4395 alpha-Synuclein Induced Reactive Gliosis

OBJECTIVES/GOALS: Reactive gliosis is a hallmark of neurodegenerative disease and is characterized by the release of pro-inflammatory cytokines and physiologic changes to glial cells. Our work identifies a novel inflammatory glial-glial cell interaction and role for mGluR5 that has the potential to provide novel insight into the mechanisms of neurodegeneration. METHODS/STUDY POPULATION: Cell Culture: Mouse primary astrocytes and microglia were isolated from P0-P3 C57BL/6 or Cx3cr1(GFP/+) mice.(1) Treatment: Glia were treated with oligomeric α-synuclein 1μg/mL or mGluR5 agonist CHPG 100 μM.(2,3) ELISA: Glia culture media was collected and analyzed according to the manufacturer. qRT-PCR: TaqMan™ probes were used according to manufacturer on extracted glia mRNA. ICC: Microglia were labeled with 1:750 Rb x Iba1 (Wako) and 1:500 Alexa Fluor 488 Gt x Rb. Phagocytosis Assay: Primary glia were treated with α-synuclein or astrocyte-conditioned culture media for 24-48hrs. For treatment of microglia with conditioned media, astrocytes were washed with PBS and fresh media was added to prevent carry over of α-synuclein to microglia. The number of fluorescent microbeads per microglia was quantified. RESULTS/ANTICIPATED RESULTS: Mouse primary cortical astrocytes simulated with α-synuclein aggregates adopt a reactive A1 phenotype independent of microglial stimulation. This A1 phenotype is characterized by release of pro-inflammatory cytokines including Complement Component 3 and the monocyte chemoattractant CCL2. Reactive astrocyte media induces a phagocytic phenotype in primary mouse microglia. Along with this, α-synuclein-directed microglial phagocytosis was attenuated with the addition of the mGluR5 agonist CHPG. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest that oligomeric α-synuclein is capable of inducing a reactive phenotype in astrocytes independent of microglia and implicate crosstalk between glia as an important mediator of inflammation and microglial phagocytosis in synucleinopathies.