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Published in final edited form as: Biogerontology. 2025 Nov 28;27(1):11. doi: 10.1007/s10522-025-10359-z Search in PMC Search in PubMed View in NLM Catalog Add to search Circadian Rhythm of Amyloid-β in the Olfactory Bulb and Cerebellum of Wild-Type and APPxPS1 Knock-In Mice Indicates a Loss of Rhythmicity in Regions More Vulnerable to Amyloid Pathology Valeria A Buzinova Valeria A Buzinova 1 Department of Molecular and Cellular Biochemistry 5 Department of the Sanders-Brown Center on Aging Find articles by Valeria A Buzinova 1, 5 , Carrie E Johnson Carrie E Johnson 1 Department of Molecular and Cellular Biochemistry 5 Department of the Sanders-Brown Center on Aging Find articles by Carrie E Johnson 1, 5 , Savannah M Turton Savannah M Turton 5 Department of the Sanders-Brown Center on Aging Find articles by Savannah M Turton 5 , Sarah E Barth Sarah E Barth 5 Department of the Sanders-Brown Center on Aging Find articles by Sarah E Barth 5 , Samantha Padgett Samantha Padgett 5 Department of the Sanders-Brown Center on Aging Find articles by Samantha Padgett 5 , M Tyler Maisel M Tyler Maisel 2 Department of Neuroscience 5 Department of the Sanders-Brown Center on Aging Find articles by M Tyler Maisel 2, 5 , Katharina Kohler Katharina Kohler 5 Department of the Sanders-Brown Center on Aging Find articles by Katharina Kohler 5 , Haleigh R Whitlock Haleigh R Whitlock 2 Department of Neuroscience Find articles by Haleigh R Whitlock 2 , Adam D Bachstetter Adam D Bachstetter 2 Department of Neuroscience 5 Department of the Sanders-Brown Center on Aging 6 Department of the Spinal Cord and Brain Injury Research Center Find articles by Adam D Bachstetter 2, 5, 6 , Sridhar Sunderam Sridhar Sunderam 3 Department of Biomedical Engineering Find articles by Sridhar Sunderam 3 , Bruce F O’Hara Bruce F O’Hara 4 Department of Biology Find articles by Bruce F O’Hara 4 , Marilyn J Duncan Marilyn J Duncan 2 Department of Neuroscience Find articles by Marilyn J Duncan 2, * , M Paul Murphy M Paul Murphy 1 Department of Molecular and Cellular Biochemistry 5 Department of the Sanders-Brown Center on Aging Find articles by M Paul Murphy 1, 5, * Author information Article notes Copyright and License information 1 Department of Molecular and Cellular Biochemistry 2 Department of Neuroscience 3 Department of Biomedical Engineering 4 Department of Biology 5 Department of the Sanders-Brown Center on Aging 6 Department of the Spinal Cord and Brain Injury Research Center * Address correspondence to: M. P. Murphy, Sanders-Brown Center on Aging, University of Kentucky, 789 S. Limestone Street, 541 Lee Todd Jr. Building, Lexington, KY, USA 40536; [email protected] ; M. J. Duncan, Department of Neuroscience, University of Kentucky, 432 Health Sciences Research Building, Lexington, KY, USA 40536; [email protected] Collection date 2025 Nov 28. PMC Copyright notice PMCID: PMC13072434 NIHMSID: NIHMS2163142 PMID: 41315136 The publisher's version of this article is available at Biogerontology Abstract Amyloid-β (Aβ) plaques are one of the primary biomarkers of Alzheimer’s Disease (AD). Other publications have reported various mechanisms regarding the clearance of Aβ, and recent studies have also investigated the relationship between daily rhythms of Aβ and AD. The intent of this study was to determine if the circadian rhythm of Aβ differed between a region that was more vulnerable to AD-related pathology (the olfactory bulbs; OB) compared to a region that is less vulnerable (the cerebellum; CER). We chose to utilize an APPxPS1 knock-in (KI) mouse strain as this strain expresses amyloid precursor protein (APP) and Aβ under control of its normal promoter as opposed to AD transgenic models that overexpress APP and, as a consequence, Aβ. Mice (N = 128, equally divided between male and female, wild type and KI) were acclimated to a 12:12 light cycle for two weeks, and tissue was collected over a 24-hour period in constant darkness. Using a unique immunoassay designed to measure human or rodent Aβ side-by-side, we confirmed a robust circadian Aβ rhythm in the mouse brain and that the OB contains more overall Aβ accumulation than the CER. The circadian Aβ rhythm was not present in the OB of the KI as compared to the WT mice. In contrast, the Aβ rhythm in the CER did not differ between genotypes. These results suggest that the loss of Aβ rhythm in disease-affected brain regions may be associated with the development of AD pathology and could have important implications for therapy. Keywords: Daily rhythm, amyloid-β precursor protein, immunoassays, aging, Alzheimer’s Disease Introduction Alzheimer’s Disease (AD) is the most common neurodegenerative disease among the elderly ( Tu et al., 2014 ). In 2022, 6.5 million individuals were diagnosed with AD, and this number is predicted to rise to 13 million by 2050 ( 2025 Alzheimer’s Disease Facts and Figures, 2025 ). One of the key neuropathological lesions that defines AD is extracellular plaques composed primarily of amyloid-β (Aβ) peptides. The appearance of toxic Aβ plaques begins decades before the onset of cognitive and functional decline, starting in cortical and limbic regions of the brain and spreading to other areas as the disease progresses. Less Aβ pathology is found in some brain regions, such as the cerebellum, compared to other regions, such as the hippocampus, cerebral cortex, and olfactory bulbs ( Calderon-Garciduenas & Duyckaerts, 2017 ; Murray et al., 2023 ). Aβ accumulation is thought to be due to decreased efficiency in brain clearance of Aβ and perhaps an increase in Aβ production ( Bloom, 2014 ; Hampel et al., 2021 ; van der Kant et al., 2020 ; Wang & Holtzman, 2020 ). Aβ is a driver of tau tangles, the other pathological hallmark in AD; however, the mechanism for this is unknown ( Murphy, 2023 ). The Aβ peptide was first isolated from the brain in 1984 and has been identified as a proteolytically processed fragment of the amyloid precursor protein (APP) encoded by a gene of the same name located on chromosome 21( Glenner & Wong, 1984 ; Hampel et al., 2021 ; Kang et al., 1987 ). APP has been a focus of AD therapeutics and continues to be studied; however, its exact biological role has yet to be determined ( Muller et al., 2017 ). After being generated as soluble monomers, Aβ peptides are found in various intermediate aggregation states (dimers, trimers, oligomers, and protofibrils) until they accumulate as extracellular plaques ( Hampel et al., 2021 ). A healthy individual produces Aβ 40 (40 amino acids long) and Aβ x-42 (42 amino acids long). Approximately 90% of Aβ 1-x is Aβ 40 , which is more soluble and thus more readily cleared from the brain, whereas the remaining 10% is Aβ x-42 , which is prone to aggregation due to the two additional hydrophobic amino acids on the carboxyl terminus. Relative changes in the proportion of Aβ x-42 in plasma or CSF can be used as a biomarker for AD, although its use in the diagnostic process is not settled ( Chouraki et al., 2015 ; Fandos et al., 2017 ; Graff-Radford et al., 2007 ; Shaw et al., 2025 ). Aβ extracted into aqueously soluble, detergent-soluble, and acid-soluble (or insoluble) fractions represents different forms of Aβ deposits in the brain, which, to some degree, correlate with the clinical status of an individual (Murphy and Levine, 2010; Niedowicz et al., 2012). Circadian rhythms are biological oscillations, with a periodicity of about 24 hours in humans, that are endogenously generated and can be synchronized (entrained) by regularly repeating environmental signals such as the daily light-dark cycle. Circadian rhythms play a role in timing the daily sleep-wake cycle ( Borbely, 1982 ). Disruptions in sleep and circadian rhythms are common in AD, but the reasons for this are unknown. The negative changes in sleep may be due to AD-pathology-induced damage to the areas that regulate the sleep-wake cycle ( Sharma et al., 2021 ). Studies show soluble APP and Aβ circulate in the cerebrospinal fluid (CSF) and blood in humans with a 24-hour rhythm and that the amplitude of this rhythm decreases with age and with increases of cerebral Aβ plaques ( Dobrowolska et al., 2014 ). Studies in AD-model mice that present Aβ-pathology have also shown that soluble Aβ in the hippocampal interstitial fluid (ISF) fluctuates with a daily rhythm that becomes disrupted as Aβ pathology develops ( Roh et al., 2012 ). Sleep and wakefulness regulate this rhythm, such that sleep deprivation during the normal rest phase prevents the expected decrease in Aβ in the hippocampal ISF ( Kang et al., 2009 ). Although it is clear that there is a daily rhythm of the Aβ peptide in the rodent hippocampal ISF and the human CSF, it is not known if other brain regions exhibit this rhythm and whether Aβ accumulation affects the rhythm. This study aimed to investigate the circadian rhythm of Aβ in two discrete brain regions: one considered more prone to developing Aβ pathology (the olfactory bulb, OB) and one considered less prone (the cerebellum, CER). The olfactory bulb was chosen as the Aβ vulnerable area as this region is not as severely affected as other areas of the limbic system or the neocortex, and the presence of large amounts of aggregated Aβ might make detecting a rhythm in soluble Aβ more challenging. To answer the question of Aβ rhythmicity, we developed a novel assay strategy to simultaneously measure the amount of Aβ in humanized APP ΔNLh/ΔNLh xPS1 P264L/P264L knock-in (KI) mice and their