investigation of the effects of innate immune activation

Investigation of the effects of Innate immune

activation on synaptic protein expression in a

Dopaminergic like neuronal cell line.

Presented by Darragh Gallagher

Parkinson's disease is a neurodegenerative disease involving the loss of dopaminergic neurons in the substantia nigra in the brain.

Background

Background Risk factors associated with Parkinson's

disease:

1) Age2) Viral infection3) Family history4) Male gender5) Environmental: Toxins, repeated head injury

Background Neurotransmission is dependent on synaptic

proteins.

Background

Virus types associated with Parkinson's disease:

1. Influenza type A2. H.I.V3. Pox 4. HCV

Background

BackgroundNumerous Viruses are

neurotropic and induce inflammation.

Secondary consequence can be Parkinsonism.

1918 Influenza pandemic concurrent with encephalitis lethargica 1916-1927 (Ravenholt and Foege, 1982).

Background Neurons express Toll Like Receptors.

Background Polyinosinic:polycytidylic acid (Poly I:C).

Immunostimulant used to simulate viral infections.

Synthetic analog of double-stranded RNA which is a molecular pattern associated with viral infection.

Recognized by TLR3 inducing NFĸB and production of cytokines.

6-hydroxydopamine (6-OHDA).

Neurotoxic synthetic organic compound.

Used with selective noradrenaline reuptake inhibitor.

Background

Expect 6-OHDA to have a retrograde loss of Tyrosine Hydroxylase -positive neurons in the substania nigra. (Debeir, Ginestet et al. 2005)

Expect to see increased levels of PSD95 because it inhibits D1 signalling by reduced D1 expression at cell surface as a consequence of enhanced constitutive endocytosis. (Porras, Berthet et al.)

Background

SH-SY5Y cells.

Sub cloned and isolated from human neuroblastoma cell line SK-N-SH.

Express Dopaminergic markers.

Background

H1: If a cell culture system is infected with a Virus or Neurotoxin it will change expression in synaptic proteins.

H0: If a cell culture system is infected with a Virus or Neurotoxin it will not change expression in synaptic proteins.

Hypothesis

To determine if viral infection in a cell culture system results in expression changes in synaptic proteins Tyrosine Hydroxylase (TH) and Post Synaptic Density 95 (PSD-95)

This will be achieved by treating SHSY5Y cells with a viral mimetic Poly I:C and a Neurotoxin 6-OHDA.

Western blotting will measure synaptic protein expression patterns.

Aim of Experiment

Experimental Design

TREATMENT 1 TREATMENT 2 TREATMENT 3 TREATMENT 4CONTROL 1

(20µl medium)POLY I:C

(20µg/ml)CONTROL 1

(20µl medium)POLY I:C

(20µg/ml)

+ + + +CONTROL 2

(20µl medium)CONTROL 2

(20µl medium)6-OHDA (20µM) 6-OHDA (20µM)

SH-SY5Y CELLS

Western Blotting

Experimental Design

CONTROLCONTROL 6-OHDACONTROL

CONTROL

CONTROL

CONTROL CONTROL CONTROL

CONTROL CONTROL CONTROLCONTROL

Poly I:C Poly I:C Poly I:C Poly I:C Poly I:CPoly I:C

6-OHDA 6-OHDA

6-OHDA 6-OHDA 6-OHDA

Experimental DesignWestern Blot procedure

Antibodies for experiment

Experiemntal Design

ANTIBODY Species cross reactivity

Molecular Weight

Source

Tyrosine Hydroxylase

Human, Mouse, Rat 50-60kDa

Rabbit

PSD-95Human, Mouse, Rat 95kDa Rabbit IgG

1. SHSY-5Y cells seeded.

2. Treatment with POLY I:C and 6-OHDA.

3. Cells lysed and proteins assessed by Bradford assay.

4. Western blot performed to detect changes in expression.

Methods