Past research projects that I contributed to at a glance:
1-) Post-doctoral Research on Neuroinflammation, Neuroninflammation and Neurodegeneration specifically focusing on ALS, FTD and AD
Research questions I continuously think about:
How come ubiquitous proteins such as SOD1 cause only neuronal death?
Why research groups stuck on transgenic animals models and why not make/utilize knock-in models?
-Well, knock in models do not display disease phenotype when the mutation is heterozygous as in humans.
-Then, why not study homozygous animals?
-Alternatively, how can we recapitulate "aging" effect in the lab that heterozygous knock-in animals also display the disease phenotype?
But first, let's understand what "genomic burden" does "aging" bring?
How does blood-brain-barrier altered in disease state? Can we use this phenomena designing drugs?
Before/while designing new drugs, can we re-purpose drugs already developed/FDA approved?
To track the effectiveness of drugs, what data/biomarkers can we extract from CSF or even postmortem tissue?
Why all genetic screens are focusing on loss-of-function suppressors but not gain-off-function suppressors?
RNA Editing: A Way to Change Genetic Code that You are Born With!
In a nutshell:
Everything in biology starts from Central Dogma: DNA codes for RNA; and RNA produces functional proteins. Sometimes, some cells of us require to change the DNA code that we are born with. Not surprisingly, one of the most complex and long-lived cells in our body, the neurons, take advantage of this mechanism quite widely. In one of my published work (Jepson et al., 2011), we explored the consequences of lowering the RNA editing mechanism used by neurons and in the second publication (Savva et al., 2012), we explored the consequences of editing enzyme’s "editing" itself. Yes, the editing protein, ADAR, edits its own RNA!!! Also please note that, RNA editing has another function besides changing the genetic code in important neuronal targets. It also opens/disrupts the perfect double-standed RNAs, which are toxic if they escape to the cytoplasm and which I study during my post-doc.
Post-transcriptional RNA modification by Adenosine to Inosine (A-to-I) editing by ADAR (Adenosine Deaminases that Act on RNA) alters the functionality of many important neuron-specific proteins such as ion channels. Deletion of the ADAR locus results in severe adult-stage behavioral abnormalities, including extreme uncoordination, seizures and a complete lack of courtship in flies; and seizures and early mortality in mice. In Jepson et al., 2011, using a novel knock-in hypomorphic allele of Drosophila ADAR coupled with cell-specific ADAR knockdown, we demonstrated that RNA editing serves a modulatory role in multiple adaptive behaviors in Drosophila. To make things more complex, ADAR also re-codes its own transcript, but the consequences of this auto-regulation in vivo are unknown. In Savva et al., 2012, we genetically engineered the ADAR gene in Drosophila so that it expresses either only re-coded ADAR transcript or the non-editable transcript. This study revealed that each form of ADAR has distinct spatial regulation and mRNA target preferences, which results in fine-tuning of neuronal transmission and organismal behavior.
Savva YA, Jepson JE, Sahin A, Sugden AU, Dorsky JS, Alpert L, Lawrence C, Reenan RA. 2012. Auto-regulatory RNA editing fine-tunes mRNA re-coding and complex behaviour in Drosophila. Journal of Biological Chemistry. 3:790. My contribution: checked the alteration of RNA editing along with Savva YA, made the initial observation for altered climbing behavior in mutant Drosophila lines, and performed behavioral analyses along with Jepson JE.
Jepson JE, Savva YA, Yokose C, Sugden AU, Sahin A, Reenan RA. 2011. Engineered alterations in RNA editing modulate complex behavior in Drosophila: regulatory diversity of adenosine deaminase acting on RNA (ADAR) targets. Nature Communications. 10:8325-37. My contribution: performed behavioral analyses along with Jepson JE.
A New Model of ALS: Precise Genetic Engineering, Characterization and Genetic Suppression
Amyotrophic Lateral Sclerosis (ALS) is a disease characterized by the degeneration and death of motor neurons, however how mutations linked to ALS initiate the decline in motor neuron function is not fully understood. I mostly blame the lack of good (non-transgenic) animal models to study ALS. Thanks to many ALS organizations, there are a lot of funding opportunities to study ALS; and thanks to Ice Bucket Challenge, ALS awareness has never been better. Speaking of funding and awareness, my PhD dissertation project started with the receipt of a pre-doctoral fellowship towards my PhD studies at Brown University from an ALS patient’s family and led to discovery of a potential pathway to target for ALS. I am very grateful for this Kirac fellowship.
After I graduated from my undergraduate studies, I committed to study RNA editing in Dr. Robert Reenan’s laboratory at Brown University. Dr. Reenan was using homologous recombination to generate RNA editing mutants in his laboratory (Pre-CRISPR era, but the same mechanism that CRISPR uses to introduce point mutations). My thesis project goals changed in an ALS conference organized by Kirac Foundation (Suna Kirac is the first businesswoman in Turkey and unfortunately an ALS patient), when Dr. Reenan and I noticed there was a lack of knock-in animal models used to study ALS pathogenesis. Thus, for my doctoral work I decided to utilize homologous recombination, that Dr. Reenan uses to study RNA editing mutants, to establish a precise model for ALS.
