Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medicine

Major Professor

Kevin Nash, Ph.D.

Committee Member

Danielle Gulick, Ph.D.

Committee Member

Alison Willing, Ph.D.

Committee Member

Craig Doupnik, Ph.D.

Keywords

FMRP, synaptic plasticity, learning and memory, Frmr1, Reelin

Abstract

Reelin is a large, extracellular matrix glycoprotein involved in neuronal migration during embryonic brain development and synaptic plasticity in the adult brain. Reelin consists of eight EGF-like repeats and undergoes natural cleavage at two sites, between repeats 2 and 3 and repeats 5 and 6, resulting in five naturally occurring fragments. The role of Reelin in the developing central nervous system has been extensively characterized, and loss of Reelin or disruption in the signaling cascade may contribute to the cognitive impairments seen in numerous neurodevelopmental or aging diseases such as schizophrenia, autism, depression, epilepsy, and Alzheimer’s disease. Reelin’s signaling potentiates glutamatergic and GABAergic neurotransmission, induces synaptic maturation, and increases AMPA and NMDA receptor subunit expression and activity. We hypothesize that a therapeutic approach could be possible for these disorders through a Reelin signaling intervention. Reelin signaling offers an advantage as a therapeutic target since it affects numerous pathways controlling basal synaptic transmission and plasticity, spine morphology, and biochemical make-up and compartmentalization of major components of synaptic function. Further investigation into the contribution of Reelin signaling during aging and in disease pathogenesis would advance our understanding of related cognitive impairment and potentially lead to new therapeutic targets. This thesis examines the association between Reelin signaling and neurodevelopmental models, and explores the potential of Reelin as a therapeutic, specifically for Fragile X Syndrome (FXS).

Reelin binds to two known receptors, very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (APOER2 or LRP8). Upon binding, it has been suggested that the receptors cluster or at a minimum, dimerize, resulting in initiation of the Reelin signaling cascade. Reelin and its receptor, ApoER2, play critical roles in prenatal brain development and postnatally in synaptic plasticity, learning, and memory. In order to examine the role of Reelin signaling in a neurodevelopmental model and explore its potential as a therapeutic, we first examined the efficacy of Reelin’s central fragment, which constitutes the EGF like repeats 3, 4, 5 and 6 (termed R3456), in initiating intracellular signaling. Immunoprecipitation experiments suggest that Reelin’s central fragment binds to ApoER2, inducing ApoER2 clustering and subsequent intracellular signaling. We developed a novel, cell-based assay of ApoER2 dimerization using a “split-luciferase” approach. Specifically, cells were co-transfected with one recombinant ApoER2 receptor fused to the N-terminus of luciferase and a second ApoER2 receptor fused to the C-terminus of luciferase. Using this assay, we directly observed basal ApoER2 dimerization/clustering in transfected HEK293T cells and, significantly, an increase in ApoER2 clustering in response to R3456. Additionally, the central fragment of Reelin activated intracellular signal transduction of ApoER2, indicated by increased levels of phosphorylation of Dab1, ERK1/2, and Akt in primary cortical neurons. Functionally, we were the first to demonstrate that injection of R3456 could rescue phenotypic deficits observed in the heterozygous reeler mouse (HRM). We observed an improvement in learning and memory tasks specifically in the hidden platform water maze and open field maze and in spatial learning and memory specifically in a contextual-based fear conditioning paradigm task. These data demonstrated that the central fragment of Reelin contributes to facilitating the Reelin intracellular signaling pathway and has potential as a therapeutic.

There is a growing literature reporting that loss of function and/or reduction of Reelin is implicated in several neurodegenerative diseases. Reelin supplementation has been demonstrated in a model of Angelman syndrome and has shown potential in recovering deficits present in learning and memory and post-synaptic dendritic function. We demonstrated recovery of spatial learning and memory and working memory in the HRM mouse via a single intracerebroventricular injection of the R3456 protein. We next wanted to explore the efficacy and potential of the R3456 protein in a mouse model of FXS.

FXS is the most common form of inherited intellectual disability and is characterized by autistic behaviors, childhood seizures, and deficits in learning and memory. FXS has a loss of function of the FMR1 gene that leads to a lack of Fragile X Mental Retardation Protein (FMRP) expression. FMRP is a translational repressor and critical for synaptic plasticity, spatial learning, and memory. FMRP is a RNA binding protein and in its absence, protein synthesis in the brain is excessive, which impairs neuronal circuitry formation and impairs higher cognitive function. Reelin as previously mentioned, plays an essential role in synaptic plasticity and neurodevelopmental processes and a reduction in Reelin signaling is implicated as a contributing factor in disease etiology in several neurological disorders. However, the role of Reelin in FXS is poorly understood. We demonstrate a baseline reduction in Reelin in Fmr1 knock-out (KO) mice, suggesting that a loss of Reelin activity may contribute to FXS cognitive impairments. We demonstrate here that Reelin signaling enhancement via a single intracerebroventricular injection of R3456 into Fmr1 KO mice can profoundly rescue cognitive deficits in hidden platform water maze and fear conditioning, as well as hyperactivity during the open field. Improvements in behavior were associated with rescued levels of the post synaptic marker PSD95 in Fmr1 KO mice when compared to controls. These data suggest that increasing Reelin signaling in FXS could offer a novel therapeutic for improving cognition in FXS.

Using the complementation luciferase assay described above, we explored various fragments and combinations of the EGF-like repeats from the R3456 protein. We identified a number of smaller fragments which could still elicit receptor dimerization, including a construct of repeats 3 and 6 (R36). We show that R36 was capable of initiating intracellular signaling in primary neuronal cultures, as shown by increased Dab1 phosphorylation and ERK phosphorylation. Furthermore, we demonstrate that a single intracerebroventricular injection of R36 protein into HRM can profoundly rescue cognitive deficits in hidden platform water maze and fear conditioning.

After demonstrating a reduction in Reelin in Fmr1 knock-out (KO) mice, and that supplementation with R3456 protein could improve Fmr1 KO cognitive deficits, we wanted to explore if our smaller Reelin fragment (R36) could elicit a similar response in the Fmr1 KO mouse. We show that Reelin signaling enhancement via a single intracerebroventricular injection of R36 protein can profoundly rescue cognitive deficits in hidden platform water maze and fear conditioning, as well as hyperactivity during the open field maze for Fmr1 KO mice.

We hypothesized that the greatest translatable aspect of R36 is related to its smaller size, and thus ability to be packaged into a recombinant adeno-associated virus (rAAV) vector. A viral vector approach would allow for a long-term expression of R36 for continued activation of the Reelin signaling pathway. Significantly, we demonstrate that a single intracerebroventricular injection of an rAAV expressing R36 presented similar cognitive rescue in HRM to our R36 protein injections. Additionally, we demonstrate the same recovery of cognitive deficits in behavioral assays when Fmr1 KO mice were given a single intracerebroventricular injection of a rAAV expressing R36. Our data suggest that a gene therapy approach directed at increasing Reelin signaling via R36, could offer a novel therapeutic approach for improving cognition in FXS. Furthermore, rAAV-R36 offers the possibility of exploring gene therapy to increase Reelin signaling in diseases with a reduction in Reelin signaling, because it offers potential long-term therapeutic effects.

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