Publications by authors named "Sebastian Kügler"

94 Publications

Dementia with Lewy bodies-associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions.

Hum Mol Genet 2021 Apr;30(3-4):247-264

Department of Neurology, University Medicine Göttingen, Göttingen 37073, Germany.

Beta (ß)-synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson's disease-related alpha (α)-synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in central nervous system (CNS) neurons in vitro and in vivo, albeit at a slower pace as compared with α-Syn. Here, we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of dementia with Lewy bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but it has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but it does not aggravate neurodegeneration. ß-Syn wild type (WT), V70M and P123H formed proteinase K-resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared with α-Syn. Under cell-free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared with WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, which are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn and is thus likely to be directly involved into the etiology of DLB. Overall, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddab036DOI Listing
April 2021

Generation and Breeding of -Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Cells 2021 Feb 27;10(3). Epub 2021 Feb 27.

Platform Degenerative Diseases, German Primate Center-Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, Germany.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells10030505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996964PMC
February 2021

Syntenin-knock out reduces exosome turnover and viral transduction.

Sci Rep 2021 Feb 18;11(1):4083. Epub 2021 Feb 18.

Department of Human Genetics, KU Leuven, Leuven, Belgium.

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-81697-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892569PMC
February 2021

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery.

Sci Rep 2021 Jan 21;11(1):1934. Epub 2021 Jan 21.

Biological Sciences, Hurvitz Brain Sciences Research Program, Sunnybrook Research Institute, Toronto, ON, Canada.

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood-brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-81046-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820310PMC
January 2021

First evaluation of a symbiotic food supplement in an allergen exposure chamber in birch pollen allergic patients.

World Allergy Organ J 2021 Jan 18;14(1):100494. Epub 2020 Dec 18.

Bencard Allergie GmbH, Munich, Germany.

Background: Allergic rhinitis/rhinoconjunctivitis is the most common immune disease worldwide, but still largely underestimated, underdiagnosed, and undertreated. Dysbiosis and reduced microbial diversity is linked to the development of allergies, and the immunomodulatory effects of pro- and prebiotics might be used to counteract microbiome dysbiosis in allergy. Adequate symbiotic (multi-strain pro-, plus prebiotic) supplementation can be suggested as a complementary approach in the management of allergic rhinitis.

Objective: The effects of the daily intake of a symbiotic food supplement (combination of and with Fructo-Oligosaccharides) for 4 months in birch pollen allergic rhinoconjunctivitis patients were investigated for the first time in an allergen exposure chamber (AEC) allowing standardised, reproducible pollen exposure before and after intake.

Methods: Eligible patients were exposed to birch pollen (8000 pollen/m³ for 120 min) at the GALEN AEC, at baseline (V1) and final visit (V3) outside the season. The Total Symptom Score (TSS) and the scores for nose, eye, bronchial system, and others were evaluated every 10 min during exposure. Other secondary endpoints were the changes in well-being, Peak Nasal Inspiratory Flow (PNIF), lung function parameters, and safety. Co-primary endpoints were differences in Total Nasal Symptom Score (TNSS) and TSS after 120 min of exposure between both visits. Temporal evolution of symptom scores were analysed in an exploratory way using linear mixed effects models.

Results: 27 patients (mean age 45 years, 15% male) completed the study. Both co-primary endpoints showed significant improvement after intake of the symbiotic. Median TNSS and TSS were decreased 50% and 80% at 120 min (adjusted p-value = 0.025 and p < 0.01 respectively).All four symptom scores and the personal well-being, improved to a clinically relevant extent over time, visible by a weaker increase in symptoms during 120 min of the final birch pollen exposure. No relevant differences were observed for PNIF, PEF, and spirometry. There were no airway obstructions or lung restrictions before and after both exposures. Late phase reactions after exposure were reduced after V3, documenting a better birch pollen tolerability of the patients. The safety and tolerability profile of the symbiotic food supplement was excellent, no adverse events (AEs) were observed.

