Investigating Viral Vector Targeting in an Organotypic Cerebellar Slice Culture- Eric Garson 2015

Investigating Viral Vector Targeting in an
Organotypic Cerebellar Slice Culture
By Eric Garson
Supervised by Dr Helen Scott and
Professor James Uney
A dissertation submitted to the University of Bristol in accordance with the
requirements of the degree of Master of Research by advanced study in Health
Sciences Research in the Faculty of Medicine and Dentistry
Date of Submission 21st
July 2015
Abstract Word Count 300
Introduction Word Count 2232
Total Word Count 10,427

of 49
Abstract
The cerebellum has been implicated in the development of neurological conditions.
Cerebellar Purkinje neurons have been implicated in a number of Ataxias for example
mutations of TRPC-3 genes. Meanwhile, Cerebellar Bergmann astrocytes, have been
implicated in the pathogenesis of Schizophrenia due to malfunctioning DAO (D-Amino Acid
Oxidase) enzymes. Gene therapy using viral vectors expressing functional genes might be
able to transduce these cells and compensate or delay the disease process. However it has
proved difficult to target Purkinje neurons and Bergmann Glia within the cerebellar cortex.
Lentiviral and Adeno-Associated viral vectors have been proposed as candidates for
targeting diseases of the central nervous system. Lentiviral vectors have been shown to
target Bergmann Glia with the manipulation of their tropism using Cathepsin K. Additionally,
Cathepsin K Inhibitor has been shown to manipulate the tropism of lentiviral vectors towards
Purkinje neurons. An Organotypic slice culture method was established to investigate the
effects of Cathepsin K and Cathepsin K inhibitors manipulation on viral vector transduction
towards targeting cerebellar cells. Wistar rat pups were anesthetised and terminally
sacrificed. Their cerebellum’s were sectioned using a McILwain tissue chopper. Slices were
cultured with AraC to prevent glial overgrowth for eight days in vitro. eGFP lentiviral and
Adeno-Associated viral vectors were combined with either Cathepsin K or Cathepsin K
Inhibitors. The solutions were pipetted on top of slices on day two in vitro. After a period in
culture selected slices were stained for Purkinje neurons or Bergmann Glia by
immunohistochemistry. Analysis showed Cathepsin K and Cathepsin K inhibitors do not alter
the tropism of lentiviral viral vectors towards Bergmann Glia or Purkinje neurons. Analysis of
Adeno-associated viral vectors transduction of cerebellar cells found similar patterns as that
obtained with lentiviral vectors. Further investigation is needed to access viral vector
manipulation by Cathepsin K and Cathepsin K Inhibitor.

of 49
Acknowledgements
My sincere thanks to Dr Helen Scott, Professor James Uney, Dr Liang-Fong Wong, Dr.
Fiona Holmes, Professor Domingo Tortonese, Jal, Anna, Seb, Darren, Jess, Hadil, Nebraz,
Aida, Esteban and the Health Sciences MRes Class of 2015.

of 49
Author’s Declaration
I declare that the work in this dissertation was carried out in accordance with the
requirements of the University’s Regulations and Code of Practice for Taught Programmes
and that it has not been submitted for any other academic award. Except where indicated by
specific reference in the text, this work is my own work. Work done in collaboration with, or
with the assistance of others, is indicated as such. I have identified all material in this
dissertation which is not my own work through appropriate referencing and
acknowledgement. Where I have quoted or otherwise incorporated material which is the
work of others, I have included the source in the references. Any views expressed in the
dissertation, other than referenced material, are those of the author.
SIGNED: ……………………………………………………………. DATE: ……………..
(Signature of student)

of 49
Contents
Abstract ..................................................................................................................................................2
Acknowledgements................................................................................................................................4
Author’s Declaration ..............................................................................................................................5
Abbreviations .........................................................................................................................................8
Introduction............................................................................................................................................9
The Cerebellum.................................................................................................................................10
Anatomy of the Cerebellum..............................................................................................................11
Purkinje Neurons ..............................................................................................................................13
Bergmann Glia...................................................................................................................................13
Lentiviral Vectors ..............................................................................................................................14
Adeno-Associated Viral Vectors........................................................................................................15
Cathepsin K .......................................................................................................................................16
Organotypic Slice Culturing...............................................................................................................16
Aim…………………………………………………………………………………………………………………………………………………17
Materials and Methods........................................................................................................................18
Reagents............................................................................................................................................19
Lentiviral Vector Production and Viral Titre .....................................................................................19
Organotypic Cerebellar Slice Culture................................................................................................20
Culture Media Preparation ...........................................................................................................20
Dissection Buffer...........................................................................................................................20
Tissue Culture Inserts....................................................................................................................21
Preparation and Maintenance of Organotypic Slices ...................................................................21
AraC...............................................................................................................................................22
Viral Transduction of Slice Cultures..................................................................................................22
Lentiviral Vector Preparation........................................................................................................22
Lentiviral Vector & Cathepsin K ....................................................................................................22
Lentiviral Vector & Cathepsin K Inhibitor .....................................................................................22
Immunohistochemistry.....................................................................................................................22
Permiabilisation ............................................................................................................................23
Block..............................................................................................................................................23
Primary Antibodies........................................................................................................................23
Secondary Antibodies ...................................................................................................................23

of 49
Mounting and Imaging..................................................................................................................23
Results...................................................................................................................................................24
Light & Fluorescent Microscopy .......................................................................................................25
Optimum Organotypic Slice Culturing Method ............................................................................26
Immunohistochemistry Results ........................................................................................................28
The Effect of Cathepsin K..............................................................................................................28
The Effect of Cathepsin K Inhibitor ...............................................................................................32
The Effect of Cathepsin K and Cathepsin K Inhibitors Prior to Lentiviral Vector Transduction....34
AAV Tropism in Organotypic Slice Cultures ......................................................................................37
Discussion.............................................................................................................................................41
Future Work......................................................................................................................................43
Conclusion ............................................................................................................................................44
References ............................................................................................................................................45

of 49
Abbreviations
AraC Cytosine-β-D-Arabinofuranoside Hydrochloride
CNS Central Nervous System
DAO D-Amino Acid Oxidase
DMEM Dulbecco’s Modified Eagle Media
DNA Deoxyribonucleic acid
DsRNA Double stranded Ribonucleic Acid
eGFP Enhanced Green Fluorescent Protein
FBS Foetal Bovine Serum
HEK293 Human Embryonic Kidney cells
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
HIV-1 Human Immunodeficiency Virus-1
LTR Long Terminal Repeats
PBS Phosphate Buffered Saline
PFA Paraformaldehyde
RNA Ribonucleic Acid
RRE Rev Response Element
TET Tetracycline Transactivator
TRPC3 Transient Receptor Potential Cation Channel 3
VSV-G Vesicular Stomatitis Virus Glycoproteins
WPRE Wood Chuck Hepatitis Post Transcriptional Response Element
X-SCID X-linked Severe Combined Immunodeficiency

of 49
Human Ataxia is a neurodegenerative disease of the cerebellum. Mutations in a number of
genes have been shown to cause ataxic pathologies, for example the TRPC-3 (Transient
Receptor Potential Cation Channel 3) gene which encodes a Calcium and Sodium ion
channel in Purkinje neurons found within the cerebellar cortex. In addition, DAO (D-Amino
Acid Oxidase) an enzyme involved in the metabolism of the neurotransmitter serine within
the brain has increased activity in the cerebellum of Schizophrenia patients particularly
within Bergmann Glia astrocytes (2-4).
The design of a gene therapy which could compensate for TRPC-3 or DAO malfunction
might alleviate the pathology and symptoms associated with Ataxia and Schizophrenia
respectfully.
Thus, the overarching aim of the project was to produce a method which could target viral
vectors viral vectors towards specific cells in the cerebellar cortex such as Purkinje neurons
and Bergmann Glia astrocytes. This would allow powerful models of human cerebellar
diseases to be established, enable the underling disease pathology to be investigated and
novel therapeutic treatments to be assessed.
Hirai and colleagues published a study showing that the addition of Cathepsin K (a
lysosomal enzyme) could shift the tropism of VSV-G lentiviral vectors towards Bergmann
Glia, in vivo. They also showed the addition of Cathepsin K Inhibitor could shift the tropism of
VSV-G lentiviral vectors towards Purkinje neurons in vivo (5).
With this in mind, my projects objectives were to develop an in vitro method for manipulating
viral vectors towards either Purkinje neurons or Bergmann Glia within the cerebellar cortex,
using either Cathepsin K or Cathepsin K Inhibitor and achieving these objectives by
Organotypic slice culturing because this would protect and conserve the integrity of the
cerebellum’s architecture and complex cell interactions.
The Cerebellum
The cerebellum unconsciously controls motor coordination, movement, balance, poise, gait
and speech. The cerebellum plays a role in emotions, cognition, behavioural awareness,
time and regulation of smooth movements. Diseases of the cerebellum show an impairment
in cognitive function and connections between other areas of the brain involved in cognitive
processes (6-11). Disorders of the cerebellum during development can lead to cognition
developmental disorders (12). The cerebellum is altered by a wide range of conditions
including Ataxia and Schizophrenia (8, 9).Ataxia predominantly affects the cerebellar cortex
and has been shown to lead to poor cognitive function such as reduced IQ and poorer

