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Outside the nervous system, members of the mitochondrial uncoupling protein (UCP) family have been proposed to contribute to control of body temperature and energy metabolism, and regulation of mitochondrial production of reactive oxygen species (ROS). However, the function of brain mitochondrial carrier protein 1 (BMCP1), which is highly expressed in brain, remains to be determined. To study BMCP1 expression and function in the nervous system, a high-affinity antibody to BMCP1 was generated and used to analyze tissue expression of BMCP1 protein in mouse. BMCP1 protein was highly expressed in heart and kidney, but not liver or lung. In the nervous system, BMCP1 was present in cortex, basal ganglia, substantia nigra, cerebellum, and spinal cord. Both BMCP1 mRNA and protein expression was almost exclusively neuronal. To study the effect of BMCP1 expression on mitochondrial function, neuronal (GT1-1) cell lines with stable overexpression of BMCP1 were generated. Transfected cells had higher State 4 respiration and lower mitochondrial membrane potential (ψm), consistent with greater mitochondrial uncoupling. BMCP1 expression also decreased mitochondrial production of ROS. These data suggest that BMCP1 can modify mitochondrial respiratory efficiency and mitochondrial oxidant production, and raise the possibility that BMCP1 might alter the vulnerability of brain to both acute injury and to neurodegenerative conditions.
BMCP1 (brain mitochondrial carrier protein 1; also referred to as UCP5), and the other mitochondrial uncoupling proteins (UCPs) are members of the superfamily of mitochondrial carriers which transport small molecules across the inner mitochondrial membrane. The first identified uncoupling protein, UCP1, dissipates the mitochondrial proton gradient by transporting H+ across the inner membrane, thereby uncoupling electron transport from ATP production (Nicholls 1977; for review see also Pecqueur et al. 2001a; Stuart et al. 2001). UCP1 has been established as a key source of fatty acid-dependent heat generation by brown adipose tissue in newborns (Nicholls and Locke 1984; Enerback et al. 1997) and has been linked to the physiological response to cold stress into adulthood (Rothwell and Stock 1979; Enerback et al. 1997). Two additional UCP family members, the ‘novel UCPs’ (UCP2 and UCP3), have significant sequence similarity to UCP1 (57% and 55%, respectively). UCP3 is found predominantly in skeletal muscle and brown adipose tissue (Boss et al. 1997), while UCP2 is expressed in multiple organs, including brain, where it is present primarily in certain hypothalamic nuclei (Fleury et al. 1997). These novel UCPs have been proposed to contribute to fatty acid metabolism and the physiological response to calorie restriction (reviewed by Pecqueur et al. 2001a; also see Stuart et al. 2001). UCP4 and BMCP1 have recently been added to the UCP family because of their sequence similarity to UCP1 (30% for BMCP1, Sanchis et al. 1998; 34% for UCP4, Mao et al. 1999), and because overexpression of these proteins in HEK293 cells was found to decrease mitochondrial membrane potential (Mao et al. 1999; Yu et al. 2000). Both show expression of message in brain, but their function in the nervous system has not been determined.
Recently, a role for UCPs in regulating mitochondrial reactive oxygen species (ROS) production has been advanced. An early study on mitochondria isolated from various tissues, employing GDP as a pharmacological inhibitor of UCP activity, indicated that inhibition of UCPs might increase H2O2 production (Negre-Salvayre et al. 1997). More recently, macrophages from UCP2-deficient mice were shown to generate higher levels of ROS (Arsenijevic et al. 2000). The site of increased ROS production was not determined. However, two additional studies indicate that UCPs specifically modulate mitochondrial ROS production. Overexpression of UCP1 in endothelial cells resulted in a specific reduction in mitochondrial ROS (Nishikawa et al. 2000), and UCP3 knockout mice exhibited enhanced superoxide radical production from mitochondria isolated from skeletal muscle (Vidal-Puig et al. 2000).
Disruption of mitochondrial energy metabolism and altered mitochondrial free radical production have been proposed to contribute to neurodegenerative diseases (Beal 1992; Rapoport et al. 1996; Melov et al. 1999; Wallace 1999). Uncoupling proteins could be a link between these two processes in the nervous system physiologically, and under injury or disease conditions. To investigate the function of BMCP1 in brain, and the role of BMCP1 in modifying neuronal mitochondrial function, we generated a high-affinity antibody to BMCP1, and evaluated BMCP1 protein expression in mouse tissues, brain slices and neural cell cultures. In addition, the effect of BMCP1 expression on mitochondrial function was investigated by generating GT1-1 hypothalamic neurons which overexpress BMCP1. Mitochondrial respiration was assessed by measuring mitochondrial oxygen consumption in BMCP1-transfected cells, and mitochondrial membrane potential (ψm), was evaluated using confocal fluorescence microscopy and the membrane potential-sensitive fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). The effect of BMCP1 expression on mitochondrial superoxide production was determined by confocal fluorescence imaging of dihydroethidium (DHE) oxidation. Our studies indicate that BMCP1 is highly expressed in neurons throughout the brain, and can modify mitochondrial respiratory function, ψm, and free radical production in neurons.