Conclusions: This first evaluation of a symbiotic food supplement in an AEC in rhinoconjunctivitis patients with or without asthma induced by birch pollen revealed a significant beneficial effect, harnessing significant improvements of symptoms and well-being while maintaining an excellent safety and tolerability profile.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.waojou.2020.100494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753943PMC
January 2021

Overshooting Subcellular Redox-Responses in Rett-Mouse Hippocampus during Neurotransmitter Stimulation.

Cells 2020 11 24;9(12). Epub 2020 Nov 24.

Zentrum Physiologie und Pathophysiologie, Institut für Neuro- und Sinnesphysiologie, Georg-August-Universität Göttingen, Universitätsmedizin Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany.

Rett syndrome (RTT) is a neurodevelopmental disorder associated with disturbed neuronal responsiveness and impaired neuronal network function. Furthermore, mitochondrial alterations and a weakened cellular redox-homeostasis are considered part of the complex pathogenesis. So far, overshooting redox-responses of MeCP2-deficient neurons were observed during oxidant-mediated stress, hypoxia and mitochondrial inhibition. To further clarify the relevance of the fragile redox-balance for the neuronal (dys)function in RTT, we addressed more physiological stimuli and quantified the subcellular redox responses to neurotransmitter-stimulation. The roGFP redox sensor was expressed in either the cytosol or the mitochondrial matrix of cultured mouse hippocampal neurons, and the responses to transient stimulation by glutamate, serotonin, dopamine and norepinephrine were characterized. Each neurotransmitter evoked more intense oxidizing responses in the cytosol of MeCP2-deficient than in wildtype neurons. In the mitochondrial matrix the neurotransmitter-evoked oxidizing changes were more moderate and more uniform among genotypes. This identifies the cytosol as an important reactive oxygen species (ROS) source and as less stably redox buffered. Fura-2 imaging and extracellular Ca withdrawal confirmed cytosolic Ca transients as a contributing factor of neurotransmitter-induced redox responses and their potentiation in the cytosol of MeCP2-deficient neurons. Chemical uncoupling demonstrated the involvement of mitochondria. Nevertheless, cytosolic NADPH- and xanthine oxidases interact to play the leading role in the neurotransmitter-mediated oxidizing responses. As exaggerated redox-responses were already evident in neonatal MeCP2-deficient neurons, they may contribute remarkably to the altered neuronal network performance and the disturbed neuronal signaling, which are among the hallmarks of RTT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9122539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760232PMC
November 2020

Patterning inconsistencies restrict the true potential of dopaminergic neurons derived from human induced pluripotent stem cells.

Neural Regen Res 2021 Apr;16(4):692-693

Department of Neurology, University Medical Center Göttingen; Center for Nanoscale Microscopy and Molecular Physiology of the Brain at Department of Neurology, University Medical Center Göttingen, Göttingen, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4103/1673-5374.295316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067935PMC
April 2021

Dopamine promotes the neurodegenerative potential of β-synuclein.

J Neurochem 2021 03 30;156(5):674-691. Epub 2020 Aug 30.

Department of Neurology, University Medicine Göttingen, Göttingen, Germany.

A contribution of α-Synuclein (α-Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related β-Synuclein (β-Syn) on these disorders remains enigmatic. β-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD β-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of β-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced β-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with β-Syn, thereby enabling structural and functional modifications. Interaction of DCH with β-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric β-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and β-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that β-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jnc.15134DOI Listing
March 2021

Homogenous generation of dopaminergic neurons from multiple hiPSC lines by transient expression of transcription factors.

Cell Death Dis 2019 11 27;10(12):898. Epub 2019 Nov 27.

Department of Neurology, University Medical Center Göttingen, Göttingen, Germany.

A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson's disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-019-2133-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881336PMC
November 2019

Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease.

Theranostics 2019 18;9(26):8127-8137. Epub 2019 Oct 18.

Biological Sciences, Hurvitz Brain Sciences Research Program, Sunnybrook Research Institute, Toronto, ON, Canada.

