Difference between revisions of "Team:Pasteur Paris/Growing Back Nerves"

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<p>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>
 
<p>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>
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<div class="block title" id="Results">
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<h1>RESULTS</h1>
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<i style="text-align: left;"><p>Achievements:<br>
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<li>Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone, creating a <b>new composite part</b></li>
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<li>Successfully co-transform E. coli with plasmid secreting NGF and plasmid expressing the secretion system, creating bacteria <b>capable of secreting NGF</b> in the medium</li>
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<li>Successfully characterized production of NGF thanks to mass spectrometry</li>
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<li>Successfully <b>observe axon growth</b> in microfluidic chip in presence of commercial NGF</li>
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<p>Next steps:<br>
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<li><b>Purify</b> secreted NGF, and characterize its effects on neuron growth thanks to our microfluidic device </li>
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<li><b>Global proof of concept</b> in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other</li>
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Revision as of 16:16, 10 October 2018

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CONTEXT

Protheses are aimed to enhance the quality of life, independence, mobility, and safety for amputees. The worldwide frequency of amputations has created an increased demand for improved prostheses technologies. Within developing countries, the part of the population with physical disabilities in demand of a prosthesis is estimated at about 0.5%. In 2050, approximatively 3 million people, in the United States, will live with a limb loss [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. Yet, 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 prosthesis, regardless of the environmental conditions or the limb position. The opportunity to record and stimulate the neuromuscular system allows the intuitive control and a better understanding of sensory perception [2].

The central nervous system (CNS), made up 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 motor or efferent neurons of the peripherical nervous system (PNS) communicate with muscle and are under voluntary control. They go from the spinal cord to the arms, hands, legs, feet and allow to transmit nerve impulses away from the CNS, leading to an action [3]. To increase the electrical signal speed, the axon is surrounded by myelin, produced by another type of cells called Schwann cells [4]. These cells twist around the axon and prevent the loss of electrical signal. After limb amputation, the main issue of the human-machine communication is due to nerves damage. Indeed, the electrical signal cannot be transmitted because the peripheral nerve cells are unable to activate target muscle from the limbs with signal from the brain [4].

OUR SOLUTION

The central idea of our project is to find a way to have motor nerves of amputees grow back and connect to our interface. Literature studies led us to think of neurotrophins as the perfect molecules to start our research on. Indeed, this family of peptides are growth factors specialized in the regulation of neuronal development, survival, plasticity and nervous system function[5].

What are neurotrophins?

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.

The ambiguity between NGF and pro-NGF.

The mature-NGF protein (or β-NGF) results from the cleavage of β-NGF from a bigger protein called pro-NGF, which contains both a pro-sequence and β-NGF (see figure 1). Both pro-NGF and β-NGF proteins are biologically active, and there is, to this date, some sort of uncertainty about the exact effect of pro-NGF.

Figure 1: Structure of the Pro-NGF gene. Adapted from Ivanisevic, 2013.

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 comes from 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 (p75NTR). 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 p75NTR. Figure 2 shows a big picture of how the two signaling pathways are thought to work (adapted from[9]).

Figure 2: NGF versus pro-NGF signaling pathways. Adapted from Wang, 2014.

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, even 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 p75NTR-dependant mechanism, and depends on the proportion of TrkA and p75NTR 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 p75NTR 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.

How to produce and secrete pro-NGF from an E. coli biofilm?

Figure 3: pro-NGF production and secretion from E. coli

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.

RESULTS

Achievements:

  • Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone, creating a new composite part
  • Successfully co-transform E. coli with plasmid secreting NGF and plasmid expressing the secretion system, creating bacteria capable of secreting NGF in the medium
  • Successfully characterized production of NGF thanks to mass spectrometry
  • Successfully observe axon growth in microfluidic chip in presence of commercial NGF

Next steps:

  • Purify secreted NGF, and characterize its effects on neuron growth thanks to our microfluidic device
  • Global proof of concept in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other

REFERENCES

  • 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 p75NTR-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.