of 49
judgment (9). Ataxia has no treatment. It is distinguished by the loss of movement,
coordination and functions associated with the cerebellum. Purkinje neurons are thought to
play a major role in pathogenesis of the disease due to mutations in the TRPC-3 gene (10,
19).
Schizophrenia is a neurological disease manifesting itself with delusions, cognitive
difficulties, speech impairments and hallucinations beginning in early adolescence. Patients
have been shown to have reduced blood flow in the cerebellum (8, 13). Lower metabolism in
the cerebellum has been observed in patients with Schizophrenia, alongside impaired
cognition (13). Furthermore poor output from Purkinje neurons has been observed in
patients with Schizophrenia (6).
Anatomy of the Cerebellum
The cerebellum is connected to the brain by three branches called cerebellar peduncles (7).
The cerebellum consists of two hemispheres connected together by the midline vermis. The
cerebellum is further split into three lobes, the anterior (top), posterior (back) and
flocculonodule which attaches to the brain stem as shown in Figure 1 (10, 14).
The cerebellar cortex is split into three layers, Granule, Purkinje cell and Molecular Layers,
as shown in Figure 2. The white matter of the cerebellum borders the Granule layer
predominantly consisting of Granule cells; this layer meets a boundary of Purkinje neurons
all aligned in a monolayer. The outmost section of the cortex is the molecular layer. The
cerebellar cortex consists of Stellate, Granule, Golgi, Bergmann Glia, Granule, Purkinje and
Basket cells, alongside Climbing and Mossy fibres from the white matter as shown in Figure
2 (10, 14).
Mossy and Climbing fibre axons enter the cerebellum propelling action potentials from the
spinal cord through the three penduncles terminating in the cerebellar cortex. Mossy fibres
synapse with Granule cell dendrites in the Granule layer. Climbing fibres synapse with
Purkinje neuron dendrites in the molecular layer as shown in Figure 2.

of 49
Figure 1 Cerebellum Anatomy. The cerebellum is split up into two hemispheres connected by the Vermis. The
cerebellum is subdivided into three lobes, the anterior (top), posterior (back) and flocculonodule lobe which sits
next to the Medulla and Pons (15).
The Granule cells have small and oval shaped cell bodies. Their axons project into the
molecular layer and form the Parallel fibres. In turn the Parallel fibres synapse with dendrites
of the Purkinje neurons in the Molecular layer. Purkinje neurons permit impulses to exit the
cerebellum’s cortex through axons (one per cell) which project through the Granule and
white matter layers eventually reaching the cerebellar nuclei, where impulses are relayed to
the rest of the brain.
Figure 2 Cerebellum Cortex Structure and Cell types. The false coloured microscope image and schematic
represent the cerebellar cortex. The dark red and black represent the climbing fibres from the white matter which
will eventually synapse with the Purkinje cell dendrites. The white matter also contains Mossy fibres which
synapse to Granule cells in the Granule layer, the blue layer, also containing Golgi cells. The Granule cells
synapse to the Purkinje neurons, large flask shaped cells in the Purkinje cell layer (the light blue monolayer). The
Purkinje cells are intertwined by smaller red cell bodies of Bergmann Glia who’s dendrites project into the
Molecular layer, the green layer. The molecular layer contains the extensive dendrites of the Purkinje neurons,
along with parallel fibres from Granule cells. The molecular layer also contains Basket and Stellate cells (16).

of 49
Purkinje Neurons
Discovered in 1827 by Jan Evangelista Purkyně, the Purkinje neuron is the main neuron of
the cerebellum. It makes up one of the five neurons of the cerebellum, along with Granule,
Basket, Golgi and Stellate neurons (17, 18).
The Purkinje neuron underpins the cerebellum’s motor learning and coordination of
controlled movements (17, 18).
Purkinje neurons have a flask shaped cell bodies and a single axon. They lie in a single cell
layer perpendicular to the cerebellar cortex. Their dendrites synapse with climbing and
parallel fibres as shown in Figure 2. In mouse and rodent studies Purkinje neuron’s structure
and shape alter during postnatal development, specifically in the 2nd
week (14, 17). Purkinje
neurons can be labelled for investigation using Ant-Calbindin D28k markers.
Disorders of Purkinje neurons have been implicated in a number of conditions including
Ataxia, tremor and dysphagia (10, 19).
Bergmann Glia
Bergmann Glial are a Proteoplasmic astrocytes. They are the principle astrocyte in the
cerebellum. Providing an adhesive or scaffolding function for Granule cells to migrate and
support nerve cells in the cerebellum. Their morphology is shared between humans, rodents
and mice. Bergmann Glia cell bodies are found in the Purkinje cell layer of the cerebellar
cortex, its fibres extend into the molecular layer and end at the pia of the cerebellar cortex,
as seen in Figure 3. The fibres can be labelled by using the GFAP (Glial Fibrillary Acid
Protein) markers. Other markers include S100 and Aquaporin-4. Astrocytes such as
Bergmann Glia may play a role in Schizophrenia development because DAO (D-Amino Acid
oxidase) expression is increased in the cerebellum, its modulation might reduce the
symptoms of Schizophrenia. (3, 4, 20, 21).

of 49
Figure 3 Bergmann Glia Morphology. A The schematic diagram shows the cells of the cerebellar cortex.
Bergmann Glia (BG) are coloured red. Their cell bodies are small and round. Their fibres extend into the
Molecular layer (ML), ending at the Pia. Bergmann Glia help provide structure and transportation of Granule cells
(GC), Parallel fibres and the support of Purkinje neurons. B The three coloured images below shown from left to
right show eGFP positive, S100 positive stains and an overlay of the Bergmann Glia stains (5, 21).
To target cerebellar cells affected by Ataxia such as Purkinje Neurons and Bergmann Glia
affected by Schizophrenia, viral vector targeting has been proposed to deliver genes which
could alter these disease cells physiology.
Lentiviral Vectors
Lentiviral vectors are a genetically modified version of the HIV-1 virus belonging to the
Lentiviradae family, which are a slow replicating complex subgroup of retroviruses. HIV-1
targets, infects, integrates and replicates its genome inside a number of cells. With these
natural properties Verma, Naldini and colleagues removed the disease causing accessory
and regulatory proteins of the HIV-1 virus to create a vector which could deliver and
integrate a genome encoding a gene or protein to cells to alter their properties and functions
(22-28).
Lentiviral vectors have a lower immunogenicity profile compared to other viral vectors such
as Herpesviruses and Adenoviral vectors (27-29). Furthermore, lentiviral vectors reduce the
chances of inflammation when transducing central nervous system tissues, additionally
expression can be sustained for long durations by either integrating or non-integrating
vectors (30).
A
B

of 49
Pertinently, lentiviral vectors can transduce non-dividing and dividing cells such as neurons
and muscle cells (27, 30-32). Adversely, lentiviral vector integration can cause insertional
mutagenesis, altering the function of the cell and lead to cancer (33, 34).
Structurally the HIV-1 GP120 and GP41 glycoproteins can be swapped for different
glycoproteins, a process known as pseudotyping, expanding or restricting the targeting of
lentiviral vector. Several pseudotypes have been borrowed both from other viruses or
genetically engineered human cellular receptors to reduce or expand their tropisms. The
most commonly used is VSV-G (Vesicular Stomatitis Virus Glycoproteins), it attaches to an
unknown binding motif of phosphatidylserine on cell plasma membranes. VSV-G easy to
concentrate by ultracentrifugation and titrate for production of lentiviral vectors.
A commonly used promoter which transcribes transgene inserts continually and at high
volume in many cell types is the CMV (Cytomegalovirus) promoter (31, 35).Tissue specific
promoters for neuronal tissues for example CAMKII and Synapsin have been developed to
boost or only transcribe inside neuronal tissues. The Wood Chuck Hepatitis Post
Transcriptional Response Element (WPRE) is a genomic sequence inserted at the 3’ end of
the transgene to enhance expression (33, 35). Lentiviral vectors can be produced by co-
transfecting HEK293T cells with plasmids containing the ingredients for lentiviral vector
production.
Enhanced Green Fluorescent Protein (eGFP) can be inserted into the transgene unit of a
lentiviral vector to create a reporter assay. Once inside a target cell, the transgene
integrates, transcribes and translates eGFP. Transduced cells will fluoresce green under
ultraviolet light and indicate lentiviral vector expression. This method permits the
development of in vitro and in vivo models to test gene therapy approaches for the
investigating the physiology and pathology of neurological diseases (30, 36).
Adeno-Associated Viral Vectors
Adeno-Associated Viral Vectors (AAV’s) are part of the Parvovirus family. As a vector, they
have a large transgene unit which can express for months if not years and since they do not
integrate they avoid insertional mutagenesis. Furthermore, they do not replicate disease
causing viruses. Multiple serotypes have been developed with differing capsid proteins and
pseudotypes which allow for expansion or restriction of cell tropism targeting. AAV’s have
been proposed for targeting stem cells in the central nervous system (37) (38).