Gene therapy can be designed to efficiently counter pathological features characteristic of neurodegenerative disorders. Here, we took advantage of the glial fibrillary acidic protein (GFAP) promoter to preferentially enhance transgene expression near plaques composed of amyloid-beta peptides (Aβ), a hallmark of Alzheimer's disease (AD), in the TgCRND8 mouse model of amyloidosis. The delivery of intravenously injected recombinant adeno-associated virus mosaic serotype 1/2 (rAAV1/2) to the cortex and hippocampus of TgCRND8 mice was facilitated using transcranial MRI-guided focused ultrasound in combination with microbubbles (MRIgFUS), which transiently and locally increases the permeability of the blood-brain barrier (BBB). rAAV1/2 expression of the reporter green fluorescent protein (GFP) under a GFAP promoter was compared to GFP expression driven by the constitutive human beta actin (HBA) promoter. MRIgFUS targeting the cortex and hippocampus facilitated the entry of rAAV1/2 and GFP expression under the GFAP promoter was localized to GFAP-positive astrocytes. Adjacent to Aβ plaques where GFAP is upregulated, the volume, surface area, and fluorescence intensity of the transgene GFP were greater in rAAV1/2-GFAP-GFP compared to rAAV1/2-HBA-GFP treated animals. In peripheral organs, GFP expression was particularly strong in the liver, irrespective of the promoter. The GFAP promoter enhanced transgene expression in proximity of Aβ plaques in the brain of TgCRND8 mice, and it also resulted in significant expression in the liver. Future gene therapies for neurological disorders could benefit from using a GFAP promoter to regulate transgene expression in response to disease-induced astrocytic reactivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/thno.36718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857057PMC
August 2020

Neuronal Trans-differentiation by Transcription Factors Ascl1 and Nurr1: Induction of a Dopaminergic Neurotransmitter Phenotype in Cortical GABAergic Neurons.

Mol Neurobiol 2020 Jan 17;57(1):249-260. Epub 2019 Jul 17.

Department of Neurology, University Medicine Göttingen, Waldweg 33, 37073, Göttingen, Germany.

Neurons with a desired neurotransmitter phenotype can be differentiated from induced pluripotent stem cells or from somatic cells only through tedious protocols with relatively low yield. Readily available cortical neurons isolated from embryonic rat brain, which have already undergone a complete neuronal differentiation process, might serve as alternative template source. These cultures consist of 85% glutamatergic and 15% GABAergic neurons, and we attempted to trans-differentiate them into dopaminergic neurons. Transcription factors Nurr1, Lmx1A and Pitx3, essential determinants of a dopaminergic cell fate during CNS development, were not sufficient to induce tyrosine hydroxylase expression in a significant number of cells. Combining Nurr1 with the generic neuronal differentiator and re-programming factor Ascl1, however, resulted in generation of neurons which express dopaminergic markers TH, AADC, VMAT2 and DAT. Only neurons of GABAergic phenotype could be trans-differentiated towards a dopaminergic neurotransmitter phenotype, while for glutamatergic neurons, this process proved to be neurotoxic. Intriguingly, GABAergic neurons isolated from embryonal midbrain could not be trans-differentiated into dopaminergic neurons by Ascl1 and Nurr1. Thus, in principle, post-mitotic embryonal neurons can serve as templates for neurons with a desired neurotransmitter phenotype. However, neurotransmitter phenotype plasticity critically depends on the differentiation history of the template neurons, which can result in relatively low yields of dopaminergic neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-019-01701-xDOI Listing
January 2020

Neuronal Redox-Imbalance in Rett Syndrome Affects Mitochondria as Well as Cytosol, and Is Accompanied by Intensified Mitochondrial O Consumption and ROS Release.

Front Physiol 2019 30;10:479. Epub 2019 Apr 30.

Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Georg-August-University Göttingen, Göttingen, Germany.

