Urbach, YK 1
Raber, KA 1
von Horsten, S 1

Institutions:

1 Friedrich-Alexander-University of Erlangen-Nuremberg, Franz-Penzoldt-Center, Experimental Therapy, Erlangen, Germany

Title of abstract : Differential effects of environmental enrichment by voluntary wheel running on Huntington’s transgenic rats as detected by automated phenotyping technology

Abstract text:

The PhenoMaster system (TSE Systems, Germany) is a modular, multi-purpose tool for the investigation of multidimensional physiobehavioral outputs of rats and is capable to automatically screen them in a home-cage-like environment for several parameters at a high temporal and spatial resolution. This novel approach allows experimenter independent monitoring of laboratory rodents and allows avoidance of stress-artifacts, higher throughput, higher sensitivity of measures (online, circadian), and combination of measurements potentially allowing multifactorial analysis and identification of novel combined behavioral and physiological dimensions.
Rats transgenic for Huntington’s disease are characterize with respect to changes of the phenotype due to environmental enrichment by voluntary wheel running.
In the present experiments a set of tgHD animals was divided into two subgroups (2 x 2 design); one group was introduced to the PhenoMaster with a running wheel and one group was screened without the wheel. Both groups where tested for 72h per experiment at the age of 4, 7, 10 and 13 months.
Screening of body weight changes revealed differences. Divided in groups with wheel and without wheel, the wild-type group with wheel have a higher body weight and the transgenic group without wheel develop a higher bodyweight. For food and fluid consumption transgenic HD rats always have a higher food and lower fluid consumption. For the activity, the results clearly show a reduction of wheel running in the tgHD rats. As for the respiratory exchange rate bimodal differences are seen. Classical behavioral tests are ongoing.
Voluntary wheel running within automated home-cage environments differentially affects wild-type and tgHD rats and modulates the phenotype.

Raber KA 1
Urbach YK 1
Stephan M 2
Nguyen HP 3
Riess O 3
von Horsten S 1

Institutions:

1 Franz-Penzoldt-Center, Section of Experimental Therapy, University of Erlangen-Nürnberg, Palmsanlage 5, 91054 Erlangen, Germany
2 Dept. of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
3 Dept. of Medical Genetics, University of Tuebingen, Calwerstr. 7, 72076 Tuebingen, Germany

Title of abstract : early detection of a behavioral phenotype in rats transgenic for huntington’s disease

Abstract text:

Huntington’s disease (HD) is an autosomal dominant, neurodegenerative disorder caused by CAG repeat expansion in the huntingtin gene. The transgenic rat model (tgHD) carrying a truncated human huntingtin fragment of 51 CAG repeats under control of the rat Htt promoter closely resembles the late-onset form of HD exhibiting the earliest aggregates at about 6 months of age. It has been shown that behavioral symptoms precede the appearance of aggregates in this model.
To further study this early behavioral phenotype in tgHD rats we performed ultrasonic vocalization, acoustic startle response, and prepulse inhibition tests in P10-17 pups. To study exploration and risk behavior the novel cage test was performed in pre-weaning 21 days old rats. In this test each animal was allowed to explore a clean cage for five minutes. Early exploration and risk taking were determined.
Exploration and risk taking were greatly altered in pre-weaning rats. Homozygous transgenic rats exhibit increased free rearing behavior and spent more time in the center and less time in the wall area of the cage compared to wild type rats. Furthermore, transgenic P10 rat pups emitted significantly less ultrasonic calls, which were also of shorter duration. Testing of sensory motor gaiting revealed a loss of prepulse inhibition at day 17 already.
Gene expression profiling and qrtPCR confirmation on P10 pups striata revealed changes in at least 8 behavior-associated genes.
These data suggest that the tgHD rat could be a useful model in treatment studies due to the early behavioral phenotype detectable at day 10 and day 21.

