Is Magnetogenetics the new Optogenetics?

Any medical student would agree that one of the most pressing issues that drives most of us to tears of desperation is “How on earth do we commit so much factual information into our memory?!Hence if someone were to claim that it was possible to zap one’s brain with mere light to selectively activate or inhibit neurons we would be among the first subscribers to this neuron function altering technique! Well the reality is that this dream has already been turned into a reality through the field of optogenetics which was discovered almost two decades back and took the world of neuroscience by storm so much so that it was bestowed with the title of ‘method of the year 2010’ by Nature.(1) Optogenetics involves the use of  light sensitive channels isolated from algae called as channel rhodopsins which are used to label neurons selectively and when light is shone onto the neurons only the labelled neurons are activated. Today this novel field has far reaching applications and has been used by scientists to create and alter memories in mice(2)(3), create false memories in the absence of any experience(4) and even suppressing depression like behaviour(5) which has a wide range of applications in the development of potential therapies for neurodegenrative disorders like Alzheimer’s and Parkinson’s disease as well as in the treatment of PTSD and Mood Disorders. However just as we start packing our bags to set off to MIT to avail of the benefits of this almost too good to be true technology that seems to be the stuff that sci-fi movies are made of we encounter a roadblock. In order for us to use this technology there is a necessity to undergo a neurosurgery to drill a hole into our cranium in order to carefully implant the fibre optic electrodes in close vicinity to the labelled neurons causing the risks of this technique to be akin to that of the Deep Brain Stimulation techniques that are currently one of the therapeutic options for diseases like Parkinson’s. The concept of magnetogenetics evolved as a means to circumvent these problems associated with the technique of optogenetics.

Magnetism and the world of Biology

Since time immemorial, humans have been awed by other animals’ remarkable feats of navigation—from arctic terns that travel quite literally from pole to pole, to sea turtles which return reliably to their natal coastlines to lay eggs after many years in the open ocean. How are these creatures able to achieve this without the aid of maps or GPS when I myself can get lost quite easily in CMC’s hospital campus? The answer lies in their unique ability of magnetoreception. The cause of this unique ‘sixth sense’ in these animals has been a topic of much study with several hypotheses being put forward.

The first plausible hypothesis is that these birds possess a light sensitive chemical based magnetoreceptor (cryptochrome) in their retinas. This concept predicts that the geomagnetic fields can influence the spin state of light-induced radical pairs and thereby modulate the outcome of biochemical reactions thereby enabling these birds to actually see magnetic field lines of the earth with their eyes. This theory could also explain how the introduction of artificial low-intensity broadband electromagnetic fields (which influence electron spins) disrupt magnetic orientation and cause the birds to lose their way.(7)

Yet another hypothesis is that they have a mechanically sensitive magnetite-based magnetoreceptor. This has been proven to exist in some species.

The best example are magnetotactic bacteria whose preferable habitat is the deeper waters at the bottom of a pond. Whenever the pond is disturbed and they float to the top of the pond they generate a chain of intracellular magnetite crystals. They employ this internal compass needle to guide their swimming along the incline of the magnetic field vector to deeper waters with favorable redox conditions. (6)

Magnetogenetics Principles

A number of groups have tried to engineer artificial magnetosensors and these systems fall into three broad categories: magneto‐thermo‐receptors which exploit radio frequency fields to cause activation of heat‐sensitive channels; force/torque‐based methods that rely on endogenously generated nanoparticles; and the expression of the iron chaperone ISCA1.(10)

Let’s explore each of these in detail-

Magneto-thermo-genetics

This approach involves using local heating of superparamagnetic nanoparticles to convert a radio-frequency (RF) magnetic signal into cell stimulation. Manganese ferrite (MnFe2O4) nano particles were targeted to cells expressing the temperature- sensitive ion channel TRPV1, and heated using a RF magnetic field. The local temperature increase opened the TRPV1 channels and caused an influx of calcium ions. The activation temperature of TRPV1 was 42 degrees celsius and it was observed that

an aqueous dispersion of MnFe2O4 nanoparticles conjugated with streptavidin and subjected to a RF

