Bioelectricity: The Missing Link in Regeneration

By Charlotte Hutchings

Regeneration occurs on several scales with species differing in their capacity to regenerate tissues, organs, and limbs (Maden, 2018). One group commonly used to study regeneration is the Planarian worms. Many planarians have the remarkable ability to regenerate their entire body after being split in two, primarily through the activity of pluripotent stem cells. Indeed, this regenerative process acts as a mechanism of asexual reproduction for the worms as well (Ivankovic et al., 2019). Although vertebrates have a lower regenerative capacity, certain groups such as urodele amphibians (newts and salamanders) are still able to regenerate complete and functional adult limbs. However, when it comes to humans and other mammals little to no regenerative capacity is observed (Beck, 2015). Understanding the mechanisms behind regeneration is not only of interest to developmental biologists but has now become a high priority in the field of regenerative medicine.

Studies of regenerative biology have often focused on the role of genetics and transcription factors. For example, pitx, a homeodomain transcription factor, has been identified as a key molecular player in the regeneration of planarians due to its roles in midline patterning and the regeneration of serotonergic neurons (Currie & Pearson, 2013). Perhaps attention on genetics is the result of historic events, such as the discovery of the DNA double helix, or sequencing of the human genome. However, to consider an organism as a code of base pairs is a dramatic over-simplification. Bioelectricity represents an alternative, and somewhat overlooked, factor involved in the determination of an organism’s morphology and physiology (Whited & Levin, 2019).

All cells, both excitable and non-excitable, are surrounded by a membrane containing various ion channels and transporters. At the simplest level, the expression and distribution of such proteins controls the bioelectrical state of a cell by controlling the movement of ions across the membrane. This movement of charge across the membrane creates a difference in voltage potential between the inside and outside of the cell. Further, cells are electrically coupled via gap jap junctions to allow bioelectrical signals to be propagated throughout the cellular network (Levin, 2009). Such signalling has emerged as an important regulator of pattern development and regeneration (Whited & Levin, 2019).

This principle is best demonstrated in model planarians. Under normal conditions, an adult worm is characterised by the presence of an anterior-posterior bioelectrical gradient whereby the head is depolarized, and the tail hyperpolarized (Durant et al., 2017). When a worm is split in two, either during asexual reproduction or experimentally, this gradient provides information to determine whether each segment regenerates a head or tail. Interestingly, the instructive ability of this bioelectrical gradient appears to be dominant over previously identified molecular gradients. Previous studies using RNAi to knockdown b-catenin, a key molecular player, results in inappropriate regeneration of a head at the posterior end of amputations (Gurley et al., 2008). However, when b-catenin RNAi is combined with the treatment using the proton pump inhibitor SCH-28020 to promote hyperpolarization, normal tail regeneration occurs (Beane et al., 2011). The suggestion that bioelectrical signalling acts upstream of gene expression is further supported by the detection of differences in membrane voltage prior to changes in gene expression during planarian regeneration (Durant et al., 2019).

As we begin to understand bioelectrical circuits and their effects on large scale patterning, the question arises whether we could manipulate bioelectrical properties to promote regeneration. The ultimate goal in regenerative medicine is to be able to regenerate functional human tissue. Given that the human genome encodes the developmental pathways required for tissue, organ

and limb formation, it does not seem unrealistic that we could re-activate such programmes after injury or amputation (Whited & Levin, 2019). Indeed, promising results have been found in animal models. In Xenopus studies, progesterone, a neurosteroid contributing to modulation of cellular bioelectrical status, is able to induce leg regeneration following amputation (Herrera-Rincon et al., 2018).

Future therapeutic approaches in regenerative medicine could be focused on modifying the bioelectrical properties of cells in order to re-activate developmental and/or repair pathways. This could include pharmacological methods, such as the hormone treatments investigated in the Xenopus, or the use of pre-existing approved ion channel modulators, a method referred to as ‘electroceuticals’ (Whited & Levin, 2019). Nevertheless, further work is required in understanding bioelectrical signalling and their downstream effects on gene expression before any human therapies can be considered.

References:

Beane, W. S., Morokuma, J., Adams, D. S. & Levin, M. (2011) A chemical genetics approach reveals H,K-ATPase-mediated membrane voltage is required for planarian head regeneration. Chemistry & Biology. 18, 77-89. doi: 10.1016/j.chembiol.2010.11.012.

Beck, C. W. (2015) Regeneration: Growth factors in limb regeneration. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001104.pub2]

Currie, K. W. & Pearson, B. J. (2013) Transcription factors lhx1/5-1 and pitx are required for the maintenance and regeneration of serotonergic neurons in planarians. Development (Cambridge). 140, 3577-3588. doi: 10.1242/dev.098590.

Durant, F., Morokuma, J., Fields, C., Williams, K., Adams, D. S. & Levin, M. (2017) Long-term, stochastic editing of regenerative anatomy via targeting endogenous bioelectric gradients. Biophysical Journal. 112, 2231-2243. doi: 10.1016/j.bpj.2017.04.011.

Herrera-Rincon, C., Golding, A. S., Moran, K. M., Harrison, C., Martyniuk, C. J., Guay, J. A., Zaltsman, J., Carabello, H., Kaplan, D. L. & Levin, M. (2018) Brief local application of progesterone via a wearable bioreactor induces long-term regenerative response in adult Xenopus hindlimb. Cell Reports (Cambridge). 25, 1593-1609.e7. doi: 10.1016/j.celrep.2018.10.010.

Ivankovic, M., Haneckova, R., Thommen, A., Grohme, M. A., Vila-Farré, M., Werner, S. & Rink, J. C. (2019) Model systems for regeneration: planarians. Development (Cambridge). 146, dev167684. doi: 10.1242/dev.167684.

Gurley, K. A., Rink, J. C. & Alvarado, A. S. (2008) β-Catenin defines head versus tail identity during planarian regeneration and homeostasis. Science (American Association for the Advancement of Science). 319, 323-327. doi: 10.1126/science.1150029.

Levin, M. (2009) Bioelectric mechanisms in regeneration: Unique aspects and future perspectives. Seminars in Cell & Developmental Biology. 20, 543-556. doi: 10.1016/j.semcdb.2009.04.013.

Levin, M., Selberg, J. & Rolandi, M. (2019) Endogenous bioelectrics in development, cancer, and regeneration: Drugs and bioelectronic devices as electroceuticals for regenerative medicine. iScience. 22, 519-533. doi: 10.1016/j.isci.2019.11.023.

Maden, M. (2018) The evolution of regeneration – where does that leave mammals? The International Journal of Developmental Biology. 62, 369-372. doi: 10.1387/ijdb.180031mm.

Whited, J. L. & Levin, M. (2019) Bioelectrical controls of morphogenesis: from ancient mechanisms of cell coordination to biomedical opportunities. Current Opinion in Genetics & Development. 57, 61-69. doi: 10.1016/j.gde.2019.06.014.

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