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Mitochondrial energy impairment and altered free radical production are believed to be important mechanisms in neurodegenerative disorders (Beal 1992; Melov et al. 1999; Wallace 1999). The mitochondrial uncoupling proteins are of special interest in this context because they appear to regulate both mitochondrial metabolic efficiency (Brand et al. 1994; Rolfe et al. 1999; Ricquier and Bouillard 2000) and free radical generation. Although this association has been shown most convincingly for UCP1, growing evidence suggests that UCP2 and UCP3 are capable of carrying out these functions as well (reviewed by Ricquier and Bouillaud 2000; Rial and Gonzalez-Barroso 2001; Stuart et al. 2001). However, although UCP2 message is found in brain, protein expression appears to be localized primarily to the hypothalamus and is present at extremely low levels (Pecqueur et al. 2001b). BMCP1, in contrast, has been shown to have a wide distribution of message in brain (Sanchis et al. 1998), and might therefore play a role throughout the nervous system. However, little is yet known about its function in the CNS.
Previous studies on BMCP1 have described expression of message, but because of lack of antibodies to BMCP1, no characterization of BMCP1 protein has been performed to date. To allow BMCP1 protein expression to be assessed, we developed and characterized an anti-mouse BMCP1 polyclonal for these studies which appeared to lack cross-reactivity with the smaller UCP1, UCP2 and UCP3 proteins, or with another more distantly related UCP homolog, UCP4. BMCP1 protein expression was abundant in brain, heart and kidney, was moderate in skeletal muscle, and was minimal or absent in adipose tissue, lung and liver. For liver, our results are consistent with the lack of BMCP1 expression in liver found on northern blot analysis of B6D2 mice (Sanchis et al. 1998), but contrast with results from a second group (Yu et al. 2000), who reported substantial BMCP1 message in liver from both C57B6 and FVB-N mice. This second group did not observe lung expression of BMCP1 in their mice, whereas Sanchis et al. (1998) documented expression in lung from both B6D2 mice and rat. These results suggest that strain differences strongly influence the pattern of tissue expression of BMCP1, and indicate that the effect of species and strain will need to be considered in evaluating the regulation of BMCP1 expression and function.
A dissociation between message and protein has been reported for UCP2, which can exhibit substantial mRNA expression without demonstrable protein synthesis (Pecqueur et al. 2001b). The lack of correlation between message and protein for UCP2 has been shown to reflect the presence of an alternative upstream ORF in the UCP2 message, also present in BMCP1, which dramatically reduces translational efficiency of UCP2 (Pecqueur et al. 2001b). However, we found a reasonable correlation between message and protein expression in the tissues examined, suggesting that steady-state levels of BMCP1 message provide a fairly reliable index of BMCP1 protein expression. However, we also found evidence that translational efficiency of BMCP1 may differ between tissues, e.g. heart versus kidney. Although our data indicate that the disparity between mRNA expression and protein synthesis may not be as profound for BMCP1 as for UCP2, it leaves open the question of whether post-transcriptional regulation of BMCP1 protein expression will be more important under conditions of stress or injury. The impact of this alternative ORF in the BMCP1 message will be an important area of future investigation.
To assess the cellular localization of BMCP1 protein expression, brain slices were evaluated for BMCP1 immunoreactivity. Strong BMCP1 immunostaining was observed in cortex, hippocampus, substantia nigra, and cerebellum, with less seen in spinal cord. Dual labeling of slices with antibodies to BMCP1 and the neuronal nuclear marker, NeuN, demonstrated that immunoreactivity of BMCP1 co-localized almost completely with cells expressing NeuN in all regions examined. In cortex and hippocampus, we observed only rare cells which expressed BMCP1 but not NeuN, suggesting that BMCP1 is primarily neuronal. This was further supported by western blot analysis of proteins from neuronal and astrocyte cultures, which showed protein expression in neurons but not astrocytes. Message for BMCP1 was also found in neurons, but not astrocytes. In brain slices, occasional NeuN-positive cells which appeared to lack BMCP1 expression were observed. It is possible that these represent neurons that do not express detectable levels of BMCP1. Further investigation of BMCP1 expression in different neuronal populations may reveal links between synaptic activity, and metabolic regulation in brain.