wild-type (WT) counterparts. The use of a knock-in mouse line allows for the study of the Aβ rhythm under normal patterns of gene regulation and permits a direct side-by-side comparison of the rhythm of the aggregation-prone human Aβ with its rodent counterpart. Methods Animals and Housing Conditions: We chose for these studies a humanized APP ΔNLh/ΔNLh xPS1 P264L/P264L knock-in (KI) line that develops age-related Aβ pathology, starting around six months of age in the brain, and has similar amyloid solubility characteristics as that found in human AD cases ( Murphy et al., 2007 ). We have also previously seen age-related changes in the onset of daytime wakefulness bouts and peak wakefulness in this mouse strain in association with age-related increases in amyloid pathology ( Duncan et al., 2012 ). This mouse strain was generated by inserting the human Aβ region, along with the Swedish ΔNL mutation, into the normal mouse APP gene ( Reaume et al., 1996 ), and these mice were crossed with a presenilin 1 (PS1) mutant line to generate the double KI ( Flood et al, 2002 ). These mice and their wild-type controls have been maintained on a hybrid CD1/129 background. This work was approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC). Mice (N = 128; ½ Male, ½ Female; ½ KI, ½ WT; Age: 13.5 ± 1.2 months) were age-matched by sex and genotype (Mean age in months, ± s.d.; Male, WT: 13.3 ± 1.4; Female, WT: 13.5 ± 1.6; Male, KI: 13.1 ± 0.7; Female, KI: 13.9 ± 0.9). The APPxPS1 KI mice have notable Aβ deposition at 12 months of age with very mild cognitive impairment ( Webster et al., 2014 ; Webster et al., 2013 ) and minimal changes in overall activity rhythms ( Duncan et al., 2012 ); at the age used in this study, the KI mice show similar activity patterns and wheel running activity as WT mice. These mice are known to exhibit Aβ plaques in their olfactory bulb starting around the age of 6 months, however, plaques are only found in the cerebellum at the age of 15 months ( Flood et al, 2002 ). The mice were acclimated to a 12:12 light:dark (LD) cycle (lights on from 7 AM until 7 PM) for two weeks. At the end of this period, the room was sealed to block all possible sources of unintended light (i.e., light leaks). On the last day of acclimation, when the lights went off at 7 PM, they remained off for the next 48 hours; for the first 24 hours of constant darkness, the mice were undisturbed. For the next 24 hours, groups of 16 mice were euthanized, and their tissue collected at three-hour intervals ( Fig. 1 ). Fig. 1. Open in a new tab Graphic representation (not to scale) of the timeline used to acclimate the mice to a 12:12 light:dark (LD) cycle to synchronize activity rhythms and the time intervals in which the mice were euthanized and their tissue collected during the takedown phase. Tissue Collection: Each group of 16 mice included 8 WT and 8 APPxPS1 KI mice, with both sexes equally represented. To avoid light pulses that might reset circadian rhythms, a containment curtain of heavy black drapes was set up outside the room to block out light when the door was opened. Mice were transferred to the nearby necropsy room within light-sealed blackout boxes. Euthanasia and tissue collection were also done in the dark, with dim red light as the only source of illumination, and the necropsy room was sealed to block possible external light sources. Tissue samples were collected and frozen on dry ice. Following tissue collection, all tissues were stored at −80°C. Aβ Extraction: Aβ was extracted into three distinct fractions: aqueously soluble Aβ, detergent-soluble Aβ and acid-soluble Aβ. To extract the most soluble pool of Aβ, 15μL of DEA buffer (0.2% diethylamine, 50 mM NaCl, plus protease inhibitor cocktail; Thermo Scientific Halt ™ Protease Inhibitor Cocktail, PIC) per mg of tissue was added. The homogenate was centrifuged at 100,000 x g for 30 minutes at 4° C. The supernatant was pipetted out and transferred to a different 1.5 mL tube. To extract detergent-soluble Aβ, RIPA buffer (radioimmunoprecipitation assay buffer: 50 mM TrisHCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1.0% Triton X-100, with PIC) was added to the tubes containing the pellets leftover from the soluble Aβ extraction, and the contents homogenized. After homogenization, the tubes were centrifuged at 20,000 x g for 30 minutes at 4° C. The supernatant was pipetted out and transferred to a different 1.5 mL tube. The pellet and supernatant were then stored at −80°C. The RIPA fraction supernatant samples were used for the assays that were utilized to measure APP. To extract the acid-soluble Aβ, 70% formic acid was added to the pellet leftover from the RIPA extraction. The same volume of buffer was used in all extractions. The