Rett syndrome (RTT), an X chromosome-linked neurodevelopmental disorder affecting almost exclusively females, is associated with various mitochondrial alterations. Mitochondria are swollen, show altered respiratory rates, and their inner membrane is leaking protons. To advance the understanding of these disturbances and clarify their link to redox impairment and oxidative stress, we assessed mitochondrial respiration in defined brain regions and cardiac tissue of male wildtype (WT) and MeCP2-deficient ( ) mice. Also, we quantified for the first time neuronal redox-balance with subcellular resolution in cytosol and mitochondrial matrix. Quantitative roGFP1 redox imaging revealed more oxidized conditions in the cytosol of hippocampal neurons than in WT neurons. Furthermore, cytosol and mitochondria of neurons showed exaggerated redox-responses to hypoxia and cell-endogenous reactive oxygen species (ROS) formation. Biochemical analyzes exclude disease-related increases in mitochondrial mass in hippocampus and cortex. Protein levels of complex I core constituents were slightly lower in hippocampus and cortex than in WT; those of complex V were lower in cortex. Respiratory supercomplex-formation did not differ among genotypes. Yet, supplied with the complex II substrate succinate, mitochondria of cortex and hippocampus consumed more O than WT. Furthermore, mitochondria from hippocampus and cortex mediated an enhanced oxidative burden. In conclusion, we further advanced the molecular understanding of mitochondrial dysfunction in RTT. Intensified mitochondrial O consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphys.2019.00479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503037PMC
April 2019

Publisher Correction: β-Synuclein-reactive T cells induce autoimmune CNS grey matter degeneration.

Nature 2019 Mar;567(7749):E15

Institute for Neuroimmunology and Multiple Sclerosis Research, University Medical Center Göttingen, Göttingen, Germany.

In this Article, owing to an error during the production process, the y-axis label of Fig. 2c should read "Number of T cells" rather than "Number of T1 cells" and the left and right panels of Fig. 4 should be labelled 'a' and 'b', respectively. These errors have been corrected online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1047-0DOI Listing
March 2019

β-Synuclein-reactive T cells induce autoimmune CNS grey matter degeneration.

Nature 2019 02 20;566(7745):503-508. Epub 2019 Feb 20.

Institute for Neuroimmunology and Multiple Sclerosis Research, University Medical Center Göttingen, Göttingen, Germany.

The grey matter is a central target of pathological processes in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. The grey matter is often also affected in multiple sclerosis, an autoimmune disease of the central nervous system. The mechanisms that underlie grey matter inflammation and degeneration in multiple sclerosis are not well understood. Here we show that, in Lewis rats, T cells directed against the neuronal protein β-synuclein specifically invade the grey matter and that this is accompanied by the presentation of multifaceted clinical disease. The expression pattern of β-synuclein induces the local activation of these T cells and, therefore, determined inflammatory priming of the tissue and targeted recruitment of immune cells. The resulting inflammation led to significant changes in the grey matter, which ranged from gliosis and neuronal destruction to brain atrophy. In humans, β-synuclein-specific T cells were enriched in patients with chronic-progressive multiple sclerosis. These findings reveal a previously unrecognized role of β-synuclein in provoking T-cell-mediated pathology of the central nervous system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-0964-2DOI Listing
February 2019

MRI-Guided Focused Ultrasound for Targeted Delivery of rAAV to the Brain.

Methods Mol Biol 2019 ;1950:177-197

Brain Sciences, Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada.

Recombinant adeno-associated viral (rAAV) vectors are a promising tool for therapeutic gene delivery to the brain. However, the delivery of rAAVs across the blood-brain barrier (BBB) and entry into the brain remains a major challenge for rAAV-based gene therapy. To circumvent this limitation, transcranial MRI-guided focused ultrasound (MRIgFUS) combined with intravenously injected microbubbles has been used to transiently and reversibly increase BBB permeability in targeted brain regions. Systemic administration of rAAVs at the time of sonication with focused ultrasound (FUS) facilitates the passage of rAAVs through the BBB and into the brain parenchyma. We and others have demonstrated that FUS-mediated rAAV delivery to the brain results in efficient transduction and transgene expression in vivo. Using this approach, the dose of intravenously injected rAAV variants that can cross the BBB can be reduced by 100 times, achieving significant transgene expression in the brain parenchyma with reduced peripheral transduction. Moreover, this strategy can be used to deliver rAAV variants that do not cross the BBB from the blood to selected brain regions. Here, we provide a detailed protocol for FUS-induced BBB permeability for targeted rAAV delivery to the brain of adult mice and rats.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-9139-6_10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546162PMC
July 2019

CX3CR1-deficient microglia shows impaired signalling of the transcription factor NRF2: Implications in tauopathies.