of 49
Cathepsin K
Hirai and colleagues recently showed Cathepsin K, a lysosomal enzyme could manipulate
lentiviral vector tropism in the cerebellar cortex (5).
There are 11 Cathepsin enzymes which make up the Cathepsin family B, H, L, S, C, K, O, F,
X, W and V. Cathepsin’s are lysosomal enzymes. They breakdown proteins and are found in
acidic areas of the cell, such as lysosomes. Their optimal effect is seen at pH 6. (5, 39-43).
A method for testing Cathepsin K manipulation of viral vector targeting in the cerebellum
requires a method which protects and preserves the architecture of the cerebellum.
Organotypic Slice Culturing
Studying rodent and mouse cerebellum properties, such as interactions and relationships
between cells in complex tissue cyto-architecture can be achieved through Organotypic slice
culturing. In particular, the delicate Purkinje cell layer requires a method which protects and
preserves its architecture for long periods. The tissue relates more closely the architecture
as seen in vivo, permitting the investigation of neurological conditions. Organotypic slice
cultures can be achieved by sacrificing an animal, obtaining the relevant neurological tissue
and sectioning by either a tissue chopper or a Vibratome. The slices are placed onto
semiporous membrane tissue culture inserts which sit on a layer of culture media and kept
above 30o
C in a sterile incubator as shown in Figure 4 (1).
The Organotypic culture method provides a better relatable tissue culture compared to
primary dissociated or immortal cell line culturing systems. Consequently, Organotypic slice
culturing is a rational approach to studying the interactions between complex cells in the
cerebellar cortex particularly Purkinje neurons and Bergmann Glia (18, 19, 44-46).
Figure 4 Tissue Culture Inserts. The tissue culture inserts floats on top a layer of media. The semiporous
membrane allows nutrients from the media to seeps through whilst simultaneously the insert still permits
oxygenation from above (1).

of 49
Furthermore, Organotypic slice culturing obtains multiple slices that can be independently
treated from each animal sacrificed per experiment. This is in keeping with reducing, refining
and replacing as best a possibly the number of animals used in research.
Together, an Organotypic slice culture model would provide an effective method for targeting
the cerebellum cortex with lentiviral and Adenoviral-Associated viral vectors. Aiming to
manipulate viral vector transduction using Cathepsin K and Cathepsin K Inhibitors and
ultimately establish a model for treating Ataxia and Schizophrenia in vitro.
Aims
To develop an in vitro Organotypic slice culture model for studying cerebellar diseases and
to use Cathepsin K or Cathepsin K Inhibitor to manipulate the tropism of lentiviral and
Adeno-Associated viral vectors towards either Purkinje neurons or Bergman Glia astrocytes..
Ultimately, these aims would allow powerful models of human cerebellar diseases to be
established, enable the underling disease process to be investigated and the development
of novel therapeutic treatments to be assessed.

of 49
Materials and Methods

of 49
Reagents
1x Phosphate Buffered Saline (PBS) (Gibco Life Scientifics), Dissection Tweezers (Inox
Biology), surgical dissection scissors (PST), 24 and 6 Well Plates (Cellstar), 0.2µm filter
(Sartorius Ministar), Original Milk Dried Milk Powder (Marvel), Triton X-100 (BDH Laboratory
supplies), Phosphate Buffered Saline Tablets (Sigma Aldrich), Methanol (Sigma Aldrich),
Donkey Horse Serum (Sigma Aldrich), Cy3 Conjugated Affinipure Donkey Anti Mouse IgG
(H+L) (Jackson Immunoresearch), Hoechst Stock 1mg/ml in H2O, Mounting Media, Rabbit
Anti-Calbindin D-28K-CB38 (SWANT), Paraformaldehyde (Sigma Aldrich), GFAP-Polyclonal
Rabbit (Glial Fibrillary Acidic Protein) 2.9g/ml (DAKO), Cy2 Conjugated Affinipure Donkey
Anti-Rabbit IgG (H+L) Green (Jackson Immunoresearch), Cy3 Conjugated Affinipure Donkey
Anti-Rabbit IgG (H+L) Red (Jackson Immunoresearch), Microscope slides Twist Frost
Ground (Fisherbrand), Microscope cover glasses (VWR), Leica DMIL, Leica EL6000 Light
Box, Leica DIRB Lens DC500, Leica DMRB and Leica DFC340FX, Water (Sigma Aldrich),
Dissecting Microscope (Leica Wild M32), D-(+) Glucose (Sigma Aldrich), Lamina Flow Hood
Safe 2020 (ThermoScientific), 37o
C Incubator (Hera Cell Heraeus), Cell Culture Inserts
(Millipore/Millicell), Filter paper 110mm thickness (Fisherbrand), Trypan Blue (Sigma
Aldrich), HBS, Cathepsin K Human, Recombinant (Enzo), Cathepsin K Inhibitor (Santa Cruz
Biotechnology), Modified Eagle Media with Earl’s salt, no glutamine, phenol-red free (GIBCO
by Life Technologies), Heat Inactivated Horse Serum (Sigma), Hank’s Balanced salt solution
without calcium, magnesium and phenol red (Life Technologies), HEPES, GlutaMAX (Life
Technologies), L-Glutamine (Life Technologies), Penicillin-Streptomycin (Sigma Aldrich),
Gey’s Balanced, salt solution (Sigma Aldrich), Isoflurane anaesthesia, Whatman filter paper
grade 5 90mm, hydrophilic PTFE- (Millipore Millicell) and McILwain Tissue Chopper (Mickle
Laboratory Engineering).
Lentiviral Vector Production and Viral Titre
HEK293T cells were maintained in DMEM (Dulbecco’s Modified Eagle Media), L-Glutamine,
Penicillin/streptomycin, FBS (Foetal Bovine Serum) and non-essential amino acids in T175
flasks and split 1 in 12 every three to four days (26).
HEK293T cells were plated at a density of 8x106
cells/dish in 12x15cm dishes.20ml of media
was added per dish (26).
The masses of each plasmid /plate were pRRL.CMV.EGFP.WPRE-10ug, pMPDLg-Prpe-
10ug, pRSv-Rev-2ug and pMD2-VSVG-3.4ug. 2M CaCl2 and sterile water (total volume/plate
1.2mL). An equal volume of 15ml 2xHBS(50 mM Hepes, 280 mM NaCl, 1.5 mM Na2HPO4
(pH7.1) had the DNA/CaCl2 mix added dropwise whilst bubbling. 30 minutes later the

of 49
solution had turned cloudy indicating successful formation of the calcium phosphate
precipitate with the DNA. 2.4ml of the solution was added to each of the 15cm plates and
spread evenly. Plates were incubated overnight at 37o
C (26).
The next day the media was changed and replenished with fresh media with Sodium
Butyrate. The media was harvested from the plates 7 hours later and stored at 4o
C
overnight. Meanwhile fresh media was added to the plates containing no sodium butyrate
(26).
The next day the harvest was centrifuged and filtered. The viral supernatant was centrifuged
at 4o
C and 6000g overnight (26).
The next day the supernatant was removed and the pellet resuspended in cold 1xPBS. The
pellet was centrifuged at 20,000rpm for 90 minutes at 4o
C. After centrifugation the TSSM
was added to the pellet and kept on ice for several hours. The pellet was resuspended in a
final volume of TSSM to give 2000 fold concentration of the volume of media harvested from
cells. The virus was aliquoted out into Eppendorf tubes and frozen at -80o
C.(26).
HEK293T cells were plated at 1x12well/plate and virus plated at 7.5x106
. A viral titre assay
was performed to access the concentration of the lentiviral vectors for further
experimentation. Media was removed from the plates. And 500μl of virus was added.
1:1000, 1:10,000 and 1:100,000 and 1:1,000,000 were prepared. Three days later the cells
were harvested from plates, and fixed with 4% PFA (Paraformaldehyde). Flow cytometry
was performed to access transduction. All lentiviral vectors were obtained at 1x109
particles /
mL (26).
Organotypic Cerebellar Slice Culture
Culture Media Preparation
Tissue culture media was prepared by adding 25mls of MEM with Earl’s salt, no glutamine,
phenol red free (Gibco by Life Technologies), 12.5ml of Heat Inactivated Horse Serum,
1.25mls 10xHanks Balanced Salt Solution without Calcium, Magnesium and phenol red (Life
Technologies), 1ml of 1M HEPES, 0.25ml of 200Mm GlutaMax (Life Technologies), 0.25mls
L-Glutamine, 1ml of Penicillin-Streptomycin, 8.5 ml autoclaved sterile water (Sigma) and
0.325g of D-Glucose. The tissue culture media was filter sterilised through a 0.2μm filter.
Tissue culture media was pre-warmed in 37o
C waterbath (47).
Dissection Buffer
Dissection buffer was prepared by adding 3.25g of D-Glucose (Sigma-Aldrich) to 500mls of
Gey’s Balanced Salt Solution (Sigma-Aldrich). The dissecting buffer was filtered through a

