iao and LeDoux, 2005). However, unlike human Cayman ataxia, the mutations in the dystonic rat model and those in the jittery and sidewinder mouse mutants are generally lethal as they probably cause more drastic changes in BNIP-H structure and quantity than the mutations observed in humans (Xiao and LeDoux, 2005). Interestingly, cerebellectomy could partially rescue the phenotype and prolong the lifespan of the dystonic rat (LeDoux et al., 1993), supporting the notion that BNIP-H does play an important role in the correct functioning of the cerebellum for movement control. Indeed, induction of dystonia upon injection of low doses of kainic acid into cerebellar vermis of mice depends on glutamatergic activation (Pizoli et al., 2002). Furthermore, the authors suggested that the cerebellar cortex plays a crucial role in this model of dystonia (Pizoli et al., 2002). These findings are consistent with our data of strong BNIP-H expression in the cerebellar cortex as detected by in situ hybridisation and immunohistochemistry (Figs 3 and 5). All these results, related to either neurotransmission or neurotoxicity, seem to point to a plausible circuitry network of control between the glutaminase, glutamate and BNIP-H. Dysregulation of such checkpoint, such as the loss of BNIP-H, could underlie the molecular basis and the etiology of cerebellar ataxia, dystonia, and possibly other related neurological disorders.
Finding the precise mode of physical interaction between BNIP-H and glutaminase is important as it allows better understanding on their functional control. We showed that such interaction is mediated by their direct binding in a constitutive manner. Our current work also shows that it involves at least two regions within the BCH domain of BNIP-H - Site I (aa 191-235) and Site II (aa 288-331) - targeting the more distal parts of the enzymatic moiety of KGA (aa 269-547). Interestingly, Site II of the BCH domain encompasses the homozygous Ser301Arg mutation associated with Cayman ataxia (Bomar et al., 2003). However, as expected from the nature of dual-site interaction, this single-point mutation alone does not affect the binding of BNIP-H to KGA (our unpublished data). On the other hand, the splicing mutation in intron 9 of the BNIP-H gene is predicted to cause a truncation of the conserved BCH domain (Bomar et al., 2003). It remains unclear if indeed this truncated form is ever expressed in patients suffering from Cayman ataxia. Further deletion studies within these regions failed to conveniently pinpoint any specific or subtle motifs, indicating that the nature of their interaction is likely to be complex. The answer to this should await high-resolution structural determination. Nonetheless, our current findings on the absolute importance of a functional BCH domain in binding and regulating KGA activity, hence glutamate homeostasis, seem to be a plausible model for further investigation on the molecular basis for this and other related neurological disorders.
In addition, our binding studies have further exemplified the repertoire of BCH domain as a dynamic protein-protein interaction device. It is now evident that different members use specific motifs to mediate their homophilic or heterophilic interactions (Low et al., 1999; Low et al., 2000b; Zhou et al., 2002; Shang et al., 2003), and also engaging Rho subfamily small GTPases to elicit cell protrusions (e.g. BNIP-2 and BPGAP1) (Zhou et al., 2005a; Shang et al., 2003) or cell rounding during apoptosis (e.g. BNIP-Sα) (Zhou et al., 2002; Zhou et al., 2005b). It has been postulated that part of the BCH domain of BNIP-H that weakly resembles the CRAL/TRIO domain (a domain that binds small lipophilic molecules) might be important for targeting specific ligand for its normal function (Bomar et al., 2003). However, this hypothesis has never been tested. Our data on the other hand revealed the unexpected role of the BCH domain in promoting intracellular trafficking and regulating the activity of a key enzyme that is important for neuronal function. Whether or not this property of trafficking is linked to its ability of targeting specific small GTPases, its homophilic or heterophilic binding or lipid binding remains to be seen. Currently, our data show that BNIP-H, unlike BNIP-2 and BNIP-S, does not interact with members of the Rho subfamily GTPases such as Cdc42, RhoA and Rac1 (our unpublished data), raising the possibility that other types of GTPases might be uniquely involved in the BNIP-H signalling pathway. A more detailed investigation is currently underway in our laboratory to probe the mechanistic control of such trafficking, in particularly the possible involvement of cytoskeletal components.
