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

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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.

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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.

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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)

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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

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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

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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

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Introduction

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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

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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.

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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).

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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).

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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

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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).

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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 30oC 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).

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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.

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Materials and Methods

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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), 37oC 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 8x106cells/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

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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 37oC (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 4oC

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 4oC 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 4oC. 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 -80oC.(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 37oC 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

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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 37oC

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.

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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 1x109particlaes/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.

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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 4oC.

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 4oC.

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 4oC.

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 4oC.

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).

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Results

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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

5x105particles/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

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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 37oC 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.

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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.

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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 5x105particles/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

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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

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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

5x105particles/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 5x105particles/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

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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 6.25x104 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

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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 5x105particles/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 5x105particles/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.

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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 5x105 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 5x105 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 6.25x104

A

B

C

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to 1x106 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 5x105particles. 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.

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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 5x105particles

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.

A B

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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 5x105particles

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

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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.

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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

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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

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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.

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Discussion

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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

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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

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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.

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investigating viral vector targeting in an organotypic cerebellar slice culture- eric garson 2015

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