Redox Biol 2019 04 6;22:101118. Epub 2019 Feb 6.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Investigación Sanitaria La Paz (IdiPaz), Instituto de Investigaciones Biomédicas "Alberto Sols" UAM-CSIC, Madrid, Spain; Department of Biochemistry, School of Medicine, Universidad Autónoma de Madrid, Spain. Electronic address:

TAU protein aggregation is the main characteristic of neurodegenerative diseases known as tauopathies. Low-grade chronic inflammation is also another hallmark that indicates crosstalk between damaged neurons and glial cells. Previously, we have demonstrated that neurons overexpressing TAU release CX3CL1, which activates the transcription factor NRF2 signalling to limit over-activation in microglial cells in vitro and in vivo. However, the connection between CX3CL1/CX3CR1 and NRF2 system and its functional implications in microglia are poorly described. We evaluated CX3CR1/NRF2 axis in the context of tauopathies and its implication in neuroinflammation. Regarding the molecular mechanisms that connect CX3CL1/CX3CR1 and NRF2 systems, we observed that in primary microglia from Cx3cr1 mice the mRNA levels of Nrf2 and its related genes were significantly decreased, establishing a direct linking between both systems. To determine functional relevance of CX3CR1, migration and phagocytosis assays were evaluated. CX3CR1-deficient microglia showed impaired cell migration and deficiency of phagocytosis, as previously described for NRF2-deficient microglia, reinforcing the idea of the relevance of the CX3CL1/CX3CR1 axis in these events. The importance of these findings was evident in a tauopathy mouse model where the effects of sulforaphane (SFN), an NRF2 inducer, were examined on neuroinflammation in Cx3cr1 and Cx3cr1 mice. Interestingly, the treatment with SFN was able to modulate astrogliosis but failed to reduce microgliosis in Cx3cr1 mice. These findings suggest an essential role of the CX3CR1/NRF2 axis in microglial function and in tauopathies. Therefore, polymorphisms with loss of function in CX3CR1 or NRF2 have to be taken into account for the development of therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2019.101118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375000PMC
April 2019

A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice.

EMBO Mol Med 2019 01;11(1)

Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany

Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the gene encoding otoferlin, might be treated by transferring wild-type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno-associated viruses (AAVs), each containing half of the otoferlin cDNA Co-injecting these dual-AAV2/6 half-vectors into the cochleae of 6- to 7-day-old otoferlin knock-out () mice led to the expression of full-length otoferlin in up to 50% of IHCs. In the cochlea, otoferlin was selectively expressed in auditory hair cells. Dual-AAV transduction of IHCs fully restored fast exocytosis, while otoferlin-dependent vesicle replenishment reached 35-50% of wild-type levels. The loss of 40% of synaptic ribbons in these IHCs could not be prevented, indicating a role of otoferlin in early synapse maturation. Acoustic clicks evoked auditory brainstem responses with thresholds of 40-60 dB. Therefore, we propose that gene delivery mediated by dual-AAV vectors might be suitable to treat deafness forms caused by mutations in large genes such as .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201809396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328916PMC
January 2019

Probing the function of glycinergic neurons in the mouse respiratory network using optogenetics.

Respir Physiol Neurobiol 2019 07 3;265:141-152. Epub 2018 Nov 3.

DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany; Institute for Neurophysiology and Cellular Biophysics, Georg-August-University Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany; Clinic for Anesthesiology, University Hospital Göttingen, 37099 Göttingen, Germany. Electronic address:

Glycine is a primary inhibitory transmitter in the ventral medullary respiratory network, but the functional role of glycinergic neurons for breathing remains a matter of debate. We applied optogenetics to selectively modulate glycinergic neuron activity within regions of the rostral ventral respiratory column (VRC). Responses of the phrenic nerve activity to the light-driven stimulation were studied in the working heart-brainstem preparation from adult glycine transporter 2 Cre mice (GlyT2-Cre), which received a unilateral injection of a Cre-dependent AAV virus into Bötzinger and preBötzinger Complex. Sustained light stimulation from the ventral medullary surface resulted in a substantial depression of the phrenic nerve (PN) frequency, which in most cases was compensated by an increase in PN amplitude. Periodic, burst stimulation with variable intervals could alter and reset respiratory rhythm. We conclude that unilateral activation of the rostral VRC glycinergic neurons can significantly affect respiratory pattern by lengthening the expiratory interval and modulating phase transition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.resp.2018.10.008DOI Listing
July 2019

Vti1b promotes TRPV1 sensitization during inflammatory pain.

