functional integrity of the secreted recombinant neurotrophin. The CNTF-ST14A cells displayed improved stress response compared with native ST14A cells under differentiation conditions, i.e., at the nonpermissive temperature of 39 degrees C and after staurosporine exposure, respectively. This effect coincided with a relatively reduced apoptosis rate and a raised metabolic activity of CNTF-ST14A cells at 39 degrees C. Neurotransplantation of CNTF-ST14A cells in the rat quinolinic acid model of Huntington's disease showed a significant and sustained decline in pathological apomorphine-induced rotations compared with parental ST14A cells. We conclude that sustained functional transgene CNTF production improves stress response as well as metabolic activity, making CNTF-ST14A cells a promising tool for neurotransplantation in the quinolinic acid model of Huntington's disease.