Regulation of serine in excitatory synapses in the mammalian brain

Abstract

D serine (DS) and L serine (LS) are important signalling molecules in the brain. DS acts as an endogenous co-agonist of NMDARs, which play a crucial role in synaptic function and plasticity. LS serves as a precursor for DS synthesis. Although neurons are the primary DS source, derived from astrocyte-produced LS, the mechanisms governing its clearance/regulation remain enigmatic. This thesis explores the topic of serine regulation, focussing on the role of transporters responsible for DS and LS uptake. Given that the astrocytes intricately envelop the excitatory synapses in the brain, it is hypothesised that the astrocytes are likely candidates to mediate the uptake of DS, after its action on the postsynaptic NMDARs. To understand the uptake phenomenon, in situ patch clamp recordings from acutely isolated mouse brain slices, focussing on the hippocampal region were conducted. Chapter 3 presents direct in situ patch clamp recordings of DS uptake into astrocytes, revealing transport-associated currents upon 10 mM DS application. Pharmacological inhibition experiments, utilising O-benzyl-L-serine and trans-4-hydroxy-L-proline, known substrate inhibitors of the sodium-dependent alanine serine cysteine transporter ASCT1 (Slc1a4), unveiled ASCT1's involvement in DS uptake into astrocytes adjacent to synapses in the stratum radiatum layer of the CA1 hippocampal region. Similar results were observed in other brain regions, including the primary somatosensory cortex, and cerebellum. These findings emphasise the central role of ASCT1 in mediating DS transport in astrocytes, thereby influencing NMDAR function and synaptic plasticity. Chapter 4 extends the investigation to LS transport at the CA3-CA1 hippocampal excitatory synapses, revealing ASCT1's involvement in this essential astrocytic function. Transport-associated currents induced by the application of 10 mM LS on stratum radiatum astrocytes sensitive to the administration of benzyl serine and trans hydroxy proline were observed. These results support the role of ASCT1 in mediating LS transport in the astrocytes. Since neurons require a constant supply of LS from the astrocytes, for DS synthesis, this result implies that astrocytic ASCT1 could mediate the LS shuttle transport. In CA1 pyramidal neurons, the transport of LS was highly sensitive to inhibition by MeAIB, implicating the involvement of system A transporters in this process. Having established the functional presence of ASCT1 in the astrocytes surrounding excitatory synapses, Chapter 5 aimed to delve into the physiological role of ASCT1 in governing the synaptic concentrations of DS and LS. Surprisingly, the addition of exogenous DS or LS did not alter NMDAR-mediated EPSC responses in the hippocampal CA3-CA1 synapses, indicating glycine binding site saturation under periods of low synaptic activity or low frequency of synaptic stimulation. Further pharmacological inhibition of ASCT1 using 10 mM benzyl serine or trans hydroxy proline did not significantly alter the NMDAR-mediated EPSC responses under low synaptic activity. These results not only highlight the complexities of agonist and co-agonist activation at excitatory synapses but also emphasise the need for further exploration into the underlying mechanisms by which ASCT1 regulates synaptic DS levels. In essence, this thesis characterises the functional presence of the astrocytic ASCT1, an elusive DS transporter pivotal in orchestrating synaptic DS dynamics. The implications of these findings resonate deeply within the realm of fundamental neuroscience and in the development of new drug targets for neurological disorders. For example, inhibiting ASCT1 may be a potential therapeutic strategy for reducing synaptic DS levels in diseases associated with DS hyperactivity, such as schizophrenia. Conversely, enhancing ASCT1 activity could be beneficial for increasing synaptic DS levels in conditions associated with DS deficiency, such as Alzheimer's disease. Show more