Elizabeth Pollina M.D., Ph.D. 

ABSTRACT

Across a lifetime, neurons must retain a remarkable level of plasticity that facilitates learning, memory, and behavior. As animals encounter new sensory stimuli and learn complex behaviors, these experiences trigger changes in the activation of the state of neurons in the brain. In turn, increased neuronal activity induces the transcription of thousands of genes, the products of which dynamically modify the cells and circuits of the brain. Neuronal activity-induced transcription is, however, a costly and risky endeavor. During transcription, the DNA is cut, unwound, and eventually resealed in a process that has the potential to create permanent mutations. How then do animals balance the benefits of elevated neuronal activity for plasticity with the risks it poses to the stability of their genetic code? The goal of this proposal is to identify the molecular mechanisms that protect neuronal genomes from damage during periods of heightened neuronal activity. In our first aim, we will identify new mechanisms that repair activity-induced DNA damage, with an eye towards future work assessing how these protective mechanisms change with age in mouse models. In our second aim, we will identify the burden of mutations that accrue during aging at activity-induced genes in different types of brain cells. These studies will identify the cell types most susceptible to damage and will highlight gene candidates with high levels of mutations that may contribute to age-associated cognitive decline. Together, our work will provide foundational knowledge of how diverse neuronal cell types maintain transcriptional control and genome stability with age and how these genome control mechanisms go awry in aging and degenerative disease.

Lay Summary: 
Our aging population is expected to develop increased incidences of neurodegenerative disease and dementia, but the molecular mechanisms that underlie these complex processes remain poorly understood. This proposal aims to understand how neuronal activity-dependent gene expression and DNA damage repair can regulate brain aging by leveraging a newly identified, activity-inducible protein complex, NPAS4:NuA4. Our work will shed light on how changing levels of activity in the brain influence the accumulation of DNA damage across neuronal genomes as we age and the consequences of this damage for brain function. These experiments will lay critical groundwork for designing targeted strategies to slow or reverse decline in the neuronal cell types most susceptible to age-dependent diseases.

Progress Report — Final (Year 2 of 2)

The goal of this project was to identify the molecular mechanisms that protect neuronal genomes from damage during periods of heightened neuronal activity. Neuronal activity induces the transcription of thousands of genes essential for learning and memory — yet it also presents a genomic risk, as activity-induced transcription is linked to DNA double-strand breaks (DSBs) at gene regulatory elements. Understanding how long-lived neurons balance transcriptional plasticity with genome stability has direct relevance to human longevity and aging therapies.

Aim 1: Mechanisms of Activity-Dependent DNA Repair

Prevailing dogma holds that post-mitotic neurons repair DSBs exclusively through non-homologous end joining (NHEJ). Using mouse models, we discovered that the homologous recombination factor RAD52 is upregulated in response to elevated neuronal activity specifically in the hippocampus, regulated transcriptionally by the NPAS4:NuA4 complex — a neuronal protein complex our lab identified as playing a key role in suppressing activity-dependent DNA damage.

RAD52 biochemically interacts with NPAS4:NuA4 subunits and co-localizes with NPAS4:NuA4 across the genome. Using genome-wide techniques, we mapped R-loop locations in activated hippocampal neurons and found substantial overlap between R-loop formation and activity-inducible DSBs bound by NPAS4 and RAD52. Our findings show that:

• Acute depletion of Rad52 in the hippocampus leads to increased 53BP1 foci, a marker of unrepaired DSBs.
• Germline Rad52 knockout (KO) mice show enhanced R-loop formation at NPAS4-bound DSB sites.
• Rad52 KO mice display reduced binding of the activity-inducible transcription factors NPAS4 and FOS to their target sites and impaired induction of activity-dependent genes in response to novel environments.
• Adult Rad52 KO mice show striking alterations in spatial learning and memory, particularly a decline in cognitive flexibility during spatial learning tasks.

Together, these findings identify a surprising role for a canonical homologous recombination factor in mammalian brain function. Based on these findings, we received our lab’s first R01 from the National Institute on Aging (NIA). A manuscript entitled “Activity-dependent induction of the homologous recombination factor RAD52 promotes hippocampal genome stability and transcriptional plasticity” is in preparation.

Aim 2: Mutational Burden at Activity-Dependent Genes Across Brain Cell Types

We expanded our mutation mapping from approximately 50 to 150 sites in germline Npas4 wild-type versus Npas4 KO mice, identifying a trend toward more insertion/deletion events in Npas4 KO mice. Over the course of the project, we transitioned our mutation mapping strategy to a sequence-capture approach coupled to rolling circle amplification — minimizing technical errors and enabling more confident mutation calling in freshly isolated brain cells. LLF funding was critical in identifying more robust methods, and we have subsequently applied for an internal grant from the WU-SMAC incorporating this new technology in the context of neurological disease models.

Future Directions

LLF support has substantially accelerated the pace of discovery for our lab. Building on this work, we have received an R01 from the NIA and have initiated a collaboration with Dr. Andrew Yoo to use microRNA-mediated reprogramming of human fibroblasts into cortical neurons — an approach that preserves age-dependent genomic and cellular features and allows modeling of late-onset neurodegenerative disease.

We have applied for a second pilot grant from the LLF and the Alzheimer’s Association to launch this work focused on human aging biology. With additional support, we will be positioned to identify novel molecular mechanisms that counteract genome damage and may thereby be targeted to enhance transcriptional fidelity and neuronal plasticity across long human lifespans.

“As our lab’s first grant, LLF funding was instrumental in allowing us to procure additional funding and build a strong research team.” — Elizabeth Pollina, Ph.D.