Borski, C. | bioRxiv Science

bioRxiv2024

AKT2 modulates astrocytic nicotine responses in vivo.

A greater understanding of the neurobiology of nicotine 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), 4{beta}2 and 7. Protein kinase B-{beta} (Pkb-{beta} 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 g/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 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 100 M nicotine sufficient for morphological changes and blocking 7 or 4{beta}2 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 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. Main PointsO_LINicotine regulates astrocytes in vivo: acute exposure boosts complexity; chronic exposure diminishes it. C_LIO_LIAKT2, expressed in astroglia, modulates morphological changes in response to nicotine and nicotine-dependent conditioned place preference. C_LI

neuroscience↗

bioRxiv2022

Identifying Optimal Neuroinflammation Treatment Using Nanoligomer Discovery Engine

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leads to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and is causally linked to a range of neurodegenerative diseases such as Alzheimers diseases (AD), Multiple Sclerosis (MS), Parkinsons diseases (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier (BBB), making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer discovery engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1{beta} or IL-1{beta}, tumor necrosis factor-alpha or TNF-, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-{kappa}{beta}) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and AI-based ranking method to design sequence-specific peptide molecules to up-or down-regulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-{kappa}{beta} (SB.201.17D.8_ NF-{kappa}{beta}1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes, and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low-dose (5mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

neuroscience↗