Stewart, GR

Institutions:

Medtronic Neuromodulation, Minneapolis, MN 55432

Title of abstract : The Unraveling of Cell Therapy for Parkinson’s Disease: Where do we go from here . . . why gene therapy trials should be listening . . . and why didn’t we learn from trophic factors?

Abstract text:

Starting in the early 1990s, biotechnology brought great promise to the treatment of neurodegenerative disease with the development and clinical translation of neurotrophic factors. Unfortunately, all these programs, ranging from CNTF for ALS to GDNF for Parkinson’s disease (PD), ended in failure, disappointment and frustration. Likewise, a wave of cell therapy has moved through the clinic, most notably for the treatment of PD, and has suffered a similar fate. Now, we are in the midst of a third wave of biotechnology in the form of gene therapy, with several ongoing trials in PD as well as other neurologic disorders.

Of interest is to look across this tremendous outpouring of scientific discovery and clinical research and ask the obvious question: what is going wrong? This talk will quickly outline the commonalities among these different forms of therapy that may have contributed to clinical failure, and look at some ways to help insure a better outcome in the future. But even if there is success in translating cell or gene therapy for PD, a second realm of concern is to examine the inherent risks and limitations in their commercialization, turning cells into pills as it were. Cell therapies, in particular, must navigate a series of barriers, from societal pressures to manufacturing to reimbursement, if they are to succeed medically and financially.

Wenning GK, Koellensperger M, Stefanova N

Institutions:

Department of Neurology
University Hospital
Anichstrasse 35
6020 Innsbruck
AUSTRIA

Title of abstract : Towards neurotransplantation in MSA: preclinical evidence

Abstract text:

Multiple system atrophy (MSA) is a fatal degenerative disorder characterized by a unique oligodendrogliopathy with cytoplasmic alpha-synuclein inclusions and secondary neuronal loss affecting striatonigral and olivopontocerebellar projections as well as central autonomic nuclei. MSA patients typically develop autonomic failure combined with L-dopa unresponsive parkinsonism (MSA-P) reflecting striatonigral degeneration or less commonly cerebellar ataxia (MSA-C). Median survival is 8-9 years regardless of presentation. Due to the lack of effective drugs targeted cell therapy involving striatal repair has been attempted to improve the response to dopaminergic stimulation. Embryonic grafts implanted into the striatum survive in the presence of oligodendroglial synuclein inclusions in a transgenic MSA model. However, more recent data show the formation of synuclein inclusions within grafted tissue. Whether this represents host-to-graft propagation as discussed in recent reports on Lewy body formation within human dopamine grafts remains to be elucidated. Functional grafts effects have sofar only been studied in a unilateral double lesion MSA-P rat model based on sequential administration of 6-OHDA and quinolinic acid into the MFB and lateral striatum. Earlier work from our group suggests that rotational responsivity to apomorphine can be restored using embryonic striatal grafts in the rat model. In contrast, more recently, striatal grafts were only partially superior to sham grafts in restoring L-Dopa responsivity of parkinson-like motor behaviour. Further work is required to define the scope of striatal cell therapy in MSA. At best it may be regarded a palliative approach improving motor function for a number of years. Complementary neuroprotective therapies will be required at the same time.

Winkler, Juergen

Institutions:

Section Molecular Neurology
University Erlangen
Germany

Title of abstract : Reduced neurogenesis in models of Parkinson disease

Abstract text:

