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1

A mifepristone-regulated adeno-associated viral vector system for regulated neurotrophic factor expression in the central nervous system

100%
Maddalena A., Tereschchenko Y., Bahr M., Kugler S.
Acta Neurobiologiae Experimentalis
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2009
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tom 69
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nr 3
Neurotrophic factors (NF) are potent molecules with great promise for treatment of devastating neurodegenerative disorders like Alzheimer´s and Parkinson´s disease. Targeted delivery of NF like NGF or GDNF by means of viral vectors to distinct populations of brain cells may signifi cantly enhance bio-availability and safety of such treatment approaches. However, gene therapy in its current understanding means to introduce e.g. a cDNA coding GDNF into neurons or astrocytes and expressing the factor from that time on continuously, without an option to tune expression levels according to individual patient’s demands or to shut down expression in case of unforeseen side effects. In order to increase effi cacy and safety of gene therapy vectors for treatments of human patients, we here describe the development of a tightly regulatable vector system based on the approved human drug, mifepristone, for applications in the CNS. A two-vector based layout allows modulating levels of the transactivator in order to achieve a very tight off-state while maintaining suffi cient potency for activation of transgene expression. We describe characterization of the vector system in cultured primary brain cells and in the rat striatum and cortex in terms of dependence of vector titres, inducing drug levels, and repeated responsiveness. We believe that such vector system will signifi cantly promote gene therapeutic applications in patients but will also have great impact on basic research applications.
2

Lack of prodomain abolishes BDNF constitutive secretion by HEK 293 cells

88%
Ziemlinska E., Skup M., Czarkowska-Bauch J., Bahr M., Kugler S.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 3
Brain-derived neurotrophic factor (BDNF) and its proBDNF precursor are released both in constitutive and activity-dependent manner. Prodomain itself proved to be necessary for BDNF targeting to regulated secretory pathway [Egan et al. (2003) Cell, Chen et al. (2005) J Neurosci] but its role in constitutive secretion is elusive. As mature BDNF (mBDNF) conveys trophic and prosurvival signals whereas proBDNF may convey growth inhibiting and death signals, an important issue arises: can we control the type of signal being triggered by BDNF? To verify this we cut off the prodomain and generated plasmid coding only for rat mBDNF. To test the constitutive mBDNF construct secretion we have chosen HEK 293 cell line. Two other plasmids coding either for proBDNF (template for both BDNF forms) or proBDNF protected from prodomain cleavage (source of proBDNF only), served as controls. BDNF secretion was evaluated with WB technique using antibodies detecting (1) both BDNF forms, (2) HA tag (mBDNF construct) and (3) MYC tag (proBDNF constructs). We found all three constructs being stably expressed in HEK cells. However, in contrast to both proBDNF constructs, which were secreted and detected in media fraction, mBDNF construct was revealed only in the cell lysate fraction, not being released to the media. This is the fi rst observation showing that mBDNF can be constitutively released only when accompanied by prodomain. Support: ASTF 211-00-2007 for EZ, Polish-German grant to MS and SK.
3

The effect of AAV1/2-mediated BDFN transgene overexpression on trkbFL and trkbTK receptor transcripts and on TrkBFL cellular lokalization in the transected spinal cord of the rat

76%
Ziemlinska E., Wiewior I., Grygielewicz P., Czarkowska-Bauch J., Kugler S., Skup M.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
|
nr S
Brain-derived neurotrophic factor (BDNF) regulates its fulllength TrkB (TrkBFL) receptor. BDNF administration to the brain or spinal cord after injury stimulates neuronal plasticity and brings some improvement of impaired functions, but a prolonged exposure of neurons to BDNF in vitro and BDNF infusions to the brain downregulate TrkBFL protein and reduce its downstream signaling, thus limiting BDNF effectiveness. In our recent study we used AAV-mediated transfer of BDNF transgene to cause long-term delivery of BDNF to isolated spinal cord transected at Th11 – Th12 segments. Three groups of rats were used: intact, spinal PBS (spPBS), and spinal AAV-BDNF (spBDNF) injected. The treatment resulted in substantial improvement of treadmill locomotion at two weeks after spinalization, but its effect weakened in time (7 weeks). The mechanism underlying this effect may arise from decreased abundance and availability of TrkBFL and its truncated forms. To verify it, we compared levels of trkbFL/trkbTK transcripts (qPCR) and evaluated TrkBFL segmental distribution (immunohistochemistry). Both transcripts decreased in the scar and in L1 – L2 segments in spPBS rats, but tended to increase in L1 – L2 in spBDNF rats (p<0.07). In L3 – L6 segments no group differences in transcripts were found. Comparison of TrkBFL and c-Myc labeling of transgene-derived BDNF revealed that: (1) caudally to the transection, TrkBFL was abundant in neurons and white matter oligodendroglia (2) c-Myc (+) or (-) neurons showed comparable intensity of TrkBFL labeling (3) neuronal TrkBFL labeling was higher in segments with BDNF excess. In summary, BDNF overproduction in isolated spinal network does not downregulate TrkB transcripts, either it alters cellular abundance and pattern of TrkBFL segmental expression. Data suggest that other aspects of TrkB-mediated signaling are responsible for weakening of functional effect of BDNF. Supported by S007/PolishGerman/2007/01 grant and EMBO fellowship (for EZ).
4

