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

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

Morphology of cells in the nervous system

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

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

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

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

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

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

Reactive astrocytes in human disease

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

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)

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.

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

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

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

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

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

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

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

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.

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

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