Introduction
Schizophrenia is a chronic, heritable brain disease with a wide array of symptoms. The underlying mechanisms are not well known, but the disease distorts a person’s perception of reality and usually manifests between the ages of 16 and 30. Schizophrenia affects roughly one percent of the population worldwide, and is influenced both by genetics and the environment. The PET scans to the right show increased microglial activity all over the brain in a schizophrenic patient, but especially in the pre-frontal lobe which is involved in planning, decision making and your personality. Microglia are the macrophages of the brain and play a role in immune attack, so their increased activity in schizophrenia causes the pre-frontal lobe to be affected.
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Synaptic pruning occurs on dendrites, specifically at dendritic spines. Dendritic spines are knob-like protrusions off of the dendrite which receive input from an axon at the synapse. During normal development, many spines and synapses are formed in childhood then selectively pruned from adolescence to adulthood. However, in schizophrenic patients there is excessive pruning of dendritic spines in regions like the prefrontal cortex and hippocampus, which decreases the number of synapses and causes schizophrenic symptoms to occur. Confocal imaging of immunofluorescently stained dendrites in wild type and schizophrenic zebrafish show a noticeable decrease in dendritic spine density and thickness in the schizophrenic case compared to the wild-type.
Last year, the C4A gene was found to be involved in dendritic pruning and schizophrenia. C4A stands for “complement component 4A” and plays a role in the classical complement pathway, which amplifies the strength of immune responses. The C4A protein has eight domains, six of which encode alpha-2-macroglobulin which is a protease inhibitor that can disrupt inflammatory cascades. The GO terms for the C4A protein show involvement in inflammatory responses, protein binding, and localization to extracellular regions. C4A is very well conserved throughout vertebrate species, as even zebrafish C4A (34% sequence identity to human C4A) contains all of the same domains. Zebrafish will be used because of their domain conservation, relatively cheap cost of maintenance, and transparency until adulthood. Zebrafish also have a rapid life cycle and a well-established pattern of development, making them an ideal candidate for high throughput screens.
From the literature, it is known that C4A variations increase the risk of schizophrenia, and that increased pruning in certain regions of the brain leads to schizophrenia. However, it is currently unknown how C4A specifically regulates dendritic spine pruning. Therefore, my primary goal is to discover how C4A regulates synaptic pruning at the dendrites specifically. I will learn more about C4A’s role in dendritic pruning by identifying domains involved in pruning, then identifying small molecules that regulate pruning and rescue pruning defects in schizophrenic zebrafish, and finally identifying C4A protein interactors that are involved in pruning. These aims will hopefully lay the groundwork for novel preventative and curative treatments for schizophrenia.
From the literature, it is known that C4A variations increase the risk of schizophrenia, and that increased pruning in certain regions of the brain leads to schizophrenia. However, it is currently unknown how C4A specifically regulates dendritic spine pruning. Therefore, my primary goal is to discover how C4A regulates synaptic pruning at the dendrites specifically. I will learn more about C4A’s role in dendritic pruning by identifying domains involved in pruning, then identifying small molecules that regulate pruning and rescue pruning defects in schizophrenic zebrafish, and finally identifying C4A protein interactors that are involved in pruning. These aims will hopefully lay the groundwork for novel preventative and curative treatments for schizophrenia.
Aim 1
To identify domains involved in dendritic spine pruning, first all C4A domains had to be identified. This was done using the SMART and Pfam databases. By looking at the homologous sequence alignment obtained from Clustal Omega, I attempted to find the most conserved stretches of amino acids between human and zebrafish C4A so that I could target the corresponding genetic sequences later for knockout using CRISPR. The most conserved stretches are likely the most important for proper C4A function. CRISPR screens will then be performed on conserved C4A domain coding regions in zebrafish embryos. The schizophrenic behavioral phenotype that I will be screening for is a decrease in pre-pulse inhibition (PPI) from the larval to adult stages of development, which is a phenotype of schizophrenia involved in reducing an organism’s startle response when a weaker stimulus is applied before a strong stimulus. Researchers have developed a video analysis program that can determine whether zebrafish have deficits in PPI, so I would use this tool in my screens to look for reduced PPI. In the zebrafish with decreased pre-pulse inhibition, live imaging of dendrites will be conducted to confirm that aberrant pruning has occurred. I expect to find zebrafish in the screen that show both schizophrenic PPI and pruning defects when certain regions of the C4A gene are knocked out.
Aim 2
After obtaining pruning mutants from the CRISPR screen, I will conduct a high-throughput chemical genomic assay on juvenile and adult wild-type and mutant zebrafish using diversity-oriented libraries. Then I’ll screen for rescued pruning defects by imaging the zebrafish dendrites, this time looking for increased dendritic spine density, meaning that less pruning has occurred. I would expect to discover some small molecules that can alter pruning in both wild type and mutant zebrafish. I would also expect the wild type juvenile zebrafish to have decreased pruning (or increased dendritic spine number) in adulthood. The treated juvenile mutants will grow to adulthood with no pruning defects. The wild-type adult will serve as a control to ensure that the small molecules with curative potential don’t have severe off-target effects. And finally it would be exciting to find treated adult mutants with rescued pruning defects. These small molecules would be promising targets for future research into finding a cure for schizophrenia.
Aim 3
In my last aim, I will identify proteins that interact with wild type and mutant C4A to regulate pruning. I will collect brain extracts from the wild-type and mutant zebrafish and then conduct tandem affinity purification with C4A and mutant C4A as the baits. After the purification and mass spec analysis, I would hope to identify a protein that interacts with mutant C4A but not wild-type C4A, although I would also expect to discover novel interactions that are shared between wild-type and mutant C4A as well.
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After identifying a unique protein that interacts with mutant C4A exclusively, I would take a similar approach as Aim 1 by conducting a CRISPR screen to knock out the gene encoding “protein B” in zebrafish embryos then look for pre-pulse inhibition deficits and increased dendritic spine pruning in the larval to adult stages of the knock-out zebrafish. This approach could be repeated on all of the C4A interactors found to see which play a role in schizophrenia and pruning as well.
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Future Directions
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