Team:UCL/Project/Developments

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<p class="major_title">OTHER PARTS OF OUR CIRCUIT</p>
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<p class="major_title">OTHER CIRCUIT COMPONENTS</p>
<p class="minor_title">Avoiding Inflammation And Supporting Neurons</p>
<p class="minor_title">Avoiding Inflammation And Supporting Neurons</p>
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Unfortunately, we did not have time to attempt to create all the parts envisioned in our original potential. However, we believe that they are theoretically significant, and so here we explain what more could be done to improve this project of ours, as we work on these improvements after the ‘WikiFreeze’ for the Regional Jamboree of the iGEM competition.  
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Unfortunately, we did not have time to attempt to create all the parts envisioned in our original potential circuit. However, we believe that they are theoretically significant, and so here we explain what more could be done to improve this project of ours, as we work on these improvements after the ‘WikiFreeze’ for the Regional Jamboree of the iGEM competition.
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The strength of our system [internal link to circuit overview] is that the microglial chassis [internal link to microglia page in background] already detect and engage [internal link to chassis page] amyloid plaques [internal link to neuropathology].
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The strength of our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">system</a> is that the <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">microglial chassis</a> already detect and engage other microglia.
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This means that our systems can create proteins in situ to improve the Alzheimer’s disease [link to Alzheimer’s disease in background] state. However, amyloid proteases such as MMP-9 [internal link to ‘degradation’] would only have a positive impact on the pathology if the ‘Amyloid Hypothesis’ [internal link to neuropathology] is correct, and there is some evidence to suggest that it may not be.  
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This means that our systems can create proteins in situ to improve the <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s disease (AD)</a> state. However, amyloid proteases such as <a href="https://2013.igem.org/Team:UCL/Project/Degradation" target="_blank">MMP-9</a> would only have a positive impact on the pathology if the <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">'Amyloid Hypothesis'</a> is correct, and there is some evidence to suggest that it may not be.
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It is thought that Alzheimer’s disease (AD) may be exacerbated into a neurodegenerative condition by the action of microglia themselves, the custodians of the brain. They can inflame the plaque area, and this damages neurons. Therefore, we propose producing a de-activating agent, such as vasoactive intestinal peptide (VIP), BioBrick with an oxidative stress promoter [internal link to detection]. This mean that our engineered microglia would activate when it detects a plaque and move towards that plaque. As it approaches, oxidative stress increases so that once near the plaque the de-activating agent would return the engineered cell and wild-type cells surrounding the plaque into their resting state, avoiding neuroinflammation. This would stop them from producing amyloid proteases such as neprilysin. However, our MMP-9 BioBrick can ensure that amyloid degradation continues (the positive action of microglia in AD) without inflammation (the negative action of microglia in AD).  
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It is thought that AD may be exacerbated into a neurodegenerative condition by the action of microglia themselves, the custodians of the brain. They can inflame the plaque area, and this damages neurons. Therefore, we propose producing a de-activating agent, such as vasoactive intestinal peptide (VIP), BioBrick with an <a href="https://2013.igem.org/Team:UCL/Project/Detection" target="_blank">oxidative stress promoter</a>. This means that our genetically engineered microglia (GEM) would activate when it detects a plaque and move towards that plaque.
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As a GEM approaches, oxidative stress increases so that once near the plaque the de-activating agent would return the GEM and wild-type microglia surrounding the plaque into their resting state, avoiding neuroinflammation. This would stop them from producing amyloid proteases such as neprilysin. However, our MMP-9 BioBrick can ensure that amyloid degradation continues (the positive action of microglia in AD) without inflammation (the negative action of microglia in AD).  
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It is also thought that AD may initiate due to cell-cycle re-entry on account of a disbalance in neurotrophin signalling [internal link to neuropathology]. Brain-derived neurotrophic factor (BDNF) is a signal that sustains neurons. If expressed by engineered microglia at plaque localities it could support dying neurons and stop other neurons progressing into an AD state.  
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It is also thought that AD may initiate due to cell-cycle re-entry on account of a disbalance in <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">neurotrophin signalling</a>. Brain-derived neurotrophic factor (BDNF) is a signal that sustains neurons. If expressed by engineered microglia at plaque localities it could support dying neurons and stop other neurons progressing into an AD state.