of 49
Bϋchner funnel vacuum with filter paper. 5-10mls of dissection buffer was decanted into a
50ml falcon tube and kept on ice for experiments (44, 47).
Tissue Culture Inserts
1ml of tissue culture media was added to wells of a 6 well plate. Tissue culture inserts
(Millipore Millicell) were placed on top of media. The plates were incubated until ready for
adding the cerebellum slices (44).
Preparation and Maintenance of Organotypic Slices
7 day old postnatal Wistar rat pups were terminally anesthetised using Isoflurane gas. The
brain was quickly removed and placed in dissection buffer (Gey’s balanced salt solution
(Sigma-Aldrich) with the addition of 3.25g of D-Glucose which had been filter sterilised) on
ice (44, 47).
The cerebellum was severed form the brain of P7 postnatal mouse pups. All rat pups were
sacrificed in accordance with the United Kingdom Animals Scientific Procedures Act (1986).
The cerebellum was placed either sagittaly or coronaly on a pre wetted sheet of Whatman
filter paper. A blade was attached to the chopping arm of a McILwain Tissue Chopper. The
thickness for cutting was set to 350μm per slice. The tissue chopper was turned on and the
platform of the chopper moved right to left slicing the cerebellum (44).
The slices were transferred into a petri dish containing 5-10mls of dissecting buffer. Under a
dissecting microscope using surgical forceps the slices were separated away from one
another. Good slices representing cerebellum architecture were placed into a separate petri
dish containing dissecting buffer (44).
The good slices were placed using a half cut Pasteur pipette into the tissue culture inserts.
Slices were evenly spaced from one another normally 3 or 4 per insert.
Depending on the experiment the slices were cultured from 3-14 days in a Hera Cell 37o
C
5% CO2 Incubator (Heraeus).Depending on the experiment, pre-prepared AraC, eGFP
Lentiviral Vectors, Adeno Associated Viral Vectors, Cathepsin K (Enzo) and Cathepsin K
Inhibitors (Santa Cruz) were added at 1x109
particles/mL and varying dilutions. Each will be
discussed later.
Photos of the slices were taken daily on the inverted light microscope to access growth,
health and viability of the slices over time. Every other day 750μl of tissue culture media was
removed and 750μl of fresh pre-warmed culture media added per well.

of 49
AraC
A stock 1M concentration of AraC (Cytosine-β-D-Arabinofuranoside Hydrochloride) was
prepared by diluting in sterile water. From this stock, aliquots of 1mM AraC was prepared.
On day two In vitro 1μl of AraC was added per well. 1μl of AraC was added every other day
with media change.
Viral Transduction of Slice Cultures
Lentiviral Vector Preparation
Lentiviral vectors at a concentration of 1x109
particlaes/mL was diluted in 1x PBS for a 1 in 2
dilution. 1μl of the solution was added per slice. Additional experiments explored the
optimum concentration of the lentiviral vector by performing a serial dilution of the lentiviral
vector in 1x PBS. The dilutions included 1 in 2, 1 in 4, 1 in 8 & 1 in16 and adding 1μl of each
respective dilution to a slice and culturing for 7-10 days In vitro .
Lentiviral Vector & Cathepsin K
A stock concentration of Cathepsin K was made at 884.62nM. The lentiviral vector was
diluted 1 in 2 with Cathepsin K to obtain a final concentration of 440nM. A 1 in 4 dilution was
prepared by adding 2μl of the lentiviral vector to 1μl of 1xPBS and 1μl of Cathepsin K for a
final concentration of 220nM.
Lentiviral Vector & Cathepsin K Inhibitor
The Cathepsin K Inhibitor was prepared by dissolving in 500µl of DMSO to obtain a 46nM/ml
stock concentration. 1µl of the stock concentration was added to 10.5µl 1xPBS for a 400nM
concentration. The lentiviral vector was diluted with Cathepsin K inhibitor for a 1 in 2 dilution
at 200nM concentration. A 1 in 4 dilution was prepared by adding 2μl of the lentiviral vector
to 1μl of 1xPBS & 1μl of Cathepsin K Inhibitor for a 100nM concentration.
Lentiviral vector transductions with or without Cathepsin K and its inhibitor were diluted as
appropriate and pipetted on top of the slices, 1μl each.
Immunohistochemistry
The slices were fixed by adding pre-prepared ice-cold 4% Paraformaldehyde (PFA) (diluted
in 1xPBS and pH’d with sodium hydroxide.) 1ml was added inside and outside the tissue
culture insert for 10 minutes. The slices were washed with ice cold tissue grade 1xPBS
(Sigma Aldrich) for 10 minutes. 1ml was added inside and outside the tissue culture insert
for ten minutes.

of 49
Ice cold 20% methanol diluted in 1xPBS (Sigma-Aldrich) was added for 5 minutes. 1ml was
added inside and outside the tissue culture insert. The slices were washed with ice cold
tissue grade 1xPBS (Sigma Aldrich) for 10 minutes.
Permiabilisation
To permeabilise the slices and allow the antibody access to the tissue, 0.5% Triton X-100
(BD laboratories) (diluted in 1xPBS) was added to the slices. 1ml was added inside and
outside the tissue culture insert and incubated overnight at 4o
C.
Block
Prior to adding the primary antibodies the slices were blocked with 1% donkey serum diluted
in 0.5% Triton X-100 (diluted in 1xPBS). Slices were incubated overnight at 4o
C.
Primary Antibodies
The slices were cut from the tissue culture membranes and placed into 1 ml of 1xPBS in a
24 well plate. Anti-Calbindin D-28k (SWANT) raised in Rabbit was diluted in 0.5%Triton X-
100 (diluted in 1xPBS) for a 1in1000 dilution. The GFAP (Glial Fibrillary Acidic Protein)
(DAKO) Polyclonal raised in rabbit was diluted either at 1 in 500 of 0.5% Triton X-100
(diluted in 1xPBS). The 1xPBS was decanted from the wells and 1ml of the Anti-Calbindin D-
28k or GFAP 0.5%Triton X-100 solutions were added. The plates were incubated at room
temperature for four hours or overnight at 4o
C.
Secondary Antibodies
The slices were washed three times in 1xPBS for 10 minutes each at room temperature. A
Cy3 Conjugated Anti-Rabbit IgG (Jackson Immunoresearch) was diluted 1 in 1000 for Anti-
Calbindin D-28k stained slices, meanwhile a 1 in 100 dilution was prepared for GFAP
stained slices. Both secondary antibodies were diluted in 0.5% Triton X-100 (diluted in
1xPBS). The secondary antibodies were added to the slices and incubated at room
temperature for four hours or overnight at 4o
C.
Mounting and Imaging
The slices were washed three times in 1xPBS for 10 minutes each at room temperature. For
the third wash, a 1 in 1000 dilution of Hoechst was added per well and incubated for a
further 10 minutes. The slices were placed onto microscope slides using forceps and on
occasion a paint brush. Excess 1xPBS was dabbed away with microscope tissue. 7-10μl of
mounting media was added per slice and a glass coverslip (VWR) placed on top.
The slices were analysed and photos taken on an inverted fluorescent microscope (Leica
DMRB and Leica DFC340FX).

