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PLoS One
2013 Jan 01;811:e78654. doi: 10.1371/journal.pone.0078654.
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The neuroplastin adhesion molecules are accessory proteins that chaperone the monocarboxylate transporter MCT2 to the neuronal cell surface.
Wilson MC
,
Kraus M
,
Marzban H
,
Sarna JR
,
Wang Y
,
Hawkes R
,
Halestrap AP
,
Beesley PW
.
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BACKGROUND: The neuroplastins np65 and np55 are two synapse-enriched immunoglobulin (Ig) superfamily adhesion molecules that contain 3 and 2 Ig domains respectively. Np65 is implicated in long term, activity dependent synaptic plasticity, including LTP. Np65 regulates the surface expression of GluR1 receptor subunits and the localisation of GABA(A) receptor subtypes in hippocampal neurones. The brain is dependent not only on glucose but on monocarboxylates as sources of energy. The. monocarboxylate transporters (MCTs) 1-4 are responsible for the rapid proton-linked translocation of monocarboxylates including pyruvate and lactate across the plasma membrane and require association with either embigin or basigin, proteins closely related to neuroplastin, for plasma membrane expression and activity. MCT2 plays a key role in providing lactate as an energy source to neurons.
METHODOLOGY/FINDINGS: Here we use co-transfection of neuroplastins and monocarboxylate transporters into COS-7 cells to demonstrate that neuroplastins can act as ancillary proteins for MCT2. We also show that Xenopus laevis oocytes contain endogenous neuroplastin and its knockdown with antisense RNA reduces the surface expression of MCT2 and associated lactate transport. Immunocytochemical studies show that MCT2 and the neuroplastins are co-localised in rat cerebellum. Strikingly neuroplastin and MCT2 are enriched in the same parasagittal zebrin II-negative stripes.
CONCLUSIONS: These data strongly suggest that neuroplastins act as key ancillary proteins for MCT2 cell surface localisation and activity in some neuronal populations, thus playing an important role in facilitating the uptake of lactate for use as a respiratory fuel.
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Figure 1. Co-transfection with np55 or np65 enables expression of MCT2 at the plasma membrane. Cos-7 cells were transiently transfected with MCT2 tagged at its N-terminus with CFP and/or np55 or np65 C-terminally labelled with YFP. The expression of np55 or np65 and MCT2 was detected by confocal microscopy. A: In single transfected cells MCT2 remains in the perinuclear region while neuroplastin is located at the plasma membrane and in the soma. B and C: In contrast, when MCT2 is transfected together with np55 or np65 both proteins are expressed at the cell surface. Cells were imaged using the Leica confocal imaging spectrophotometer system (TCS-SP2) attached to a Leica DMIRBE inverted epifluorescence microscope.
doi:10.1371/journal.pone.0078654.g001
Figure 3. Xenopus oocytes express neuroplastin that facilitates MCT2 expression at the plasma membrane. A: shows the results of RT- PCR performed on mRNA extracted from Xenopus laevis oocytes, liver and thymus as described in the Experimental section. Primers for basigin (Bas), embigin (Emb) and neuroplastin (Np) were used as indicated. B: shows the expression of MCT2 in oocytes with or without antisense mRNA for basigin or neuroplastin detected by immunofluorescence microscopy. Antisense treatment reduces expression at the plasma membrane (PM) but increases intracellular expression (IC). The scale bar is 50 mm. C: shows parallel data for MCT2 expression in a crude membrane fraction derived from the oocyte and detected by SDS-PAGE and Western blotting. Here, scrambled (Scram) antisense RNA is used as an additional control. D: presents parallel data for the transport of [14C]-L-lactate into oocytes determined over 5 min. Error bars represent the S.E.M. of the number of oocytes indicated.
doi:10.1371/journal.pone.0078654.g003
Figure 4. MCT2 immunoreactivity in 40 mm sagittal sections through the adult rat cerebellar cortex reveal Purkinje cell immunoreactivity. A: MCT2 immunoreactivity visualised by peroxi- dase reaction product is deposited in the Purkinje cell dendrites in the molecular layer (ml), Purkinje cell somata in the Purkinje cell layer (pcl), and weakly in the granular layer (gl). B: Anti-MCT2 immunofluorescence staining is prominent in the granular layer (gl). C: Anti-MCT2 immunofluorescence staining outlines Purkinje cell somata in the Purkinje cell layer (pcl) and their proximal dendrites in the molecular layer (ml): the distal dendritic arbour is either weakly stained or unreactive. Scale bar = 50 mm.
doi:10.1371/journal.pone.0078654.g004
Figure 5. MCT2 is selectively expressed by Purkinje cell subsets. A: Levels of MCT2 immunoreactivity vary between Purkinje cell somata of the Purkinje cell layer (pcl) from strong (white arrowhead), to weak (black arrow), to none (black arrowhead). No boundaries are seen in the granular layer (gl). B: Clear boundaries are seen in the molecular layer (ml) between regions that express high levels of MCT2 immunoreactivity and others that are either weakly reactive or unreactive (arrows). C: A transverse section through lobule VIa of the adult rat cerebellar cortex double immunofluorescence labelled for zebrin II/aldolase C (green) and MCT2 (red), showing an array of alternating Purkinje cell stripes. The stripes of MCT2 immunoreactivity alternate with those of zebrin II/aldolase C. Scale bars: A and C=50 mm, B=100 mm. doi:10.1371/journal.pone.0078654.g005
Figure 2. FRET measurements demonstrate a close association between MCT2 and np55 and np65.COS-7 cells were transiently co-transfected with the MCT2nCFP, np55cYFP and npc65-YFP constructs indicated (where n and c refer to the N- or C- terminal location of the CFP/YFP tag. Live cell imaging with determination of FRET was performed as described under “Methods”. Data are presented as means ± S.E.M. for 26, 22 and 26 separate cells respectively.
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