Glutamate Receptors in Bone

Introduction
Glutamate not only functions as an amino acid building block of proteins, but also as a neurotransmitter in the central nervous system. It acts as a signalling molecule activating various receptors on different cells. Glutamate acts on two receptor classes, ionotropic receptors (ligand gated ion channels), and metabotropic receptors (G-protein coupled receptors).

Ionotropic receptors can be divided in three main groups; AMPA, NMDA and Kainate (according to their pharmacological or electrophysiological properties). These receptors get their name from antagonists that act on them to deactivate their function. For example, NMDA (N-methyl-D-Aspartate) is a selective antagonist that acts on the thus named NMDA receptor. Activation of these glutamate ionotropic receptors in the central nervous system results in basal excitatory synaptic transmission and synaptic plasticity. Synaptic plasticity, the changing in strength of synapse (connections) between two neurons, results in long-term potentiation or depression of nerve cell function. It is believed that this synaptic plasticity in the hypocampus is responsible for cellular memory and learning.

Glutamate ionotropic receptors have a similar structure to other ionotropic receptors such as GABA. Effectively, they are heterodimers made up of four trans-membrane subunits, TMI – TMIV. All subunits have a hydrophobic centre, and an extracellular N-terminal, which forms a ligand binding domain between TMI and TMIII. TMII forms a loop within the membrane defining the N and C terminals. The C-terminal is intracellular.

Extensive diversity exists within these receptors. Variation can be a result of both pre and post transcriptional modification. Pre-transcription diversity results from the fact that the same subunit can be transcribed by different genes in different cells. Post-transcriptional variation is brought about by pre-mRNA modifications either via splicing or enzymatic modification. Variation in receptor subunits enables such receptors to be localized in different cell types and therefore have varied functions, such as in bone.

A nomenclature system was produced to identify subunits from various glutamate ionotropic receptors and the genes coding for each of the subunits. The finalized nomenclature was agreed upon by the IUHPAR (International Union of Basic and Clinical Pharmacology) in 2008.

Bone Remodelling
Without disregarding the importance of AMPA and Kianate receptors, I would like to focus on NMDA receptors, as most research in glutamate receptors in bone has been focused on the function of NMDA in bone.

Bone is a very diverse organ and has a variety of functions, from being a scaffold to which muscles attach allowing locomotion, to supporting body weight and protecting organs as well as providing a mineral reservoir that responds to the physiological requirements. Being a load-bearing organ the skeleton responds to load-bearing demands by continual remodelling.

Bone remodelling involves resorption (remodelling of bone to reduce its volume) of bone lining cells by osteoclasts followed by infiltration and differentiation of osteoblast precursors into mature osteoblasts which produce a protein, called osteoid. This mineralises to form bone. After matrix deposition, osteoblasts either undergo apoptosis or differentiate into osteocyes, which have various functions from bone formation, maintenance and calcium homeostasis.

Continual remodelling is tightly controlled by signalling molecules that regulate the activity of bone cells. Damage to this homeostatic mechanism causes a variety of bone disease such as osteoporosis, affecting 1 in 3 females and 1 in 5 males. Conditions such as osteoporosis pose a financial burden on the health services therefore a lot of money is invested in studying the mechanisms leading to development of such conditions.

It is suggested that calcium flux through glutamate receptors in bone induces a memory/learning function to load-bearing bones, regulating the rate by which bones remodel themselves to make up for the damage and stress caused by day-to-day load-bearing demands. As discussed earlier, the central nervous system has been the traditional model for studying the NMDA receptors. Recent studies have been using the knowledge of the function of these receptors on the CNS and apply it to bone to assess the function of such receptors on bone remodelling and load-bearing capabilities.

NMDA Receptor (NMDAR) Structure
The NMDA receptor is a heterodimer composed of two NR1 (glycine binding) and 2 NR2 (glutamate binding) subunits. There are also NR3a and NR3b (accessory) subunits present in the complex. A magnesium ion is present in the pore between NMDAR1 and NMDAR2 subunits in NMDAR, producing a voltage-dependent block, which needs to be removed for the channel to open.