samples were thawed on wet ice, and protein concentration was estimated by standard BCA assay (Thermo Scientific Pierce ™ BCA Protein Assay Kit). Prior to assay, the DEA extract was neutralized by the addition of a 1/10 volume of 1.0 M Tris-HCl solution (pH=6.2), and the FA extract was neutralized by 1:20 dilution in 1.0 M Tris-Base / 0.5 M Na 2 HPO 4 . Aβ ELISA: After extracting Aβ from the brain tissue, two assay strategies were performed via ELISA to measure soluble Aβ 1-x and Aβ x-42 protein levels in the olfactory bulbs and cerebellum. Two 384-well plates (Immunolon 4HBX) were coated with Aβ specific antibodies ( Fig. 2 ). To measure Aβ 1-x , the plate was coated (1.0 μg/well) with antibody (Ab) 32.4.1 on the top half and antibody (Ab) 42.5 on the bottom. Ab32.4.1 specifically binds rodent Aβ and, thus, would measure Aβ extracted from the WT mice; Ab42.5 binds human Aβ, which would measure Aβ extracted from the APPxPS1 KI mice. To measure Aβ x-42 , a second plate was entirely coated with Ab2.1.3, which can bind to both rodent and human Aβ x-42 at the C-terminal end ( Eckman et al., 2023 ; Kukar et al., 2005 ; Levites, Das, et al., 2006 ; McGowan et al., 2005 ). The plates were then blocked with Synblock ™ (Bio-Rad), sealed, and stored in a desiccator at 4°C until samples were ready to be loaded. Before loading standards and samples, the plate was washed twice with 1X PBST (phosphate buffered saline with tween; pH 7.4; 200mg KCl, 8.12g NaCl, 200mg KH 2 PO 4 , 1.14g Na 2 HPO 4 , 0.5% Tween-20) and washed twice with 1X PBS (phosphate buffered saline; phosphate buffered saline with tween; pH 7.4; 200mg KCl, 8.12g NaCl, 200mg KH 2 PO 4 , 1.14g Na 2 HPO 4 ). Recombinant Aβ (rPeptide) standards and samples were diluted in antigen capture (AC) buffer (pH 7.0; 0.1M NaH 2 PO 4 , 0.1M Na 2 HPO 4 , 0.5g NaN 3 , 2mM EDTA, 0.4M NaCl, 10g BSA, 0.5g CHAPS) and then loaded in the same layout for both plates ( Fig. 3 ). Fig. 2. Open in a new tab Top: Graphic demonstrating the different assay strategies utilizing specific antibodies to measure Aβ. (A) Ab42.5 is specific to the N-terminal end of human Aβ, whereas Ab32.4.1 is specific to the N-terminal end of rodent Aβ; Ab2.1.3 is specific to the C-terminal end of Aβ x-42 , and thus can bind to both human and rodent Aβ x-42 (the C-terminal ends of rodent and human Aβ x-42 are identical, whereas there are different amino acids at position 5, 10, and 13). Bottom: Graphs demonstrating the specificity of the antibodies utilized in both assay strategies to measure human/rodent total Aβ (1-x) and human/rodent Aβ 42 (x-42). Left Panel: Ab32.4.1 specifically binds to rodent Aβ; Middle Panel: Ab42.5 specifically binds human Aβ; Right Panel: Ab2.1.3 will bind Aβ x-42 from either humans or rodents, but not shorter peptides (such as Aβ 40 ). Ab2.1.3 is used as the detection antibody in the first two panels, and 4G8 used as detection in the final panel; 2-3 replicates were performed for each peptide concentration. Fig. 3. Open in a new tab Diagram representing the 384-well plate layout for the ELISA assays (Aβ 1-x shown; to measure Aβ x-42 , the same plate layout was executed, but Ab2.1.3 coated the entire plate). After capture, Aβ was detected using biotinylated 4G8, which binds equally to both human and rodent Aβ. In both cases the amount of peptide was determined relative to recombinant Aβ peptide. In this way, both rodent and human Aβ can be measured in all mice simultaneously, in two brain regions, over a 24-hour period. The next day, biotinylated 4G8 (Biolegend) was diluted (1 μg/mL) into detection buffer (DB; pH 7.0; 0.1M NaH 2 PO 4 , 0.1M Na 2 HPO 4 , 20mg thimerosal, 2mM EDTA, 0.4M NaCl, 10g BSA). We used biotinylated 4G8 for detection in both assay strategies to better facilitate inter-assay comparison. The contents of the wells were discarded and washed twice with 1X PBST and twice with 1X PBS. After washing, 100μL of biotinylated 4G8 solution was added to each well. The plates were sealed and placed on a rocker at room temperature (RT) for 2 hours. The contents of the plates were discarded, and the plates were washed three times with 1X PBST and three times with 1X PBS. A diluted (0.1 μg/mL) neutravidin-HRP (Thermo Scientific) solution was prepared in DB, and 100μL was added to each well. Following incubation for 30 minutes at RT, plates were washed four times with 1X PBST and then four times with 1X PBS. A developing solution was made by adding a 1:1 ratio of hydrogen peroxide solution (H 2 O 2 ) and peroxidase substrate (TMB) (Thermo Scientific); 50μL of this solution was added to each well, and then developed for up to 30 minutes at RT. After the plates developed, 50μL of stop solution (0.6% o -phosphoric acid) was added to each well, and the plates were