Pain 2019 Feb;160(2):508-527

Somatosensory Signaling and Systems Biology Group, Max-Planck Institute of Experimental Medicine, Goettingen, Germany. Ms. Barry is now with Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom. Ms. Abdelaziz is now with Oncophysiology Group, Max-Planck Institute of Experimental Medicine, Goettingen, Germany.

Sensitization of the transient receptor potential ion channel vanilloid 1 (TRPV1) is critically involved in inflammatory pain. To date, manifold signaling cascades have been shown to converge onto TRPV1 and enhance its sensitization. However, many of them also play a role for nociceptive pain, which limits their utility as targets for therapeutic intervention. Here, we show that the vesicle transport through interaction with t-SNAREs homolog 1B (Vti1b) protein promotes TRPV1 sensitization upon inflammation in cell culture but leaves normal functioning of TRPV1 intact. Importantly, the effect of Vti1b can be recapitulated in vivo: Virus-mediated knockdown of Vti1b in sensory neurons attenuated thermal hypersensitivity during inflammatory pain without affecting mechanical hypersensitivity or capsaicin-induced nociceptive pain. Interestingly, TRPV1 and Vti1b are localized in close vicinity as indicated by proximity ligation assays and are likely to bind to each other, either directly or indirectly, as suggested by coimmunoprecipitations. Moreover, using a mass spectrometry-based quantitative interactomics approach, we show that Vti1b is less abundant in TRPV1 protein complexes during inflammatory conditions compared with controls. Alongside, we identify numerous novel and pain state-dependent binding partners of native TRPV1 in dorsal root ganglia. These data represent a unique resource on the dynamics of the TRPV1 interactome and facilitate mechanistic insights into TRPV1 regulation. We propose that inflammation-related differences in the TRPV1 interactome identified here could be exploited to specifically target inflammatory pain in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/j.pain.0000000000001418DOI Listing
February 2019

Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.

Hum Mol Genet 2019 01;28(1):31-50

Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany.

Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddy326DOI Listing
January 2019

Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Exp Neurol 2018 11 1;309:79-90. Epub 2018 Aug 1.

Dept. of Neurology, University Medical Center Göttingen, Waldweg 33, 37073 Göttingen, Germany. Electronic address:

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.expneurol.2018.07.017DOI Listing
November 2018

Correction to: Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model.

Cell Death Dis 2018 May 25;9(6):634. Epub 2018 May 25.

Development and Maintenance of the Nervous System, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Correction to: Cell Death Dis. (2016) 7, e2359; https://doi.org/10.1038/cddis.2016.263 ; published online 08 September 2016.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-018-0636-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970264PMC
May 2018

Pathophysiological Consequences of Neuronal α-Synuclein Overexpression: Impacts on Ion Homeostasis, Stress Signaling, Mitochondrial Integrity, and Electrical Activity.

Front Mol Neurosci 2018 7;11:49. Epub 2018 Mar 7.

Department of Neurology, University Medical Center Goettingen, Göttingen, Germany.