NMDA Receptor Function
All AMPA, NMDA and Kiane receptors require glutamate to be activated, but NMDA also requires a co-agonist, glycine. Glutamate is released and binds to postsynaptic glutamate binding site on the NR2 subunit. Glycine binds to a glycine binding site on the NR1 subunit. On binding of both agonists, the postsynaptic membrane is excited which causes it to depolarise and removes the blocking Mg2+. In turn this allows the channel to open, allowing more Na+ and Ca2+ to enter than K+ to exit. Therefore, activation requires both binding of agonists and a positive change in the trans-membrane potential brought about by expulsion of magnesium ions that block the channel from the outside. Together these activate the channel and allow an ion flux through the pore.

Presence and Function of NMDA Receptors in Bone
Eight variants of the NR1 subunit were identified, all produced by alternative splicing of exon 3 in NMDAR1 gene (GRIN1), (Spencer et al., 2007). Four isoforms of the NR2 submit were also identified in invertebrates. On the other hand the cytoplasmic domain is highly adaptable via enzymatic modification via phosphatise and kinase action. The C-terminal domain also interacts with scaffolding proteins and adaptors to keep the receptor structure intact. Locating different scaffold proteins, adapters and subunits led to the idea that NMDA receptors might be located at places other than the CNS and directed research into the presence of NMDAR in bone.

Various studies have been carried out to localise NMDAR in bone and identify its functions. NMDAR has been found present in bone by localising GLAST, a glutamate transporter in osteoblasts and osteocytes, suggesting that glutamate might be used as a signalling molecule in bone. Different glutamate receptor subunits were also located in bone using in-situ hybridisation and immunochemistry. Furthermore, PSD-95, an NMDA scaffold protein normally found in the CNS, was also located in bone (A.J. Patton et al., 1998).

Further in-vitro research established that antagonists, such as MK801 (a channel blocker), inhibited osteoclast development in osteoblast/osteoclast co-cultures (Peet, Grabowski et al., 1999, Merle, Itzstein et al., 2003), while also inhibiting osteoblast mineralisation (Taylor, 1999). More antagonist studies showed that NMDA antagonists on mature osteoclasts inhibited bone resorption (Chenu, Serre et al., 1998), and that MK-801 treatment during early stages of OB culture resulted in the inhibition of matrix mineralization (Taylor, 2002; Hinoi, Fujimori et al., 2003; Ho, Tsai et al., 2005).

Other studies showed that inhibition of glutamate release from osteoblasts increased the number of apoptotic cells in culture, and inhibited osteoblasts differentiation in co-cultures (Genever and Skerry, 2001). On the other hand, inhibition of glutamate release from osteoclasts increased the resorptive activity of the same cells.

These studies did not only produce data to account for the presence of NMDA receptors in bone but also showed that NMDA receptors have a role in bone development, mineralisation and differentiation. Electrophysological studies on glutamate receptors now suggest a “neural control” of metabolism in bone cells. Glutamate containing nerve fibres in close proximity or in contact with, bone cells indicate “glutametric neurotransmission” between nerve and bone cells (Chenu, 2002).

Although NMDA receptors have received much attention it is important to note that other glutamate receptors have also been identified in bone. Therefore a question posed was, what is the source of glutamate in bone? Research shows that osteoblasts release glutamate in a coordinated method, similarly to and at the same levels at which glutamate is released in neuronal cells (Genver & Skerry, 2001). Fluorescently labelling cells with dyes such as FMI-43 also showed presence of glutamate containing vesicles in osteoblasts.

Current Research into NMDAR Function in Bone
Research is now moving from in-vitro analysis to in-vivo studies, where mice models are used to analyse changes in morphological parameters, such as bone volume and mineral density when the functional NMDA receptor is knocked out in bone cells. Moving research into in-vivo analyses is a very important step towards generating data relevant to medical applications. On-going research at the University of Sheffield suggests changes in bone volume of load-bearing long bones lacking functional NMDAR.

The physiological role of glutamate has been difficult to establish because full knockout animals have lethal phenotypes while most chemical antagonists administered in-vivo have significant side effects on the CNS. To counter this, a Cre Lox P system which allows targeted deletion of NMDA in bone cells is used. This can be done by, for example, knocking down the region coding for the NR1 (glycin binding) subunit within the GRIN1 gene. The octeocalcin promoter (Oc-Promoter) is used as a target for Cre recombinase in osteoblasts. Being solely present in all osteoblasts makes the Oc-promoter a selective means to knocking out GRIN1 in only targeted cells. If the NMDAR receptor is to be knocked out in other cell types, such as osteoclasts, an osteoclast specific promoter (such as the TRAP promoter) is used as a target. Offspring which contain both the loxP-flanked target locus plus the Cre gene will express the Cre gene in the desired cell type, and the resulting recombination between the loxP sites in these cells results in tissue-specific inactivation of the target locus of GRIN1, in this case NR1 locus. Oc-Cre+/+ mice are crossed with GRIN1 flox/flox (floxed meaning; flanked by lox) mice to obtain mice of a knockout geneotype (Cre+GRIN flox/flox ) and controls (Cre -/- GRIN1 flox/flox). Once knockout and control mice are obtained, bone morphometric parameters can be analysed and compared.