At present, we also show that retinoic-acid-induced differentiation of P19 cells was accompanied by the activation of BNIP-H gene expression in developing and differentiating neurons, suggesting that BNIP-H expression is also regulated at cellular level during the early stages of brain development. This is consistent with the BNIP-H mRNA being developmentally regulated in the hippocampus and cerebellum (Xiao and LeDoux, 2005). Interestingly, the Purkinje cell layer known for its GABAergic neurons appeared to express BNIP-H although glutaminase is known to be absent in Purkinje cells of the adult rodent brain. This implies that BNIP-H could have functions other than just the relocalisation of glutaminase in certain cellular systems or developmental stages.
BNIP-H relocalises KGA to neurite terminals independently of mitochondria. PC12 cells were untransfected (A) or transfected with the indicated epitope-tagged expression plasmids (B-E) and stimulated with NGF for 24 hours. (A) Cells were visualised under bright field to indicate the neurite-outgrowth induced after NGF treatment. (B-E) PC12 cells overexpressing the indicated proteins were fixed, permeabilised and detected by appropriate anti-FLAG or anti-HA, followed by appropriate fluorophore-conjugated secondary antibodies and confocal microscopy detection as described in the Materials and Methods. Mitochondria were stained with MitoTracker dye. Representative images are shown either in their individual colour, merged or in bright fields. Arrows in E indicate the neurite terminals to which KGA had been relocalised by BNIP-H. However, there is no increase in the concentration of mitochondria in these sites. Bars, 20 μm (A-D); 40 μm (E).
Taken together, we have identified and confirmed KGA as a novel target of BNIP-H in the brain where their interaction could play an important role in reducing the steady state levels of glutamate in the cells. Based on the biochemistry of glutamate and the pathophysiological outcomes arising from the loss of BNIP-H in human and mice or rat models of ataxia and dystonia as discussed above, our findings could present a novel paradigm for regulating the homeostasis of glutamate synthesis important for proper neurotransmission and/or neuronal cell growth. The immediate challenge now is to determine both the in vivo levels of glutaminase activity and glutamate in the neurons of the mouse or rat models with defective BNIP-H function, as well as their effects on neurotransmission and neurotoxicity. Following this, they should be subjected to genetic or biochemical means of lowering the activity of glutaminase (e.g. by conditional knockout of KGA or inhibitory peptide from BNIP-H) or to `dampen' the excessive glutamate from uncontrolled glutamatergic activation or neuroexcitotoxicity.
Brains from adult female rats or cultured mammalian cells (Neuro2A and PC12) were homogenised in lysis buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 5 mM sodium orthovanadate, 5 mM glycerol 2-phosphate and a mixture of protease inhibitors) using a Dounce tissue grinder (Wheaton) with Pestle A and B. The homogenate was centrifuged and the supernatant was carefully collected. Glutathione-Sepharose beads (Amersham Biosciences) coated with 100 μg of bacterially expressed GST or GST-BNIP-H were mixed with the lysate and incubated overnight at 4°C with gentle shaking. As a control, glutathione-Sepharose beads coated with 100 μg GST-BNIP-H were mixed with lysis buffer and processed the same way. The beads were then extensively washed with lysis buffer. The bound proteins were eluted with lysis buffer containing 4 M urea, resolved by SDS-PAGE and visualised by silver staining (Bio-Rad). Protein bands excised from SDS-polyacrylamide gels were reduced, alkylated, and then in-gel digested with trypsin (Shevchenko et al., 1996). The extracted peptides were analysed on a matrix-assisted laser desorption/ionisation mass spectrometer equipped with a time-of-flight analyser (MALDI-TOF; Voyager STR BioSpectrometry workstation, Applied System). The obtained mass fingerprints were then used to perform a MS-Fit search on NCBI databases to identify the protein.
The FLAG and HA expression vectors were from Ed Manser (Institute for Molecular and Cell Biology, Singapore). The KGA cDNA was a generous gift from Norman P. Curthoys (Colorado State University, Fort Collins; GenBank accession number AF327434) (Holcomb et al., 2000). KGA full-length cDNA or various fragments were generated through polymerase chain reaction (PCR) with appropriate primers containing either a BamHI or XhoI restriction site for cloning into the FLAG- and HA-pXJ40 expression vectors. Full-length BNIP