Hayk Davtyan^1,2, Jean Paul Chadarevian^1,2, Jonathan Hasselmann^1,2, Ghazaleh Eskandari-Sedighi¹, Sherry Lin-Koch⁴, Alina L Chadarevian^2,3, Joia Kai Capocchi¹, Duc Duong⁵, Jasmine Nguyen¹, Christina Tu^1,2, Sepideh Kiani Shabestari³, Fang Wu⁵, Anantharaman Shantaraman⁵, Kimiya Mansour², William Carlen-Jones¹, Michael Mgerian³, Katia Deynega¹, Tau En Lim¹, Alan Khoi Mai¹, Lauren Le¹, Ani Agababian³, David A Hume⁶, Clare Pridans⁷, Elizabeth Head¹, Sandeep Robert Datta⁴, Nicholas T Seyfried⁵, Mathew Blurton-Jones^1,2,3

Hematopoietic stem cell transplantation (HSCT) in combination with microglia depletion is increasingly being examined as a potential therapy for neurological disorders. The premise of this approach is that HSCT-derived monocytes (MN) may infiltrate the brain and differentiate into “microglia-like” cells. However, induced pluripotent stem cells derived microglia (iMG) provides a potential alternative source of therapeutic cells. As many questions remain regarding the similarities and differences between microglia and monocytes, we utilized a xenotransplantation-compatible model that lacks endogenous microglia (hFIRE mice) to examine the transcriptional and functional properties of human iMG and MN. iMG and MN from four patients were transplanted into adult hFIRE brains and four months later behavioral testing was performed. Brains were then examined via spatial RNA sequencing, proteomics, histological, and biochemical approaches. Despite four months of brain residence and near complete chimerism, human iMG and MN continued to exhibit many important differences. In particular, we found that MN, but not iMG, induced a chronic proinflammatory state associated with significant levels of astrogliosis, demyelination, synaptic loss, and behavioral impairment. Taken together, these results demonstrate the critical role of ontogeny on myeloid cell function within the brain and provide important implications for the development of CNS-wide microglial replacement therapies.

¹Institute for Memory Impairments & Neurological Disorders, University of California, Irvine, CA, USA

²Stem Cell Research Center, University of California, Irvine, CA, USA

³Department of Neurobiology & Behavior, University of California, Irvine, CA, USA

⁴Harvard Medical School, Boston, MA, USA

⁵Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA

⁶Mater Research Institute, University of Queensland, Brisbane, Australia

⁷Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

Charles Hoeffer^1,2, Andrew M. Lombardi¹, Mina Griffioen^1,2, Helen Wong², Ryan Milstead^1,2,3, Curtis Borski², Erin Shiely^1,2, Myra E. Bower ^1,2, Emily Schmitt¹, Lauren LaPlante², Marissa A. Ehringer^1,2, Jerry Stitzel^1,2.

Improved understanding of nicotine neurobiology is needed to reduce or prevent chronic addiction, ameliorate detrimental nicotine withdrawal effects, and improve cessation rates. Nicotine binds and activates two astrocyte-expressed nicotinic acetylcholine receptors (nAChRs), α4β2 and α7. Protein kinase B-β (Pkb-β or Akt2) expression is restricted to astrocytes in mice and humans and is activated by nicotine. To determine if AKT2 plays a role in astrocytic nicotinic responses, we generated astrocyte-specific Akt2 conditional knockout (cKO) and full Akt2 KO mice. For in/ex vivo studies, we examined mice exposed to chronic nicotine for two weeks in drinking water (200 μg/mL) or following acute nicotine challenge (0.09, 0.2 mg/kg) after 24 hrs. Our in vitro studies used cultured mouse astrocytes to measure nicotine-dependent astrocytic responses. Sholl analysis was used to measure glial fibrillary acidic protein responses in astrocytes. Our data show that wild-type (WT) mice exhibit increased astrocyte morphological complexity during acute nicotine exposure, with decreasing complexity during chronic nicotine use, whereas Akt2 cKO mice showed enhanced acute responses and reduced area following chronic exposure. In culture, we found that 100μM nicotine was sufficient for morphological changes and blocking α7 or α4β2 nAChRs prevented observed morphologic changes. We performed conditioned place preference (CPP) in Akt2 cKO mice which revealed reduced nicotine preference in cKO mice compared to controls. Finally, we performed RNASeq experiments comparing nicotine- and LPS-mediated gene expression identifying robust differences between these two astrocytic stimuli. These findings show the importance of nAChRs and AKT2 signaling in the astrocytic response to nicotine.

¹Department of Integrative Physiology, University of Colorado at Boulder; ²Institute for Behavioral Genetics, University of Colorado at Boulder. Boulder, CO, 80303 USA.