Prostheses aim to enhance the quality of life, independence, mobility, and safety of amputees. The worldwide frequency of amputations has created an increased demand for improved prostheses technologies. Within developing countries, the population with physical disabilities in demand of a prosthesis is estimated at about 0.5%. There are currently almost 2 million people living with limb loss in the United States. In 2050, this amount will exceed the 3 million people.^[1].

Over the past decade, many types of prostheses have been created. One of them is the myoelectric prosthesis, which captures the electromyography (EMG) signal of residual limb muscle through surface electrodes on the skin. These kinds of robotic prostheses allow amputees to recover some autonomy and to accomplish simple everyday gestures. However, they are still limited by the number of controllable moves and by the lack of sensory feedback ^[1].

Today, the direct attachment of the prosthesis to the skeleton introduces the concept of osseointegration. This kind of prosthesis provides the patient with precise and reliable control of the new limb, regardless of the environmental conditions or the position. The opportunity to record and stimulate the neuromuscular system allows intuitive control and a better understanding of sensory perception ^[2].

The central nervous system (CNS), composed of the brain and the spinal cord, aids to integrate, influence and coordinate activities across the whole organism. The neural tissues bring information from one region of the body to another by sending electrical signals along the axon ^[3]. The efferent motor neurons of the peripheral nervous system (PNS) communicate with muscles and are under voluntary control. They go from the spinal cord to the arms, hands, legs and feet and allow to transmit nerve impulses away from the CNS, leading to an action ^[3]. To increase the electrical signal speed, axons are surrounded by myelin, produced by another type of cells called Schwann cells. These cells twist around the axon and prevent the loss of the electrical signal ^[4]. After limb amputation, the main issue of the human-machine communication is due to the damage inflicted to nerves. Indeed, the electrical signal cannot be transmitted because peripheral nerve cells are no longer able to activate target muscles or relay sensory information from the limbs back to the brain.

The first neurotrophin to be discovered was the Nerve Growth Factor (NGF). It was described in 1952 by Rita Levy Montalcini and Viktor Hamburger^[6]. Since then, many other neurotrophins were discovered (BDNF, NT-3, NT-4, NT-6)^[7] and much progress has been achieved towards understanding how they work. Yet, NGF is still considered a “prototype neurotrophin”[5] and is generally used as an example to describe their function. For this reason, we chose to clone this polypeptide into our bacteria, creating the first component of the NeuronArch project.

Indeed, if there is no doubt that β-NGF is a neurotrophic factor, it has still not been determined exactly whether pro-NGF is a neurotrophic or an apoptotic factor^[8]. Many articles are still in contradiction on this matter and part of the reason is the biological pathways by which both proteins act on neurons. Two receptors are involved in their signaling: tropomyosin-related kinase A (TrkA) and the p75 neurotrophin receptor (p75^NTR). The two proteins (proNGF and β-NGF) are able to bind both receptors, but it is globally accepted that β-NGF has a higher affinity for TrkA and pro-NGF has a higher affinity for p75^NTR. Figure 2 shows a big picture of how the two signaling pathways are thought to work (adapted from[9]).

At this point, it might seem that choosing to clone β-NGF in the bacteria of our interface would be a better idea than pro-NGF. Yet, a few other information led us to think the other way around.
First, despite of the existing controversy, pro-NGF has been proven to exhibit a neurotrophic activity on some neural cells, even though it is not as strong as mature β-NGF (there is a fivefold activity difference)^[10]. This neurotrophic activity is likely generated by a p75^NTR-dependant mechanism, and depends on the proportion of TrkA and p75^NTR in the neural cells^[11]. Moreover, TrkA is mainly expressed in three types of neurons: peripheral sensory neurons, sympathetic neurons and basal forebrain cholinergic neurons, whereas p75^NTR is more evenly dispersed in different types of neurons^[9]. Thus, expressing pro-NGF in our case seems like a good choice.
Furthermore, several articles support the idea that the pro-sequence of NGF facilitates, and is even absolutely necessary, for the proper folding of the protein when cloned into bacteria, which is particularly relevant as this protein contains three disulfide bonds and is particularly difficult to express using synthetic biology while keeping its function^[12],^[13].

