Alzheimer's disease (AD) is a progressive neurological disorder that causes the gradual loss of memory, thinking skills, and ability to perform everyday functions. It is the most common cause of dementia, accounting for 60-80% of dementia cases. It is characterized by the buildup of amyloid beta plaques and tau tangles in the brain which impair communication between neurons and lead to their death.
The disease progresses from mild cognitive impairment to a severe loss of cognitive and functional abilities.
Over the years, a number of hypotheses have been proposed to understand AD. However, the amyloid beta and acetylcholinesterase hypotheses have driven much of Alzheimer's disease drug development and research (Du et. al, 2018).
▪ Amyloid beta hypothesis: This posits that the accumulation of amyloid beta peptide plaques in the brain initiates a pathogenic cascade leading to neuronal death and dementia. The plaques are thought to disrupt cell signaling and cause inflammation driving disease progression. Therapies targeting amyloid beta such as monoclonal antibodies aim to reduce plaques (Du et. al, 2018).
▪ Cholinergic hypothesis: This proposes that reduced synthesis of the neurotransmitter acetylcholine is a key factor in memory loss and cognitive decline in Alzheimer's. Acetylcholinesterase breaks down acetylcholine, so inhibitors of this enzyme help increase acetylcholine levels and modestly improve symptoms. Drugs like donepezil and rivastigmine follow this approach (Du et. al, 2018).
These two hypotheses form the basis of Project Forget-Me-Not.
Introduction to the gut-brain axis
The gut has important bidirectional relationships with many other organs and systems in the body, mediated through metabolic, hormonal, immune, and neural signaling.The enteric nervous system (ENS) is one of the main divisions of the nervous system and is also known as the "second brain" or the "gut brain"
The gut-brain axis refers to the biochemical signaling that takes place between the gastrointestinal tract and the central nervous system. It consists of bidirectional communication via neural, endocrine, and immune pathways (Appleton et.al,2018).
The gut and brain are connected by the vagus nerve, which links the billions of microbes residing in the digestive tract to the brain.
The gut microbiome can affect the brain and behavior by producing neurotransmitters like serotonin, dopamine, and gamma-aminobutyric acid (GABA), as well as short-chain fatty acids.
The brain can influence the gut by releasing neurotransmitters and hormones that regulate gastrointestinal functioning. For example, stress can alter gut motility and increase intestinal permeability.
An imbalance in gut microbes (dysbiosis) has been associated with brain-related conditions like anxiety, depression, autism spectrum disorder, and Parkinson's disease.
Conversely, probiotic supplementation may have positive effects on mood, stress response, and cognitive function by modulating the gut-brain axis.
The gut-brain axis allows for bidirectional communication via neural, hormonal, immunological, and metabolic mechanisms to maintain homeostasis and influence brain function and behavior.
Understanding this axis is shedding light on how gut microbes may be leveraged for brain health. Recently, it emerged that the gene APOE — variants of which each confer a different risk of Alzheimer’s disease — has a role in modulating this gut–brain communication (Seo et. al,2023).
The gut-brain axis in Alzheimer’s disease
The gut-brain axis (GBA) describes a bidirectional communication between the gut and brain; this implies that there are several hypotheses describing the onset of AD arising from the gut (Kowalski et. al, 2019).
▪ Bacterial amyloids produced in the gut, such as curli from E. coli, can prime the immune system and lead to increased immune response and inflammation in the brain. Exposure to curli-producing bacteria in rats led to increased α-syn deposition and inflammation in the brain.
▪ Bacterial lipopolysaccharides (LPS) found in increased levels in AD brains can activate inflammatory pathways and promote amyloid fibril formation. LPS activates TLRs on microglia, leading to neuroinflammation.
▪ Intestinal inflammation and gut barrier dysfunction, marked by increased fecal calprotectin, allows bacterial products like LPS to translocate into circulation and contribute to neuroinflammation.
▪ Altered gut microbiota composition and small intestinal bacterial overgrowth can increase intestinal permeability ("leaky gut"), allowing translocation of bacteria and inflammatory mediators into circulation.
▪ In the brain, neuroinflammation around amyloid plaques is mediated by activated microglia. Chronic systemic inflammation from the gut may prime microglia, making them more reactive to amyloids like Aβ.