of 49
Light & Fluorescent Microscopy
Prior to modulating lentiviral vector transduction to identify the optimal expression within the
cerebellar slice cultures, I performed a serial dilution of the eGFP lentiviral vector in 1xPBS.
A P7 rat cerebellum was sectioned and good slices placed on to tissue culture inserts and
incubated. The cerebellar slices were incubated overnight. On day 2 in vitro, the lentiviral
vector dilutions were pipetted on top of the individual slices at a concentration of
5x105
particles/mL and a volume of 1μl per slice.
As shown in Figure 5A, B and C the more dilute the lentiviral vector, the less expression of
the eGFP was observed. The optimal dilution of the lentiviral vector was identified as 1 in 2,
because undiluted lentiviral vector bleached the slices and was unworkable for future
experiments with Cathepsin K or Cathepsin K Inhibitor. Additionally, the lower dilutions of the
lentiviral vector prevented analysis of target cells and cerebellar cortex structure.
Overall, the optimum dilution for the lentiviral vector to express within Organotypic slices was
a 1 in 2 dilution, equivalent to 5x105
particles when adding 1μl per slice. These result were
carried forward into all future experiments.
Figure 5 Analysis of lentiviral vector expression in Organotypic slice culture. A Shows a serial dilution of
the eGFP lentiviral vector diluted in 1xPBS, images were acquired at 4x magnification. The less diluted the
lentiviral vector is the less expression of the eGFP is observed. B Shows higher magnification photos of eGFP

of 49
lentiviral vector illustrating the bleaching effect of undiluted lentiviral vector compared to a 1 in 2 dilution. C
Shows three representative Organotypic slices showing the location of eGFP expression predominantly within the
white matter of the cerebellum and the edges of the cerebellar slices, their respective light microscopy images
are underneath. (All images were taken at x2.5 magnification).
Optimum Organotypic Slice Culturing Method
After achieving an optimum concentration of the lentiviral vector for transducing target cells, I
established the optimum culturing conditions for assessing the manipulation of lentiviral
vector tropism. This meant a method and timescale which would allow for the addition of
lentiviral vectors to the slices, the addition of Cathepsin K and Cathepsin K Inhibitor, time for
the lentiviral vector to transduce and express within target cell types and the flexibility for
manipulating lentiviral vectors using Cathepsin K or Cathepsin K inhibitors towards Purkinje
neurons or Bergmann Glia implicated in Ataxia and Schizophrenia pathogenesis
respectively.
After varying the timescale, culturing conditions, the volume of culture media necessary to
replenish the slices, the number of slices per insert and the incubation period between
adding new culture media had all been extensively experimented with. I arrived at an
optimised method of 8 days in vitro culture method. This involved sectioning the slices on
day 1, adding the lentiviral vector with or without Cathepsin K or Cathepsin K inhibitor on day
2, replenishing the media every other day by removing 750μl of old culture media and
replacing with fresh 750μl of 37o
C pre warmed culture media. By day 5 the slices had begun
to show expression of the lentiviral vector and on day 8 I could fix and permeabilise the
slices in preparation for immunohistochemistry. All stages were documented with light and
fluorescent microscopy during the method, accessing health and quality of the slices as
shown in Figure 6.
The establishment of an 8 day in vitro culturing method permitted the addition of viral vectors
and Cathepsin K or Cathepsin K inhibitors to access potential altered transduction towards
Purkinje neurons or Bergmann Glia astrocytes in the cerebellar cortex.

of 49
Figure 6 Optimal conditions for Organotypic cerebellar culturing. A shows a representative cerebellar slice
during the course on an 8 day in vitro experiment. The light microscope images show the cerebellar slices
flattening out over the tissue culture insert and the outgrowth of cells from the edges of the slices. B shows a
comparison between representative slices cultured with AraC addition or without AraC addition at a concentration
of 1mM addition every other day. (All images were taken at x2.5 magnification).
Once establishing an optimum culture method, for the objective of manipulating viral vectors
targeting towards Purkinje Neurons and Bergmann Glia implicated in Ataxia and
Schizophrenia respectively, I began preliminary immunohistochemistry staining for GFAP
expressing Bergmann Glia and Anti-Calbindin-D28k expressing Purkinje neurons by firstly
fixing the slices in 4% PFA and permeablising the slices in 0.5% Triton X-100. I
subsequently added primary antibody incubations of GFAP or Anti-Calbindin D28k, washed,
followed by a Cy3 secondary antibody incubation, culminating in a final with Hoechst to stain
cell nuclei.
Preliminary results (not shown), illustrated poor staining with GFAP. As a consequence, the
immunohistochemistry procedure was changed. Firstly, the primary and secondary
antibodies were diluted in 0.5% triton X-100 instead of 1xPBS; additionally the
concentrations of primary antibodies were changed to a 1 in 500 dilution for GFAP and a 1 in
1000 for Anti-Calbindin D28k primary antibodies. Meanwhile the Cy3 antibody dilutions were
changed to 1 in 1000 for Anti-Calbindin D28k stained slices and 1 in 100 for GFAP stained
slices.

of 49
Furthermore, during the fixation, the slices were washed with 1xPBS for 10 minutes rather
than a quick wash after being in 4% PFA for 10 minutes. Another possibility for the poor
staining of Bergmann Glia with GFAP was thought to be glial overgrowth over the cerebellar
slices during in vitro culturing on the tissue culture inserts. To reduce Glial overgrowth, I
added 1μl of AraC at a concentration of 1mM to culture media every other day, alongside
culture media replenishment. AraC induces apoptosis in mitotic cells by interfering with DNA
synthesis. As seen in Figure 6B, the addition of AraC during the course of the in vitro
culturing of Organotypic slices reduced glial overgrowth.
These optimised changes enhanced the staining of both GFAP and Anti-Calbindin D28k
stained slices. As a consequence, all subsequent experiments followed this procedure
helping to identify the target cells in the cerebellum and providing robust fluorescent imagery
for determining whether Cathepsin K and Cathepsin K inhibitor could alter the transduction
of eGFP expressing lentiviral vectors towards Purkinje neurons or Bergmann Glia, as a step
towards targeting these cell types with lentiviral vectors which could silence the expression
of TRPC3 or DAO in Ataxia and Schizophrenia respectfully.
Immunohistochemistry Results
The Effect of Cathepsin K
After establishing an optimal culturing method which obtained healthy Organotypic slice
cultures capable of effective staining using Anti-Calbindin D28k and GFAP antibodies at
optimal dilutions for the investigation of manipulation of viral vector targeting by Cathepsin K
and Cathepsin K Inhibitor altering the tropism of viral vectors to either Purkinje neurons and
Bergmann Glia. I began to investigate whether Cathepsin K enzymes might manipulate the
tropism of eGFP lentiviral vectors towards Bergmann Glia astrocytes, as they are implicated
in the development of Schizophrenia.
Thus, I obtained sectioned Organotypic slice cultures and used the optimal culturing
procedure previously described. A 1 in 2 dilution of an eGFP expressing lentiviral vector
solution was prepared at a concentration of 5x105
, alongside a 1 in 2 dilution of eGFP
lentiviral vector diluted in Cathepsin K at a concentration of 5x105
particles/mL. Additionally a
1 in 4 dilution of eGFP lentiviral vector was diluted 1 in 4 with Cathepsin K at a concentration
ranging from 6.25x104
to 1x106
particles / slice. The respective solutions were pipetted on
top of separate sets of slices. All had 1μl added per slice on day 2 in vitro. The slices were
incubated for a total of 8 days with AraC added every other day at a concentration of 1mM.
The slices fixed with 4% PFA and permeabilised in 0.5% Triton X-100. The slices were
blocked in 1% donkey horse serum. The slices were stained for Bergmann Glia by

of 49
incubating in 1 in 500 dilution of GFAP diluted in 1xPBS. The slices were washed and Cy3
secondary antibody added at a 1 in 100 dilution. The slices were washed and a Hoechst
stain culminated in staining the nuclei. The slices were analysed on an inverted fluorescent
microscope.
Figure 7A and B shows a comparison between GFAP stained cerebellar slices transduced
with eGFP lentiviral vector diluted with or without Cathepsin K enzymes. Figure 8A and B
shows the difference of diluting the lentiviral vector at 1 in 4 with Cathepsin K. The Cathepsin
K treated slices show no difference in lentiviral vector tropism compared to slices not treated
with Cathepsin K. The lentiviral vector appears to have transduced in similar areas of the
slice and not definitively Bergmann Glia astrocytes. Additionally, GFAP stains all glia not just
Bergmann Glia, therefore any conclusions that lentiviral vectors can only transduce and
express within Bergmann Glia is unsupportable. However it might be possible to suggest the
lentiviral vector has transduced the Granule layer of the cerebellum cortex. As it is noticeable
the lentiviral vector appears to express predominantly in the Granule layers of the
cerebellum cortex.
Overall the expression shown in Figure 7B suggests Cathepsin K cannot alter the
transduction of eGFP expressing lentiviral vectors towards Bergmann Glia. However might
have an impact by manipulating transduction towards cells in the Granule layer of the
cerebellar cortex

of 49
Figure 7 Effect of Cathepsin K on lentiviral vector tropism towards Bergmann Glia. A shows a
representative Organotypic cerebellar slice transduced with eGFP lentiviral vectors at a concentration of
5x10
5
particles/mL particles/mL stained for GFAP markers. It also includes the individual stains for Hoechst,
GFAP and eGFP. B shows a representative Organotypic cerebellar slice transduced with eGFP lentiviral vectors
diluted in Cathepsin K at concentration of 5x10
5
particles/mL particles / mL at a dilution of 1 in 2. The comparison
shows Cathepsin K has no effect on the tropism of eGFP lentiviral vectors. All images were taken at 5x
magnification.
A
B

