Person: Srikanth, Priya
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Srikanth
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Priya
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Srikanth, Priya
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Publication Cell-type Dependent Alzheimer's Disease Phenotypes: Probing the Biology of Selective Neuronal Vulnerability(Elsevier, 2017) Muratore, Christina; Zhou, Constance; Liao, Meichen; Fernandez, Marty; Taylor, Walter M.; Lagomarsino, Valentina N.; Pearse, Richard; Rice, Heather C.; Negri, Joseph; He, Amy; Srikanth, Priya; Callahan, Dana; Shin, Taehwan; Zhou, Monica; Bennett, David A.; Noggle, Scott; Love, J. Christopher; Selkoe, Dennis; Young-Pearse, TracySummary Alzheimer's disease (AD) induces memory and cognitive impairment in the absence of motor and sensory deficits during its early and middle course. A major unresolved question is the basis for this selective neuronal vulnerability. Aβ, which plays a central role in AD pathogenesis, is generated throughout the brain, yet some regions outside of the limbic and cerebral cortices are relatively spared from Aβ plaque deposition and synapse loss. Here, we examine neurons derived from iPSCs of patients harboring an amyloid precursor protein mutation to quantify AD-relevant phenotypes following directed differentiation to rostral fates of the brain (vulnerable) and caudal fates (relatively spared) in AD. We find that both the generation of Aβ and the responsiveness of TAU to Aβ are affected by neuronal cell type, with rostral neurons being more sensitive than caudal neurons. Thus, cell-autonomous factors may in part dictate the pattern of selective regional vulnerability in human neurons in AD.Publication Comparison and Optimization of hiPSC Forebrain Cortical Differentiation Protocols(Public Library of Science, 2014) Muratore, Christina; Srikanth, Priya; Callahan, Dana G.; Young-Pearse, TracySeveral protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual SMAD inhibition differentiation protocols, manual vs. AggreWell aggregate formation, plating substrates, neural progenitor cell (NPC) isolation methods, NPC maintenance and expansion, and astrocyte co-culture. The embryoid aggregate protocol, using a Matrigel substrate, consistently generates a high yield and purity of neurons. NPC isolation by manual selection, enzymatic rosette selection, or FACS all are efficient, but exhibit some differences in resulting cell populations. Expansion of NPCs as neural aggregates yields higher cell purity than expansion in a monolayer. Finally, co-culture of iPSC-derived neurons with astrocytes increases neuronal maturity by day 40. This study directly compares commonly employed methods for neuronal differentiation of iPSCs, and can be used as a resource for choosing between various differentiation protocols.Publication Schizophrenia-Relevant DISC1 Interruption Alters Wnt Signaling and Cell Fate in Human iPSC-Derived Neurons(2015-07-09) Srikanth, Priya; Sahin, Mustafa; Harwell, Corey; Morrow, EricThe advent of human induced pluripotent stem cell (iPSC) technology has allowed for unprecedented investigation into the pathophysiology of human neurological and psychiatric diseases. Use of human iPSC-derived neural cells to study disease is complicated by the genetic heterogeneity of cell lines and diversity of differentiation protocols. Here, I address issues surrounding neuropsychiatric disease modeling with human iPSCs. Dozens of published protocols exist to differentiate iPSCs into forebrain neuronal cultures. Among the factors that distinguish these methods are: use of small molecules, monolayer vs. aggregate culture, choice of plating substrates, method of NPC isolation, and glial co-culture. Each of these factors is evaluated here, creating a resource that directly compares a variety of differentiation procedures. The most efficient and reproducible method was an embryoid aggregate differentiation protocol, including aggregate plating onto a Matrigel substrate, enzymatic neural rosette selection, and neuronal dissociation and plating onto Matrigel. This optimized protocol is used to model a schizophrenia-relevant mutation in human neural cells. Genetic and clinical association studies have identified disrupted-in-schizophrenia 1 (DISC1) as a strong candidate risk gene for major mental illness. DISC1 was initially associated with mental illness upon the discovery that its coding sequence is interrupted by a balanced chr(1;11) translocation in a Scottish family, in which the translocation cosegregates with psychiatric disorders. I investigate the functional and biochemical consequences of DISC1 interruption in human neurons using TALENs or CRISPR-Cas9 to introduce DISC1 frameshift mutations into iPSCs. I show that disease-relevant DISC1 targeting results in decreased DISC1 protein expression by nonsense-mediated decay, increases baseline Wnt signaling in neural progenitor cells, and causes a shift in neural cell fate. DISC1-dependent Wnt signaling and cell fate changes can be reversed by antagonizing the Wnt pathway during a critical window in neural progenitor development. These experiments suggest that DISC1-disruption increases Wnt signaling, which alters the balance and identity of neural progenitors, thereby subtly modifying cell fate. These studies evaluate the use of multiple differentiation procedures in neural disease modeling, shed light on the roles of DISC1 during human brain development, and further our understanding of the pathogenesis of major mental illness.Publication Shared effects of DISC1 disruption and elevated WNT signaling in human cerebral organoids(Nature Publishing Group UK, 2018) Srikanth, Priya; Lagomarsino, Valentina; Muratore, Christina; Ryu, Steven C.; He, Amy; Taylor, Walter M.; Zhou, Constance; Arellano, Marlise; Young-Pearse, TracyThe development of three-dimensional culture methods has allowed for the study of developing cortical morphology in human cells. This provides a new tool to study the neurodevelopmental consequences of disease-associated mutations. Here, we study the effects of isogenic DISC1 mutation in cerebral organoids. DISC1 has been implicated in psychiatric disease based on genetic studies, including its interruption by a balanced translocation that increases the risk of major mental illness. Isogenic wild-type and DISC1-disrupted human-induced pluripotent stem cells were used to generate cerebral organoids, which were then examined for morphology and gene expression. We show that DISC1-mutant cerebral organoids display disorganized structural morphology and impaired proliferation, which is phenocopied by WNT agonism and rescued by WNT antagonism. Furthermore, there are many shared changes in gene expression with DISC1 disruption and WNT agonism, including in neural progenitor and cell fate markers, regulators of neuronal migration, and interneuron markers. These shared gene expression changes suggest mechanisms for the observed morphologic dysregulation with DISC1 disruption and points to new avenues for future studies. The shared changes in three-dimensional cerebral organoid morphology and gene expression with DISC1 interruption and WNT agonism further strengthens the link between DISC1 mutation, abnormalities in WNT signaling, and neuropsychiatric disease.