15 research outputs found
A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell–Cell Induced Protein Secretion
Recent evidence suggests that the
extracellular protein milieu
is much more complex than previously assumed as various secretome
analyses from different cell types described the release of hundreds
to thousands of proteins. The extracellular function of many of these
proteins has yet to be determined particularly in the context of three-dimensional
tissues with abundant cell–cell contacts. Toward this goal,
we developed a strategy of dual SILAC labeling astrocytic cultures
for in silico exclusion of unlabeled proteins from serum or neurons
used for stimulation. For constitutive secretion, this strategy allowed
the precise quantification of the extra-to-intracellular protein ratio
of more than 2000 identified proteins. Ratios covered 4 orders of
magnitude indicating that the intracellular vs extracellular contributions
of different proteins can be variable. Functionally, the secretome
of labeled forebrain astrocytic cultures specifically changed within
hours after adding unlabeled, “physiological” forebrain
neurons. “Nonphysiological” cerebellar hindbrain neurons,
however, elicited a different, highly repulsive secretory response.
Our data also suggest a significant association of constitutive secretion
with the classical secretion pathway and regulated secretion with
unconventional pathways. We conclude that quantitative proteomics
can help to elucidate general principles of cellular secretion and
provide functional insight into the abundant extracellular presence
of proteins
Single-dose ethanol intoxication causes acute and lasting neuronal changes in the brain
Alcohol intoxication at early ages is a risk factor for the development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, we used stable-isotope-labeled mice combined with quantitative mass spectrometry to screen more than 2,000 hippocampal proteins, of which 72 changed synaptic abundance up to twofold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and ankyrin-G. Based on these candidate proteins, we found acute and lasting molecular, cellular, and behavioral changes following a single intoxication in alcohol-naive mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila flies. This study introduces mitochondrial trafficking as a process implicated in reward learning and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior
TRI to Communicate: Public Knowledge of the Federal Toxics Release Inventory
Investigate public knowledge and use of information from the U.S. Toxics Release Inventory (TRI), the most internationally prominent and well-regarded environmental information program, providing industrial facility chemical release and transfer data since 1989. Copyright (c) 2007 Southwestern Social Science Association.