GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3Ј-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14 -29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of ϳ3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

Purpose: We assessed whether perioperative circulating tumor DNA (ctDNA) could be a biomarker for early detection of molecular residual disease (MRD) and prediction of postoperative relapse in resected non–small cell lung cancer (NSCLC). Experimental Design: Based on our prospective, multicenter cohort on dynamic monitoring of ctDNA in lung cancer surgery patients (LUNGCA), we enrolled 950 plasma samples obtained at three perioperative time points (before surgery, 3 days and 1 month after surgery) of 330 stage I–III NSCLC patients (LUNGCA-1), as a part of the LUNGCA cohort. Using a customized 769-gene panel, somatic mutations in tumor tissues and plasma samples were identified with next-generation sequencing and utilized for ctDNA-based MRD analysis. Results: Preoperative ctDNA positivity was associated with lower recurrence-free survival (RFS; HR = 4.2; P < 0.001). The presence of MRD (ctDNA positivity at postoperative 3 days and/or 1 month) was a strong predictor for disease relapse (HR = 11.1; P < 0.001). ctDNA-based MRD had a higher relative contribution to RFS prediction than all clinicopathologic variables such as the TNM stage. Furthermore, MRD-positive patients who received adjuvant therapies had improved RFS over those not receiving adjuvant therapy (HR = 0.3; P = 0.008), whereas MRD-negative patients receiving adjuvant therapies had lower RFS than their counterparts without adjuvant therapy (HR = 3.1; P < 0.001). After adjusting for clinicopathologic variables, whether receiving adjuvant therapies remained an independent factor for RFS in the MRD-positive population (P = 0.002) but not in the MRD-negative population (P = 0.283). Conclusions: Perioperative ctDNA analysis is effective in early detection of MRD and relapse risk stratification of NSCLC, and hence could benefit NSCLC patient management.

Syndecan-4, a member of the syndecan gene family of proteoglycans, is an important regulator of bFGF signaling. In particular, bFGF-dependent regulation of cell growth and migration has been linked to syndecan-4 cytoplasmic domain-mediated interactions. Screening of a yeast two-hybrid library with a cytoplasmic domain of rat syndecan-4 identified a novel binding partner, here termed synectin. Synectin is highly homologous to semaphorin F binding protein semcap1, glucose 1 transporter binding protein glut1cbp, and RGS-GAIP/neuropilin-1 binding protein GIPC. Overexpression of synectin in ECV304 cells in culture led to a dose-dependent inhibition of migration while not affecting cell adhesion or growth rate. We conclude that synectin is involved in syndecan-4-dependent interactions and may play a role in the assembly of syndecan-4 signaling complex.