Narcolepsy Type 1 (Narcolepsy with Cataplexy) is a neurological sleep disorder categorized by rapid onset rapid eye movement (REM) sleep. Patients report excessive daytime sleepiness [1] and frequently need naps and stimulant medication to function in day-to-day life. It can be caused by a mutated version of the prepo-orexin (HCRT) gene which acts as a precursor to neurotransmitter peptides hypocretin A and hypocretin B. These peptides are expressed by specific neurons that function to control the circadian rhythm throughout the brain. Despite extensive research that hypocretin neuropeptides are associated with sleep/wake cycles in the brain, it is unclear how HCRT regulates circadian rhythm.
In this study, we hope to elucidate how peptides encoded by HCRT and their binding to HCRTR1 and HCRTR2 contribute to the proper functioning of the brain's circadian rhythm. A Danio Rerio mode will be used because it has a well-studied circadian rhythm, small brains, and the orthology of the HCRT gene and potential stimulatory mechanism. We hypothesize that HCRT functions in circadian rhythm by up/down-regulating expression of other genes involved in neurotransmitter synthesis
AIM 1: Identify conserved amino acid sequences necessary for ligand binding
Hypothesis: I hypothesize that mutants with the lowest binding affinity to HCRTR1 and/or HCRTR2 will have the biggest differences in sleep/wake cycle duration and frequency.
Rationale: Identifying amino acids that impact binding to HCRTR1 and HCRTR2 can give insight into how mutations of HCRT may impact neuronal activation.
Approach: Align Hypocretin A and Hypocretin B domains in HCRT gene and identify conserved amino acids. Generate synthetic peptides mutated in the conserved AAs. Identify mutants with low binding affinity using a radiolabeled competition binding assay to HCRTR1 and/or HCRTR2 Use CRISPR/Cas9 to generate a mutant library at loci where there was low binding affinity to HCRTR1 and/or HCRTR2 and assess the circadian rhythm phenotype of the of these mutants.
AIM 2: Identify differentially expressed genes activated in neurons
Hypothesis: I hypothesize that knocking out identified differentially expressed genes in activated neurons will highly impact circadian rhythm.
Rationale: By knocking out different domains in the HCRT we will be able to distinguish differential gene expression in activated neurons caused by Orexin A and/or Orexin B.
Approach: First, I will perform scRNA seq on Hypocretin A, Hypocretin B knockout, and wt mice on mature fish’s dorsolateral hypothalamus tissue after light and dark treatment for each homozygous mutant line of HCRT+wt. I will then knock out and analyze the phenotype of fish where gene expression changed in cells where a C-Fos marker was highly expressed.
AIM 3: Identify protein interactions of differentially expressed genes activated in neurons that are related to circadian rhythm.
Hypothesis: I hypothesize that STRING analysis will reveal key proteins that are responsible for neuronal activation of the neurons involved in the circadian rhythm Rationale: By tagging the identified differentially expressed proteins we will be able to further capture protein-protein interactions involved in neuropeptide signaling during circadian rhythm.
Approach: A TurboID tag will be fused to proteins that were differentially expressed in Aim 2 and showed alterations in circadian rhythm phenotype. After processing, STRING and Pathway Enrichment analysis of proteins in close proximity of these proteins will be performed and connected to other known neurotransmission and regulation-related genes.
In this study, we hope to elucidate how peptides encoded by HCRT and their binding to HCRTR1 and HCRTR2 contribute to the proper functioning of the brain's circadian rhythm. A Danio Rerio mode will be used because it has a well-studied circadian rhythm, small brains, and the orthology of the HCRT gene and potential stimulatory mechanism. We hypothesize that HCRT functions in circadian rhythm by up/down-regulating expression of other genes involved in neurotransmitter synthesis
AIM 1: Identify conserved amino acid sequences necessary for ligand binding
Hypothesis: I hypothesize that mutants with the lowest binding affinity to HCRTR1 and/or HCRTR2 will have the biggest differences in sleep/wake cycle duration and frequency.
Rationale: Identifying amino acids that impact binding to HCRTR1 and HCRTR2 can give insight into how mutations of HCRT may impact neuronal activation.
Approach: Align Hypocretin A and Hypocretin B domains in HCRT gene and identify conserved amino acids. Generate synthetic peptides mutated in the conserved AAs. Identify mutants with low binding affinity using a radiolabeled competition binding assay to HCRTR1 and/or HCRTR2 Use CRISPR/Cas9 to generate a mutant library at loci where there was low binding affinity to HCRTR1 and/or HCRTR2 and assess the circadian rhythm phenotype of the of these mutants.
AIM 2: Identify differentially expressed genes activated in neurons
Hypothesis: I hypothesize that knocking out identified differentially expressed genes in activated neurons will highly impact circadian rhythm.
Rationale: By knocking out different domains in the HCRT we will be able to distinguish differential gene expression in activated neurons caused by Orexin A and/or Orexin B.
Approach: First, I will perform scRNA seq on Hypocretin A, Hypocretin B knockout, and wt mice on mature fish’s dorsolateral hypothalamus tissue after light and dark treatment for each homozygous mutant line of HCRT+wt. I will then knock out and analyze the phenotype of fish where gene expression changed in cells where a C-Fos marker was highly expressed.
AIM 3: Identify protein interactions of differentially expressed genes activated in neurons that are related to circadian rhythm.
Hypothesis: I hypothesize that STRING analysis will reveal key proteins that are responsible for neuronal activation of the neurons involved in the circadian rhythm Rationale: By tagging the identified differentially expressed proteins we will be able to further capture protein-protein interactions involved in neuropeptide signaling during circadian rhythm.
Approach: A TurboID tag will be fused to proteins that were differentially expressed in Aim 2 and showed alterations in circadian rhythm phenotype. After processing, STRING and Pathway Enrichment analysis of proteins in close proximity of these proteins will be performed and connected to other known neurotransmission and regulation-related genes.
Specific Aims Drafts:
specific_aims_v2.docx | |
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specific_aims_v3.doc | |
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specific_aims_v4.docx | |
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Specific Aims Final Version:
giehtbrockspecificaims05102024finaldraft__3_.docx | |
File Size: | 8 kb |
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giehtbrockspecificaims05102024finaldraft__3___2_.pdf | |
File Size: | 70 kb |
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This page was produced as an assignment for Genetics 564, a capstone course at UW-Madison.