Stefan Jean-Pierre Haas

Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells

The lipophilic dye PKH26 that binds irreversibly to cell membranes has been used to label various cell types in vitro prior to transplantation in order to recognize grafted cells posttransplantationally in the host tissue by fluorescence microscopy. The purpose of the present study was to optimize immunocytochemical staining procedures for PKH26-containing specimens in cell culture and after transplantation into rat brain. We demonstrated that freeze-thawing allowed for proper immunostaining of intracellular epitopes whereas PKH26-labelling was preserved. PKH26-labelled donor cells were detectable at least up to 4 months after transplantation in the host brain.

Efferent Projections of the Anterior and Posterodorsal Regions of the Medial Nucleus of the Amygdala in the Mouse

The efferent projections of the anterior and posterodorsal part of the medial nucleus (MePD) in the mouse were studied by means of anterograde axonal tracing using biotinylated dextran amine. The MePD axons ran mainly via the stria terminalis and to a lesser extent via the ventral amygdalofugal pathway. The projections to the forebrain were broadly distributed and varied from very strong to scant. The most significant connections were destined to the bed nucleus of the stria terminalis in which all parts of the medial division were innervated by MePD neurons. Moderate projections reached the limbic striatum (nucleus accumbens), olfactory tubercle and the lateral septal nucleus. The substantia innominata was also innervated by the MePD, and especially the projection to its ventral portion was substantial. The profuse innervation of the medial preoptic nucleus and medial preoptic area indicated significant involvement of the MePD in sexual behavior. Many hypothalamic nuclei were innervated but to a different extent. The very strong innervation of the ventral premammillary nucleus further indicated the involvement of the MePD in the neuronal circuitry for sexual behavior. Substantial projections also reached the anterior hypothalamus and tuber cinereum, while the connections to the lateral hypothalamus were widespread but showed moderate density. MePD strongly innervated the ventrolateral part of the ventromedial hypothalamic nucleus and moderately its remaining parts. The neurosecretory hypothalamic nuclei and the arcuate nucleus contained only a few MePD terminals. The thalamic innervation was very scant and reached the lateral habenular nucleus and the nuclei of the midline. The mesencephalic connections were moderate to sparse and projected to the mesolimbic dopaminergic groups in the ventral tegmental area, the pars lateralis and the dorsal tier of the substantia nigra pars compacta, the periaqueductal gray and the dorsal raphe nucleus. The present results principally resembled data known in other rodent species; however, the efferents of the MePD often differed in extent and/or topical distribution.

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

Parkinsons 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.

Dopaminergic differentiation of the Nurr1-expressing immortalized mesencephalic cell line CSM14.1 in vitro

The use of neural stem cells as grafts is a potential treatment for Parkinsons disease, but the potential of stem cells to differentiate into dopaminergic neurones requires investigation. The present study examined the in vitro differentiation of the temperature‐sensitive immortalized mesencephalic progenitor cell line CSM14.1 under defined conditions. Cells were derived from the mesencephalic region of a 14‐day‐old rat embryo, retrovirally immortalized with the Large T antigen and cultured at 33 °C in DMEM containing 10% fetal calf serum (FCS). For differentiation, the temperature was elevated at 39 °C and FCS was reduced (1%). Using histology, immunocytochemical detection of the stem cell marker Nestin and the neuronal marker MAP5 and, in addition, Western blotting to determine the presence of neurone‐specific enolase and the neurone nuclei antigen we demonstrated a differentiation of these cells into neuronal cells accompanied by a decrease in Nestin production. In Western blots, we detected the orphan nuclear receptor Nurr1 in these cells. This was followed by a time‐dependent up‐regulation of the enzymes tyrosine hydroxylase and aldehyde dehydrogenase 2 characteristic of mature dopaminergic neurones. Our in vitro model of dopaminergic cell differentiation corroborates recent in vivo observations in the developing rodent brain.

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

An Erratum has been published for this article in Journal of Neuroscience Research 75: 861, 2004.

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.

Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour

Neural stem cell transplantation is a promising strategy for the treatment of neurodegenerative diseases. To evaluate the differentiation potential of human neural progenitor cells (hNPCs) as a prerequisite for clinical trials, we intracerebrally transplanted in vitro expanded fetal mesencephalic hNPCs into hemiparkinsonian rats. On postnatal day one (P1), 17 animals underwent a unilateral intraventricular 6‐hydroxydopamine injection into the right lateral ventricle. At P3, animals (n = 10) received about 100 000 hNPCs (1 µL) in the right striatum. Five weeks after birth, animals underwent behaviour tests prior to fixation, followed by immunohistochemistry on brain slices for human nuclei, glial fibrillary acidic protein, S100β, neuronal nuclei antigen, neuron‐specific enolase and tyrosine hydroxylase. Compared with the apomorphine‐induced rotations in the lesioned‐only group (7.4 ± 0.5 min−1), lesioned and successfully transplanted animals (0.3 ± 0.1 min−1) showed a significant therapeutic improvement. Additionally, in the cylinder test, the lesioned‐only animals preferred to use the ipsilateral forepaw. Conversely, the lesioned and transplanted animals showed no significant side bias similar to untreated control animals. Transplanted human nuclei‐immunoreactive cells were found to survive and migrate up to 2000 µm into the host parenchyma, many containing the pan‐neuronal markers neuronal nuclei antigen and neuron‐specific enolase. In the striatum, tyrosine hydroxylase‐immunoreactive somata were also found, indicating a dopaminergic differentiation capacity of transplanted hNPCs in vivo. However, the relative number of tyrosine hydroxylase‐immunoreactive neurons in vivo seemed to be lower than in corresponding in vitro differentiation. To minimize donor tissue necessary for transplantation, further investigations will aim to enhance dopaminergic differentiation of transplanted cells in vivo.

Expression of Tgfβ1 and Inflammatory Markers in the 6-hydroxydopamine Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by loss of midbrain dopaminergic (mDA) neurons in the substantia nigra (SN). Microglia-mediated neuroinflammation has been described as a common hallmark of PD and is believed to further trigger the progression of neurodegenerative events. Injections of 6-hydroxydopamine (6-OHDA) are widely used to induce degeneration of mDA neurons in rodents as an attempt to mimic PD and to study neurodegeneration, neuroinflammation as well as potential therapeutic approaches. In the present study, we addressed microglia and astroglia reactivity in the SN and the caudatoputamen (CPu) after 6-OHDA injections into the medial forebrain bundle (MFB), and further analyzed the temporal and spatial expression patterns of pro-inflammatory and anti-inflammatory markers in this mouse model of PD. We provide evidence that activated microglia as well as neurons in the lesioned SN and CPu express Transforming growth factor β1 (Tgfβ1), which overlaps with the downregulation of pro-inflammatory markers Tnfα, and iNos, and upregulation of anti-inflammatory markers Ym1 and Arg1. Taken together, the data presented in this study suggest an important role for Tgfβ1 as a lesion-associated factor that might be involved in regulating microglia activation states in the 6-OHDA mouse model of PD in order to prevent degeneration of uninjured neurons by microglia-mediated release of neurotoxic factors such as Tnfα and nitric oxide (NO).

Quinolinic acid lesions of the caudate putamen in the rat lead to a local increase of ciliary neurotrophic factor.

When applied prior to excitotoxic lesions, ciliary neurotrophic factor (CNTF) has been shown to be neuroprotective. However, data concerning the endogenous CNTF content of the intact rat striatum are rare and have not until now been available for the quinolinic acid (QA)‐lesioned striatum. Therefore, we investigated the CNTF content in the QA‐lesioned rat striatum for at least 1 month using immunohistochemistry and Western blot analysis. In lesioned striata a neuronal loss was observed by Nissl staining and by a reduction of NeuN‐immunoreactive cells, whereas increased glial fibrillary acidic protein immunoreactivity showed a gliotic reaction. With CNTF immunohistochemistry we found that in the QA‐lesioned striatum CNTF was increased over time, whereas it was not detectable in intact and sham‐lesioned striata. CNTF‐immunoreactive cells had the morphology of protoplasmatic astrocytes. Furthermore, quantitative Western blotting demonstrated that the content of CNTF protein from striatal lysates containing 1 mg of whole protein 1 month after QA lesioning (2.76 ± 1.71 ng) was significantly increased (P < 0.05, U‐test) compared with sham‐lesioned hemispheres (0.68 ± 0.25 ng) and intact controls (0.55 ± 0.25 ng). We conclude that CNTF content is correlated with glial scar formation and suggest that our results may be of relevance to cell grafting strategies for the treatment of Huntingtons disease.

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-β 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.