of 49
Figure 8 The effect of Cathepsin K at a 1 in 4 dilution on lentiviral vector tropism manipulation towards
Bergmann Glia astrocytes. A and B show two representative Organotypic cerebellar slice cultures transduced
with a 1 in 4 dilution lentiviral vector with Cathepsin K at a concentration ranging from 6.25x10
4
to 1x10
6
particles
/ slice. At a lower concentration the Cathepsin K enzyme does not alter the tropism of the lentiviral vector towards
Bergmann Glia, however might be manipulate the lentiviral vector towards the Granule layer of the cerebellar
cortex (all images taken at 5x magnification).
A

of 49
The Effect of Cathepsin K Inhibitor
After establishing lentiviral vector transduction cannot be manipulated towards Bergmann
Glia Astrocytes using Cathepsin K, I aimed to establish a method to test whether Cathepsin
K Inhibitor could manipulate the transduction of eGFP lentiviral vectors towards transducing
Purkinje neurons. As Purkinje neurons are implicated in the development of Ataxia
pathogenesis due to the mutation of TRPC3 genes. This model could test the lentiviral
vector targeting of Purkinje neurons and consequently begin accessing treatments for
Ataxia.
The experiment was designed by sectioning P7 rat cerebellums and culturing using tissue
inserts. A 1 in 2 dilution of eGFP lentiviral vector at a concentration of 5x105
particles/mL was
prepared by diluting in 1xPBS, alongside another solution eGFP lentiviral vector diluted 1 in
2 with Cathepsin K Inhibitor at a concentration of 5x105
particles/mL. 1μl of the eGFP
lentiviral vector was diluted in 1xPBS was added to one set of cerebellar slices. Whilst 1μl of
eGFP lentiviral vector diluted in Cathepsin K Inhibitor was pipetted on top of another set of
cerebellar slices. Each set of cerebellar slices was replenished with fresh tissue culture
media every other day with the addition of 1μl AraC at a concentration of 1mM. Light and
fluorescent microscope images were taken to observe the health of the slices. By day 8 in
vitro the slices were fixed and immunostained using Anti-Calbindin D28k neuronal marker.
Figure 9A and B shows a comparison between Anti-Calbindin D28k stained cerebellar slices
transduced either with eGFP lentiviral vectors diluted with or without Cathepsin K inhibitor.
Figure 9B shows the addition of Cathepsin K Inhibitor does not manipulate the tropism of
lentiviral vectors towards transducing Purkinje neurons, this is because there is little co-
staining of eGFP in Anti-Calbindin D28k positively stained Purkinje neurons.

of 49
Figure 9 The effect of Cathepsin K inhibitor on manipulating lentiviral vector tropism towards Purkinje
neurons. A shows a representative Organotypic cerebellar slice transduced with eGFP lentiviral vector only at a
concentration of 5x10
5
particles / mL (x5 magnification). B shows a representative Organotypic cerebellar slice
transduced with eGFP lentiviral vectors diluted with Cathepsin K inhibitor at a concentration of 5x10
5
particles /
mL (far left x5 & far right x10 magnification). C shows a representative Organotypic slice culture transduced with
a lentiviral vector diluted with Cathepsin K Inhibitor at a 1 in 4 dilution and at concentration ranging from 6.25x10
4
A
B
C

of 49
to 1x10
6
particles / slice (x5 magnification). All slices were stained with Anti-Calbindin D28k. Individual Hoechst,
eGFP and Anti-Calbindin D28k stains are included.
The overall conclusion is Cathepsin K Inhibitor does not influence the tropism of eGFP
expressing lentiviral vectors towards Purkinje neurons.
The Effect of Cathepsin K and Cathepsin K Inhibitors Prior to Lentiviral
Vector Transduction
Establishing Cathepsin K and Cathepsin K inhibitors did not manipulate eGFP expressing
lentiviral vectors towards Bergmann Glia or Purkinje neurons respectively, I hypothesised
what influence the addition of Cathepsin K or Cathepsin K Inhibitor might have prior to the
addition of the eGFP expressing lentiviral vector and whether this might influence their
tropism.
To investigate, I sectioned and cultured cerebellar slices, placing slices on tissue culture
inserts for the beginning of an in vitro culture. I diluted Cathepsin K or Cathepsin K inhibitors
in 1xPBS and pipetted 1μl on top of two sets of selected slices. I cultured the slices
overnight. On day 2 in vitro I added 1μl of eGFP lentiviral vectors diluted 1 in 2 with 1xPBS
at a concentration of 5x105
particles. By day 8 in vitro slices were fixed and immunostained
using Anti-Calbindin D28k neuronal marker and GFAP Glial marker.
Figure 10A and B shows GFAP stained cerebellar slices cultured with eGFP lentiviral vector
diluted in 1xPBS added on day 2 in vitro, compared against cerebellar slices cultured with
Cathepsin K diluted in 1xPBS added on day 1 in vitro and the addition of eGFP lentiviral
vector added on day 2 in vitro.
Figure 11A and B shows Anti-Calbindin D28k stained cerebellar slices cultured with eGFP
expressing lentiviral vector diluted in 1xPBS added on day 2 in vitro, compared against
cerebellar slices cultured with Cathepsin K inhibitor diluted in 1xPBS added on day 1 in vitro
and the addition of eGFP expressing lentiviral vectors added on day 2 in vitro.
The addition of Cathepsin K on day 1 in vitro and the addition of the lentiviral vector on day 2
in vitro as opposed to diluting the lentiviral vector directly with Cathepsin K and adding it to
the cerebellar slices on day 2 in vitro as shown in Figure 10 Illustrates Cathepsin K addition
on day 1 in vitro does not alter the tropism of the eGFP lentiviral vector towards Bergmann
Glia astrocytes. However might alter the transduction towards Granule cells.

of 49
Figure 10 The effect of Cathepsin K addition one day prior to lentiviral vector addition. A shows a
representative cerebellar slice transduced only with eGFP lentiviral vectors at a concentration of 5x10
5
particles
particles / mL (x10 magnification). B shows a representative cerebellar slice with Cathepsin K addition on day 1
in vitro and eGFP lentiviral vector added on day 2 in vitro (far left x10, top right x20 magnification and bottom right
x40 magnification). Both slices were stained with GFAP primary antibodies for Bergmann Glia astrocytes. The
white boxes indicate where images were magnified from.
AB

of 49
Figure 11 The effect of Cathepsin K addition one day prior to lentiviral vector addition. A shows a
representative cerebellar slice transduced with eGFP lentiviral vectors at a concentration of 5x10
5
particles
particles / mL (x5 magnification) added on day 2 in vitro. B Shows the difference of adding Cathepsin K Inhibitors
on day 1 in vitro and subsequently adding lentiviral vector on day 2 in vitro (far left x5, top right x10, bottom right
x40 magnification). One or two Purkinje neurons are co-stained, but not enough to suggest a manipulation of
lentiviral vector tropism. The white boxes indicate where the magnified images come from. The white arrow
points towards co-staining cells.
Meanwhile the addition of Cathepsin K inhibitor on day 1 in vitro and eGFP lentiviral vector
on day 2 in vitro, as opposed to diluting the lentiviral vector directly with Cathepsin K inhibitor
and adding it onto the slices on day 2 as shown in Figure 11B. Illustrates that the addition of
Cathepsin K a day earlier prior to lentiviral vector addition does not alter the tropism of the
eGFP lentiviral vector tropism towards Purkinje neurons.
A
B
HOECHST eGFP CALBINDIN
HOECHST eGFP CALBINDIN

of 49
AAV Tropism in Organotypic Slice Cultures
In addition to understanding the manipulation of lentiviral vector tropism in the cerebellar
cortex by Cathepsin K and Cathepsin K inhibitors in Organotypic slice cultures, I also wished
to compare the alteration of tropism using Adeno-Associated viral vectors (AAV’s). As these
vectors might have a better transduction capacity, which for the delivery of genes to alter the
physiology of target cells such as Purkinje neurons and Bergmann Glia affected by Ataxia
and Schizophrenia might be of significance to developing treatments.
I sectioned cerebellar slice cultures and incubated on semiporous tissue culture inserts. On
day 1 in vitro I added AAV2-7 and AAV2-9 to two sets of slices at a concentration of 1.1
x1013
/ml. 1ul was added per slice for each set. The slices were cultured for 8 days in vitro
with the replenishment of tissue culture media every other day with AraC to prevent Glial
overgrowth at a concentration of 1mM.On day 8 in vitro the slices were fixed in 4% PFA and
permeabilised in 0.5% Triton X-100. The slices were blocked in 1% donkey horse serum
A selection of the slices were stained for Bergmann Glia using GFAP primary antibody at a
dilution of 1 in 500, while a selection of slices were stained for Purkinje neurons using Anti-
Calbindin D28k at a dilution of 1 in 1000. Both were washed and stained with Cy3
secondary antibodies at a dilution of 1 in 100 for the GFAP stained slices, while 1 in 1000 for
the Anti-Calbindin D28k stained slices. The slices were washed and the nuclei stained using
Hoechst. The slices were analysed using fluorescent microscopy.
Figure 12 shows the comparison of AA2-7 transduced Organotypic slice cultures either
stained with GFAP or Anti-Calbindin D28k. It shows the AAV2-7 eGFP expressing
predominantly in the white matter of the cerebellum in Figure 12A. Meanwhile the Anti-
Calbindin D28k staining has picked out a large number of Purkinje neurons within the
cerebellar cortex. The higher magnification images illustrate small number of Purkinje
neurons co-staining as shown in Figure 12B.