Back in 2009, when I started my PhD thesis project, SOD1 was the most common cause of ALS, there were no non-transgenic animal models to study ALS pathogenesis, and the CRISPR/Cas9 system was not available. To address concerns about the dosage of mutant SOD1 in ALS pathogenesis, and the lack of a knock-in ALS model, we genetically engineered SOD1 point mutations into the endogenous locus of Drosophila SOD1; and analyzed the molecular, biochemical and behavioral alterations that arise from these mutations. Contrary to previous transgenic models, we recapitulated ALS-like phenotypes without over-expressing the mutant protein.
First, I introduced four human disease-causing point mutations into the endogenous Drosophila SOD1 locus via ends-out homologous recombination (This was pre-CRISPR era). Then, I studied these knock-in flies for known altered cellular mechanisms in neurodegeneration. Drosophila carrying specific SOD1 mutations, which cause a rapid disease progression in ALS patients, exhibited neurodegeneration, locomotion deficit, and a shortened life span in a mutant-SOD1-dosage responsive manner. (Read more about the first part of my PhD project at Sahin et al., 2017 and Held et. al., Submitted). Next, I undertook an unbiased approach to understand the ALS disease progression by performing a detailed expression profile from mutants using RNA sequencing. Lastly, I performed a large-scale genetic screen to identify suppressor mutations that can reverse lethality resulting from severe SOD1 ALS mutations. From this screen, I discovered a potential target for ALS-which is being developed by Brown University’s Technology Transfer Office.
I believe my thesis work represents a significant step forward in our understanding of the molecular mechanisms and suppressors of mutant SOD1 and the benefits of a precisely controlled system. The basis of my PhD work served as a foundation for successfully funded Brown University Biomed Innovations, ALSA and Target ALS grants, an industry collaboration with Biogen, and internal collaborations at Brown University.
Sahin A, Held A, Major P, Bredvick K, Achilli TM, Kerson AG, Wharton K, Stilwell G, Reenan RA. 2017. Human SOD1 ALS mutations in a Drosophila knock-in model cause severe phenotypes and reveal dosage-sensitive gain and loss of function components. Genetics. 205(2):707-723. This is the main chapter of my PhD Thesis. My contribution to this manuscript: generated four fly lines, performed all molecular and behavioral experiments, wrote the manuscript and constructed the figures.
Held A, Major P, Sahin A, Reenan RA, Lipscombe D, Wharton KA. 2017. Motor circuit dysfunction preceding motor neuron degeneration in a dSod1-ALS model can be alleviated by BMP signaling. Submitted. This is a follow up manuscript for my Genetics publication. My contribution: generated the SOD1 knock in fly lines and performed preliminary neuromuscular junction analyses.
dsRNA as a Source of Neurodegeneration, Neuroninflammation and Neuroinflammation
As a postdoctoral researcher at the Albers Lab, I continue to investigate the candidate neurodegeneration pathways that I identified in the ALS model I developed during my PhD; and cytoplasmic dsRNA, which is a potential product of lack of "promiscuous RNA editing" that I studied as a side project during my PhD. Different than my PhD thesis, for my postdoctoral work I use multiple model systems and concentrate on multiple neurodegenerative diseases. Specifically, I study dsRNA mediated neurodegeneration and neuroinflammation in mouse models and post mortem tissue from Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s Disease (AD) and Frontotemporal Dementia (FTD) using imaging and molecular methods.
The pathogenic processes underlying neurodegenerative disease are multifactorial. At present, it is not fully understood which disease alterations are casual and which are secondary responses. In recent years RNA metabolism-related proteins have been linked to neurodegenerative diseases such as ALS, AD and FTD. Moreover, loss of function in the majority of dsRNA binding proteins cause neurodegeneration, and activated innate immunity is a hallmark shared by almost all neurodegenerative diseases. On the other hand, accumulation of cytoplasmic double stranded RNA (dsRNA) in the context of viral infection has long been known to invoke immune response, including secretion of type 1 interferons, and cause neurodegeneration. The first publication I contributed to at the Albers Lab (Rodriguez et al., 2018) demonstrates that accumulation of cytoplasmic dsRNA is present in ALS postmortem brains. In this publication, we study dsRNA-caused neuronal death in cell culture, mouse model and postmortem tissue. Our results reveal that cytoplasmic dsRNA is very stable and after reaching a certain threshold it is sufficient to trigger neuron death through the type 1 interferon pathway. In neurodegenerative diseases, dsRNA accumulation -as a direct byproduct of age-exacerbated genomic instability, expansion of heterochromatin regions, or activation of transposable elements- may independently or synergistically lead to innate immune activation, neuronal dysfunction, and eventual death.
My work currently focuses on:
The scope of cytoplasmic dsRNA accumulation in other neurodegenerative diseases.
Potential therapeutical ways of interrupting dsRNA mediated neuroninflammation.
Mapping the sources of cytoplasmic dsRNA via dsRNA sequencing.
Understanding the scope/pathways of Neurodegeneration, Neuroninflammation and Neuroinflammation in single cell level via imaging.
Rodriguez S, Schrank BR, Sahin A , Al-Lawati H, Constantino I, Benz E, Fard D, Albers AD, Cao L, Gomez AC, Ratti E, Cudkowicz M, Frosch MP, Talkowski M, Sorger P, Hyman BT, Albers MW. 2018. Genome-encoded Cytoplasmic Double-Stranded RNAs, Found in C9ORF72 ALS-FTD Brain, Provoke Propagated Neuronal Death. Submitted, available at BioRxiv 248328; doi: My Contribution: Performed IHC on ALS patient postmortem samples and cytoplasmic dsRNA isolation via IP.