magnetic field (40 MHz, 8.4 G) heats up at an initial rate of 0.62 degrees Celsius/second, a field strength that satisfies the FDA requirements for RF fields applied during MRI. The cells of interest were genetically made to express the engineered membrane protein marker AP-CFP-TM. This protein marker contains a transmembrane domain (TM) of the platelet-derived growth factor, an extracellular fluorescent protein (CFP) and a biotin acceptor peptide (AP) that is enzymatically biotinylated to bind the streptavidin-conjugated nanoparticle. In order to prove that the heating effect was localised to the vicinity of the nano particles and that there is a definite change in temperature due to the magnetic field the streptavidin coated nano particles were attached to fluorophores (DyLight549) which acted as nanoscale thermometers and showed a reduction in the intensity of fluorescence within 15s of application of the RF field. The influx of calcium due to the opening of the TRPV1 channels was proved in the HEK293 cells where when intra- cellular calcium concentration was measured using the genetically encoded Fo ̈rster resonance energy transfer (FRET)-based calcium sensor, Troponin extra large (TN-XL)23 within 15 s of applying the RF magnetic field (40 MHz, 8.4 G) the cytosolic calcium concentration increased from about 100 nM to 1.6 mM. The increase was found to be caused by calcium influx through thermally activated TRPV1 channels, because cells with nanoparticles but without TRPV1 channels, and cells with TRPV1 channels but without nanoparticles, did not show any calcium influx upon application of the same RF magnetic field.

The  calcium influx results in a neuronal depolarization that is sufficient to elicit an action potential, which is necessary for the control of neuronal function. The scientists measured changes in the membrane potential of hippocampal neurons that expressed TRPV1 and were labelled with nanoparticles, using the voltage-sensitive dye ANNINE624. Immediately after applying the RF magnetic field, the ANNINE6 fluorescence intensity decreased as the membrane temperature rose and several small membrane voltage spikes followed by an action potential type depolarization was registered. They also showed how Caenorhabditis elegans whose brains were labelled with the particles had an aversive response and reversal of direction of locomotion in a magnetic field consistent with their reflex to avoid a heat stimulus while unlabelled worms showed no such response when exposed to the same magnetic field. In essence, this experiment established the remote control of ion channels in cells using RF magnetic-field heating of nanoparticles. (8)

Along the same lines a set of experiments were performed in vertebrates by the Anikeeva laboratory (9) that induced calcium influx in HEK cells, action potentials in primary hippocampal neurons and neuronal activation in deep brain areas in vivo in mice. While the size and elemental composition of artificial nanoparticles permit the generation of heat with greater precision, the nanoparticles must still be delivered by injection, which risks tissue damage, and their dispersion over time. The ideal system would therefore require genetically encoded nanoparticles. 

This challenge was addressed by a set of experiments that used a chimeric ferritin tethered to TRPV1 via a GFP nanobody. (11)In vertebrates, the ferritin supercomplex is made of 24 subunits of both light and heavy chains that enclose an iron oxide nanoparticle. This particle, which is ~6 nm in size, is predominantly composed of ferrihydrite but may also contain magnetite (Fe3O4) and maghemite (Fe2O3) phases.(12) The scientists validated that magnetic fields could cause the ferritin tethered TRPV1 to be opened causing a calcium influx and further activation of a calcium responsive genetic element that caused the release of human pro insulin. (11) They also showed that a similar construct introduced into the VMH(ventromedial hypothalamus) could stimulate remote magnetic field mediated activation of glucose sensing neurons in the hypothalamus thereby increasing plasma glucose and glucagon, lowering insulin levels and stimulating feeding when the animals were placed close to the electromagnetic coil of an MRI machine.(13)

Torque based magnetogenetics

An alternative to generation of heat by an oscillating RF field is using a strong magnetic gradient that exerts a force on a magnetic particle

This was what another set of scientists aimed to recreate by  a novel magnetogenetic actuator that they called Magneto2.0.(14)They hypothesised that when fused to a mechanosensitive channel called TRPV4, a paramagnetic protein would enable magnetic torque to tug open the channel to depolarize cells. The addition of a plasma membrane trafficking signal enhanced the prototype channel’s membrane expression causing increase in the calcium influx. Mice expressing Cre recombinase under control of the dopamine receptor 1 promoter which is expressed in approximately half of the medium spiny neurons (MSNs) of the striatum were injected with adenovirus vectors carrying Magneto2.0 and it was shown that not only was there increased neural firing in deep brain regions in response to magnetic fields but this could also be translated to control of mammalian reward behaviour that is regulated by dopaminergic signalling. This was done by subjecting the mice to a real time place preference (RTPP) assay where they could choose between a magnetized arm, lined with eight permanent NdFeB magnets delivering a magnetic field gradient of 250–50 mT, and a non-magnetized arm. It was observed that Magneto2.0 expressing mice showed a significant preference for the magnetized arm in contrast to WT mice which exhibited no such preference.(14)

The last among the three proposed techniques of magnetogenetics involved the use of iron chaperone protein ISCA1 to form a magnetic protein biocompass in conjugation with the light‐sensitive molecule cryptochrome (CRY4) (15) however further studies have failed to replicate the results(16).