α-Synuclein (α-Syn) is intimately linked to the etiology of Parkinson's Disease, as mutations and even subtle increases in gene dosage result in early onset of the disease. However, how this protein causes neuronal dysfunction and neurodegeneration is incompletely understood. We thus examined a comprehensive range of physiological parameters in cultured rat primary neurons overexpressing α-Syn at levels causing a slowly progressive neurodegeneration. In contradiction to earlier reports from non-neuronal assay systems we demonstrate that α-Syn does not interfere with essential ion handling capacities, mitochondrial capability of ATP production or basic electro-physiological properties like resting membrane potential or the general ability to generate action potentials. α-Syn also does not activate canonical stress kinase Signaling converging on SAPK/Jun, p38 MAPK or Erk kinases. Causative for α-Syn-induced neurodegeneration are mitochondrial thiol oxidation and activation of caspases downstream of mitochondrial outer membrane permeabilization, leading to apoptosis-like cell death execution with some unusual aspects. We also aimed to elucidate neuroprotective strategies counteracting the pathophysiological processes caused by α-Syn. Neurotrophic factors, calpain inhibition and increased lysosomal protease capacity showed no protective effects against α-Syn overexpression. In contrast, the major watchdog of outer mitochondrial membrane integrity, Bcl-Xl, was capable of almost completely preventing neuron death, but did not prevent mitochondrial thiol oxidation. Importantly, independent from the quite mono-causal induction of neurotoxicity, α-Syn causes diminished excitability of neurons by external stimuli and robust impairments in endogenous neuronal network activity by decreasing the frequency of action potentials generated without external stimulation. This latter finding suggests that α-Syn can induce neuronal dysfunction independent from its induction of neurotoxicity and might serve as an explanation for functional deficits that precede neuronal cell loss in synucleopathies like Parkinson's disease or dementia with Lewy bodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2018.00049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845890PMC
March 2018

Pharmacological targeting of GSK-3 and NRF2 provides neuroprotection in a preclinical model of tauopathy.

Redox Biol 2018 04 6;14:522-534. Epub 2017 Nov 6.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Investigación Sanitaria La Paz (IdiPaz), Instituto de Investigaciones Biomédicas Alberto Sols UAM-CSIC, Madrid, Spain; Department of Biochemistry, Faculty of Medicine, Autonomous University of Madrid, Madrid, Spain. Electronic address:

Tauopathies are a group of neurodegenerative disorders where TAU protein is presented as aggregates or is abnormally phosphorylated, leading to alterations of axonal transport, neuronal death and neuroinflammation. Currently, there is no treatment to slow progression of these diseases. Here, we have investigated whether dimethyl fumarate (DMF), an inducer of the transcription factor NRF2, could mitigate tauopathy in a mouse model. The signaling pathways modulated by DMF were also studied in mouse embryonic fibroblast (MEFs) from wild type or KEAP1-deficient mice. The effect of DMF on neurodegeneration, astrocyte and microglial activation was examined in Nrf2 and Nrf2 mice stereotaxically injected in the right hippocampus with an adeno-associated vector expressing human TAU and treated daily with DMF (100mg/kg, i.g) during three weeks. DMF induces the NRF2 transcriptional through a mechanism that involves KEAP1 but also PI3K/AKT/GSK-3-dependent pathways. DMF modulates GSK-3β activity in mouse hippocampi. Furthermore, DMF modulates TAU phosphorylation, neuronal impairment measured by calbindin-D28K and BDNF expression, and inflammatory processes involved in astrogliosis, microgliosis and pro-inflammatory cytokines production. This study reveals neuroprotective effects of DMF beyond disruption of the KEAP1/NRF2 axis by inhibiting GSK3 in a mouse model of tauopathy. Our results support repurposing of this drug for treatment of these diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2017.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681345PMC
April 2018

A MicroRNA124 Target Sequence Restores Astrocyte Specificity of gfaABCD-Driven Transgene Expression in AAV-Mediated Gene Transfer.

Mol Ther Nucleic Acids 2017 Sep 6;8:13-25. Epub 2017 Apr 6.

Center of Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Goettingen, Germany; Department of Neurology, University Medical Center Goettingen, Waldweg 33, 37073 Goettingen, Germany.