Our project focuses on mediating neuroinflammation in AD via the gut brain axis. Mechanism of neuroinflammation (Alkasir et.al, 2017):
Microglia and astrocytes play central roles in driving the neuroinflammation associated with Alzheimer's disease pathology. Microglia are capable of existing in a protective anti-inflammatory state (M2) or a destructive proinflammatory state (M1) (Lukiw et. al, 2016) (Minter et.al, 2016). In Alzheimer's, microglia become chronically activated into the M1 phenotype through multiple pathways. Amyloid plaques directly stimulate microglial activation via engagement of receptors like TLRs and RAGE. Microglia are also activated by pathogens, toxins and cellular debris that reach the brain when the blood-brain barrier is disrupted (Calsaro et. al, 2016). This chronic activation leads microglia to overproduce inflammatory cytokines like TNF-α and IFNγ, while also impairing their ability to clear amyloid plaques through phagocytosis (Boutajangout et. al,2013). The sustained inflammation creates a neurotoxic environment, damaging neurons, reducing synaptic plasticity, and accelerating neurodegeneration (Minter et.al, 2016). Astrocytes also become activated by amyloid deposits, responding by releasing their own inflammatory factors. Chronic astrocyte activation further degrades the blood-brain barrier, allowing entry of more blood-borne inflammation-driving molecules and cells. While transient microglial activation may be an adaptive response to clear early amyloid species, mutations in proteins like TREM2 in late stage Alzheimer's prevent this beneficial pathway (Wiatrak et. al,2022). Soluble amyloid oligomers can directly damage neurons before plaque formation, but they cannot activate microglial responses alone. Targeting the chronic overactivation of microglia and astrocytes has emerged as a strategy to dampen neurotoxic inflammatory cascades in Alzheimer's (Cattaneo et. al, 2017). Modulating systemic inflammation through the gut-brain axis may also attenuate neuroinflammation by preventing the priming and constant stimulation of microglia. Understanding the complex interplay between innate immune cells, amyloid species, inflammation pathways and neuronal integrity is key to developing effective interventions for Alzheimer's disease (Wiatrak et. al, 2022).
Engineered Probiotics for early AD intervention
Conversely, engineered probiotics can be used as early therapeutic intervention for various hard to treat neurodegenerative diseases (Mancuso et.al, 2018).
▪ Bacterial amyloids produced in the gut, such as curli from E. coli, can prime the immune system anProbiotics like Lactobacillus and Bifidobacterium strains can enhance gut barrier integrity, reduce inflammation, and protect against neuroinflammation and neurodegeneration in animal studies. Some clinical studies show probiotics improve cognitive scores in AD patients (Frasca et.al,2016).
▪ Antibiotics that treat small intestinal bacterial overgrowth and pathogenic gut bacteria, like rifaximin, can improve gastrointestinal and even motor symptoms in Parkinson's disease patients (Fasano et.al,2013).
▪ Fecal microbiota transplantation to restore diversity and healthy microbiota profiles shows promise in animal models and some neurological disorders, but human trials are still needed for AD (Evrensel et.al,2016).
▪ Dietary interventions like increased fiber, prebiotics, anti-inflammatories, and reduced saturated fats can beneficially reshape the gut microbiome. A Mediterranean-style diet high in plants, fish, nuts and healthy fats may lower AD risk (Perez et.al,2017).
▪ Multi-modal therapies that combine probiotics, diet, exercise and other lifestyle factors may provide maximum benefit in maintaining cognitive health and preventing Alzheimer's progression (Pistollato et.al,2016).
CRISPR as the next frontier of gut microbiome editing
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has potential to revolutionize microbiome engineering through its ability to precisely edit microbial genomes.Nobel laureate and researcher behind the technology, Dr. Jennifer Doudna is now pursuing gut microbiome editing using CRISPR to alleviate early childhood asthma (TED Audacious project). Dr. Doudna explains how her team at the Innovative Genomics Institute is pioneering a brand new field of science -- precision microbiome editing -- that uses CRISPR in an effort to solve seemingly insurmountable problems like asthma and climate change.
CRISPR was first discovered in the genome of marine bacteria. When faced with a viral threat, bacterial cells developed an immune response by capturing and copying DNA fragments of viruses. This allowed bacteria to recognize subsequent attacks and cleave the viral DNA to stop the viral infection. It was also discovered that the Cas enzyme was responsible for DNA cleavage. This defense mechanism was later leveraged by Doudna and Charpentier, who could target a specific DNA sequence and isolate it using the CRISPR-Cas9 system (Doudna et. al, 2014)
CRISPR enables accurate targeting of genes in specific bacterial strains, unlike antibiotics that indiscriminately kill pathogens and beneficial microbes. It also facilitates rapid generation of libraries of thousands of edited microbial strains for screening studies. Careful testing ensures edits are specific, with minimal off-target effects.
CRISPR-edited microbes may lead to novel therapeutics like smart probiotics, antimicrobials, diagnostics, and improved production strains. It can also help elucidate connections between the microbiome and disease by allowing high-throughput screening of gene knockouts.
Team NCSU hosted some of the foremost startups in the CRISPR field when we were in our initial design stages.
Introduction to CRISPR
Locus Biosciences
Broad Institute’s Dr.Jesse Boehm
Intellia Therapeutics
Looking ahead, CRISPR could enable combination editing in microbes to optimize industrial strains and probiotics. It may also lead to more targeted antimicrobials that eliminate pathogens while sparing beneficial microbes. CRISPR is expanding the horizons of microbiome engineering.
Team NCSU's BBa_K4943005 is a dual plasmid induced CRISPR/Cas9 system adapted to edit the Clostridium genus. Our proof of concept edits C. butyricum to upregulate the short chain fatty acid butyrate, that is reaching the brain via the gut-brain axis in AD. If administered at the Mild Cognitive Impairment stage could help alleviate symptoms of AD.
This is a modular platform for iGEM teams to work with non model gut commensals. We hope that this will introduce a CRISPR based precision microbiome editing part for Therapeutics projects at iGEM to innovate upon.
The future looks bright for CRISPR editing to harness the microbiome for human health and sustainability.
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