Leukocyte common antigen-related receptor protein tyrosine phosphatases-comprising LAR, PTPδ, and PTPσ-are synaptic adhesion molecules that organize synapse development. Here, we identify glypican 4 (GPC-4) as a ligand for PTPσ. GPC-4 showed strong (nanomolar) affinity and heparan sulfate (HS)-dependent interaction with the Ig domains of PTPσ. PTPσ bound only to proteolytically cleaved GPC-4 and formed additional complex with leucine-rich repeat transmembrane protein 4 (LRRTM4) in rat brains. Moreover, single knockdown (KD) of PTPσ, but not LAR, in cultured neurons significantly reduced the synaptogenic activity of LRRTM4, a postsynaptic ligand of GPC-4, in heterologous synapse-formation assays. Finally, PTPσ KD dramatically decreased both the frequency and amplitude of excitatory synaptic transmission. This effect was reversed by wild-type PTPσ, but not by a HS-binding-defective PTPσ mutant. Our results collectively suggest that presynaptic PTPσ, together with GPC-4, acts in a HS-dependent manner to maintain excitatory synapse development and function.PTPσ | glypican | LRRTM4 | synaptic cell adhesion | heparan sulfate

BackgroundA number of reports have described the protective effects of ginsenoside Rg1 (Rg1) in Alzheimer's disease (AD). However, the protective mechanisms of Rg1 in AD remain elusive.MethodsTo investigate the potential mechanisms of Rg1 in β-amyloid peptide-treated SH-SY5Y cells, a comparative proteomic analysis was performed using stable isotope labeling with amino acids in cell culture combined with nano-LC-MS/MS.ResultsWe identified a total of 1,149 proteins in three independent experiments. Forty-nine proteins were significantly altered by Rg1 after exposure of the cells to β-amyloid peptides. The protein interaction network analysis showed that these altered proteins were clustered in ribosomal proteins, mitochondria, the actin cytoskeleton, and splicing proteins. Among these proteins, mitochondrial proteins containing HSD17B10, AARS2, TOMM40, VDAC1, COX5A, and NDUFA4 were associated with mitochondrial dysfunction in the pathogenesis of AD.ConclusionOur results suggest that mitochondrial proteins may be related to the protective mechanisms of Rg1 in AD.

BackgroundThe ginsenoside Rb1 (Rb1) is the most abundant compound in the root of Panax ginseng. Recent studies have shown that Rb1 has a neuroprotective effect. However, the mechanisms underlying this effect are still unknown.MethodsWe used stable isotope labeling with amino acids in cell culture, combined with quantitative mass spectrometry, to explore a potential protective mechanism of Rb1 in β-amyloid-treated neuronal cells.ResultsA total of 1,231 proteins were commonly identified from three replicate experiments. Among these, 40 proteins were significantly changed in response to Rb1 pretreatment in β-amyloid-treated neuronal cells. Analysis of the functional enrichments and protein interactions of altered proteins revealed that actin cytoskeleton proteins might be linked to the regulatory mechanisms of Rb1. The CAP1, CAPZB, TOMM40, and DSTN proteins showed potential as molecular target proteins for the functional contribution of Rb1 in Alzheimer's disease (AD).ConclusionOur proteomic data may provide new insights into the protective mechanisms of Rb1 in AD.

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Cav3.1 T‐type Ca2+ channels play pivotal roles in neuronal low‐threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Cav3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Cav3.1 channel has been poorly investigated. In this work, we analyzed rat Cav3.1 protein expressed in HEK‐293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Cav3.1 phosphorylation map which includes the reported mouse Cav3.1 phosphosites. Site‐directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho‐silent Cav3.1 mutants expressed in Xenopus oocytes showed that the phospho‐silent mutation of the N‐terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho‐silent mutations of the C‐terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage‐dependent gating properties. In contrast, the phosphomimetic Asp mutations of Cav3.1 on the N‐ and C‐terminal Ser residues reversed the effects of the phospho‐silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Cav3.1 at the N‐ and C‐terminal regions play crucial roles in the regulation of the channel activity and voltage‐dependent gating properties.