Micro Computed Tomography (µCT)
The main method used to analyse bone morphometric architecture is Micro Computed Tomography, an enclosed X-ray system where a fixed X-ray source is passed through the specimen and projected onto a detector.

As the specimen rotates, consecutive images are combined to build a three-dimensional block of data showing the X-ray snapshot at each point within the specimen. This enables computed measurements of distances between different points within the rotating sample that are used to render a 3D model. Pixel densities from the X-ray image produced are used to compute the bone mineral densities, while the volume (area of mineralized tissue) of bone is calculated from the area of the density histogram produced.

Understanding the morphological consequences of targeted deletion of glutamate receptors on bone not only reinforces the molecular basis of the receptor mechanism and function but also helps address the question as to why bone cells require excitatory neurotransmission as a means of cellular communication, such as found in the CNS. It is argued, amid speculation, that such signalling pathways have a direct influence on bone function. What is certain is that mechanical loading of bone has a direct influence on bone remodelling rates and evidence exists for memory function in bone that has a regulatory function in bone remodelling. Studies into the effects of mechanical loading on bone show a direct relationship with expression of glutamate receptors (Spencer & Genver, 2003).

Prevailing Questions
In-vivo research on glutamate receptors in bone is still at its infancy and a lot of questions are still unanswered. Firstly, long bones have much higher load-bearing capabilities than flat bones, such as skulls, and this has a direct influence on the rate of bone remodelling that occurs between different types of bone. Therefore, remodelling regulation is specific to bone type, and so might the expression of glutamate receptors be. Moreover, long bones and flat bones have different developmental origins. Long bones develop via endochondrial ossification, which occurs when hyaline cartilage is replaced by bone tissue, while skulls develop into layers forming a rigid structure. Such differences result in different bone morphologies, and therefore NMDA may have different roles, or functions at different rates in different bones. Moreover, we need to question if the subunit being knocked out in the receptor is actually required for NMDR function in bone cells. Since, the NMDARs were traditionally analysed in the nervous system, knockout mice were designed to knockout NMDAR in nerve cells and it is still questionable whether knockout models have the same knockout effect in bone. Further analysis is therefore required, from GRIN1 mRNA assays to test if NMDAR is being knocked out to calcium flux assays to assess whether knockout cells can still import calcium ions via different channels other than the type being knocked out.

Conclusion
By understanding how load-bearing bones maintain and regulate their remodelling mechanisms, we can better understand how this can go wrong and produce deterioration in the bone mineral density, such as in osteoporosis. The hope of being able to regulate bone remodelling mechanisms to make up for bone exposure to damage and avoid bone mineral density deterioration is an exciting prospect.

References
Spencer, G., C. McGrath, et al. (2007). “Current perspectives on NMDA-type glutamate signalling in bone.” The International Journal of Biochemistry & Cell Biology 39(6): 1089-1104.

Patton, A. J., Genver, P. G. et al. (1998). “Expression of N-Methyl-D-Aspartate-Type Receptor by Human and Rat Osteoblasts and Osteoclasts Suggests a Novel Glutamate Signalling Pathway in Bone”. Bone 22:645-649.

Stephenson, F.A. (2006) “Structure and trafficking of NMDA and GABAa receptors”. Biochemical Society Transactions. 34: 877-881.

Buckwalter, J.A. et al. (1995). “Bone Biology. Part II: Formation, Form, Modeling, Remodeling and Regulation of Cell Function.” The Journal of Bone & Joint Surgery, 77-A 1276-1283.

Peet, D. M., Grabowski, et al (1999). “The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation”. FASEB J. 13, 2179-2185.

Chenu,C. Et al. (1998), Glutamate receptors are expressed in bone cells and are involved in bone resorption. Bone, 22, 295-299.

MRC Centre for Synaptic Plasticity, University of Bristol, Bristol. Glutamate Receptors.
Available at: http://www.bristol.ac.uk/synaptic/receptors/