ABSTRACT
CRIPT, the cysteine-rich PDZ-binding protein, binds to the third PDZ domain of PSD-95 (postsynaptic density protein 95) family proteins and directly binds microtubules, linking PSD-95 family proteins to the neuronal cytoskeleton. Here, we show that overexpression of a full-length CRIPT leads to a modest decrease, and knockdown of CRIPT leads to an increase in dendritic branching in cultured rat hippocampal neurons. Overexpression of truncated CRIPT lacking the PDZ domain-binding motif, which does not bind to PSD-95, significantly decreases dendritic arborization. Conversely, overexpression of a full-length CRIPT significantly increases the number of immature and mature dendritic spines, and this effect is not observed when CRIPT∆PDZ is overexpressed. Competitive inhibition of CRIPT binding to the third PDZ domain of PSD-95 with PDZ3-binding peptides resulted in differential effects on dendritic arborization based on the origin of respective peptide sequence. These results highlight multifunctional roles of CRIPT during development and underscore the significance of the interaction between CRIPT and the third PDZ domain of PSD-95.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Disks Large Homolog 4 Protein/physiology , Hippocampus/cytology , Neuronal Plasticity/physiology , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Motifs , Animals , Binding, Competitive , Cells, Cultured , Dendritic Spines/physiology , Dendritic Spines/ultrastructure , Gene Knockdown Techniques , Microtubules/metabolism , Microtubules/ultrastructure , Protein Binding , Protein Interaction Mapping , RNA Interference , RNA, Small Interfering/genetics , RatsABSTRACT
Dyshomeostasis of amyloid-ß peptide (Aß) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer's disease. Aß appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer's disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aß-induced depression. Mechanistically, Aß triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aß-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aß-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aß signaling.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Cognition Disorders/physiopathology , PTEN Phosphohydrolase/physiology , Synaptic Transmission/physiology , Alzheimer Disease/complications , Amyloid beta-Peptides/toxicity , Animals , Cognition Disorders/complications , Disease Models, Animal , Gene Knock-In Techniques , Mice , Mice, Transgenic , PDZ Domains/genetics , PDZ Domains/physiology , PTEN Phosphohydrolase/antagonists & inhibitors , PTEN Phosphohydrolase/genetics , Primary Cell Culture , Rats , Synaptic Transmission/drug effectsABSTRACT
The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers.