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<p class="minor_title">Vasoactive Intestinal Peptide</p>
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Zeocin is a glycopeptide antibiotic capable of killing most bacteria, fungi, yeast, plant, and animal cells by intercalating DNA and inducing double strand breakage. This makes zeocin resistance an ideal selective maker for our project, which involves both bacterial and mammalian chassis. The product of the ''Sh ble'' gene, isolated from the bacterium Streptoalloteichus hindustanus <a href="http://www.ncbi.nlm.nih.gov/pubmed/2450783" target="_blank">(Gatignol et al. 1988)</a>, confers zeocin resistance to transfected/transformed cells. Sh ble is a small binding protein with strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA.
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This is a 28 amino acid long secreted neuropeptide. It stimulates heart contractility, vasodilation and glycogenolysis, muscle relaxation in the gastrointestinal tract and lowers arterial blood pressure and relaxes the smooth muscle of trachea, stomach and gall bladder <a href="http://www.ncbi.nlm.nih.gov/pubmed/6129023" target="_blank">(Fahrenkrug and Emson 1982)</a>. In the brain, it plays a key role in circadian rhythm control in the hypothalamus. It has been suggested as a therapeutic as  it is also known to have a neuroprotective role. Studies have shown that VIP prevents activated microglia inducing the neuroinflammatory conditions that engender and may drive neurodegeneration <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Neuroprotective+effect+of+vasoactive+intestinal+peptide+(VIP)+in+a+mouse+model+of+Parkinson%E2%80%99s" target="_blank">(Delgado and Ganea March 2003)</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Vasoactive+intestinal+peptide+prevents+activated+microglia+neurodegeneration+under+inflammatory+conditions%3Ainduced" target="_blank">(Delgado and Ganea Jan 2003)</a>.
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Oxidative stress via free radical production increases with proximity to senile plaques <a href="http://www.ncbi.nlm.nih.gov/pubmed/10863548" target="_blank">(Colton et al., 2000)</a>. The microglia immune response around plaques also increases oxidative stress. Therefore, we have designed a promoter which will initiate transcription in response to oxidative stress to ensure the production of key proteins only in the plaques’ locales. This promoter is an improvement of a yeast minimal promoter (<a href="http://parts.igem.org/Part:BBa_K105027" target="_blank">cyc100</a>) already in the registry. NF-κB is a transcription factor which translocates to the nucleus under oxidative stress <a href="http://www.ncbi.nlm.nih.gov/pubmed/12730877" target="_blank">(Shi et al., 2003)</a>, and binds to the sequence GGGAATTT <a href="http://www.ncbi.nlm.nih.gov/pubmed/19435890" target="_blank">(Park et al., 2009)</a>. Thus, by placing this site upstream of the yeast minimal promoter, we created a novel mammalian promoter which initiates transcription in response to oxidative stress.
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This is a secreted, 252 amino acid long neurotrophic protein. it is made in the endoplasmic reticulum and secreted from dense core vesicle. it’s assortment into these vesicles is aided by the enzyme  carboxypeptidase E. Decreased levels of BDNF have been associated with Alzheimer’s, depression and epilepsy - and so this would be a very useful medical BioBrick. It mainly mediates it effects through membrane protein TrkB. BDNF promotes the neurons’ survival, growth, differentiation and maintenance. It is active at the communicative connections between neurons (synapses), where it helps sustain the synapse and facilitate changes in that synapse’s strength over time, in response to experience. This is called ‘synaptic plasticity’, and is believed to play a key role in learning and memory. A decrease in BDNF could engender cell cycle re-entry <a href="http://www.ncbi.nlm.nih.gov/pubmed/20436277" target="_blank">(Frade & Lopez-Sanchez 2010)</a> and the senile plaques in AD can disrupt synapses, meaning that they receive less BDNF.
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MMP-9, also known as gelatinase B, is most commonly known for its role in breaking down the extracellular matrix. Naturally it is secreted in its inactive form and must be cleaved by other proteases, but our GEM are are meant to produce just the active form. It has been shown by Yan et al. that MMP-9 is the only known endogenous protease that degrades both fibrillar and soluble forms of amyloid-β peptide (Aβ). This satisfies the <a href="https://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">'Amyloid Cascade Hypothesis'</a> as well as theories that see plaques as <a href="https://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">neuroprotective</a>, and soluble Aβ as the real threat. MMP-9 is expressed at low basal levels in microglia and may keep plaque size in dynamic equilibrium <a href="http://www.ncbi.nlm.nih.gov/pubmed/16787929" target="_blank">(Yan et al. 2006)</a>. Over producing it in (inactive) GEM could greatly improve both soluble and insoluble Aβ clearance. MMP-9 must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could cause damage to brain tissue if, for example, injected into the brain.
 
