MRes Poster University of Bristol - Eric Garson 2015
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Transcript of MRes Poster University of Bristol - Eric Garson 2015
Investigation of viral vector targeting in organotypic cerebellar slice culture Eric Garson, Helen Scott & James Uney. Regenerative Medicine Laboratory, School of Clinical Sciences, University of Bristol
Introduction • The cerebellum unconsciously controls movement and coordination. In addition to involvement in
development and cognition. These functions are determined by Purkinje neurons ,the principle
neuron of the cerebellum and Bergmann Glia ,the principle astrocytes of the cerebellum. Both are
found in the cerebellum cortex made up of three layers Granule, Purkinje Cell and Molecular layers.
Bergmann Glia cell bodies are found in the Purkinje cell layer. Purkinje neurons underpin the
cerebellum’s motor learning and coordination ability. While Bergmann Glia provide a scaffold and
adhesive for Granule cells to migrate into the Molecular layer.
• Numerous conditions have been associated with the cerebellum including Ataxia and Schizophrenia.
A form of human Ataxia caused by the TRPC3 (Transient Receptor Potential Cation Channel 3)
mutation found in Purkinje neurons leading to mutations in calcium and sodium channels. Whereas,
Schizophrenia causes malfunction of the DAO (D-Amino Acid Oxidase) enzyme found in Bergmann
Glia leading to the inability to metabolism the serine neurotransmitter. DAO activity is increased and
has been associated with symptoms and pathology of Schizophrenia.
• Gene therapy using viral vectors might overcome the barriers of targeting cerebellar cells and
compensate for TRPC-3 or DAO malfunction.
• In addition, it has been reported that Cathepsin K (a lysosomal enzyme) manipulates lentiviral vector
tropism towards Bergmann Glia. While its inhibition manipulates lentiviral vector tropism towards
Purkinje neurons both in vivo. Meanwhile, organotypic slice culturing provides a method to access
these factors because they retain the cyto-architecture and interactions of the cerebellum cortex,
permit immunostaining of Bergmann Glia and Purkinje neurons and permit assessment of viral vector
tropism and manipulation by Cathepsin K or Cathepsin K inhibitor.
Aims and Objectives The overall aim was to develop an optimised in vitro organotypic slice culture model for studying and
targeting cerebellar diseases. This would be achieved by using Cathepsin K or Cathepsin K Inhibitor to
manipulate the tropism of lentiviral vectors towards either Bergmann Glia astrocytes or Purkinje
neurons. Ultimately, these aims would realize a model for human cerebellar diseases, develop an
understanding of the underling disease processes involved and the prospect of developing novel
therapeutic treatments.
The specific objectives were to:
• Immunostain for Purkinje neurons and Bergmann Glia astrocytes.
• Manipulate lentiviral vector tropism towards Bergmann Glia astrocytes using Cathepsin K.
• Manipulate lentiviral vector tropism towards Purkinje neurons using Cathepsin K Inhibitors.
• Assess Cathepsin K or Cathepsin K Inhibitor addition 1 day before lentiviral vector addition.
• Access Adeno-Associated viral vector 2-9 (AAV2-9) transduction.
Methods
Results
-AraC
+AraC
Day 1 Day 8 Day 3 Day 2
Conclusions An optimised 8 day in vitro organotypic cerebellar slice culturing system achieved on tissue culture inserts permitted viral vector
expression beginning on day 5 in vitro with an optimum dilution/concentration at 5x105 particles/slice. Meanwhile, 1mM AraC
stopped Glia overgrowth for successful GFAP immunostaining. Successful immunostaining for Bergmann Glia and Purkinje
neurons using GFAP and Anti-Calbindin D-28k respectively was achieved. However manipulation of lentiviral vector tropism
towards Bergmann Glia using Cathepsin K and manipulation using Cathepsin K inhibitor towards Purkinje neurons was
unsuccessful because of no co-staining observation. AAV2-9 was successfully assessed using GFAP and Anti-Calbindin D28k.
References
1. Gahwiler, B. H., et al. 1997.Trends in Neurosciences 20: 471-477
2. Goenawan, H., et al. 2012. Journal of Neurovirology 18: 521-531
3. Pham, A. H., et al. 2014. Methods in enzymology 547: 111-129
4. de Mendoza, T. H., et al . 2011. Jove-Journal of Visualized Experiments: 5
5. Deglon, N.,et al. 2005. Journal of Gene Medicine 7: 530-539.
6. Burnet, P., et al. 2008. Mol Psychiatry 13: 658-660.
7. Becker, E. B. 2015. Cerebellum.
Figure 1: Optimisation and maintenance of cerebellar slice culture procedure
Figure 2: 1mM AraC Prevents Glial Overgrowth
Figure 3: Cathepsin K does not manipulate lentiviral vector tropism towards Bergmann Glia
Figure 4: Inhibition of Cathepsin K does not manipulate lentiviral vector tropism towards Purkinje neurons
Figure 5: Cathepsin K addition one day prior to lentiviral vector tropism addition does not manipulate lentiviral vectors towards Bergmann Glia
Figure 6: Cathepsin K Inhibitor addition one day prior to lentiviral vector addition does not manipulate lentiviral vectors towards Purkinje neurons
Figure 7: Assessment of AAV2-9 shows transduction in white matter
- Cathepsin K + Cathepsin K
- Cathepsin K Inhibitor + Cathepsin K Inhibitor
GFAP Overlay Anti-Calbinidn D28K Overlay
Anti-Calbindin D28 K Overlay
GFAP Overlay
500.000 μm
500.000 μm 500.000 μm
500.000 μm 500.000 μm
500.000 μm
500.000 μm
Day 1 Day 3 Day 2 Day 4 Day 5 Day 8