Treating Schizophrenia: Keepin’ it ReelinBradley Van Rooy, Jessica Dewey, Tracy Dinh, Kate Geschwind, Willie Meder, Colleen Smith, Jonah Widmer, Kristen Woodhouse

Schizophrenia: A Chronic Psychotic Syndrome. 1,2

•Patients falsely interpret reality• Severely debilitating symptoms include delusions, hallucinations,

and impaired social and communicative functioning•2.4 million patients in America each year

May be caused by decreased expression of RELN gene 3,4,5

•RELN protein product, reelin, causes appropriate neuronal positioning•50% less reelin in schizophrenic patients•Due to hypermethylation of RELN gene

RELN Transcription Factor: T-box brain 1 is the protein product of Tbr16,7,8

• Less Tbr1 expression means less reelin•More Tbr1 means more reelin

Reeler mice (Mus musculus) as a model organism for schizophrenia9,10,11,12

•Deleterious RELN gene disrupts reelin signaling pathway•Heterozygous mice have 50% less reelin

Our experiment seeks to increase RELN transcription in reeler mice•Deliver transcription activator to mouse brains using exosome

treatment• The goal is to increase RELN expression, improve neural connectivity,

and achieve therapeutic alleviation of schizophrenic symptoms

RELN: An ancestral character for the Eutheria clade.13 • Maximum parsimony

phylogenetic tree shows evolutionary relationship between humans, mice

• Orthologs of Homo sapiens RELN have been located in 86 mammals.

• H. sapiens RELN is conserved in 9 of these mammals, including Mus musculus.14

• Research methods applied in M. musculus may eventually be applied to orthologous species.

• Large number of RELN paralogs exist in every species that carries RELN.

pUC19 Plasmid contains all necessary coding regions. 21,34

• rep region causes replication.• bla region (Apr) confers ampicillin resistance as screening system.• lacZ gene, its promoter, lac repressor binding site as reporter system.• Tbr1 inserted at multiple cloning site.

Animal research must be ethical. 35

• Clear purpose of benefit and justified as a “last resort” measure• Animal welfare maintained• Follow IACUC guidelines for animal treatment as per www.iacuc.org• Humane surgical procedures; multiple surgeries approved

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(2005).Gene expression profiling during the embryonic development of mouse brain using an oligonucleotide-based microarray system. Molecular brain research, 136(1), 231-254. 33. Sato, N., Fukushima, N., Chang, R., Matsubayashi, H., &Goggins, M. (2006). Differential and Epigenetic Gene Expression Profiling Identifies Frequent Disruption of the RELN Pathway in Pancreatic Cancers. Gastroenterology, 130(2), 548-565, 34. pUC19 vector. (n.d.). Retrieved from http://www2.le.ac.uk/departments/genetics/genie/images/project-related-images/pUC19.jpg/view, 35. American Psychological Association. (2012, February 24).Guidelines for ethical conduct in the care and use of nonhuman animals in research. Retrieved from https://www.apa.org/science/leadership/care/guidelines.aspx?item=1, 36. "Trials of War Criminals before the Nuremberg Military Tribunals under Control Council Law No. 10", Vol. 2, pp. 181-182. Washington, D.C.: U.S. Government Printing Office, 1949. Accessed at http://www.hhs.gov/ohrp/archive/nurcode.html , 37. Department of Health, Education, and Welfare. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research Sess. (1979) Washington, D.C. IMAGE CREDITS: Experimental design images retrieved from: http://pegstookey.legacycentered.com/files/2013/03/Brain-clipart.png, http://pixabay.com/p-296581/?no_redirect, http://sweetclipart.com/multisite/sweetclipart/files/dna_black.png, http://upload.wikimedia.org/wikipedia/commons/0/07/Protein_HBZ_PDB_1jeb.png, http://www.clker.com/cliparts/B/o/K/L/E/9/cho-cell-petri-dish2-hi.png, http://www.ipharmd.net/images/blue_filled_syringe.png, http://anniehalliday.com/biopics/composite/microarray.jpg. Phylogenetic Tree adapted from http://www2.le.ac.uk/departments/genetics/genie/images/project-related-images/pUC19.jpg/image_preview. Plasmid image adapted from http://filebox.vt.edu/users/chagedor/biol_4684/Methods/transformed.gif. Tbr1 gene image adapted from http://www.ncbi.nlm.nih.gov/gene/10716.

Future research may investigate alternative human therapies.

•Alternative genes and gene products•Alternative brain regions•Environmental triggers•Extension of research towards effects of RELN gene

• Differentiation of neurons• Neurogenesis in the hippocampus• Effects on other neuropathologies

•Additional research on animals more closely related to humans (pigs, monkeys, etc.)•Eventual research with human patients36,37

• Informed consent and required ability to stop research• Avoid unnecessary harm and maximize benefits

Will increasing RELN transcription have a therapeutic benefit?

•Poor neuronal orientation is only one aspect of complex disease pathology•Possible procedural errors:

• Isolation of Tbr1, amplification during PCR, plasmid recombination, or transformation of E. coli• DCDE may not be suitable carrier for Tbox brain 1 due to

unforeseen size or other molecular qualities• Problematic brain tissue culturing or removal

•Failure to target correct RELN paralog may complicate schizophrenic pathology

Experimental Design

References

BIOL 2002 – Section 1; Team 10; Spring 2014

Tbr1 Gene11,412 bp