Technical Reference #2019

2019.

A Pathologic Cascade Leading to Synaptic Dysfunction in a-Synuclein-Induced Neurodegeneration David A. Scott; Iustin Tabarean; Yong Tang; Anna Cartier; Eliezer Masliah; and Subhojit Roy, University of California San Diego, Journal of Neuroscience, 30(2019), (2010)
Link To Paper

Abstract:
Several neurodegenerative diseases are typified by intraneuronala-synuclein deposits synaptic dysfunction and dementia. While even modest a-synuclein elevations can be pathologic the precise cascade of events induced by excessive a-synuclein and eventually culminating in synaptotoxicity is unclear. To elucidate this we developed a quantitative model system to evaluate evolving a-synucleininduced pathologic events with high spatial and temporal resolution using cultured neurons from brains of transgenic mice overexpressing fluorescent-human-a-synuclein. Transgenic a-synuclein was pathologically altered over time and overexpressing neurons showed striking neurotransmitter release deficits and enlarged synaptic vesicles; a phenotype reminiscent of previous animal models lacking critical presynaptic proteins. Indeed several endogenous presynaptic proteins involved in exocytosis and endocytosis were undetectable in a subset of transgenic boutons (“vacant synapses”) with diminished levels in the remainder suggesting that such diminutions were triggering the overall synaptic pathology. Similar synaptic protein alterations were also retrospectively seen in human pathologic brains highlighting potential relevance to human disease. Collectively the data suggest a previously unknown cascade of events where pathologic a-synuclein leads to a loss of a number of critical presynaptic proteins thereby inducing functional synaptic deficits.

Materials & Methods:
Cell cultures from transgenic mice. The PDGF-h-a-syn:GFP mice (C57/B6 background) used in this study have been described previously (Rockenstein et al. 2005). Hippocampal neurons were obtained from brains of heterozygous postnatal (P0 –P2)a-synuclein:GFP transgenic pups. Pups were screened using a “GFP flashlight” (Nightsea) that made the GFP1 pups glow. Nontransgenic littermates were used as controls. For all cell biology experiments dissociated cells were plated at a density of 100000 cells/cm2 in poly-D-lysine-coated glass-bottom culture dishes (Mattek) and maintained in Neurobasal/B27 media (Invitrogen) supplemented with 0.5mM glutamine. All animal studies were performed in accordance with University of California guidelines. Immunofluorescence studies were performed as previously described (Roy et al. 2008). Briefly cultured neurons were fixed with paraformaldehyde/120mMsucrose rinsed several times and stained with the appropriate antibodies. Alexa 488 594 and 647 dyes (Invitrogen) were used as secondary antibodies.

Microscopic Technique
Immunofluorescence imaging, inverted motorized epifluorescence microscopy

Cell Type(s)
Hippocampal neurons