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This is a small non-inflammatory chemokine that induces chemotaxis [internal link to chemotaxis page] in macrophages. Microglia originate from a macrophage lineage. It elicits its effect through cell surface chemokine receptor CXCR3. Plaques already attract microglia, but this is partly due to local microglial activation. De-activated GEM will not produce many chemokines, so in order attract more GEM (as well as native microglia) to the plaque site, in order to speed up Aβ clearance.
 
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Latest revision as of 03:21, 5 October 2013

OTHER CIRCUIT COMPONENTS

Avoiding Inflammation And Supporting Neurons

Unfortunately, we did not have time to attempt to create all the parts envisioned in our original potential circuit. However, we believe that they are theoretically significant, and so here we explain what more could be done to improve this project of ours, as we work on these improvements after the ‘WikiFreeze’ for the Regional Jamboree of the iGEM competition.

The strength of our system is that the microglial chassis already detect and engage other microglia.

This means that our systems can create proteins in situ to improve the Alzheimer’s disease (AD) state. However, amyloid proteases such as MMP-9 would only have a positive impact on the pathology if the 'Amyloid Hypothesis' is correct, and there is some evidence to suggest that it may not be.

It is thought that AD may be exacerbated into a neurodegenerative condition by the action of microglia themselves, the custodians of the brain. They can inflame the plaque area, and this damages neurons. Therefore, we propose producing a de-activating agent, such as vasoactive intestinal peptide (VIP), BioBrick with an oxidative stress promoter. This means that our genetically engineered microglia (GEM) would activate when it detects a plaque and move towards that plaque.

As a GEM approaches, oxidative stress increases so that once near the plaque the de-activating agent would return the GEM and wild-type microglia surrounding the plaque into their resting state, avoiding neuroinflammation. This would stop them from producing amyloid proteases such as neprilysin. However, our MMP-9 BioBrick can ensure that amyloid degradation continues (the positive action of microglia in AD) without inflammation (the negative action of microglia in AD).

It is also thought that AD may initiate due to cell-cycle re-entry on account of a disbalance in neurotrophin signalling. Brain-derived neurotrophic factor (BDNF) is a signal that sustains neurons. If expressed by engineered microglia at plaque localities it could support dying neurons and stop other neurons progressing into an AD state.

Vasoactive Intestinal Peptide

This is a 28 amino acid long secreted neuropeptide. It stimulates heart contractility, vasodilation and glycogenolysis, muscle relaxation in the gastrointestinal tract and lowers arterial blood pressure and relaxes the smooth muscle of trachea, stomach and gall bladder (Fahrenkrug and Emson 1982). In the brain, it plays a key role in circadian rhythm control in the hypothalamus. It has been suggested as a therapeutic as it is also known to have a neuroprotective role. Studies have shown that VIP prevents activated microglia inducing the neuroinflammatory conditions that engender and may drive neurodegeneration (Delgado and Ganea March 2003)(Delgado and Ganea Jan 2003).

Brain-Derived Neurotrophic Factor

This is a secreted, 252 amino acid long neurotrophic protein. it is made in the endoplasmic reticulum and secreted from dense core vesicle. it’s assortment into these vesicles is aided by the enzyme carboxypeptidase E. Decreased levels of BDNF have been associated with Alzheimer’s, depression and epilepsy - and so this would be a very useful medical BioBrick. It mainly mediates it effects through membrane protein TrkB. BDNF promotes the neurons’ survival, growth, differentiation and maintenance. It is active at the communicative connections between neurons (synapses), where it helps sustain the synapse and facilitate changes in that synapse’s strength over time, in response to experience. This is called ‘synaptic plasticity’, and is believed to play a key role in learning and memory. A decrease in BDNF could engender cell cycle re-entry (Frade & Lopez-Sanchez 2010) and the senile plaques in AD can disrupt synapses, meaning that they receive less BDNF.

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