Localization of amyloid beta peptides to locus coeruleus and medial prefrontal cortex in corticotropin releasing factor overexpressing male and female mice

Abstract

A culmination of evidence from the literature points to the Locus Coeruleus (LC)-Norepinephrine system as an underappreciated and understudied area of research in the context of Alzheimer's Disease (AD). Stress is a risk factor for developing AD, and is supported by multiple clinical and preclinical studies demonstrating that amplification of the stress system disrupts cellular and molecular processes at the synapse, promoting the production and accumulation of the amyloid beta (Aβ₄₂) peptide. Stress-induced activation of the LC is mediated by corticotropin releasing factor (CRF) and CRF receptors exhibit sex-biased stress signaling. Sex differences are evident in the neurochemical, morphological and molecular regulation of LC neurons by CRF, providing a compelling basis for the higher prevalence of stress-related disorders such as AD in females. In the present study, we examined the cellular substrates for interactions between Aβ and tyrosine hydroxylase a marker of noradrenergic somatodendritic processes in the LC, and Dopamine-β-Hydroxylase (DβH) in the infralimbic medial prefrontal cortex (ILmPFC) in mice conditionally overexpressing CRF in the forebrain (CRFOE) under a Doxycycline (DOX) regulated tetO promoter. CRFOE was sufficient to elicit a redistribution of Aβ peptides in the somatodendritic processes of the LC of male and female transgenic mice, without altering total Aβ₄₂ protein expression levels. DOX treated groups exhibited lysosomal compartments with apparent lipofuscin and abnormal morphology, indicating potential dysfunction of these Aβ₄₂-clearing compartments. In female DOX treated groups, swollen microvessels with lipid-laden vacuoles were also observed, a sign of blood-brain-barrier dysfunction. Finally, sex differences were observed in the prefrontal cortex, as females responded to DOX treatment with increased frequency of co-localization of Aβ₄₂ and DβH in noradrenergic axon terminals compared to vehicle treated controls, while male groups showed no significant changes. We hypothesize that the observed sex differences in Aβ₄₂ distribution in this model of CRF hypersignaling is based on increased responsivity of female rodent CRF_R1 in the LC. Aβ₄₂ production is enhanced during increased neuronal activation, therefore, the excitation of DOX treated female CRFOE LC neurons projecting to the mPFC may exhibit more frequent co-localization with Aβ due to increased neuronal activity of noradrenergic neurons.

Keywords: Adrenergic receptors; Amyloid; Dopamine-β-hydroxylase; Norepinephrine; Stress.

Figure 1.. Co-localization of Amyloid and TH in LC of CRFOE Mice.

Confocal fluorescence micrographs showing TH (green) co-localized with Aβ₄₂ (red) in the LC of vehicle or DOX treated CRFOE male and female mice. TH was visualized using tetramethyrhodamine-5-isothiocyanate (TRITC)-conjugated donkey anti-rabbit while MOAB2 was visualized using fluorescein-isothiocyanate (FITC)-conjugated donkey anti-mouse. Arrow heads point to individually labeled antigens while arrows indicate co-localization. Panels A-C show representative images of each fluorphore (A,B) and merged (C) for male vehicle treated mice, while areas of colocalization (yellow) are displayed on the prime and double prime panels to the right of panel C. Panels a-c show representative images of DOX-treated male mice, with two additional panels showing a higher magnification of regions of colocalization (yellow). Panels D-F show representative images of female vehicle treated mice, with three higher magnification panels of the merged image (F). The corresponding lower case panels (d-f) show representative images of DOX-treated female mice, with two higher magnification panels of the merged image (f). Scale bar 25 μm.

Figure 2.. Co-localization of Amyloid, TH and GFAP in the LC of CRFOE mice.

Confocal fluorescence micrographs showing TH (green) co-localized with Aβ₄₂ (cyan) and GFAP (red) in the LC of vehicle or DOX treated CRFOE male and female mice. Arrows and arrow heads point to individually labeled antigens while asterisks indicate co-localization. Yellow represents the colocalization of GFAP and TH, while dark blue shows colocalization of Aβ₄₂ and TH, fiiscia indicates colocalization of TH, Aβ₄₂ and GFAP. Panels A-D show the labeling of individual fluorophores (A-C) and a merged image taken from male vehicle treated mice, with additional higher magnification images of co-localization labeled prime through triple prime. Representative images of TH-immunoreactivity (ir), Aβ₄₂-ir and GFAP-ir and merged from DOX treated male mice are shown in panels a-d. Higher magnification shown in the prime and double prime panels show areas of co-localization. Panels E-H show representative micrographs taken from vehicle treated mice, and are shown at higher magnification in the prime and double prime panels. Panels e-h show representative micrographs of TH-ir, GFAP-ir and Aβ₄₂-ir taken from female DOX treated mice. Higher magnification images from the merged panel (prime and double prime) show regions of colocalization. Scale bar 25 μm.

