Mitigating neuroinflammation : new potential therapeutics and targets

Neurodegenerative disorders, from chronic diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) to more acute injuries such traumatic brain injury (TBI), present a particular challenge to public health as they lack treatments and have devastating consequences. Across the variety of central nervous system (CNS) diseases and injuries, inflammation is a hallmark and potential target for effective therapies. Microglia, the immune cells of the brain, normally perform homeostatic maintenance roles in the brain; however, in cases of injury and disease, they are often dysfunctional and produce excessive quantities of pro-inflammatory proteins that exacerbate conditions. This makes microglia a prime target for intervention. As there is a current lack of drugs available to treat brain injuries and diseases, repurposing clinically approved medications may be the fastest route for new drugs to reach patients. Understanding cellular mechanisms and proteins that mediate inflammation are valuable for the development of new therapies. Overall, this thesis aims to evaluate several drug classes, including incretins and Rho-associated coiled-coil containing kinase (ROCK) inhibitors, which have potential for repurposing from their approved uses to treating neurodegenerative disorders. Herein, novel cellular mechanism and proteins that induce neuroinflammation are also investigated.

In this thesis, the incretins, which are endogenous insulin regulating hormones currently used to treat diabetes, and ROCK inhibitors, clinically used for glaucoma, are evaluated in different injury and disease models to understand their ability to affect brain health. With these studies, we evaluate microglia as a primary source of inflammation and utilize both cellular and rodent models of neuroinflammation. Our cellular studies leverage the potent inflammatory properties of lipopolysaccharide (LPS) to mimic in vivo neuroinflammation observed in our rodent models of mildmoderate traumatic brain injury (mTBI), which are also used throughout this thesis. We apply pharmacologic or genetic manipulations to understand how pathology progresses with the intervention, assessing proinflammatory protein production and microglial activation via enzyme-linked immunosorbent assays (ELISA), electrochemiluminescence, and immunochemistry, among other methods.

Incretin effects on the brain are the focus of Papers I-III. In Paper I, we investigate the pharmacokinetics and efficacy of PT302, a new slow-release formulation of the GLP-1 mimetic, Exenatide. Exenatide was initially approved for clinical use as a twice daily subcutaneously administered drug. We demonstrate steady-state plasma levels of Exenatide formulated as PT320 for up to three weeks after a single subcutaneous injection, with brain penetration reaching up to 2 to 3% of peak plasma concentrations, which is reasonable for a peptide-based drug and appears to be within its pharmacologically relevant range. Notably, PT302 administered at a dose that is translatable to human use is anti-inflammatory in the mouse brain, as we demonstrated the drug’s ability to decrease microglial activation following a mTBI injury. Behavioral deficits were also mitigated by PT302. For the next study (Paper II), we also utilized the mTBI model to investigate efficacy of a novel monomeric incretinbased triagonist, which showed similar effects as PT302 at mitigating behavioral deficits and inducing cellular signaling pathways that are neuroprotective. Additionally, I introduce a primary microglia (PMg) cell model for neuroinflammation and show potent anti-inflammatory potential for the triagonist. The last of the incretin studies (Paper III) utilizes cellular models of neurodegeneration and neuroinflammation to understand the physiological roles in the nervous system of the GLP-1 metabolite, GLP-1(9-36). This work has relevance as GLP-1(9-36) circulates in the bloodstream long after its insulinotropic parent peptide is inactivated. Our work indicates this metabolite, indeed, has physiologic roles in nervous system cells, including microglia. In this study, I utilized the relatively new immortalized (IMG) mouse microglial cell line as a model system for PMg.

The Nogo-signaling system, known for its neuronal plasticity restricting properties via RhoA and ROCK1 and ROCK2 activation, has recently been implicated in microglial inflammation processes. In Paper IV, we sought to elucidate the roles that RhoA and ROCK play in influencing the inflammatory process. We show that ROCK inhibitors potently inhibit LPS induced inflammation via a variety of mechanisms in both primary and IMG microglia. In parallel to the inquiry of ROCK influence on inflammation, this study serves as a “proof of concept” for the utility of using IMG cells in preclinical drug development and screening.

Lastly, in Paper V, we investigated the effects of microglial conditional deletion of a potent endogenous activator of RhoA/ROCK signaling in nervous system cells, Nogo. I developed a novel mouse model to perform this genetic manipulation and applied a moderate TBI injury, via a controlled cortical impact (CCI) injury to exposed brain tissue. Mice with conditional deletion of microglial Nogo (MinoKO mice) show signs of decreased microglial and astrocytic activation following CCI. CCI injured mice with microglial specific Nogo deletion exhibited hyperactivity among other phenotypes post-injury. MinoKO mice did not exhibit asymmetric motor function one week post-CCI as their control cohorts did, thereby providing further evidence of microglial Nogo negatively influencing recovery.

Overall, the studies within this thesis provide strong evidence for the utility of using incretins to treat neurodegenerative conditions. In addition, the current studies elucidating novel roles of RhoA, ROCK, and Nogo in microglial-induced inflammation add to the growing list of potential drug targets that may mitigate disease pathology.

List of scientific papers

I. Bader, M., Choi, H.-I., Rubovitch, V., Li, Y., Kim, H.K., Glotfelty, E., Pick, C.G., Lecca, D., Hoffer, B.J., Kim, D.S., Tweedie, D., Greig, N.H., Rachmany, L. (2018). Pharmacokinetics and efficacy of PT302, a sustained-release Exenatide formulation, in a murine model of mild traumatic brain injury. Neurobiology of Disease. 124, 439–453.
https://doi.org/10.1016/j.nbd.2018.11.023

II. Li, Y., Glotfelty, E.J., Namdar, I., Tweedie, D., Olson, L., Hoffer, B.J., DiMarchi, R.D., Pick, C.G., Greig, N.H. (2020). Neurotrophic and neuroprotective effects of a monomeric GLP-1/GIP/Gcg receptor triagonist in cellular and rodent models of mild traumatic brain injury. Experimental Neurology. 324, 113113.
https://doi.org/10.1016/j.expneurol.2019.113113

III. Li, Y., Glotfelty, E.J., Karlsson, T., Fortuno, L. V., Harvey, B.K., Greig, N.H. (2021). The metabolite GLP-1 (9-36) is neuroprotective and anti-inflammatory in cellular models of neurodegeneration. Journal of Neurochemistry. 159, 867–886.
https://doi.org/10.1111/jnc.15521

IV. Glotfelty, E.J., Tovar-Y-Romo, L.B., Hsueh, S., Tweedie, D., Li, Y., Harvey, B.K., Hoffer, B.J., Karlsson, T., Olson, L., Greig, N.H. (2023). The RhoA-ROCK1/ROCK2 pathway exacerbates inflammatory signaling in immortalized and primary cells microglia. Cells. 12, 1367.
https://doi.org/10.3390/cells12101367

V. Glotfelty, E.J., Hsueh, S., Bedolla, A., Karlsson, T., McGee, A., Luo, Y., Greig, N.H., Olson, L. (2023). Microglial Nogo delays recovery following traumatic brain injury in mice. [Manuscript]