We then evaluated the subcellular location of BMCP1 protein. Despite evidence that the original uncoupling protein, UCP1, is localized to the inner mitochondrial membrane, a mitochondrial location for other UCP homologs is less well established. A recent study in yeast found that UCP3 was primarily extramitochondrial (Winkler et al. 2001), although these authors proposed that this might be an artifact of overexpression of UCPs in yeast. However, other members of the mitochondrial carrier protein family localize to peroxisomes instead of mitochondria (Weber et al. 1997). Using both subcellular fractionation and immunocytochemistry to determine whether BMCP1 co-localized with cytochrome c, we confirmed that in cultured neurons, BMCP1 protein is localized to mitochondria. Our results also indicated that BMCP1 does not undergo cleavage during mitochondrial import, despite the presence of an N-terminal sequence that appears to be required for mitochondrial targeting (Yu et al. 2000).
To allow analysis of the functional effects of BMCP1 expression on neuronal mitochondria, a neuronal line (GT1-1) was used to generate cells with stable overexpression of BMCP1. Polarigraphic studies of O2 consumption were performed on digitonin-permeabilized GT1-1 cells. We employed this approach instead of using isolated mitochondrial preparations because digitonin-permeabilization results in mitochondria with better respiratory coupling (Becker et al. 1980; Moreadith and Fiskum 1984), and because of the difficulty in isolating sufficient mitochondria from cell cultures. We found that cells overexpressing BMCP1 demonstrated a greater degree of uncoupling (state 4 respiration) than either the vector control cell line, or the parental GT1-1 line. As it is known that lipids, including non-esterified fatty acids, can activate mitochondrial uncoupling by other UCPs (Nicholls 1977; Nicholls and Locke 1984; reviewed by Rial and Gonzalez-Barroso 2001), we evaluated the effect on state 4 respiration of removing lipids. GT1-1 cells expressing BMCP1 showed enhanced uncoupling (state 4 respiration). Fatty acid-free BSA was added to remove endogenous lipid activators of BMCP1, and decreased state 4 respiration to a greater extent in BMCP1 cells than in the vector control cells. Conversely, BMCP1-mediated uncoupling of mitochondrial respiration was increased by low concentrations of the unsaturated fatty acid, linoleic acid (3 µm). Although this concentration of linoleic acid is somewhat higher than that required to activate UCP1 in isolated mitochondria, which is 100 nm (Gonzalez-Barroso et al. 1998), in digitonin-permeabilized cells, fatty acids are likely to be incorporated into other cellular membranes, including the plasma membrane, thereby decreasing the effective concentration at the mitochondrial membrane. Uncoupling proteins 1–3 are also regulated (inhibited) by purine nucleotides ATP, GTP, ADP and GDP through a high-affinity nucleotide binding site (reviewed by Klingenberg and Echtay 2001). ATP is believed to be the principal ligand. We did not explore the effect of purine nucleotides on BMCP1 in the current studies, however, because of the difficulty in removing endogenous ATP from the nucleotide binding site, which would result in ‘masking’ of the binding site and underestimation of the inhibitory effect of added nucleotides (Klingenberg and Echtay 2001). In addition to fatty acids and purine nucleotides, a number of other physiological regulators of UCP1, UCP2 and UCP3 have been identified (Echtay et al. 2000, 2001). However, factors which regulate BMCP1 remain to be determined. Future studies on BMCP1 will focus on the physiological and pharmacological regulation of BMCP1 activity.
Mitochondria in GT1-1 cells transfected with BMCP1 had a slight decrease in mitochondrial membrane potential (ψm), but no drop in ATP levels. Recently, the idea has been advanced that extremely polarized mitochondria generate substantially more ROS than slightly depolarized mitochondria (Skulachev 1998), i.e. under conditions where the mitochondrial are slightly depolarized, ATP production would still be supported, but superoxide and/or H2O2 production would be decreased. In keeping with this idea, neurons transfected with BMCP1 had lower levels of superoxide radical production than controls. The modest overexpression of BMCP1 we achieved in transfected GT1-1 cells was sufficient to abolish the majority of superoxide production which derived from mitochondria. This is consistent with a previous study on UCP1-transfected endothelial cells which also found that mitochondria superoxide production could be suppressed by relatively low levels of UCP expression (Nishikawa et al. 2000)
In summary, our data suggest that BMCP1 is neuronal protein that is widely expressed throughout the CNS, and is localized to neuronal mitochondria. We found that BMCP1 can lower mitochondrial membrane potential without affecting cellular ATP levels, and can enhance uncoupling of mitochondrial respiration in a manner that appeared to be lipid-dependent. Finally, expression of BMCP1 decreased mitochondrial superoxide radical production in neurons. Taken together, these data suggest that BMCP1 might play a role in modifying neuronal mitochondrial function, although whether BMCP1 contributes to damage or rescue of CNS tissue after injury remains an open question, and will be the focus of future studies.