In vivo transduction of spinal cord cells in adult, spinalized rats, with adeno-associated viral vectors:long-term efficiency and specificity of AAV1/2-hSYN and AAV5-mCMV, encoding eGFP

76%
Platek R., Ziemlinska E., Czarkowska-Bauch J., Kugler S., Schachner M., Skup M.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
|
nr 1
We compared the efficiency and specificity of in vivo transduction of spinal cord cells in adult, spinalized rats, with adeno-associated viral vectors: AAV1/2 and AAV5, with human synapsin (hSYN) and murine cytomegalovirus (mCMV) promoters, respectively. Both AAV vectors carried eGFP transgen, and were injected bilaterally to the lumbar L1 segment immediately after spinal transection at the Th10/11. At 5-6 weeks postlesion (1) the distribution and extent of eGFP expressing cells and fibers and (2) their phenotype (immunohistochemical identification; IHC) were determined. To evaluate virus expansion we compared distribution of eGFP signal at the microscopical reconstructions (parasagittal sections). A comparison between serotypes showed, that caudorostral range of cells expressing eGFP was comparable (AAV1/2 – 6.8 mm; AAV5 – 8 mm), with a core of transduced cells (AAV1/2 – 4.2/4.6 mm; AAV5 – 3.4/3.8 mm), surrounding the injection site. Fibers emerging from AAV1/2 -transduced cells reached the lesion border, many of them entered the lesion and occasionally went across the scar, whereas fibers of AAV5-transduced cells faded in a proximity of 300 µm to it. Dorsoventrally, cellular eGFP signal was detected in a gray matter of the subjects transduced with both serotypes, whereas only AAV5 - mCMV transduced cells also in a white matter. Morphology of eGFP expressing cells indicated that both serotypes transduced interneurons and large neurons of Lamina IX. IHC documented that AAV5 and, to a lesser extent, AAV1/2, transduced cholinergic cells (VAChT), whereas none of the transduced neurons were GABAergic (GAD67) or glutamatergic (VGLUT2). AAV5 transduced also glial cells, some identified as astrocytes (GFAP). In conclusion, both vectors efficiently transduce neurons in spinal animals; mCMV promoter drives eGFP expression also in glia. Support: Polish-German Project PBZ-MIN-001/P05/13, S007/PN/2007/01and statutory grant to Nencki Institute.
5

L1 overexpression in rats with complete spinal cord transection downregulates phosphacan and upregulates synaptophysin, GAP43 and Adcy1 expression

76%
Platek R., Grycz K., Wieckowska K., Czarkowska-Bauch J., Kugler S., Schachner M., Skup M.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
|
nr Suppl.1
Recovery after spinal cord injury requires neuronal remodeling which is regulated by cell adhesion molecules (CAMs) and chondroitin sulfate proteoglycans (CSPGs). CSPG may be potentially both inhibitory and supportive of regenerative plasticity. To verify whether chronic (5 weeks) L1 CAM overexpression in transected spinal cord of the rat, proven to promote recovery in mice, affects CSPG phosphacan and markers of synaptic plasticity, adeno-associated viral vector encoding L1 protein (AAV5-L1) was injected into L1 lumbar segment, immediately after transection at Th10/11. Control group received AAV5-EGFP. AAV5-L1 transduced neurons and astrocytes below the lesion, resulting in 170-fold increase in L1 mRNA level at low thoracic segments (Th
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