read at a wavelength of 450nm. The assay displayed good inter-assay variability metrics [Coefficients of Variation (CV): Ab32.4.1, 14.6%; Ab42.5, 16.8%; Ab2.1.3, vs. rodent Aβ, 11.2%; Ab2.1.3, vs. human Aβ, 12.1%]. These antibodies and methods have been described in detail elsewhere, by ourselves and others ( Das et al., 2003 ; Das et al., 2001 ; Das et al., 2006 ; Eckman et al., 2023 ; Holler et al., 2012 ; Kukar et al., 2005 ; Levites, Das, et al., 2006 ; Levites, Jansen, et al., 2006 ; Murphy et al., 2007 ), although they have not been previously applied in this format. Generating standard curves and analyzing the data involved utilizing software applications SigmaPlot ™ (v15), SPSS ™ (v27), and Cosinor.Online ( Molcan, 2023 ). APP ELISA: The APP ELISA used was as described, and validated by protein-level confirmation by Western blot, in a recently published study ( Turton et al., 2025 ), and was modified from ( Holler et al., 2012 ). The RIPA fraction samples were thawed on ice at RT and diluted 1:1000 with 1X PBS. Recombinant APP (Invitrogen) was used as a standard. Standards were serially diluted starting at 2,000 pg/μL; 100μL of standard or diluted samples were loaded into their respective wells on a 384-well plate (layout as above in Figure 2 ). The plates were then covered with a plate seal and placed at 4°C overnight. Following this step, plates were washed and blocked with Synblock ™ , washed (twice with 1X PBST/twice with 1X PBS), and then probed with 1 μg/mL of biotinylated 22C11 monoclonal antibody (Invitrogen) in DB. This antibody binds to rodent and human APP. The remaining steps were the same as described in the Aβ ELISA above (the CV for this assay was 12.7%). Results Starting with the DEA soluble fraction, we measured the amount of Aβ 1-x and Aβ x-42 in the olfactory bulbs and cerebellum. Whereas Ab32.4.1 and Ab42.5 will only detect human or rodent Aβ 1-x , respectively, Ab2.1.3 will capture both rodent and human Aβ x-42 with approximately the same affinity because the C-terminus of the peptide has the same epitope in both cases. As expected, there was more Aβ 1-x and Aβ x-42 in the region more prone to Aβ pathology (the olfactory bulb) compared to the region that is considered to be relatively spared in AD (the cerebellum) ( Fig. 4 ). Note that the absolute value for Aβ x-42 may be higher than the value for Aβ 1-x since they were measured on separate plates using different antibodies, or because the Aβ x-42 assay might detect some amount of the α-secretase derived p3 peptide (Aβ 17-42 ). A regional difference was also seen in the RIPA (detergent-soluble Aβ) fraction (Mean, s.d.: CER, Aβ 1-x : 127.260 fmol/mg ± 51.969; Aβ x-42 : 145.113 ± 64.605; OB, Aβ 1-x : 260.329 fmol/mg ± 181.715; Aβ x-42 : 306.081 fmol/mg ± 167.178; p<0.000001, OB vs. CER, for both Aβ measures). We did not detect an appreciable signal in the FA (acid-soluble Aβ) fraction in these regions, in this age group. Additionally, when looking at the Aβ x-42 data measurements, a marginally significant sex difference (sex * genotype, p < 0.05, F = 6.115) was noticed between the APPxPS1 KI males and females. In the olfactory bulbs of the APPxPS1 KI female mice, there was slightly more DEA soluble Aβ x-42 present compared to the males of the same genotype (356 ± 27 fmol/mg vs. 304 ± 27 fmol/mg, respectively) . This sex difference was not seen in the other fractions, and no sex differences were detected in rhythms. Although we have previously noted sex differences in Aβ, sleep, and rhythms in this mouse line ( Turton et al., 2025 ) and the 5xFAD mouse ( Sethi et al., 2015 ), it is unclear if this small difference in the OB is meaningful (see ( Johnson et al., 2024 ) for a discussion of this topic). Fig. 4. Open in a new tab There was less DEA soluble Aβ in the cerebellum (CER; Mean Aβ 1-x : 67.334 fmol/mg ± 1.932, Mean Aβ x-42 : 251.886 ± 9.223) compared to the olfactory bulb (OB; Mean Aβ 1-x : 156.850 fmol/mg ± 4.742, Mean Aβ x-42 : 500.905 fmol/mg ± 16.243), a result replicated with both antibody combinations. The results shown, relative to the OB, are from the collective data from all mice, specifically samples from the DEA fraction, with individual data points represented by the gray circles; error bars shown are standard errors. ***= p<0.001. When the collective data from both tissues from all mice across all time points were examined ( Fig. 5 ), they showed that the DEA soluble Aβ followed a circadian rhythm. This rhythm troughed at 10:43 AM, corresponding to the previous light phase, and peaked at 10:43 PM, which would have occurred during the previous dark phase. We wanted to know if APP also followed a similar circadian rhythm, considering that soluble APP has been found to circulate in a diurnal pattern