Neurogenesis persists in the adult mammalian brain. One of the most exciting ideas for repair is that one might be able to harness the adult brain’s endogenous capacity for cell renewal. To stimulate adult neurogenesis, we choose a dopamine receptor agonist (DRA). These compunds have a potent effect to increase proliferation and olfactory bulb neurogenesis. Furthermore, we showed that the application of EGF and FGF-2 increases dopaminergic neurogenesis in the olfactory bulb, promote migration of newly-generated neuroblasts into the lesioned striatum, however fail to obtain a dopaminergic phenotype. Both studies indicate that exogenous applied compounds are capable to induce cellular repair mechanisms.
The second hypothesis was that neurogenic regions are more susceptible to protein aggregation disorders than other brain regions. We determined neurogenesis in transgenic mouse models for synucleinopathies: (1) In mice that expresses high levels of wild-type human -synuclein in neurogenic regions neuronal integration of progenitors is impaired. (2) A53T -synuclein transgenic mice showed a more pronounced decrease in olfactory neurogenesis. (3) Using a conditional tet-regulatable model of PD expressing human -synuclein we demonstrated, that that blocking synuclein expression restores adult neurogenesis and may offer new therapeutic targets. These findings suggest that pathways associated with -synuclein aggregation interfere with the generation and integration of newly generated neurons in neurogenic regions. Consecutively, we hypothesize that impaired neurogenesis in the olfactory bulb and the hippocampus may be the underlying structural substrate for pre-motor symptoms early in the course of PD such as olfactory deficits, depression and cognitive deficits.

M. Anderova 1,3
I. Prajerova1,2
P. Honsa 1
and A. Chvatal 1-3

Institutions:

1 Department of Cellular Neurophysiology - Laboratory of Neurobiology, Inst. Exp. Med., ASCR;
2 Dept. Neurosci. and
3 Center for Cell Therapy and Tissue Repair 2nd Medical Faculty, Charles University; Prague, Czech Republic

Title of abstract : The role of Sonic hedgehog and Wnt-7a in neural stem cell differentiation in vitro

Abstract text:

Sonic hedgehog (Shh) and Wnt-7a are secreted morphogens involved in ongoing neurogenesis of the adult brain. GFP-labeled P0 mouse neural stem cells expressing either Shh (Shh/GFP) or Wnt-7a (Wnt-7a/GFP) were used to study their effect on neural stem cell proliferation and differentiation in vitro; cells expressing only GFP (WT/GFP) were used as a control. Eight days after the onset of differentiation the cells were analyzed for expression of cell specific markers and receptors using immunohistochemical and Western blot analyses. The cell membrane properties were characterized using the patch-clamp technique. Both Shh and Wnt-7a increased the expression of neuronal marker MAP-2, however, only Wnt-7a also increased the expression of DCX and beta-III tubulin, when compared to control. In WT/GFP cells electrophysiological analysis revealed a neuron-like current pattern in MAP-2-, DCX- or beta-III tubulin-positive cells, with a mean Vm of -77 mV and IR of 876 MOhms, voltage-dependent KA, KDR and TTX-sensitive Na+ currents (INa). In Shh/GFP cells the number of cells expressing the neuron-like current pattern was significantly decreased when compared to control. In Wnt-7a/GFP cells the neuron-like current pattern was predominant and also the INa occurrence was increased Based on our immunohistochemical data we can conclude that both Shh and Wnt-7a increase the expression of neuronal markers in differentiated neural stem cells compared to WT/GFP cells. However, electrophysiological analysis revealed an increased incidence of neuron-like current patterns only in Wnt-7a/GFP cells.
Supported by GACR 305/06/1316, GAUK62/2006/C/2.LF, AVOZ50390512, LC554 and 1M0538.

Prajerova I. 1,2
Anderova M. 1,3
Honsa P. 1
Machon O. 4
and Chvatal A. 1-3

Institutions:

1 Department of Cellular Neurophysiology - Laboratory of Neurobiology, Institute of Experimental Medicine, ASCR;
2 Department of Neuroscience, 2nd Med. Faculty, Charles University;
3 Center for Cell Therapy and Tissue Repair, Charles University;
4 Department of Transcriptional Regulation, Institute of Molecular Genetics, ASCR; Prague, Czech Republic

Title of abstract : Immunohistochemical and electrophysiological analysis of D6/GFP-neural stem/progenitor cells during in vitro differentiation and after transplantation into the injured rat brain.