of 49
Figure 12 The effect of AAV2-7 transduction on cerebellar slices. A shows a representative slice culture
transduced with eGFP expressing AAV2-7 stained with GFAP primary antibodies (image taken at 5x
magnification). B shows a representative slice culture transduced with eGFP expressing AAV2-7 stained with
Anti-Calbindin D28k primary antibodies (image taken at x5 magnification). The white boxes indicate where
magnified images have come from.
I was unable to compare these control slices against the addition of Cathepsin K or
Cathepsin K Inhibitor because of limited project time.
However, I repeated the same protocol for Figure 12 above for Organotypic slices
transduced with AAV2-9, stained with either GFAP primary antibodies at a dilution of 1 in
500 or Anti-Calbindin D28k primary antibodies at a dilution of 1 in 1000, as seen in Figure
13. In addition to a representative slice transduced with AAV2-9 diluted 1 in 2 with Cathepsin
A
B

of 49
K, to observe whether the enzyme might alter the tropism of the AAV2-9 towards Bergmann
Glia, as seen in Figure 14.
Figure 13 The effect of transducing Organotypic slice cultures with AAV2-9. A shows a representative slice
culture transduced AAV2-9 and stained with Anti-Calbindin D28k primary antibodies. B shows a representative
slice transduced with AAV2-9 and stained with GFAP primary antibodies. Images were taken at x5 magnification.
B

of 49
Figure 14 The effect of Cathepsin K on the tropism of AAV2-9. The representative Organotypic slice culture
shows Cathepsin K has no effect on manipulating the transduction of AAV2-9 towards Bergmann Glia.
Overall, Figure 13 shows the transduction of cerebellar slices with AAV2-7 and AAV2-9 is
remarkably different compared to lentiviral vector transduction. The AAV’s appear to
transduce predominantly the white matter of the cerebellum and express throughout the
slices more diffusely. In comparison, Figure 14 shows Cathepsin K does not alter the tropism
of AAV2-9 towards Bergmann Glia, as no co-staining is apparent.

of 49
The objective of this project was to develop an Organotypic cerebellar slice culture to test
the ability of lentiviral and Adeno-Associated viral vectors to transduce target cells in the
cerebellar cortex. Specifically Purkinje neurons affected by Ataxia and Bergmann Glia
affected by Schizophrenia. This would pave the way towards developing treatments for the
aforementioned diseases.
The project accomplished obtaining Organotypic slice cultures from P7 post-natal rat
cerebellums and successfully sectioning them using a McILwain tissue chopper. The tissue
culture insert method permitted long term culture and retention of cerebellum cortex
architecture. Additionally this provided the capacity to perform immunohistochemistry for the
staining of target cells. In total this process took on average two to two half weeks per
experiment. The in vitro culture system permitted the addition of eGFP expressing lentiviral
and Adeno-Associated viral vectors.
I showed that an optimum dilution of 1 in 2 dilution for eGFP lentiviral vectors (equivalent to
5x105
particles/slice) would facilitate the expression needed to transduce cerebellar slices
successfully after 5 days in vitro. Furthermore, the optimum dilution permitted the addition of
Cathepsin K and Cathepsin K inhibitors for evaluating manipulation of viral vector tropism.
I demonstrated that the addition of AraC at a concentration of 1mM was able prevent Glia
overgrowth whilst permitting Glia cells to survive within the slices for investigation of
Bergmann Glia physiology within the cerebellar cortex. Moreover, AraC permitted
accessibility to the astrocytes for effective staining with GFAP primary antibodies to
effectively stain Glia.
After multiple strategies were explored, I showed that an 8 day in vitro culture on semiporous
tissue culture inserts permitted the time necessary for eGFP lentiviral and Adeno Associated
Viral Vectors expression. Furthermore, this provided a timescale to explore the effects of
Cathepsin K and Cathepsin K inhibitors, potentially manipulating viral vector tropism.
I illustrated that Bergmann Glia could be stained for using GFAP primary antibodies, whilst
Purkinje neurons could be stained for using Anti-Calbindin D-28k primary antibodies.
Overlaid images of cerebellar slices with the expression of eGFP lentiviral or Adeno-
Associated viral vectors, Hoechst and Anti-Calbindin D-28k or GFAP stain provided a means
to access whether viral vectors could be targeted towards Purkinje neurons to study Ataxia
or towards Bergmann Glia to study Schizophrenia.
Unfortunately, the addition of Cathepsin K was unable alter the tropism of eGFP lentiviral
vectors towards Bergmann Glia Meanwhile, the addition of Cathepsin K Inhibitors was

of 49
unable to manipulate the tropism of eGFP lentiviral vectors towards Purkinje neurons.
Possible reasons, could be poor physical access to the target cells, the wrong type of
pseudotype receptor or the method used to target the cells in the first place. Possible
avenues for overcoming might include using different pseudotypes on the viral vectors such
as Rabies, Mokola and Ross River Virus glycoproteins, using a gene gun or injecting the
slices directly with a fine syringe.
Meanwhile I was able to illustrate that, AAV2-7 and AAV2-9 can transduce cells of the
cerebellum. Both showed a marked difference in expression of eGFP compared to the
lentiviral vectors within the Organotypic slices. Due to project time constraints I was unable
to further access the effects of Cathepsin K and Cathepsin K inhibitors altering the tropism of
the AAV2-7 viral vectors towards Purkinje neurons or Bergmann Glia. However I did show
that AAV2-9 tropism is not altered to Bergmann Glia due to Cathepsin K addition. Overall the
AAV results showed restricted transduction within the slices, particularly in the grey matter.
Future Work
Additionally, using more selective antibodies such as S100, to distinguish the differences
between Bergmann Glia from other GFAP expressing cells. Meanwhile using Anti-Calbindin
D-28k and Paravalbumin to determine Purkinje neurons, compared against Golgi, Basket
and Stellate neuronal cells. Purkinje neurons stain with Anti-Calbindin D-28k and
Paravalbumin. Staining with NeuN might have identified whether the lentiviral vector was
definitely transducing Granule cells.
A comparison between the effectiveness of sectioning with a Vibratome verses a McILwain
tissue chopper might have produced higher numbers of usable cerebellar slices.
Additionally, testing Cathepsin K at acidic conditions might have increased lentiviral vector
transduction of Bergmann Glia. Alongside, testing other Cathepsin enzymes from the 11
strong Cathepsin family

of 49
Conclusion
Organotypic slice culture of sectioned Post-natal 7 day old rat cerebellums provides an in
vitro model for accessing the targeting of lentiviral and Adeno-Associated viral vectors.
Further investigation is needed to access whether Cathepsin’s can alter the tropism of viral
vectors targeting cells in the cerebellar cortex.