in human CSF and blood ( Dobrowolska et al., 2014 ). We also wanted to explore whether changes in APP, the precursor to Aβ, could explain the observed differences in Aβ rhythms. The ELISA protocol we used to achieve our results was developed in the lab using a biotinylated 22C11 monoclonal antibody that measures rodent and human APP ( Turton et al., 2025 ). However, in contrast to Aβ, APP did not show a circadian rhythm in these two brain regions, indicating that the differences in Aβ cannot simply be accounted for by changes in the amount of its precursor. Fig. 5. Open in a new tab (a) For the DEA soluble Aβ curve, the time of the lowest point of the rhythm is ZT +3.73 (10:43 AM clock time), and acrophase is ZT +15.73 (10:43 PM clock time), mesor is 248.4 fmol/mg, amplitude is 39.7 fmol/mg. Test for zero amplitude, F[2,125] = 3.87, p < 0.03. (b) RIPA soluble (detergent-soluble) APP. APP does not have a rhythm when examined in the same tissue samples (test for zero amplitude, F[2,113] = 0.02, p > 0.9). In (a) and (b) , the solid line shows the overall mean (mesor) and the dotted lines show the 95% CIs around the mean; square symbols show the means for each combined group of mice (n=16), ± the 95% CIs. ** = p < 0.01 relative to overall mean. Breaking down the data into subgroups separated by brain region and genotype, we found similar circadian rhythms of DEA-soluble Aβ in the cerebellum of both the WT and APPxPS1 KI mice, and these rhythms showed a phase delay compared to the rhythm in the olfactory bulb of the WT mice ( Fig. 6 ). The mechanism underlying this phase difference is unknown. However, the Aβ levels in the olfactory bulb of the APPxPS1 KI mice were arrhythmic. This outcome was the same using either antibody combination ( Fig. 7 ). Detergent-soluble and acid-soluble Aβ were also extracted from the olfactory bulb and cerebellar tissues via RIPA and FA, respectively. Similar to the data collected from the DEA soluble Aβ fraction, data collected from the RIPA fraction was analyzed in four subgroups based on genotype (WT or APPxPS1 KI) and brain region (olfactory bulb or cerebellum) in both assay strategy measurements (Aβ 1-x and Aβ x-42 ). Unlike the DEA soluble Aβ fraction findings, the detergent-soluble fraction did not exhibit an overall Aβ rhythm, except for in the APPxPS1 KI cerebellum of both assay measurements (p < 0.05) ( Supplemental Figures 3 – 4 ; Supplemental Tables 3 – 4 ). Only background signal was detected from the FA fraction ( not shown ). Fig. 6. Aβ Rhythms (1-x) from the OB and CER as measured by ELISA. Open in a new tab Soluble Aβ 1-x , from the DEA fraction, values broken out by region (olfactory bulb; OB vs. cerebellum; CER) and genotype (WT vs. KI). WT mice show a daily rhythm in both the OB and CER; in contrast, Aβ levels do not display a rhythm in the OB in the KI mice (a region that is more AD vulnerable), but show a rhythm similar to WT mice in the CER (a region that is considered less AD vulnerable); the phase and amplitude of the Aβ rhythm is similar between WT and KI CER; a comparison of the WT OB to the CER indicates a phase shift of ~4.5 hours (OB acrophase = ~2:30 pm; CER acrophase = ~7 pm). The solid line shows the overall mean (mesor) and the dotted lines show the 95% CIs around the mean; square symbols show the means for each combined group of mice (n=16), ± the 95% CIs. Cosinor data generated by cosinor.com ( Molcan, 2023 ). Graphs including individual data points and corresponding circadian rhythm values can be viewed in supplemental data ( Supplemental Fig. 1 ; Supplemental Table 1 ). Fig. 7. Aβ Rhythms (x-42) from the OB and CER as measured by ELISA. Open in a new tab Soluble Aβ x-42 , from the DEA fraction, values broken out by region (olfactory bulb; OB vs. cerebellum; CER) and genotype (WT vs. KI). WT mice show a circadian rhythm in both the OB and CER; the KI mice, in contrast, did not exhibit a rhythm in Aβ levels in the OB (a region that is more AD vulnerable), but show a circadian rhythm similar to WT mice in the CER (a region that is considered less AD vulnerable). In the CER, the phase and amplitude of the Aβ rhythm is similar between WT and KI mice; the rhythm in the CER is delayed by ~4.5 hours compared to the OB (OB acrophase = ~2:30 pm; CER acrophase = ~7 pm). The solid line shows the overall mean (mesor) and the dotted lines show the 95% CIs around the mean; square symbols show the means for each combined group of mice (n=16), ± the 95% CIs. Cosinor data generated by cosinor.com ( Molcan, 2023 ). Graphs including individual data points and corresponding circadian rhythm values can be viewed in supplemental data ( Supplemental Fig. 2 ; Supplemental Table 2 ). Although we did not find an overall APP rhythm in the combined