Abstract text:

D6 is a promoter/enhancer of mDach1 gene, which is involved in the development of neocortex including ventricular zone and hippocampus, and is expressed in the proliferating neural stem/progenitor cells of the cortex (Machon et al., 2002). Embryonic neural stem/progenitor cells were isolated from E12 mouse embryos of D6-GFP transgenic mice, in which the expression of GFP is driven by D6 promoter/enhancer. The differentiation potential of these cells was first characterized in vitro. Electrophysiological and immunohistochemical analysis revealed two distinct cell populations. Large flat cells forming an underlying layer expressed GFAP and/or nestin. Smaller cells with multiple long processes expressed neuronal markers beta-III tubulin, MAP-2 or DCX. These cells with an average membrane potential of -54 mV, a membrane resistance of 2326 MOhms and a membrane capacitance of 9.8 pF displayed voltage-dependent A-type K+-channels, delayed outwardly rectifying K+-channels and Na+-channel, which was blocked by TTX. To study their survival and differentiation potential in vivo, D6-GFP neural stem/progenitor cells were transplanted into the intact tissue or into the site of the photochemical lesion (a model of thrombotic stroke). One week after the transplantation the cells survived and expressed markers of mature neurons (NeuN-, NF68-, beta-III tubulin- and MAP2- positive cells). Based on these data, D6/GFP-cells could provide a suitable tool for studying cell survival, migration and differentiation under pathological conditions.
Supported by GACR305/06/1316, AVOZ50390512, LC554, 1M0538 and GAUK62/2006/C/2.LF.

Anderova M. 1,3
Prajerova I. 1,2,
Honsa P. 1
and Chvatal A. 1-3

Institutions:

1 Department of Cellular Neurophysiology - Laboratory of Neurobiology, Inst. Exp. Med., ASCR;
2 Dept. Neurosci. and
3 Center for Cell Therapy and Tissue Repair 2nd Medical Faculty, Charles University; Prague, Czech Republic

Title of abstract : The role of Sonic hedgehog and Wnt-7a in neural stem cell differentiation in vitro

Abstract text:

Sonic hedgehog (Shh) and Wnt-7a are secreted morphogens involved in ongoing neurogenesis of the adult brain. GFP-labeled P0 mouse neural stem cells expressing either Shh (Shh/GFP) or Wnt-7a (Wnt-7a/GFP) were used to study their effect on neural stem cell proliferation and differentiation in vitro; cells expressing only GFP (WT/GFP) were used as a control. Eight days after the onset of differentiation the cells were analyzed for expression of cell specific markers and receptors using immunohistochemical and Western blot analyses. The cell membrane properties were characterized using the patch-clamp technique. Both Shh and Wnt-7a increased the expression of neuronal marker MAP-2, however, only Wnt-7a also increased the expression of DCX and beta-III tubulin, when compared to control. In WT/GFP cells electrophysiological analysis revealed a neuron-like current pattern in MAP-2-, DCX- or beta-III tubulin-positive cells, with a mean Vm of -77 mV and IR of 876 MOhms, voltage-dependent KA, KDR and TTX-sensitive Na+ currents (INa). In Shh/GFP cells the number of cells expressing the neuron-like current pattern was significantly decreased when compared to control. In Wnt-7a/GFP cells the neuron-like current pattern was predominant and also the INa occurrence was increased Based on our immunohistochemical data we can conclude that both Shh and Wnt-7a increase the expression of neuronal markers in differentiated neural stem cells compared to WT/GFP cells. However, electrophysiological analysis revealed an increased incidence of neuron-like current patterns only in Wnt-7a/GFP cells.
Supported by GACR 305/06/1316, GAUK62/2006/C/2.LF, AVOZ50390512, LC554 and 1M0538.