The problems with magnetogenetics

As tantalising as the idea of remotely controlling cell expression and neuronal action potential with a non invasive magnetic field based approach sounds the technology as it stands today in its nascent stage is indeed fraught with obstacles that need to be overcome to bring it into mainstream scientific research and stand a chance to be a worthy successor to replace optogenetics.

Firstly it is unclear how the techniques that rely on genetically encoded ferritin nanoparticles, actually work.  Our current knowledge of the ferritin moiety indicates that it lacks the magnetic properties to activate either a mechanical or temperature‐sensitive channel. For instance, the force generated by a single ferritin nano‐particle, which contains about 4,500 iron atoms, in a 50 mT field with a gradient of 6.6 T/m is just 7 × 10−23 N, well below the 2 × 10−13 N required to open known mechanoreceptors.(17)

Hence further attempts to replicate these experiments independently as well as further research into the localisation, shape and magnetic properties of the nanoparticle within the ferritin supercomplex and the thresholds for receptor activation becomes necessary. A recent article proposed certain mechanisms that could explain how the ferritin moiety’s magnetic properties cause channel opening which include – the complex superparamagnetic interactions between adjacent ferritin particles could generate magnetic fields strong enough to open the channels, the diamagnetic interaction between ferritin and the channels and the low Young’s modulus of nerve membranes could cause deformation of the ion channels and cell membrane and their mechanical opening by means of magnetic fields, application of a polarizing magnetic field cause the magnetic spins to align in the direction of the  field which lowers the entropy of the spin ensemble and in an adiabatic process where there is no exchange of heat with the environment, this change in spin ensemble entropy has to be compensated for by the exchange of energy between the spin ensemble and the magnetite particle lattice, resulting in the change of temperature of the particle and lastly it is a fundamental tenet of quantum mechanics that magnetic moment m of a particle is proportional to mechanical angular momentum L of that particle, m = ɣ·L, where ɣ is the gyromagnetic ratio and as per the Einstein-de Haas effect a reversal of a magnetic moment of a sample by an applied magnetic field has to be accompanied by a corresponding change in mechanical angular momentum of that sample which is converted to rotational kinetic energy that is eventually transferred to the magnetite particle lattice and the environment through friction causing an increase in temperature and thereby opening the ion channels.(18)

Secondly it takes more time to activate channels via magnetic fields which requires seconds to work  as compared to optogenetics that can switch on or off cells in milliseconds. Efficiency and speed could be improved in a number of ways. To date, most systems use a fusion protein of human light and heavy‐chain ferritin. However certain mutant forms of ferritin are able to load more iron and consequently have a greater magnetic susceptibility. A potential candidate is the heavy‐chain ferritin from the thermophilic bacterium Pyrococcus furiosus that triples iron loading(19). There is also scope to co‐opt ferritins from magnetite generating species such as chitons, to further enhance the magnetic properties of the system. Similarly, future incarnations of magnetogenetic sensors may incorporate temperature‐ or mechano‐sensitive channels with lower thresholds of activation, such as TRP channels from infrared‐sensing snakes or vampire bats.

The third disadvantage to magneto genetics is the much higher cost of infrastructure needed to develop such a system while in comparison optogenetics requires a much lesser investment for the light source that is relatively inexpensive. This problem can only be surmounted by improving the efficacy of the coil systems that is used in magneto genetics.

