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H2020

SorCSbalance Report Summary

Project ID: 659927
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - SorCSbalance (Role of the sorting receptor SorCS1 in controlling excitation/inhibition balance in neural circuits.)

Reporting period: 2015-05-01 to 2017-04-30

Summary of the context and overall objectives of the project

Controlling the precise balance between excitation and inhibition (E/I balance) is critical for information processing in the brain. A perturbed E/I balance has been implicated in the etiology of a wide range of neuropsychiatric disorders. The factors that dictate the balance between excitatory and inhibitory synaptic transmission are still poorly defined, but trans-synaptic interactions between adhesion molecules such as neurexins and neuroligins are thought to be important. We had strong experimental evidence supporting the idea that SorCS1 is a pivotal regulator of synaptic abundance of adhesion molecules, however it was still unclear whether SorCS1 can regulate neuronal function or not. Considering that SorCS1 has also been associated with a variety of neural disorders, including autism, schizophrenia, and Alzheimer’s disease, here I hypothesized that SorCS1 controls E/I balance in the brain by regulating the synaptic abundance of cell surface receptors.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Recently the host laboratory has shown that sorting receptor SorCS1 regulates synaptic recruitment of diverse synaptic receptors, such as neurotransmitter receptors and cell adhesion molecules (see Neuron 87, 764-780; Savas et al., 2015). To tackle the first aim I used a conditional knockout (KO) mouse for SorCS1 (SorCS1flox/flox), to acutely remove SorCS1. By using mouse cortical neurons prepared from SorCS1flox/flox mice and transfected with Cre recombinase to reduce SorCS1 levels and SEP (superecliptic pHluorin)-tagged Neurexin1, I assessed the surface levels of Neurexin. I found that upon loss of SorCS1 axonal surface levels of Neurexin-1 are decreased. Interestingly, if expression of SorCS1 was reduced the dendritic surface levels of Neurexin-1 were increased. These results show that SorCS1 controls the balance of Neurexin-1 in the axon and dendrites. Additional work is required to clarify the role of SorCS1 on the balance of Neurexin in axons and dendrites. To further analyze whether SorCS1 controls the surface expression of other synaptic receptors I performed immunostainings for surface AMPA and GABA receptors. By using cortical neurons electroporated with GFP and Cre recombinase I found that surface and synaptic levels of GluA1 and GluA2 were decreased in SorCS1 KO neurons (Fig.1 K-P). In contrast, surface and synaptic levels of the β2/3 subunit of the receptor GABA were not affected by SorCS1 loss (Fig.1 Q-S). Altogether these results indicate that SorCS1 is necessary to maintain surface abundance of diverse synaptic cell surface receptors.
A major aspect raised by the previous findings is to understand whether SorCS1 controls synaptic function in vitro and in vivo (aim 2). To test whether SorCS1 controls synapse number, I electroporated mouse cortical cells from SorCS1flox/flox mice with GFP and Cre recombinase and then I quantified the density of excitatory and inhibitory synapses. I found that loss of SorCS1 does not affect the density of puncta positive for the excitatory synaptic markers (Fig.1 G and H), but decreases the density of puncta positive for the inhibitory synaptic markers (Fig.1 I and J). Then, I tested whether an altered abundance of synaptic receptors and decreased number of inhibitory synapses affects synaptic transmission. We recorded spontaneous miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) from somatosensory layer 5 pyramidal neurons in acute slices from SorCS1flox/flox and Emx1-Cre:SorCS1flox/flox mice to measure basal synaptic transmission (SorCS1flox/flox mice were crossed with Emx1-Cre transgenic mice to specifically decrease SorCS1 expression in principal cortical neurons). The frequency, but not the amplitude, of mEPSCs and mIPSCs was significantly decreased in Emx1-Cre:SorCS1flox/flox cortical neurons comparing with control cortical neurons (Fig. 1 A-C). This result shows that loss of SorCS1 in vivo impairs synaptic transmission.
To further explore this data, namely to understand whether this defect was caused by a pre-synaptic or postsynaptic impairment in neuronal function, we moved to an in vitro system. We recorded mEPSCs and mIPSCs from cortical neurons prepared from SorCS1flox/flox mice and electroporated with GFP and Cre, but also from non-electroporated neurons in Cre-electroporated cultures, to assess whether the effect of SorCS1 loss on spontaneous synaptic transmission is cell-autonomous (postsynaptic) or non-cell-autonomous (presynaptic). Frequency of mEPSCs and mIPSCs, but not amplitude, was decreased in SorCS1flox/flox in Cre-electroporated neurons in comparison with GFP-electroporated cells (Fig.1 D-F). We also found no change in the frequency of non-electroporated in comparison to GFP-electroporated cells (Fig.1 D-F), strongly suggesting that the effect of SorCS1 loss on mPSC frequency is cell-autonomous. The decrease in inhibitory synapse density aforementioned fully explains the reduced mIPSC frequency in SorCS1 KO cells,

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The work I have performed in the scope of this project was essential to identify the sorting receptor SorCS1 as a major regulator of synapse function in the brain. These findings constitute a major breakthrough, because they support the idea that a single endosomal sorting receptor might have a pivotal role in adjusting the composition and function of synapses. This fellowship was an important asset that has allowed me to broaden our knowledge about SorCS1 biology and its role in the central nervous system. By suggesting that SorCS1 is a pleiotropic player required to sustain synapse composition, our results support the idea that we can manipulate SorCS1 expression to restore defects in neurotransmission underlying several brain diseases. Indeed, SorCS1 and synaptic surface receptors have all been linked to diseases in which the function of synapses is perturbed, including autism, schizophrenia, and Alzheimer's disease, which are also characterized by defects in neurotransmission, and an imbalance in synaptic E/I. In my current work, I am therefore dissecting whether perturbed SorCS1 levels and disease-associated SorCS1 mutations affect E/I balance in neural circuits. These experiments will give insights into the mechanisms underlying synaptic diseases, which may ultimately allow for the design of therapeutic interventions.

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