of 49
References
1. Gahwiler, B. H., M. Capogna, D. Debanne, R. A. McKinney, and S. M. Thompson.
1997. Organotypic slice cultures: a technique has come of age. Trends in
Neurosciences 20: 471-477.
2. Becker, E. B. 2015. From Mice to Men: TRPC3 in Cerebellar Ataxia. Cerebellum.
3. Burnet, P., S. Eastwood, G. Bristow, B. Godlewska, P. Sikka, M. Walker, and P.
Harrison. 2008. D-Amino acid oxidase (DAO) activity and expression are increased in
schizophrenia. Mol Psychiatry 13: 658-660.
4. Ono, K., Y. Shishido, H. K. Park, T. Kawazoe, S. Iwana, S. P. Chung, R. M. Abou El-
Magd, K. Yorita, M. Okano, T. Watanabe, N. Sano, Y. Bando, K. Arima, T. Sakai, and
K. Fukui. 2009. Potential pathophysiological role of D-amino acid oxidase in
schizophrenia: immunohistochemical and in situ hybridization study of the expression
in human and rat brain. J Neural Transm 116: 1335-1347.
5. Goenawan, H., and H. Hirai. 2012. Modulation of lentiviral vector tropism in
cerebellar Purkinje cells in vivo by a lysosomal cysteine protease cathepsin K.
Journal of Neurovirology 18: 521-531.
6. Hoppenbrouwers, S. S., D. J. Schutter, P. B. Fitzgerald, R. Chen, and Z. J.
Daskalakis. 2008. The role of the cerebellum in the pathophysiology and treatment of
neuropsychiatric disorders: a review. Brain Res Rev 59: 185-200.
7. Glickstein, M., and K. Doron. 2008. Cerebellum: connections and functions.
Cerebellum 7: 589-594.
8. Baldacara, L., J. G. Borgio, A. L. Lacerda, and A. P. Jackowski. 2008. Cerebellum
and psychiatric disorders. Rev Bras Psiquiatr 30: 281-289.
9. Steinlin, M. 2008. Cerebellar disorders in childhood: cognitive problems. Cerebellum
7: 607-610.
10. Crossman, A., and D. Neary. 2010. Neuroanatomy. An Illustrated Colour Text.
Churchill and Livingstone.
11. Ito, M. 2002. Historical review of the significance of the cerebellum and the role of
Purkinje cells in motor learning. Ann N Y Acad Sci 978: 273-288.
12. Steinlin, M. 2008. Cerebellar Disorders in Childhood: Cognitive Problems.
Cerebellum 7: 607-610.
13. Andreasen, N. C., and R. Pierson. 2008. The role of the cerebellum in schizophrenia.
Biol Psychiatry 64: 81-88.
14. Manto, M., and M. Pandolfo. 2002. The Cerebellum And Its Disorders, Cambridge
University Press.

of 49
15. Knierim, J. Department of Neuroscience. John Hopkins University.,
http://neuroscience.uth.tmc.edu/s3/chapter05.html.
16. Deerinck, T. 2008. Cerebellum. National Centre for Imaging Microscopy and Imaging
Research, http://ncmir.ucsd.edu/gallery/index.shtm.
17. Tanaka, M. 2009. Dendrite formation of cerebellar Purkinje cells. Neurochem Res 34:
2078-2088.
18. Tam, W. Y., C. K. Leung, K. K. Tong, and K. M. Kwan. 2011. Foxp4 is essential in
maintenance of Purkinje cell dendritic arborization in the mouse cerebellum.
Neuroscience 172: 562-571.
19. Lu, H.-x., H. Levis, Y. Liu, and T. Parker. 2011. Organotypic slices culture model for
cerebellar ataxia: Potential use to study Purkinje cell induction from neural stem cells.
Brain Research Bulletin 84: 169-173.
20. Molofsky, A. V., R. Krencik, E. M. Ullian, H. H. Tsai, B. Deneen, W. D. Richardson, B.
A. Barres, and D. H. Rowitch. 2012. Astrocytes and disease: a neurodevelopmental
perspective. Genes Dev 26: 891-907.
21. Xu, H., Y. Yang, X. Tang, M. Zhao, F. Liang, P. Xu, B. Hou, Y. Xing, X. Bao, and X.
Fan. 2013. Bergmann glia function in granule cell migration during cerebellum
development. Mol Neurobiol 47: 833-844.
22. Blomer, U., L. Naldini, I. M. Verma, D. Trono, and F. H. Gage. 1996. Applications of
gene therapy to the CNS. Human Molecular Genetics 5: 1397-1404.
23. Blomer, U., L. Naldini, I. M. Verma, D. Trono, and F. H. Gage. 1996. Stable
transduction of neurons in the adult brain by an HIV-derived lentiviral vector. Society
for Neuroscience Abstracts 22: 324-324.
24. Blomer, U., L. Naldini, T. Kafri, D. Trono, I. M. Verma, and F. H. Gage. 1997. Highly
efficient and sustained gene transfer in adult neurons with a lentivirus vector. Journal
of Virology 71: 6641-6649.
25. Bukovsky, A. A., J. P. Song, and L. Naldini. 1999. Interaction of human
immunodeficiency virus-derived vectors with wild-type virus in transduced cells.
Journal of Virology 73: 7087-7092.
26. Dull, T., R. Zufferey, M. Kelly, R. J. Mandel, M. Nguyen, D. Trono, and L. Naldini.
1998. A third-generation lentivirus vector with a conditional packaging system.
Journal of Virology 72: 8463-8471.
27. Naldini, L., U. Blomer, F. H. Gage, D. Trono, and I. M. Verma. 1996. Efficient
transfer, integration, and sustained long-term expression of the transgene in adult rat
brains injected with a lentiviral vector. Proceedings of the National Academy of
Sciences of the United States of America 93: 11382-11388.

of 49
28. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D.
Trono. 1996. In vivo gene delivery and stable transduction of nondividing cells by a
lentiviral vector. Science 272: 263-267.
29. Hall, K. M., T. L. Horvath, K. Cornetta, R. Abonour, and E. F. Srour. 2003. Detection
of reverse transcribed DNA in quiescent human bone marrow CD34+cells following
retroviral transduction. Blood 102: 995A-996A.
30. Davidson, B. L., and X. O. Breakefield. 2003. Viral vectors for gene delivery to the
nervous system. Nature Reviews Neuroscience 4: 353-364.
31. Froelich, S., A. Tai, and P. Wang. 2010. Lentiviral vectors for immune cells targeting.
Immunopharmacology and Immunotoxicology 32: 208-218.
32. Banasik, M. B., and P. B. McCray, Jr. 2010. Integrase-defective lentiviral vectors:
progress and applications. Gene Therapy 17: 150-157.
33. Shaw, A., and K. Cornetta. 2014. Design and Potential of Non-integrating Lentiviral
Vectors. Journal of Biomedicines. 14-35.
34. Schambach, A., D. Zychlinski, B. Ehrnstroem, and C. Baum. 2013. Biosafety
Features of Lentiviral Vectors. Human Gene Therapy 24: 132-142.
35. Sakuma, T., M. A. Barry, and Y. Ikeda. 2012. Lentiviral vectors: basic to translational.
Biochemical Journal 443: 603-618.
36. Deglon, N., and P. Hantraye. 2005. Viral vectors as tools to model and treat
neurodegenerative disorders. Journal of Gene Medicine 7: 530-539.
37. Murlidharan, G., R. J. Samulski, and A. Asokan. 2014. Biology of adeno-associated
viral vectors in the central nervous system. Front Mol Neurosci 7: 76.
38. Mingozzi, F., and H. Buning. 2015. Adeno-Associated Viral Vectors at the Frontier
between Tolerance and Immunity. Front Immunol 6: 120.
39. Brix, K., A. Dunkhorst, K. Mayer, and S. Jordans. 2008. Cysteine cathepsins: cellular
roadmap to different functions. Biochimie 90: 194-207.
40. Lecaille, F., D. Bromme, and G. Lalmanach. 2008. Biochemical properties and
regulation of cathepsin K activity. Biochimie 90: 208-226.
41. Repnik, U., V. Stoka, V. Turk, and B. Turk. 2012. Lysosomes and lysosomal
cathepsins in cell death. Biochim Biophys Acta 1824: 22-33.
42. Turk, V., V. Stoka, O. Vasiljeva, M. Renko, T. Sun, B. Turk, and D. Turk. 2012.
Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim
Biophys Acta 1824: 68-88.
43. Berdowska, I. 2004. Cysteine proteases as disease markers. Clin Chim Acta 342: 41-
69.

of 49
44. de Mendoza, T. H., B. Balana, P. A. Slesinger, and I. M. Verma. 2011. Organotypic
Cerebellar Cultures: Apoptotic Challenges and Detection. Jove-Journal of Visualized
Experiments: 5.
45. Ghoumari, A. M., I. Dusart, M. El-Etr, F. Tronche, C. Sotelo, M. Schumacher, and E.
E. Baulieu. 2003. Mifepristone (RU486) protects Purkinje cells from cell death in
organotypic slice cultures of postnatal rat and mouse cerebellum. Proceedings of the
National Academy of Sciences of the United States of America 100: 7953-7958.
46. Gogolla, N., I. Galimberti, V. DePaola, and P. Caroni. 2006. Staining protocol for
organotypic hippocampal slice cultures. Nature Protocols 1: 2452-2456.
47. Pham, A. H., and D. C. Chan. 2014. Analyzing mitochondrial dynamics in mouse
organotypic slice cultures. Methods in enzymology 547: 111-129.

Investigating Viral Vector Targeting in an Organotypic Cerebellar Slice Culture- Eric Garson 2015

Recommended

Recommended

More Related Content

What's hot

What's hot (6)

Similar to Investigating Viral Vector Targeting in an Organotypic Cerebellar Slice Culture- Eric Garson 2015

Similar to Investigating Viral Vector Targeting in an Organotypic Cerebellar Slice Culture- Eric Garson 2015 (20)

Investigating Viral Vector Targeting in an Organotypic Cerebellar Slice Culture- Eric Garson 2015