tissues, similar to the detergent-soluble Aβ fraction, a significant rhythm was present in the APPxPS1 KI cerebellum (p < 5.0E-5) but not in the WT cerebellum or in either genotype’s olfactory bulbs ( Supplemental Fig. 5 ; Supplemental Table 5 ). A statistical analysis confirmed a significant difference in APP levels by genotype in both regions in which WT mice had more APP than the APPxPS1 KI mice ( Supplemental Fig. 6 ). Note that the APP in the WT mice is endogenous rodent APP, while the APPxPS1 KI mice express a partially humanized APP ( Reaume et al., 1996 ). Looking specifically at the male WT mice, there was slightly more APP in the olfactory bulbs than in the cerebellum. However, it was the opposite in the female WT mice in which they had less APP in the olfactory bulbs than in the cerebellum. A repeated measures ANOVA found a marginally significant region*sex interaction (p < 0.045), where males had slightly more APP in the OB, and females had more APP in the cerebellum ( Supplemental Fig. 6 ). Discussion The primary objective of this study was to further examine the circadian rhythm of the Aβ peptide in the brain by determining if this rhythm differed between a region relatively more affected by AD (the olfactory bulb) and a less affected region (the cerebellum). Using a novel assay strategy to evaluate APPxPS1 KI mice and WT controls side by side, we could directly compare the daily Aβ rhythm in both sexes. A key rationale for the use of this line was that it is a good mimic of the natural spread of amyloid pathology, and a close match for characteristics of human amyloid pathology ( Murphy et al., 2007 ). This line develops Aβ deposits in the olfactory bulb well in advance of the cerebellum ( Flood et al., 2002 ). We found that the circadian rhythm of DEA-soluble Aβ at 13-14 months of age was essentially the same between the KI and WT mice in the cerebellum, but the Aβ rhythm was lost in the olfactory bulb of the KI mice. The finding of a robust overall rhythm for Aβ in the most soluble fraction (DEA), but not in less soluble fractions (RIPA), could be due to more soluble Aβ being easier to clear from the brain via the glymphatic system. Our results show that DEA-soluble Aβ levels peak in the evening when mice exhibit the highest levels of wakefulness and a trough in the morning when they exhibit the highest levels of sleep. This pattern is consistent with reports that Aβ and other metabolic wastes accumulate in the ISF of the hippocampus during wakefulness and undergo clearance during sleep via the glymphatic movement of fluid ( Iliff et al., 2012 ; Iliff et al., 2013 ; Xie et al., 2013 ). However, circadian changes in Aβ production might also be important because it is increased by neuronal activity that is generally higher during wakefulness. This led us to investigate whether there might be regional differences in circadian rhythms of APP, the precursor for Aβ. Although APP did not have an overall rhythm when the data from both genotypes and brain regions were combined, there was a significant circadian rhythm in APP in the cerebellum of the APPxPS1 KI mice. Since there was no genotype difference in APP expression in the OB that could explain the genotype difference in the OB Aβ rhythm, some factor other than availability of APP must account for this difference. It is also possible that the disruption of the circadian rhythm in disease-affected regions might be due to changes in APP cleavage by either β-secretase or γ-secretase, or regional-specific changes in Aβ clearance, as both enzymes may have a putative rhythm ( Ma et al., 2016 ). Resolving this mechanism will require detailed studies, in both humans and in animal models of the disease. Our results also showed that there was indeed more Aβ present in the olfactory bulb than in the cerebellum, supporting previous reports of brain regional vulnerability in the development of AD pathology. Previous studies have demonstrated that the olfactory bulb has substantial pathology in other AD mouse models ( Murray et al., 2023 ; Son et al., 2021 ), including the one used in this study ( Flood et al., 2002 ), in which amyloid pathology in the OB appears as early as 6 months (i.e., long before older age that we assessed). In human AD patients, the loss of the sense of smell is a frequent symptom associated with neurodegeneration ( Fatuzzo et al., 2023 ; Marin et al., 2018 ). The cerebellum is one of the last brain regions to be affected by AD, and it shows considerably less Aβ than other regions ( Abrahamson et al., 2022 ; Calderon-Garciduenas & Duyckaerts, 2017 ; Murray et al., 2023 ; Son et al., 2021 ). ELISA measures of Aβ correlate well with both plaque counts and area of Aβ immunostaining in both humans and mice ( Murphy et al., 