In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Abstract

Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high-and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensorydeprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development. mass spectrometric proteomics | somatosensory cortex | experience-dependent plasticity | synaptic protein dynamics S ensory information is encoded in the brain by activation of specific neuronal circuits that operate via chemical synapses. Important sensory experiences are stored by strengthening activated synapses in the circuit, a process known as "experience-dependent plasticity" (1). When animals are deprived of sensory experience during development, for example, by trimming rodent whiskers from birth to 30 d after birth, synaptic strength and mature synaptic morphology are attenuated, suggesting that the low activity in the deprived circuits interferes with normal development (2-7). However, the molecular changes that alter these synaptic properties in response to experience remain unclear. Synaptic activation has been shown to promote regulated and highly localized effects on synaptic proteins such as local translation, recruitment, and targeted degradation (8-11), and this spatiotemporal control of protein availability is required for long-term plasticity and synaptic maturation, which are fundamental in memory formation and storage and ultimately for an organism's survival (12-14). The need for highly controlled protein availability is highlighted in studies of neurological diseases, which often result from the disruption of protein availability at the synapse (15-17). ...

Cellular diversity of the somatosensory cortical map plasticity

“…whole tissue punch vs synaptoneurosomes, respectively, the molecules linked to brain disorders also differ substantially in the protein classes they encompass ( Figure 7). For example, cytoskeletal proteins make up a large portion of the diseaserelated targets identified by Butko et al (Butko et al, 2013) and transcription factors are not found, whereas the reverse Figure 7. Experience-dependent plasticity and select brain disorders share common molecular targets.…”

“…Because RNA translation into protein is not a linear process, and controlled by posttranscriptional regulatory mechanisms (Keene, 2007), proteomic studies will be required to mechanistically address the molecular pathways associated with expression of plasticity. Currently the only proteomics study available in barrel cortex was performed by Butko and colleagues (Butko et al, 2013), who either clipped or brushed the whisker pads of rats for a period of 30 days, starting from P4, and subsequently prepared synaptoneurosomes from barrel cortex. Using tandem mass spectrometry Butko and colleagues identified systematic downregulation of translation for various ion channels (e.g.…”

“…We therefore asked whether EDP-related transcripts and proteins identified in the studies by Vallès et al (Vallès et al, 2011) and Butko et al (Butko et al, 2013) are differentially regulated in neurological disorders. We used the Disease and Gene Annotations (DGA) database (Peng et al, 2013), which entails documented relationships between genes and diseases/disorders (such as altered protein or mRNA levels, singlenucleotide polymorphisms and mutations).…”

“…Experience-dependent plasticity and select brain disorders share common molecular targets. Top: Neurological disorders associated with differentially expressed transcripts after exposure to an enriched environment (left, data from (Vallès et al, 2011)) or proteins (right, data from (Butko et al, 2013)); genes are clustered by disorder. Bottom: Protein classes of transcripts (left) or proteins (right) associated with neurological diseases (Based on Panther Database).…”

“…http://dx.doi.org/10.1101/201293 doi: bioRxiv preprint first posted online Both Vallès (Vallès et al, 2011) and Butko (Butko et al, 2013) and their colleagues have examined, through in situ hybridization or immunostainings, the distribution of a subset of identified targets across cortical lamina, revealing tight spatial restrictions on their expression in cellular populations. Such post-hoc approaches however are of low throughput, and future studies should strive to obtain lamina-specific tissues (much like in Belgard et al, 2011;Kole et al, 2017aKole et al, , 2017b and use these for subsequent processing and analysis.…”