Experimentally restricting transgene expression exclusively to astrocytes has proven difficult. Using adeno-associated-virus-mediated gene transfer, we assessed two commonly used glial fibrillary acidic protein promoters: the full-length version gfa2 (2,210-bp human glial fibrillary acidic protein [GFAP] promoter) and the truncated variant gfaABCD (681-bp GFAP promoter). The capacity to drive efficient, but also cell-type specific, expression of the EGFP in astrocytes was tested both in vitro in rat primary cortical cultures as well as in vivo in the rat striatum. We observed an efficient, but not entirely astrocyte-specific, gfa2-driven reporter expression. gfaABCD exhibited a weaker activity, and most importantly, off-target, neuronal expression of the transgene occurred in a larger fraction of cells. Therefore, we explored the potential of a microRNA (miR)-specific target-sequence-based approach for abolishing off-target expression. When miR124 target sequences were incorporated into the 3' UTR, neuronal gene expression was effectively silenced. However, unexpectedly, the insertion of an additional sequence in the 3' UTR clearly diminished transgene expression. In conclusion, the gfaABCD promoter on its own is not sufficient to specifically target transgene expression to astrocytes and is not well suited for AAV-based gene targeting, even if short promoter sequences are required. The combination with a miR de-targeting sequence represents a promising experimental strategy that eliminates off-target, neuronal expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtn.2017.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476465PMC
September 2017

Long-Term Assessment of AAV-Mediated Zinc Finger Nuclease Expression in the Mouse Brain.

Front Mol Neurosci 2017 23;10:142. Epub 2017 May 23.

Department of Neurology, University Medical Center GöttingenGöttingen, Germany.

Gene editing tools like TALENs, ZFNs and Crispr/Cas now offer unprecedented opportunities for targeted genetic manipulations in virtually all species. Most of the recent research in this area has concentrated on manipulation of the genome in isolated cells, which then give rise to transgenic animals or modified stem cell lines. Much less is known about applicability of genetic scissors in terminally differentiated, non-dividing cells like neurons of the adult brain. We addressed this question by expression of a pair of ZFNs targeting the murine cathepsin D gene in CNS neurons by means of an optimized AAV viral vector. We show that ZFN expression resulted in substantial depletion of cathepsin D from neuronal lysosomes, demonstrating a robust gene deletion. Importantly, long-term ZFN expression in CNS neurons did not impair essential neuronal functionality and did not cause inflammation or neurodegeneration, suggesting that potent genetic scissors can be expressed safely in the mouse brain. This finding opens up new venues to create novel research models for neurodegenerative disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2017.00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440507PMC
May 2017

The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease.

PLoS Biol 2017 03 3;15(3):e2000374. Epub 2017 Mar 3.

CEDOC, NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal.

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2000374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336201PMC
March 2017

Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model.

Cell Death Dis 2016 09 8;7(9):e2359. Epub 2016 Sep 8.

Development and Maintenance of the Nervous System, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF's neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF's effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cddis.2016.263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059866PMC
September 2016

Lentiviral Delivery of miR-133b Improves Functional Recovery After Spinal Cord Injury in Mice.

Mol Neurobiol 2017 08 13;54(6):4659-4671. Epub 2016 Jul 13.

Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, 08554, USA.

Based on the observation that microRNA (miRNA) 133b enhances regeneration after spinal cord injury in the adult zebrafish, we investigated whether this miRNA would be beneficial in a mammalian system in vitro and in vivo. We found that infection of cultured neurons with miR-133b promotes neurite outgrowth in vitro on an inhibitory substrate consisting of mixed chondroitin sulfate proteoglycans, when compared to infection with green fluorescent protein (GFP) for control. In vivo, viral infection of the injured adult mouse spinal cord at the time of injury at and in the vicinity of the lesion site enhanced expression of miR-133b. Measurements of locomotor recovery by Basso Mouse Scale (BMS) showed improvement of recovery starting at 4 weeks after injury and virus injection. This improvement was associated with downregulation of the expression levels of Ras homolog gene family member A (RhoA), chondroitin sulfate proteoglycans, and microglia/macrophage marker in the spinal cord as assayed 6 weeks after injury. Potential inhibitory molecules carrying consensus sequences for binding of miR-133b were identified in silico and verified in a reporter assay in vitro showing reductions in expression of RhoA, xylosyltransferase 1 (Xylt1), ephrin receptor A7 (Epha7), and purinergic receptor P2X ligand-gated ion channel 4 (P2RX4). These results encourage targeting miR-133 for therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-016-0007-zDOI Listing
August 2017