TMEM119-positive microglia were increased in the brains of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that has been reported to be affected by inflammatory cells, such as microglia and macrophages, through the concept of non-cell autonomous neuronal death. Resident microglia in the human brain and monocyte-derived macrophages (MoDM) infiltrating in tissues are difficult to distinguish. Therefore, the effects of microglia and MoDMs in ALS remain poorly understood. This study aimed to investigate the role of resident microglia and MoDMs in the pathogenesis of ALS using postmortem brain and spinal cord samples. The samples used for immunohistochemical analysis included 11 cases of sporadic ALS and 11 age-matched controls. We stained the cells with TMEM119 to detect resident microglia and CCR2 to detect MoDMs. In ALS cases, TMEM119-immunopositive resident microglia were abundant in the motor cortex and subcortical white matter (SWM) of the motor area, whereas CCR2-immunopositive MoDM was similar to control cases. In addition, the mean density of CD68-immunopositive cells in the SWM significantly correlated with the mean density of pTDP-43-positive GCIs. These results suggest that resident microglial activation plays an important role in the cerebral pathogenesis of ALS and may provide novel therapeutic strategies to target excessive activation of resident microglia in ALS.

DOPAnization of tyrosine in α-synuclein by tyrosine hydroxylase leads to the formation of oligomers.

Parkinson's disease is a progressive neurodegenerative disorder characterized by the preferential loss of tyrosine hydroxylase (TH)-expressing dopaminergic neurons in the substantia nigra. Although the abnormal accumulation and aggregation of α-synuclein have been implicated in the pathogenesis of Parkinson's disease, the underlying mechanisms remain largely elusive. Here, we found that TH converts Tyr136 in α-synuclein into dihydroxyphenylalanine (DOPA; Y136DOPA) through mass spectrometric analysis. Y136DOPA modification was clearly detected by a specific antibody in the dopaminergic neurons of α-synuclein-overexpressing mice as well as human α-synucleinopathies. Furthermore, dopanized α-synuclein tended to form oligomers rather than large fibril aggregates and significantly enhanced neurotoxicity. Our findings suggest that the dopanization of α-synuclein by TH may contribute to oligomer and/or seed formation causing neurodegeneration with the potential to shed light on the pathogenesis of Parkinson's disease.

Wide expression of type I Na+-phosphate cotransporter 3 (NPT3/SLC17A2), a membrane potential-driven organic anion transporter.

Membrane potential (Δψ)-driven and Cl(-)-dependent organic anion transport is a primary function of the solute carrier family 17 (SLC17) transporter family. Although the transport substrates and physiological relevance of the major members are well understood, SLC17A2 protein known to be Na(+)-phosphate cotransporter 3 (NPT3) is far less well characterized. In the present study, we investigated the transport properties and expression patterns of mouse SLC17A2 protein (mNPT3). Proteoliposomes containing the purified mNPT3 protein took up radiolabeled p-aminohippuric acid (PAH) in a Δψ- and Cl(-)-dependent manner. The mNPT3-mediated PAH uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDs) and Evans blue, common inhibitors of SLC17 family members. The PAH uptake was also inhibited by various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs (NSAIDs) and urate. Consistent with these observations, the proteoliposome took up radiolabeled urate in a Δψ- and Cl(-)-dependent manner. Immunohistochemistry with specific antibodies against mNPT3 combined with RT-PCR revealed that mNPT3 is present in various tissues, including the hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, apical membrane of follicle cells in the thyroid, bronchiole epithelial cells in the lungs, and astrocytes around blood vessels in the cerebrum. These results suggested that mNPT3 is a polyspecific organic anion transporter that is involved in circulation of urate throughout the body.

Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets.

Nucleotides are stored in the dense granules of platelets. The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation. However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood. The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized. We hypothesized that the vesicular nucleotide transporter (VNUT) is also involved in the vesicular storage of nucleotides in platelets. In this article, we present three lines of evidence that VNUT is responsible for the vesicular storage of nucleotides in platelets and that vesicular ATP transport is crucial for platelet function, detection and characterization of VNUT activity in platelets isolated from healthy humans and MEG-01 cells, RNA interference experiments on MEG-01 cells, and studies on nucleotide transport and release with a selective inhibitor.

Vesicular nucleotide transport: a brief history and the vesicular nucleotide transporter as a target for drug development.

Neurons and neuroendocrine cells store nucleotides in vesicles and release them upon stimulation, leading to intercellular purinergic signaling. The molecular machinery responsible for the vesicular storage of nucleotides was a long standing enigma, however, recently the transporter involving in the process was identified. This article summarizes the history of vesicular storage of nucleotides and the identification of the vesicular nucleotide transporter (VNUT) responsible for the process. The significance of VNUT as a drug target to control purinergic chemical transmission is also discussed.

Type 1 sodium-dependent phosphate transporter acts as a membrane potential-driven urate exporter.

SLC17A1 protein (NPT1) was the first identified member of the SLC17 phosphate transporter family, and is known to mediate Na(+)/inorganic phosphate (Pi) co-transport when expressed in Xenopus oocytes. Although this protein was suggested to be a renal polyspecific anion exporter, its transport properties were not well characterized. The clean biochemical approach revealed that proteoliposomes comprising purified NPT1 as the only protein source transport various organic anions such as urate, p-aminohippuric acid (PAH), and acetylsalicylic acid (aspirin) in a membrane potential (Δψ)-driven and Cl(-) -dependent manner. Human NPT1 carrying an SNP mutation, Thr269Ile, known to increase the risk of gout, exhibited 32% lower urate transport activity compared to the wild type protein, leading to the conclusion that NPT1 is the long searched for transporter responsible for renal urate excretion. In the present article, we summarized the history of identification of the urate exporter and its possible involvement in the dynamism of urate under physiological and pathological conditions.

Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter.

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Δψ)- and Cl(-)-dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na(+)-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl(-)-dependent manner at the expense of an electrochemical gradient of protons, especially Δψ, across the membrane. The Δψ- and Cl(-)-dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.