The loss of neuropil in the DLPFC of SCZ, specifically decreased dendritic spine density, may predict alterations in pre- and post-synaptic microdomains and a paralleled decrease in PSD proteins. Likewise, a likely point of convergence for genetic risk factors to exert their effect on NR function and synaptic plasticity is through altered PSD protein composition and/or interactions72, 75, 78, 88. Thus, based on this evidence, one might predict that synaptic protein levels would be decreased in the DLPFC of SCZ subjects, while the protein composition, interactions and/or posttranslational modifications of PSD proteins would be altered.
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Several research groups, including ours, have investigated the transcription and/or translation of synaptic genes in the prefrontal cortex using a variety of techniques,
including real time PCR, in situ and Western blot, and micro array. Despite this intense
scrutiny, no consensus has emerged as to the existence or direction of changes in synaptic protein expressions levels between disease and control tissues (Note, the term expression
is used here, and throughout the text, interchangeably with level and amount and is not
meant to imply that transcription is the sole determiner of mRNA or protein amounts in a cell). Presynaptic mRNAs were once widely believed to be decreased in the DLPFC of SCZ subjects95, although a recent review of studies from the last decade shows that altered expression of presynaptic mRNAs and proteins has not been consistently
observed 96. As for PSD protein and mRNA expression, Table 1-1 reviews the findings of some of the most recent studies investigating PSD95, NR1, NR2A and NR2B mRNA and protein levels in the PFC of SCZ patients97-105. This table highlights the lack of
consistency in mRNA findings, as measured by in-situ hybridization and real-time PCR
(qPCR), with most studies finding either a decrease or no change in transcriptions. Western blot (WB)
investigations report more consistent results, with all but one study finding no difference in protein levels for PSD95 and NR
subunits. Decreased spine density however, suggests that total amounts of both pre- and postsynaptic- proteins should be
decreased in the DLPFC of SCZ subjects.
Table 1-1. Translation and transcription of PSD95 and
NMDA receptor subunits have been repeatedly measured in the PFC of schizophrenia by several methods. This table summarizes some of the more prominent studies from the last 15 years. Technique: Tech.: Technique used, WB: Western Blot, IS: in situ hybridization, RT-PCR: Real Time PCR. Area: the brain area used as reported in the manuscript: FC: Frontal cortex, PFC: Prefrontal cortex, DL-PFC:
Dorsolateral prefrontal cortex, BA 9: Brodmann Area 9. :
There are two main reasons that likely contribute to these conflicting and inconsistent results: 1) mRNA and protein levels could be differentially regulated.
The largest discrepancy in the literature is the difference between mRNA and proteins levels. Differences in the regulation of transcription and translation as well as degradation of mRNA and proteins are prime candidates to explain these differences. Currently, technologies for quantifying mRNA are superior to those for protein in terms of multiplexing, accuracy and throughput. However, mRNA amounts may not reflect protein amounts. 2)Lack of spatial resolution. One advantage histological studies have
over expression technologies such as real-time PCR and Western blot, is the ability to resolve the unit being quantified, e.g. spines, in space. The ability to differentiate between cortical layers, neuronal subtypes and synaptic compartments (e.g. pyramidal neuron vs inter neuron and spine vs axon terminal) was essential to the successful investigation of total neuron number, pyramidal cell volume, dendritic branching and spine density. Thus, the signals of differences in mRNA or protein amount between SCZ and control tissues that are localized to a specific layer, neuronal type or synaptic compartment could be lost in the noise of a homogenized tissue sample. The introduction of laser-capture and linear amplification strategies have mitigated this issue somewhat, allowing for
quantification of mRNAs in discreet cortical layers and specific neuronal populations106-
108. Laser-capture – mRNA quantification strategies, however, are still unable to asses
protein amount, partitioning or post-translational modifications.
Considering that the observation of decreased dendritic spines was made in specific cortical layers and on/about a discreet part of the neuron, the synapse, it is not surprising that investigations with current strategies have observed either no difference between SCZ or control or small inconsistent findings. Thus, there is a need to overcome the technical challenges described above, as identifying the molecular correlates of decreased spine density is likely essential for linking genetic risk to NR hypofunction. The observation of these correlates requires an approach with advancements over traditional strategies that allows for multiplex quantification of synaptic proteins, with high precision and accuracy and spatial resolution for synaptic microdomains, cortical layers and neuronal subtypes.
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Several groups have turned to some combination of subcellular fractionation, immunoprecipitation and/or mass spectrometry (MS) based proteomics to qualify and quantify synaptic microdomain and receptor complex proteomes in animal tissues109-111. Unfortunately, MS proteomic strategies as currently configured are ill suited for
addressing this specific question in human tissues. In the next section I provide a brief review of the more popular quantitative MS proteomics strategies and propose a more targeted approach to overcome some of the technical hurdles described above.