Figure 3.. DOX Treated Mice show significant increases in co-localization in TH and Aβ in LC.

A. Representative electron micrographs of LC sections taken from vehicle treated male mice showing TH-ir peroxidase labeling co-localized with Aβ-ir gold conjugated, silver enhanced puncta. B-C. Representative electron micrographs of LC sections taken from DOX treated male mice showing TH-ir peroxidase labeling co-localized with high intensity Aβ-ir. D. Electron micrograph taken from vehicle treated female of TH-ir soma (TH-s) that is Aβ-ir. Interestingly, Aβ is localized to lysosomal compartments that are morphologically intact and with typical shaped vacuole (v) and mitochondria (m). In contrast, the micrograph shown in panel E. is taken from a DOX treated female that shows a TH-ir soma visualized with lysosomal compartments that are both Aβ-ir and morphologically distinguished by the presence of lipofuscin. F. Electron micrograph showing a blood vessel in a vehicle treated female CRFOE mice that is Aβ-ir and shows normal morphology of endothelium (end), basal lamina (B) and pericyte (p). G. Electron micrograph showing a blood vessel in DOX treated female CRFOE mice that is Aβ-ir and shows normal morphology of endothelium (end), but with abnormal morphology of a pericyte (p) enclosed by the basal lamina (B), that is Aβ-ir and dense with lipid-laden vacuoules. Arrow heads point to Aβ labeled with gold-conjugated anti-mouse that has been intensified with silver, visualized as dense punctate deposits. Arrows point to synaptic specializations. H. Semi-quantitative analysis of frequency of co-localization of TH and Aβ in the LC of vehicle and DOX treated male and female mice. Significant increases in frequency of co-localization were observed in male and female DOX treated groups (p<0.05). Scale bar 0.5 μm.

Figure 4.. Complex Alterations in Protein Expression in the LC.

A. Similar intensity bands migrated between 125-90kDa for APPα detection and 68kDa for BACE-1 under vehicle and DOX treated CRFOE mice. Significant differences in signal intensity of APP-β were detected between male and female DOX treated groups. Similar intensity bands migrated to 62, 79, and 50kDa for TH, p-Tau 396 and CRF_R1, respectively. Quantification of western blot APPα (B) and BACE1 (D) bands revealed no significant differences. Similarly, there were no significant differences between gender or treatment (vehicle or DOX) for TH (H), p-Tau396 (F) and CRFRI (G). There were significant differences in the intensity of the APP-β (C) bands between DOX treated males and DOX treated females.

Figure 5.. CRF_R1 positively correlates to BACE1 and Tau-p at 396 Site, while NE positively correlates to Aβ₄₂ levels in the LC.

A. Quantification of ELISA directed against NE or B. Aβ₄₂ showed no overall changes in protein expression levels detected by the respective ELISAs. In line with previous findings, there was a significant positive correlation between Aβ₄₂ expression and NE expression (E). C. Correlation and linear regression analyses revealed a significant relationship between BACE-1 expression and APP-β. Surprisingly, no significant relationships were observed between CRFR1 and Aβ₄₂ expression (D), or between Aβ₄₂ expression and GFAP (F). However, GFAP was strongly positively correlated to both CRFR1 (G), as well as phosphorylation of tau at serine 396 site (H). **, ***, **** denote p<0.01, p<0.001, and p<0.0001, respectively.

Figure 6.. DOX treatment increases co-localization of Amyloid and DβH in CRF OE female mice.

Confocal fluorescence micrographs showing DβH (green) co-localized with Aβ₄₂ (red) in the ILmPFC of vehicle or DOX treated CRFOE male and female mice. DβH was visualized using tetramethyrhodamine-5-isothiocyanate (TRITC)-conjugated donkey anti-rabbit while MOAB2 was visualized using fluorescein-isothiocyanate (FITC)-conjugated donkey anti-mouse. Arrows and arrow heads point to individually labeled antigens while asterisks indicate co-localization. Panels A-D show individually labeled fluorophores (A,B), merged (C) and higher magnification (D) micrographs taken from male vehicle treated mice. Panels a-d show DβH-ir and Aβ₄₂ (a,b), merged (c), and high magnification micrographs of male DOX treated mice. Panels E-H are representative micrographs taken from vehicle treated female mice, while panels e-h were taken from DOX treated female mice. Panels D and d show higher magnification of regions of colocalization for vehicle and DOX treated female mice, respectively. Scale bar 25 μm.

