L-Phenylalanyl-L-glutamine

Currently TAR DNA binding protein 43 (TDP-43) pathology, underlying Amyotrophic Lateral Sclerosis (ALS), remains poorly understood which hinders both clinical diagnosis and drug discovery efforts. To better comprehend the disease pathophysiology, positron emission tomography (PET) and multi-parametric magnetic resonance imaging (mp-MRI) provide a non-invasive mode to investigate molecular, structural, and neurochemical abnormalities in vivo. For the first time, we report the findings of a longitudinal PET-MR study in the TDP-43A315T ALS mouse model, investigating disease-related changes in the mouse brain. 2-deoxy-2-[18F]fluoro-D-glucose [18F]FDG PET showed significantly lowered glucose metabolism in the motor and somatosensory cortices of TDP-43A315T mice whereas metabolism was elevated in the region covering the bilateral substantia nigra, reticular and amygdaloid nucleus between 3 and 7 months of age, as compared to non-transgenic controls. MR spectroscopy data showed significant changes in glutamate + glutamine (Glx) and choline levels in the motor cortex and hindbrain of TDP-43A315T mice compared to controls. Cerebral blood flow (CBF) measurements, using an arterial spin labelling approach, showed no significant age- or group-dependent changes in brain perfusion. Diffusion MRI indices demonstrated transient changes in different motor areas of the brain in TDP-43A315T mice around 14 months of age. Cytoplasmic TDP-43 proteinaceous inclusions were observed in the brains of symptomatic, 18-month-old mice, but not in non-symptomatic transgenic or wild-type mice. Our results reveal that disease- and age-related functional and neurochemical alterations, together with limited structural changes, occur in specific brain regions of transgenic TDP-43A315T mice, as compared to their healthy counterparts. Altogether these findings shed new light on TDP-43A315T disease pathogenesis and may prove useful for clinical management of ALS.

The peptide transporter PEPT2 mediates transmembrane uptake of small peptides. So far, its expression has not been evidenced in the gastrointestinal tract. We have investigated peptide transport activity in the neuromuscular layers of the gastrointestinal tract by using the fluorescent tracer-dipeptide beta-Ala-Lys-Nepsilon-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Whole-mount preparations from mouse, rat, and guinea pig stomach and small and large intestine were incubated with Ala-Lys-AMCA in the presence or absence of the uptake-inhibitors L-histidine, D-phenylalanyl-L-alanine (D-Phe-Ala), glycyl-L-sarcosine (Gly-Sar), glycyl-L-glutamine (Gly-Gln), benzylpenicillin, and cefadroxil. Fluorescence microscopy revealed that Ala-Lys-AMCA specifically accumulated in both ganglionic layers of the enteric nervous system (ENS) in all regions and species studied. This could be inhibited by Gly-Sar, D-Phe-Ala, Gly-Gln, and cefadroxil, but not by free histidine and benzylpenicillin, indicating uptake via PEPT2. Accordingly, dipeptide uptake was completely abolished in PEPT2-deficient mice. Reverse transcriptase-polymerase chain reaction analysis detected a PEPT2-specific transcript in extracts from the ganglionic ENS layers of mouse small and large intestine, further proving that enteric dipeptide transport activity is specifically mediated via PEPT2. The cellular site of dipeptide uptake was immunohistochemically localized to enteric glial cells and tissue-resident macrophages. In addition, dipeptide uptake occurred in a neurochemically defined subset of neurons in the guinea pig ENS. Our results constitute the first functional evidence for dipeptide transport activity in the ENS. PEPT2-mediated dipeptide transport in enteric glia could contribute to the clearance of neuropeptides in the ENS. In addition, the fluorophore-coupled dipeptide uptake via PEPT2 is a novel vital marker for glial cells in the ENS.

H(+)/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H(+)/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (K(m,app)) of 0.806+/-0.161 mmol l(-1). Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H(+)/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)(+) RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated D-Phe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H(+)/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0 degrees C for the former and 25 degrees C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.