1. The identification of novel markers for reactive astrocytes A1 & A2 subtype makers

2. Classification of cells in the nervous system
The nervous system is made up of two groups of cells:
Neurons - responsible for the transmission of nerve impulses, structural unit of the nervous system.
Glial cells- provide functional and nutritional support to neurons:
Astrocytes : Reactive & Non-Reactive
Oligodendrocytes
Microglia
Ependymal cells

3. Morphology of cells in the nervous system

4. Functions of astrocytes
Trophic support for neurons
Promote formation and function of neurons
Synaptic pruning by phagocytosis
Homeostatic support functions

5. After brain injury & disease…
Astrocytes undergo a transformation called “reactive astrocytosis”
Upregulation of many genes and glial scar formation occurs after acute CNS injury
Transformed to become reactive astrocytes which can support or hinder CNS recovery

6. Subtypes of reactive astrocytes
induced by neuroinflammation
destructive to neuron synapses
A2:
induced by ischemia
upregulate neurotrophic factors (protective)

7. The role of A1 astrocytes in human CNS diseases
Previous research investigated whether A1 astrocytes are present in neuroinflammatory and neurodegenerative diseases
Microglia are found in neurodegenerative diseases (microglia activates A1 astrocytes as mentioned earlier)
The Complement component 3 (C3) gene is also highly expressed in neurodegenerative/inflammatory diseases
To identify the presence of C3 expressing A1 astrocytes

8. Background - Methods In situ hybridisation and immunochemistry
Post-mortem tissues from patients with:
Alzheimer’s disease
Huntington’s disease
Parkinson’s disease
Amyotrophic Lateral Sclerosis
Multiple Sclerosis

9. Background - Results
GFAP- and S100β-positive astrocytes that were C3-positive were located in regions traditionally associated with each disease
qPCR analysis confirmed the presence of C3 expression in post-mortem tissue samples
In human AD, about 60% of GFAP-positive astrocytes in the prefrontal cortex were positive for C3
Conclusion: A1 reactive astrocytes are present in most major neurodegenerative diseases

10. Reactive astrocytes in human disease

11. Background - Conclusions
A1 reactive astrocytes form as a pathological response of the CNS to LPS- induced neuroinflammation, acute CNS injury or neurodegenerative diseases
Microglia play an important role in inducing A1 astrocytes formation via secretion of Il-1α, TNF and C1q
A1 astrocytes:
secrete neurotoxins that induce neuronal and oligodendrocyte death
Also activate components of complement that drive synapses degeneration
A1 astrocytes are abundant in neurodegenerative diseases, suggesting a contribution to disease progression

12. Experimental aims
To investigate gene expression in astrocytes and other glials cells in both a healthy and injured state
Determine the co-localisation of probes with fibroblast growth factor receptor 3 (fgfr3)

13. Experimental method - Mouse model and probes
Mice were injected with a single dose of 5mg/kg of the endotoxin lipopolysaccharide (LPS)from Escherichia coli O55:B55 of their spinal cord to induce neuroinflammation.
After 24 hours they were culled.
Synthesis of probes which were the genes to be investigated (Amigo2, Aspg, Cd109, C3, GFAP, iigp1, LCN2, Serping1, Slc10a6, Steap4, Tgm1, Ugt1a1)
Reason why these probes were chosen: These probes are associated with synthesis of compounds released by reactive astroyctes in the neuronal cells.

14. Experimental method - Staining protocol
DIG labelled probes diluted into hybridisation buffer incubated with section
Slides washed with in MABT to permeabilize samples
Sections were blocked and re-washed with PBS buffer before overnight storage.
Addition of Tyramide reagent and Hoechst preparation
Fixed with DAKO fluorescent mounting medium.
Sections imaged via confocal microscopy

15. Single chromogenic in-situ with NeuN and Oligo 2 antibody
Each probe has 2 slides, one for olig2 the other NeuN.
Hybridisation buffer added with fgfr3 probes coverslip and incubated at 65 °C oven overnight with plenty of 65 °C Wahs Buffer in the bottom of chamber.
Washing of slides with MABT followed by the addition of blocking buffer
Rewashing of slides with PBS and application of olig2 or NeuN antibodies

16. Double RNA fluorescent in-situ hybridisation
DIG labelled probe and FITC labelled probe were diluted in hybridisation buffer before being incubated with the sections
Sections were washed and FITC POD-conjugated antibody was applied
Slides were washed with PBS and FITC Tyramide fluorescent amplification reagent was added
Sections rewashed with PBS and blocked with the addition of diluted DIG POD-conjugated antibody solutions
Addition of CY3 Tyramide Fluorescent Amplification Reagent and treatment of Hoechst in PBS, fixed with DAKO fluorescent mounting medium

17. Expected results…
The gene expression of tested probes including CLCF1, C2, iigp1, LCN2, Ugt1a1,C109 in reactive astrocytes in different condition of the cell (especially during injured state)
Co-localization of at least one of the probes with fgfr3

18. Results - single chromogenic in-situ hybridization
Probe/Cell Condition
Clcf1 C3
iigp1
LCN2
Ugt1a1
C109
Healthy
Expressed
Increased expression
Not expressed
(Oligodendrocytes) Expressed in other neuronal cells -
Injured
Highly Expressed
Slightly increased expression
Not expressed (Oligodendrocytes), Expressed more in other neuronal cells
Not expressed(Oligodendrocytes) Expressed in other neuronal cells

19. Interpretation Of Results
All the probes including C3, LCN2, iigp1 and utg1a1 were similarly expressed in both control and LPS injured tissues while Clcf1 was visibly higher in the injured tissue.
Results indicate that the Clcf1 probe is the most suitable marker for detecting reactive cells during CNS injury.
Interpretation Of Results

20. Results - co-localization of probes with fgfr3
Probe/Cell Condition
Clcf1 C3
LCN2
Ugt1a1
C109
Healthy
Negative -
Injured

21. Interpretation of Results
None of the probes showed colocalization with the fibroblast growth factor receptor 3.
The probes are unable to localise to and replace the fgfr3.
Suggestive to be due to the use of two stains, making identification of positive cells difficult.

22. Future work
To repeat experiments focusing on optimising the staining protocol
To investigate the underlying mechanisms of the obtained experiments results
To use a different set of probes to identify the co-localization with fgfr3

23. Thank you for your attention!