Considering all this information, we finally chose to clone pro-NGF in our interface.

The composition of our final composite biobrick BBa_K2616000 is detailed in the PARTS submenu of this wiki.

Concretely, it expresses two main proteins:

• pro-NGF, linked to HlyA, a type I secretion system export signal in E. coli. Between the two, we added a TEV protease cleavage site.
• TEV protease, a protein from Tobacco Etch Virus that recognizes a specific sequence and cleaves it. We also linked the TEV protease to the same export signal.

Once exported from the cell, the TEV protease can cleave the pro-NGF from HlyA and free the pro-neurotrophin in the external medium.

• P. F. Pasquina, B. N. Perry, M. E. Miller, G. S. F. Ling, and J. W. Tsao, “Recent advances in bioelectric prostheses,” Neurol. Clin. Pract., vol. 5, no. 2, pp. 164–170, Apr. 2015.
  
  
• Y. Li and R. Brånemark, “Osseointegrated prostheses for rehabilitation following amputation,” Unfallchirurg, vol. 120, no. 4, pp. 285–292, Apr. 2017.
  
  
• A. Farley, C. Johnstone, C. Hendry, and E. McLafferty, “Nervous system: part 1,” Nurs. Stand., vol. 28, no. 31, pp. 46–51, Apr. 2014.
  
  
• “Schwann Cells,” PubMed Health. [Online]. Available: https://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0025728/. [Accessed: 27-Sep-2018].
  
  
• L. Ivanisevic and H. U. Saragovi, “Neurotrophins,” in Handbook of Biologically Active Peptides, Elsevier, 2013, pp. 1639–1646.
  
  
• S. Cohen, R. Levi-Montalcini, and V. Hamburger, “A nerve growth-stimulating factor isolated from sarcom AS 37 and 180,” Proc. Natl. Acad. Sci. U. S. A., vol. 40, no. 10, pp. 1014–8, Oct. 1954.
  
  
• S. Razavi, G. Nazem, M. Mardani, E. Esfandiari, S. Esfahani, and H. Salehi, “Neurotrophic factors and their effects in the treatment of multiple sclerosis,” Adv. Biomed. Res., vol. 4, no. 1, p. 53, 2015.v
  
  
• M. Fahnestock, G. Yu, and M. D. Coughlin, “ProNGF: a neurotrophic or an apoptotic molecule?,” 2004, pp. 101–110.
  
  
• H. Wang et al., “The Nerve Growth Factor Signaling and Its Potential as Therapeutic Target for Glaucoma,” Biomed Res. Int., vol. 2014, pp. 1–10, 2014.
  
  
• M. Fahnestock et al., “The nerve growth factor precursor proNGF exhibits neurotrophic activity but is less active than mature nerve growth factor,” J. Neurochem., vol. 89, no. 3, pp. 581–592, 2004.
  
  
• L. Howard, S. Wyatt, G. Nagappan, and A. M. Davies, “ProNGF promotes neurite growth from a subset of NGF-dependent neurons by a p75^NTR-dependent mechanism,” Development, vol. 140, no. 10, pp. 2108–2117, 2013.
  
  
• A. Rattenholl, H. Lilie, A. Grossmann, A. Stern, E. Schwarz, and R. Rudolph, “The pro-sequence facilitates folding of human nerve growth factor from Escherichia coli inclusion bodies,” Eur. J. Biochem., vol. 268, no. 11, pp. 3296–3303, 2001.
  
  
• M. Kliemannel et al., “The mature part of proNGF induces the structure of its pro-peptide,” FEBS Lett., vol. 566, no. 1–3, pp. 207–212, May 2004.