Figure 7.. DOX treated female mice show significant increases in co-localization in DβH and Aβ in PFC.

A. Representative electron micrograph of ILmPFC sections taken from vehicle treated female mice showing DβH-ir peroxidase labeling co-localized with Aβ-ir gold conjugated, silver enhanced puncta. B. Representative electron micrograph of ILmPFC sections taken from DOX treated female mice showing DβH-ir peroxidase labeling co-localized with high intensity Aβ-ir. C. Representative electron micrograph of ILmPFC sections taken from vehicle treated male mice showing DβH-ir peroxidase labeling co-localized with Aβ-ir. D-E. Representative electron micrographs of ILmPFC sections taken from DOX treated male mice showing DβH-ir peroxidase labeling co-localized with high intensity Aβ-ir. F. Semi-quantitative analysis of frequency of co-localization of DβH and Aβ in the ILmPFC of vehicle and DOX treated male and female mice. Significant increases in frequency of co-localization were observed in female DOX treated groups (p<0.05). G. Semi-quantitative analysis of distribution of the Aβ peptide in pre- and post- synaptic compartments of the ILmPFC. Arrow heads point to Aβ labeled with gold-conjugated anti-mouse that has been intensified with silver, visualized as dense punctate deposits. Scale bar 0.5 μm.

Figure 8.. DOX treatment in CRFOE mice does not significantly alter expression of noradrenergic or APP-associated Proteins in the PFC.

A. Similar intensity bands migrated between 125-90kDa for APPα, APPβ cleavage products and 68kDa for BACE-1 under vehicle and DOX treated CRFOE mice. B. Quantification of western blot APP-α, C. APP-β, D. BACE1, E. NET and F. DβH bands revealed no significant differences between groups.

Figure 9.. NE positively correlates with Aβ₄₂ in the PFC of Mice.

Panels A and B show the quantification of ELISA directed against NE (A) or Aβ₄₂ (B). There were no overall changes in protein expression levels detected by the ELISA directed against NE, or Aβ₄₂ As expected, we confirmed a strong positive correlation between BACE-1 expression and APP-β fragments, shown in panel C. While NE and Aβ₄₂ levels did not change between groups, there was a significant positive correlation between NE and Aβ₄₂ expression levels in the PFC, shown in panel D. **** denotes p<0.001

Figure 10.. Sex differences in mPFC of DOX treated CRFOE may be explained by Sex biased signaling of the CRF_R1.

A. CRFOE results in increased frequency of co-localization of Aβ peptides and TH in male and female mice treated with DOX, likely through mechanisms related to the activation of CRF_R1 B. CRF_R1 exhibits sex-biased signaling, likely rendering female mice treated with DOX more vulnerable to stress-related dysfunction. Male rodent CRF_R1 preferentially binds β-arrestin, resulting in the internalization of receptors from the cell surface, effectively terminating responsivity to stress during periods of prolonged exposure. Female rodent CRFR1 is more likely to bind the stimulatory G-protein subunit alpha (Gα_s), resulting in downstream signaling cascades that promote the excitation of LC neurons. Thus, female rodent LC neurons expressing CRFR1 have enhanced responsivity to stress, potentiating responses during prolonged stress exposure, potentially rendering them more vulnerable to stress-related dysfunction (Bangasser, Curtis et al. 2010, Valentino, Bangasser et al. 2013, Reyes, Bangasser et al. 2014, Bangasser, Wiersielis et al. 2016). C. Previous studies from our lab localized Aβ peptides to DβH immunoreactive terminals of the ILmPFC, findings that were further confirmed in the present study utilizing CRFOE mice. DβH is the NE synthesizing enzyme that resides in the synaptic vesicles of NE axon terminals. D. We observed increased frequency of co-localization between DβH and Aβ peptides in the mPFC in female CRFOE mice treated with DOX, but not in male CRFOE treated with DOX. Based on the increased responsivity of female rodent CRF_R1 in the LC, it is possible that neuronal activation of projections to the mPFC exhibit increased co-localization of these peptides due to alterations in activity on a circuit-level. This notion is supported by the concept that Aβ42 production is enhanced during increased neuronal activation (Cirrito, Yamada et al. 2005, Bero, Yan et al. 2011), therefore the excitation of DOX treated female CRFOE LC neurons projecting to the mPFC may exhibit more frequent co-localization with Aβ due to increased neuronal activity of noradrenergic neurons.