Conclusion

The potential applications of magneto genetics is far ranging and diverse.From using it to generate localised and targeted hyperthermia in order to kill cancer cells or to cause release of human pro insulin from non beta cells, to selectively stimulating neural circuits in the brain in a less invasive manner as compared to optogenetics, which can have far ranging implications in the treatment of various neuro degenerative and psychiatric disorders the future does seem promising for this novel technique of manipulating cell biology and pathology. Such a system might eventually rival optogenetics as the pre‐eminent tool in neuroscience however at present there are many technical obstacles in the path to it being used in mainstream clinical practice. However further dedicated research may find solutions to the present obstacles and pave the path for the evolution of this  promising technique to being used to treat some of the most daunting diseases that challenge modern medicine.

references 

(1) Method of the Year 2010. Nat Methods 8, 1 (2011). https://doi.org/10.1038/nmeth.f.321

(2)Liu, X., Ramirez, S., Pang, P. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012). https://doi.org/10.1038/nature11028

(3) Ramirez S, Liu X, Lin PA, et al. Creating a false memory in the hippocampus. Science. 2013;341(6144):387-391. doi:10.1126/science.1239073

(4) Vetere, G., Tran, L.M., Moberg, S. et al. Memory formation in the absence of experience. Nat Neurosci 22, 933–940 (2019). https://doi.org/10.1038/s41593-019-0389-0

(5) Ramirez, S., Liu, X., MacDonald, C. et al. Activating positive memory engrams suppresses depression-like behaviour. Nature 522, 335–339 (2015). https://doi.org/10.1038/nature14514

(6) Nordmann GC, Hochstoeger T, Keays DA. Magnetoreception-A sense without a receptor. PLoS Biol. 2017;15(10):e2003234. Published 2017 Oct 23. doi:10.1371/journal.pbio.2003234

(7) Engels, S., Schneider, NL., Lefeldt, N. et al. Anthropogenic electromagnetic noise disrupts magnetic compass orientation in a migratory bird. Nature 509, 353–356 (2014). https://doi.org/10.1038/nature13290

(8) Huang H, Delikanli S, Zeng H, Ferkey DM, Pralle A. Remote control of ion channels and neurons through magnetic-field heating of nanoparticles. Nat Nanotechnol. 2010;5(8):602-606. doi:10.1038/nnano.2010.125

(9) Chen R, Romero G, Christiansen MG, Mohr A, Anikeeva P. Wireless magnetothermal deep brain stimulation. Science. 2015;347(6229):1477-1480. doi:10.1126/science.1261821

(10) Nimpf S, Keays DA. Is magnetogenetics the new optogenetics?. EMBO J. 2017;36(12):1643-1646. doi:10.15252/embj.201797177

(11) Stanley SA, Gagner JE, Damanpour S, Yoshida M, Dordick JS, Friedman JM. Radio-wave heating of iron oxide nanoparticles can regulate plasma glucose in mice. Science. 2012;336(6081):604-608. doi:10.1126/science.1216753

(12) Quintana C, Cowley JM, Marhic C. Electron nanodiffraction and high-resolution electron microscopy studies of the structure and composition of physiological and pathological ferritin. J Struct Biol. 2004;147(2):166-178. doi:10.1016/j.jsb.2004.03.001

( 13) Stanley SA, Kelly L, Latcha KN, et al. Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism. Nature. 2016;531(7596):647-650. doi:10.1038/nature17183

(14) Wheeler MA, Smith CJ, Ottolini M, et al. Genetically targeted magnetic control of the nervous system. Nat Neurosci. 2016;19(5):756-761. doi:10.1038/nn.4265

(15) Qin S, Yin H, Yang C, et al. A magnetic protein biocompass. Nat Mater. 2016;15(2):217-226. doi:10.1038/nmat4484

(16) Pang K, You H, Chen Y, et al. MagR Alone Is Insufficient to Confer Cellular Calcium Responses to Magnetic Stimulation. Front Neural Circuits. 2017;11:11. Published 2017 Mar 16. doi:10.3389/fncir.2017.00011

(17) Meister M. Physical limits to magnetogenetics. Elife. 2016;5:e17210. Published 2016 Aug 16. doi:10.7554/eLife.17210

(18) Barbic M. Possible magneto-mechanical and magneto-thermal mechanisms of ion channel activation in magnetogenetics. Elife. 2019;8:e45807. Published 2019 Aug 2. doi:10.7554/eLife.45807

(19) Matsumoto Y, Chen R, Anikeeva P, Jasanoff A. Engineering intracellular biomineralization and biosensing by a magnetic protein. Nat Commun. 2015;6:8721. Published 2015 Nov 2. doi:10.1038/ncomms9721