ISR-Dependent Regulation of Translation in Neurons During Synaptic Plasticity
Abstract
The integrated stress response (ISR) is a core pathway for maintaining cellular proteostasis and a key regulator of translation in processes beyond the cellular response to stress. For example, the ISR regulates developmental axonogenesis, learning and memory, and synaptic plasticity in the brain. While these processes are supported by a large body of evidence, much less is understood about where and when ISR dependent translation is occurring in both specific cell-types in the brain and within the neurons themselves. One barrier to uncovering ISR roles in health and disease is the challenge of monitoring its activity. The transient nature of regulatory phosphorylation events and a lack of antibodies that effectively label the major ISR effector, ATF4, using immunohistochemical (IHC) approaches make the development of reporter systems to monitor ISR activity a desirable option to circumvent these challenges. Here, I present my efforts to develop and characterize fluorescent protein-based reporters to visually monitor ISR-dependent translation with cellular and subcellular resolution depending on the system used. To monitor ISR-dependent local translation in neurons, I modified the single molecule imaging of nascent peptides (SINAPS) platform to be translationally upregulated during synaptic plasticity and ISR activation. SINAPS utilizes SunTag and MS2 stem loop signal amplification technologies to visually track both mRNA and active translation of the reporter construct (Wu et al., 2016). The modified SINAPS Plasticity Reporter Suite was packaged in lentiviral vectors and delivered to primary hippocampal cultures. Compounds were used to stimulate synaptic plasticity or ISR activation followed by quantification of reporter puncta. The ultimate goal of this system was to utilize it for targeted knockdown of genes suspected of being involved in ISR-dependent translation. Although the reporters expressed in primary neurons, this project was abandoned due to difficulty optimizing signal-to-noise characteristics and a variety of methodological constraints that are discussed below. Using the dual-color fluorescent reporter of cellular ISR activity called selective phospho-eIF2α ORF tracking light (SPOTlight), the Calakos Lab identified striatal cholinergic interneurons (CINs) to exhibit a population wide need for elevated ISR activity in otherwise healthy neurons (Helseth et al., 2021). I found that the elevated ISR state in CINS was regulated by activity-dependent PERK kinase signaling using an inhibitory designer receptor exclusively activated by designer drugs (DREADD) followed by IHC. AAV-mediated delivery of the SPOTlight reporter brainwide revealed that other neuronal classes with similar action potential firing patterns did not share the elevated ISR signaling characteristic seen in striatal CINs. To expand the capabilities of the SPOTlight reporter, we developed a transgenic mouse line allowing for Cre recombinase dependent expression of SPOTlight. This resource enables selective visualization of ISR-regulated functional activity across genetically defined cell populations body-wide. To characterize the DIO-SPOTlight transgenic mouse line, it was crossed with the pan-neuronal Cre line (Nestin-Cre) where I investigated how pervasive the elevated ISR activity is in cholinergic neurons throughout the brain and spinal cord. I found that most cholinergic neuron populations (13/19) exhibited elevated ISR activity compared to non-cholinergic neighboring neurons using SPOTlight fluorescence. Identifying a class of neurons with elevated ISR states challenges the pervasive model that ISR activation is transient and an indication of poor cellular health. Indeed, runaway or disrupted ISR activity is associated with a variety of neurological disorders and is a desirable target for therapeutic options. However, if future therapies are intended to suppress ISR activity, detailed understanding of how these treatments could negatively impact cholinergic neuron function is necessary. Finally, the basally elevated ISR activity in cholinergic neurons establishes a conceptual precedent for the possibility that other classes of neurons may share this characteristic, an important avenue for future investigations.
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Oliver, Matthew Lyles (2025). ISR-Dependent Regulation of Translation in Neurons During Synaptic Plasticity. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/32787.
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