Abstract
The neuronal glycine transporter GlyT2 is an essential regulator of glycinergic neurotransmission that recaptures glycine in presynaptic terminals to facilitate transmitter packaging in synaptic vesicles. Alterations in GlyT2 expression or activity result in lower cytosolic glycine levels, emptying glycinergic synaptic vesicles and impairing neurotransmission. Lack of glycinergic neurotransmission caused by GlyT2 loss-of-function mutations results in Hyperekplexia, a rare neurological disease characterized by generalized stiffness and motor alterations that may cause sudden infant death. Although the importance of GlyT2 in pathology is known, how this transporter is regulated at the molecular level is poorly understood, limiting current therapeutic strategies. Guided by an unbiased screening, we discovered that E3 ubiquitin ligase Ligand of Numb proteins X1/2 (LNX1/2) modulate the ubiquitination status of GlyT2. The N-terminal RING-finger domain of LNX1/2 ubiquitinates a cytoplasmic C-terminal lysine cluster in GlyT2 (K751, K773, K787 and K791), and this process regulates the expression levels and transport activity of GlyT2. The genetic deletion of endogenous LNX2 in spinal cord primary neurons causes an increase in GlyT2 expression and we find that LNX2 is required for PKC-mediated control of GlyT2 transport. This work identifies, to our knowledge, the first E3 ubiquitin-ligases acting on GlyT2, revealing a novel molecular mechanism that controls presynaptic glycine availability. Providing a better understanding of the molecular regulation of GlyT2 may help future investigations into the molecular basis of human disease states caused by dysfunctional glycinergic neurotransmission, such as hyperekplexia and chronic pain.
Highlights
Glycine acts as an inhibitory neurotransmitter in the central nervous system (CNS), playing a fundamental role in neuronal circuits of the central auditory pathway, receptive fields in the retina and spinal cord sensitive pathways
We explored whether LNX1-p70, a shorter isoform with no E3 ligase activity (Fig. 1A), interacts with GlyT2, as this isoform contains the PDZ domain of LNX1/2 (PDZ2) domain that was predicted to bind the transporter
This study provides compelling novel evidence demonstrating that the RING-finger E3 ubiquitin ligases LNX1 and LNX2 are functional regulators of the neuronal glycine transporter GlyT2
Summary
Glycine acts as an inhibitory neurotransmitter in the central nervous system (CNS), playing a fundamental role in neuronal circuits of the central auditory pathway, receptive fields in the retina and spinal cord sensitive pathways. LNX1 and LNX2, two of the five members of the family, are expressed in neurons and other cell types in the nervous system[28] and present high structural homology with one RING and four consecutive PDZ domains (Fig. 1A) These PDZ domains promote the interaction with many neuronal substrates such as NUMB28, c-Src[29] or PKCα30, as well as the presynaptic active zone proteins CAST31, ERC1, ERC2 and LIPRIN-αs[32], which has led to suggest that LNX1/2 may modulate synapse maturation and neurotransmission[33,34]. We show that ablation of LNX2 in spinal cord neurons abolished the effect of PKC on the expression of GlyT2, suggesting that PKC is a key controller of LNX1/2 activity and its effect on GlyT2 expression Taken together, these findings indicate that LNX1/2 can modulate presynaptic glycine recapture by regulating GlyT2 ubiquitination levels and expression, suggesting that these proteins may play a role in controlling inhibitory glycinergic neurotransmission strength in the CNS
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