2007 ). However, since the primary changes in circadian rhythms of expression were observed here in the most soluble form of extractable Aβ, it seems highly unlikely that there would be associated circadian rhythms in plaque density, as plaques consist of densely aggregated, less soluble Aβ. Nonetheless, our results are the first to show quantitative evidence that the APPxPS1 KI mouse line exhibits selective vulnerability to Aβ accumulation in the olfactory bulbs relative to the cerebellum, suggesting that this line might be a good model for studying the role of olfactory bulb pathology in AD. Whether this pattern holds in other regions more or less vulnerable to Aβ accumulation remains to be seen. After examining the circadian rhythm in the pooled data, we divided the data into four subgroups by region and genotype. We found that in three subgroups, WT olfactory bulb, WT cerebellum, and APPxPS1 KI cerebellum, DEA-soluble Aβ followed a circadian rhythm. When comparing the respective regions between genotypes, the cerebellar rhythms of WT and KI mice are highly similar, consistent with this region being very little affected by AD pathology. The rhythm observed in the olfactory bulb of WT mice is not present in the olfactory bulb of KI mice and, thus, is consistent with this region being more strongly affected by AD. As an individual with AD pathology ages, soluble Aβ and Aβ deposits increase in a region specific manner ( van der Kant et al., 2020 ). Changes in pathology are correlated with changes in in the amplitude and phase of circadian rhythms ( Hollis et al., 2022 ), and APP related fragments show corresponding age-related changes in the CSF ( Dobrowolska et al., 2014 ). These results also establish a starting point to understand the baseline for circadian rhythms of Aβ in WT and APPxPS1 KI mice in these regions, potentially helpful information as the genetically modified genes in this model are driven by their endogenous promoters ( Duncan et al., 2012 ; Flood et al., 2002 ; Murphy et al., 2007 ; Reaume et al., 1996 ). While the olfactory bulb and cerebellum of WT mice exhibited a circadian rhythm of DEA-soluble Aβ, these two brain regions of WT mice did not show a circadian rhythm of APP. Surprisingly, only the cerebellum of the APPxPS1 KI mice exhibited a circadian rhythm of APP levels. Further investigation will have to be conducted to determine why a rhythm was observed for APP only in the cerebellum of the KI mice and if this has any relevance to AD. APP is not known to exhibit a circadian rhythm at the transcriptional level ( Hollis et al., 2025 ; Ma et al., 2016 ), although it is possible that regional differences exist or that a post-transcriptional process might be involved, although not much is known regarding species specific differences in APP regulation. It is possible that the APP rhythm is related to the difference in control of APP expression between the APPxPS1 KI and WT mice in these regions. For instance, the generation of the original APP KI founders ( Reaume et al., 1996 ) from which the current line is descended ( Flood et al., 2002 ; Murphy et al., 2007 ) involved replacing both exonic and intronic sequences of the mouse APP gene in order to introduce the human Aβ sequence. It is possible that this event altered or removed a regulatory element involved in controlling regional expression in the mouse brain. For example, there is a putative androgen response element present in intron 15 that may have been affected by gene targeting ( Sato et al., 2021 ). Conclusion This study of aged wild type and APP-PS1 KI mice of both sexes extends previous reports showing that soluble Aβ follows a circadian rhythm in the human CSF and mouse hippocampal ISF ( Dobrowolska et al., 2014 ; Roh et al., 2012 ). Here, we demonstrate that soluble Aβ also exhibits a circadian rhythm in two neuroanatomically defined regions (olfactory bulbs and cerebellum) that exhibit a differential sensitivity to AD-related neuropathology. Furthermore, our study shows that the OB, which is more sensitive to AD neuropathology, fails to exhibit a circadian rhythm of soluble Aβ in the APP-PS1 KI mice at an age when amyloid pathology is abundant. Determining the potential root contributions of circadian disruption to amyloid expression may improve the understanding of AD pathology and lead to strategies for its alleviation. Supplementary Material Supplementary NIHMS2163142-supplement-Supplementary.docx (9.5MB, docx) Acknowledgements Supported by NIH grants AG068215 and AG068215-03S1 (MPM, MJD, SS, BFO, and ADB). CEJ was supported by T32 AG078110. This publication was also supported by the University of Kentucky Neuroscience Research Priority Area (NRPA). 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