Cholecystokinin-expressing GABAergic (CCK-GABA) neurons are perisomatic inhibitory cells that have been argued to regulate emotion and sculpt the network oscillations associated with cognition. However, no study has selectively manipulated CCK-GABA neuron activity during behavior in freely-moving animals. To explore the behavioral effects of activating CCK-GABA neurons on emotion and cognition, we utilized a novel intersectional genetic mouse model coupled with a chemogenetic approach. Specifically, we generated triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FL-hM3Dq (CCK-GABA/hM3Dq) mice that expressed the synthetic excitatory hM3Dq receptor in CCK-GABA neurons. Results showed that clozapine-N-oxide (CNO)-mediated activation of CCK-GABA neurons did not alter open field (OF) or tail suspension (TS) performance and only slightly increased anxiety in the elevated plus maze (EPM). Although CNO treatment had only modestly affected emotional behavior, it significantly enhanced multiple cognitive and memory behaviors including social recognition, contextual fear conditioning, contextual discrimination, object recognition, and problem-solving in the puzzle box. Collectively, these findings suggest that systemic activation of CCK-GABA neurons minimally affects emotion but significantly enhances cognition and memory. Our results imply that CCK-GABA neurons are more functionally diverse than originally expected and could serve as a potential therapeutic target for the treatment of cognitive/memory disorders.

BackgroundPyroglutamate‐3Aβ (pGlu3Aβ) is a toxic N‐terminally truncated and modified form of Aβ that leads to faster aggregation and seeding of plaques, making it an important target in Alzheimer’s disease. Anti‐amyloid antibodies, e.g., bapineuzumab, are associated with Amyloid‐Related Imaging Abnormalities (ARIA) involving vasogenic edema and microhemorrhages in AD patients, especially in ApoE4 carriers. Previously, we demonstrated reductions in plaques and cognitive deficits using an anti‐pGlu3Ab mAb, 07/2a. Here, we tested a novel CDC‐mutant version (07/2a‐k, murine precursor to PBD‐C06) to avoid C1q activation to reduce vascular‐related inflammation associated with anti‐amyloid antibodies.MethodTo estimate an effective therapeutic dose, 12 mo‐old APP/PS1dE9 mice were treated weekly (i.p.) for 5 weeks with 150, 300 or 600 µg 07/2a‐k or IgG2a (n=10‐12/group). General Ab and pGlu3Aβ levels were quantified by ELISA and immunohistochemistry. Next, 16 mo‐old APP/PS1dE9; hApoE4 mice were treated weekly (i.p.) for 15 weeks with 350 µg of 07/2a‐k, 3D6‐L (a murine analog of bapineuzumab), or IgG2a, while hApoE4 mice were treated with PBS (n=10‐13 mice/group). Subsequently, cognitive testing was performed over 3 weeks using the Spatial Novelty Y Maze (SNYM), Novel Object Recognition (NOR), and the Barnes Maze followed by euthanasia.ResultFive weekly treatments with 07/2a‐k in the dosing study did not alter general and pGlu3 Aβ biochemical levels; however, hippocampal pGlu3Aβ plaques were significantly reduced (300 µg, p<0.0005; 600 µg, p<0.005). In the therapeutic study, 3D6‐L reduced Aβx‐42 levels (p<0.009) by ELISA but not Abx‐40 levels. 07/2a‐k did not significantly alter Aβx‐42 or Abx‐40 levels. Both antibodies improved performance in the NOR test (07/2a‐k, p<0.05; 3D6‐L, p=0.05) and showed a non‐significant trend for improvement in the SNYM and Barnes maze compared to IgG2a controls. Macro‐hemorrhages were visible in 33% of 3D6‐L treated mice but not in 07/2a‐k treated mice.ConclusionTreatment with the 3D6‐L mAb, a murine analog of bapineuzumab, lowered cerebral Aβ levels and improved cognition but induced macro‐hemorrhages in aged APP/PS1dE9;hApoE4 mice, while treatment with 07/2a‐k, the precursor to PBD‐C06, did not alter general Ab levels but improved cognition in the absence of macro‐hemorrhages. Further analyses are underway to quantify cerebral pGlu3Ab levels, plaques, CAA, microhemorrhages and gliosis. (NIH 1RF1AG058657‐CAL)

BackgroundThe development of immunotherapies against Aβ is burdened by clinical adverse events (AEs) observed as amyloid‐related imaging abnormalities (ARIA) on magnetic resonance imaging scans. The vascular inflammatory AEs have been reported in AD clinical trials administrating certain anti‐Aβ plaque‐binding antibodies and are thought to represent cerebral microhemorrhages and vasogenic edema. ARIA incidence appears apolipoprotein (APOE) genotype‐dependent, wherein APOE E4 allele carriers are at higher risk when compared to non‐carriers. With hopes to robustly model patients most at risk for the vascular complications of anti‐Aβ immunotherapy, we selected a relatively new transgenic mouse model bearing the human APOE E4 gene. The model has not yet been extensively characterized at multiple age points. To assess the face validity of the mouse model, we are conducting a battery of histological and biochemical tests.MethodsTo evaluate age‐ and sex‐related pathological changes, we collected brain and blood plasma from euthanized male and female APP/PS1dE9;hAPOE4 (APP/E4) mice across 3 ages: 8‐, 12‐ and 16‐months. General‐ and pGlu3‐Aβ levels were quantified using enzyme‐linked immunoassays (ELISA). Prussian Blue hemosiderin staining was used to examine microhemorrhages.ResultsImmunoassays of brain homogenates exhibited significantly higher insoluble Aβx‐40, Aβx‐42 and pGlu3Aβ levels in 16‐month‐old mice compared to 8‐ and 12‐month‐old mice. Moreover, the 16‐month‐old females demonstrated significantly higher levels of Aβx‐40 and pGlu3Aβ than age‐matched males. Hemosiderin staining was observed in all 12‐ and 16‐month mice, however no significant differences in abundance was found between the 2 age points. Immunostainings for plaque deposition of various Aβ species, vascular amyloid, dystrophic neurites and gliosis are ongoing.ConclusionsAge‐ and sex‐dependent increases in Aβ deposition were revealed in the APP/E4 mouse model. Preliminary evidence for microhemorrhage was observed. Subsequent analyses will better characterize the translational credibility of APP/E4 as a model for future nonclinical immunotherapies targeting Aβ. (1RF1AG058657‐CAL)