Physiological and molecular characterisation of circadian rhythms in red blood cells

Circadian rhythms allow organisms to actively anticipate, as opposed to passively reacting to, the changes of the 24-hour cycle of a day and a night. These rhythms are generated within our cells, and until recently, the consensus was that they were generated by specific clock genes. The finding, reported three years ago by two of the applicants, that circadian rhythms are also found in isolated human red blood cells (RBCs) was therefore a very remarkable one, because RBCs lack a cell nucleus and therefore do not have any genes. In that report, the protein peroxiredoxin (PRX) was found to alternate rhythmically between an oxidised and a reduced state. Two other applicants have recently found a similar oscillation in the electrophysiological properties of the cell membrane and the cytoplasm that it encloses, using a method called dielectrophoresis (DEP). These two groups have now joined together with the ambition of gaining a greater understanding of how these oscillations are created and connected.

We will sequentially incubate human RBCs in the presence of chemicals that specifically inhibit different groups of biologically active proteins: Protein kinases, which add phosphate groups to other proteins, protein phosphatases, which remove them again, and ion channels, which control the flow of ions across cell membranes and therefore regulate the electrochemical properties of the membrane. Every cell has multiple members of each group, but RBCs, being very specialised cells, have an unusually limited repertoire, and there are chemicals available that act on multiple members of a group, and others that act specifically on a single type. In each case, we will investigate the effect of each chemical both on PRX status and on electrochemical properties. We will interpret a significant effect on the oscillation on both readouts as an indication of a joint mechanism, whereas if it only affects one of them, that protein at least will only affect one of them. This will allow us to identify which proteins within these groups in the RBCs are involved in circadian rhythmicity. We will then proceed to a method known as patch-clamping, which allows us to study a single ion channel molecule within the cell in the presence or absence of an inhibiting chemical.

The outcomes of this research will help us understand how RBCs keep time. These mechanisms may or may not be applicable to other cells. However, RBCs are crucially important for survival, and a greater understanding of how they function may be of benefit to haematology and potentially improved understanding and treatment of malaria, a disease with a strong rhythmic component.

Multiple biological processes display circadian rhythms controlled by endogenous mechanisms. Current models suggest that this process is driven by the rhythmic expression of clock genes. The recent report that circadian rhythms in redox status are present in purified human red blood cells (RBCs) challenges this paradigm, asking important mechanistic questions. We used dielectrophoresis (DEP) to investigate the electrical parameters of RBCs using human blood samples, and found near-24 h cyclic activity in ionic conductance across the membrane, and an antiphasic rhythmicity in cytoplasmic ionic content. We hypothesised that RBC rhythms would be sensitive to the generic kinase inhibitor, staurosporine; investigation showed an increase in the circadian period of the electrophysiological oscillation caused by this drug, indicating that kinases play a role in this non-transcriptional clock. This proposal is based on the hypothesis that the post-translational rhythms observed in RBCs are modulated by protein phosphorylation and dephosphorylation. We aim to establish which kinase/s and/or phosphatase/s underlie and sustain the circadian clock, which ion channel(s) facilitate(s) the circadian rhythm observed by DEP, and whether electrophysiological rhythms are required for redox oscillations in isolated human RBCs, and vice versa. Ion channels, kinases, and phosphatases in RBCs are limited in number. We will examine the effect of various drugs in order to identify those that affect rhythmic behaviours in DEP and/or redox status. This will provide insight into coupling mechanisms between membrane electrophysiology and RBC metabolism. Inhibitor effects will be investigated sequentially through the same iterative process. Generic inhibitors will be followed by others specific to isoforms. Further investigation using patch clamping (for channel candidates) or other agents such as activators once a change (is observed will examine the direct or indirect effects on ion channels.

The proposed project will provide impact benefiting not only the academic sector, but also to the public sector, commercial private sector, third sector and the wider public. The major envisioned areas of impact are the following:

Shedding new light on a basic biological mechanism

The initial report of gene-independent circadian rhythms in RBCs by the Cambridge co-applicants received considerable attention both in the scientific community and in news media. We anticipate that, with the proposed continuation of the investigation, there will be a demand for continued educational activities explaining our findings and their implications. The investigators will be working with the press offices of the universities to enable them to publicise our activities and new findings through classical and new media channels, as well as public fora such as science festivals. Recent examples of such participation includes a Brazilian Globo Reporter documentary, viewed by millions, and the Shuffle Festival (London). Future engagements will be tailored according to the progress of the project and suitable opportunities arising. This will include a bid for participation in the Royal Society Exhibition.

Time scale: First public presentations within 12 months.

Dielectrophoresis of RBCs or whole blood as a circadian phase marker

The standard marker of circadian phase is melatonin, which may be sampled and measured from blood, saliva, or (indirectly through its excretion product 6-sulphatoxymelatonin) from urine. Melatonin assays are a very precise and important tool, but have the drawback of not being able to provide an instant answer, as well as the use of a radioimmunoassay involving radioactive iodine. We envision a potential use of dielectrophoresis as a marker of circadian phase that will be faster, cheaper, and safer, and (although not possible with saliva or urine) require a much smaller blood volume.

We can envision the possibility of developing this method commercially with whole blood rather than purified RBCs, which would add greatly to its utility. If the results from the initial phase of our investigation are encouraging for such a development, we will seek development funding and consider partnering with a commercial organisation. Beneficiaries of such as development would involve both basic researchers in human and animal chronobiology and clinicians needing to determine circadian phase for a broad number of different diagnoses.

Timeline: Initiation of development phase after 12 months.

A greater understanding of RBC biology: Implications for haematology research, development, and ultimately clinical practise

The exploration of hitherto unknown and unexpected rhythmic properties of RBC will have important potential impact. Whilst the research proposed in this application is entirely basic in nature, its successful completion will open a novel avenue of investigation. Clinical research settings will be required to establish whether the same rhythms are present in vivo. Whilst that work will not have a major impact on basic research, any RBC rhythms detected cannot be disentangled from entraining cellular and humoral signals, the presence of such rhythms will be of importance to researchers, developers, and ultimately clinicians within the haematology field, in particular with respect to a greater understanding and potentially new treatment avenues for malaria, which is strongly rhythmic in its manifestations.

Timeline: After completion of project