Garcia J, Hahn M, Timmer M, Papazoglou A, Nikkhah G

Institutions:

Dept. of Stereotactic Neurosurgery, Laboratory of Molecular Neurosurgery, Neurocentre, University Hospital Freiburg. Breisacher Str. 64, 79106 Freiburg, Germany

Title of abstract : Distinctive pattern of anatomical maturation and functional integration of dopaminergic progenitor cells following transplantation in a rat model of Parkinson’s disease

Abstract text:

The restorative capacity of dopaminergic (DA) transplants is determined by DA neurogenesis, migration,TH-positive fibre reinnervation, host-derived anatomical and functional influences. Here we examined the impact of graft-host interactions on the survival and functional capacity of DA progenitor cells in rats with unilateral 6-hydroxydopamine lesions. DA transplant-induced functional recovery was observed in postural balancing reactions already after 10 days and in stepping behaviour after 13 days, , and later, after 16 days, in the amphetamine-induced rotation test. Three distinct patterns of functional recovery could be observed at 6 to 9 weeks post-transplantation. Firstly, behavioural improvements in drug-induced rotational asymmetry, stepping and skilled forelimb behaviour were directly related to DA neuron survival and TH-positive fibre reinnervation. Secondly, recovery in postural balancing reactions was closely related to a specific developmental time window of donor age, e.g. only seen in embryonic day (E) 13 (9-10mm) and E14 (11-12 mm) grafts. Finally, no functional graft effects were seen in the table lift test. Interestingly, DA neuron graft survival, TH-positive fibre outgrowth and graft volumen were significantly influenced by the developmental time window in which the DA progenitor cells were dissected from the ventral mesencephalon, i.e. from E12 (7-8mm), E13 (9-10mm), E14 (11-12 mm) or E15 (13-14 mm)old rat embryos.

These data highlight the complexity of DA graft-host interactions and provides novel insights into the dynamics of DA progenitor graft-mediated functional recovery in animal models of Parkinson’s disease.

Schmidt NO 1; Hansen, K 1; Laurent LC 2; Schwartz P 3; McKercher S 4; Lee JP 4/5; Lamszus K 1; Westphal M 1; Loring, J 2; Mueller FJ 2,6

Institutions:

1. Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
2. Center for Regenerative Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
3. Children’s Hospital of Orange County Research Institute, Orange, California 92826, USA.
4. Stem Cell & Regeneration Program, Center for Neuroscience and Aging Research, Burnham Institute for Medical Research, La Jolla, California 92037, USA.
5. Department of Pediatrics, University of California San Diego, School of Medicine, La Jolla, California 92093, USA.3.
6. Center for Psychiatry and Psychotherapy, University Hospital Schleswig Holstein, Niemannsweg 147, D-24105 Kiel, Germany

Title of abstract : The Adult Human Brain Contains a Unique Population of Highly Motile Neural Progenitors

Abstract text:

Neural stem and progenitor cells have been shown to be useful for cell replacement and neuroprotective strategies in various neuropathological conditions. While the adult human brain contains populations of neural progenitors, their lineage identity, their role in normal brain function, and their clinical potential remain to be evaluated. We obtained highly proliferative human neural progenitors (HANSE cells) from surgical specimens obtained at amygdalohippocampectomies (n=10) and resection of cerebral arteriovenous malformations (n=2). Different anatomical regions were prepared separately and a total of 39 different cultures were established. (mean patient age: 32 years, range 24-46). HANSE cells expressed stem cell markers like nestin, SOX2, BMI1, and musashi , and displayed multipotent differentiation and targeted migration patterns in glioblastoma xenograft models. Comprehensive functional genomics profiling of mRNA and microRNA phenotypes was interpreted in the context of diverse pluripotent and multipotent stem cell preparations (n=219). Unsupervised machine learning algorithms for global sample clustering and prediction of differentially-active protein networks were used to a) identify cell lineage relationships and b) define molecular pathways responsible for unique cell type functions. Our data indicate that the adult human brain contains a unique population of neural progenitor cells with significant responsiveness to brain malignancy. We found a surprising heterogeneity among genome-wide transcriptional phenotypes of in vitro neural progenitor cell preparations that are currently believed to be sufficiently identified with the term “neural stem cells”. Our findings put into question current prevalent stem cell identification strategies such as marker based selection or commonly used in vitro assays.