Figure 1: Differential regulation of trophic factors in the aged choroid plexus: A Age-related transcriptional level changes in the neurotrophic factors (NTF) in the choroid plexus (CP) of males B Age-related transcriptional level changes in the NTF’s in CP of females. QIAcuity dPCR analysis showed that the copy number of neurotrophic factors VGF, MDK, BDNF, EPO, and EPOR was significantly reduced in the old CP of both Males (Figure-1a) and Females. VEGF sowed a gender-specific expression; the concentration was unchanged in males, whereas in females, it showed an age-dependent decline in the copy number. C The mean pixel intensity of immunohistochemical analysis showed an increased VEGF staining in the aged CP of males and D females. VEGF localization is particularly increased outside CP vasculature of both genders. E VEGF (Green)-Isolectin B4 (Red) co-labeling indicates VEGF protein expression in and out of the CP vasculature of males and F females. Scalebar-100µm **P < 0.01, *** P < 0.001, **** P < 0.0001
Neurotrophic factor expression is significantly altered in the aged choroid plexus.
Neurotrophic factors are highly expressed and secreted in the CP and other brain regions. CP secretes several trophic factors into CSF and circulates it to different brain regions [2]. Here, we studied the gender-specific expression patterns of major neurotrophic factors in the murine CP. To address the age-dependent transcriptional level changes in murine CP, we first analyzed neurotrophic factor gene expression patterns at three different time points: 5–6 m.o. (from now on referred to as mature adult), 11–12 m.o. (middle-aged) and 18–24 m.o. (aged). Further, we confirmed transcript levels using digital droplet PCR. Most of the trophic factors examined showed transcriptional dysregulation in the old CP; some were gender specific. First, we analyzed the transcription pattern of the VEGF gene, which is actively involved in the maintenance of the CP vasculature and has a role in the permeability of blood vessels. VEGF showed a differential transcriptional pattern in males and females. In male CP, the relative gene expression showed a consistent VEGF gene expression throughout the lifespan (Supplementary Fig. 1a), and the ddPCR gene expression analysis further confirmed that in males, VEGF mRNA transcription is not affected by aging (Fig. 1A). However, in females, VEGF mRNA expression is significantly (P < 0.001) reduced with age (Fig. 1B), and the reduction is evident from middle age (P < 0.01) onwards (Supplementary Fig. 1b). We used immunohistochemical analysis to determine whether these gene expression changes are mediated at the protein level. Interestingly, unlike gene expression changes, VEGF protein is more highly expressed(P < 0.0001) in the aged CP than in adults in both males (Fig. 1C) and females (Fig. 1D) We also found that VEGF localization was not limited to the choroid vasculature. The expression was also observed in the basal membrane of epithelial cells or stroma (Fig. 1E&1F).
Next, we investigated the expression pattern of Midkine (MDK), another trophic factor highly expressed in the CP. MDK is known to provide neurotrophic support and neurite outgrowth [39]. MDK mRNA transcription showed a significant reduction in the aged male (P < 0.001) and female (P < 0.0001) mice (Supplementary Fig. 1A and 1B). The decline in MDK gene expression starts in middle age and continues throughout the later stages of life. ddPCR gene expression (Figure-1A & 1B) also confirmed the reduction of MDK transcripts in CP of older males (P < 0.01) and female mice (P < 0.0001). Then, we turned our attention to BDNF, which is widely expressed in the CNS and is involved in neuronal survival, development, and synaptic plasticity [40, 41, 42]. QIAcuity dPCR analysis showed a lower copy number of BDNF (Fig. 1A) in CP than other trophic factors, indicating reduced transcription of BDNF mRNA in the CP. However, an age-dependent reduction is evident in BDNF mRNA levels (Supplementary Figs. 1A and B) in the CP of both males (P < 0.0001) and females(P < 0.001). Another important trophic factor, VGF, showed an age-dependent reduction in VGF gene transcription (Fig. 1A&B) in males (P < 0.001) and females (P < 0.0001). Interestingly, females exhibit a higher copy number than males. Erythropoietin (EPO), a neurotrophic and neuroprotective cytokine, exhibits a significantly lower copy number (Figure-1A &B) in the CP of aged males (P < 0.001) and female mice (P < 0.0001). EPO acts through its classical receptor, the erythropoietin receptor (EPOR). EPOR copy number is significantly decreased (Figure-1A &B) in the aged CP of both males (P < 0.0001) and females (P < 0.0001), indicating reduced EPO signaling in the later stages of life.
Figure 2: Insulin-like growth factor expression is reduced in aged choroid plexus. A, IGF transcription was significantly altered in the aged CP of males and B females. IGF1 and IGF2 mRNA Copy numbers showed a significant reduction in both genders, indicating reduced gene expression. IGF1R expression showed a gender-specific expression, IGF1R expression was unchanged in the males and reduced in the females. IGFBP4 and IGFBP7 in the females showed a reduction in copy number, indicating a reduction in the gene expression. D IGF2 (Red)-IsolectinB4 (Green) co-labeling showed that IGF2 is absent in choroidal vasculature (Fig. 2d). E The co-labeling study using plectin (Green) showed that IGF2 (Red) is expressed in the basolateral side of choroidal epithelial cells of male mice. C The reduction of IGF2 Protein expression is evident from the mean pixel intensity of IHC images. Scalebar-100µm**P < 0.01, *** P < 0.001, **** P < 0.0001
IGF expression decreased in the choroid plexus of aged mice.
Insulin-like growth factors (IGFs) are essential growth-promoting peptides that act as endocrine, paracrine, and autocrine factors. IGF signaling plays a crucial role in controlling aging and life span in invertebrates [43, 44]. To analyze IGF signaling in aged mice, we first examined the mRNA of the IGF1 gene. The relative expression of IGF1 mRNA showed differential expression patterns in the aged males and females. We found a significant down-regulation (P < 0.0001) of IGF1 mRNA expression in the CP of aged females. Age-related changes were significant (P < 0.0001) from middle age onwards. ddPCR (Fig. 2b) (P < 0.0001) gene expression confirmed the altered transcription of IGF1 mRNA in aged females. Even though the relative expression (Supplementary Fig. 1C) appeared unchanged in male CP, absolute gene expression showed (Fig. 2A) a significant reduction (P < 0.001) in IGF1 transcript levels.
IGF-II is a significant growth factor in the brain and is involved in memory consolidation. IGF-II transcription in the brain is mainly localized at the CP and leptomeninges [45]. Quantitative PCR analysis showed an age-dependent reduction in IGF-II gene transcription in the CP of both males(P < 0.001) and females (P < 0.0001) (Supplementary Fig. 1C & 1D). The reduction was evident from middle age onwards. QIAcuity ddPCR further confirmed the reduced concentration of IGF-II transcripts in aged CP of males (P < 0.01) and females (P < 0.0001) (Fig. 2A&2B). Further, we focused on age-related protein-level changes in IGF- II expression in the CP epithelium and vasculature. The co-labeling study showed that in adult CP, IGF-II protein is expressed in the epithelial cells, particularly in the basal membrane of epithelial cells (Fig. 2E & Supplementary Fig. 3B). The vasculature of the CP is free from IGF-II expression (Fig. 2D & Supplementary Fig. 3A). Natural aging dramatically reduced IGF-II protein expression (Fig. 2C& Supplementary Figure C) in the choroid epithelium of both males(P < 0.0001) and females (P < 0.002).
IGF1 and IGF2 act through the IGF receptor, and the IGF1R gene expression showed a differential expression in both males and females (Fig. 2A&B). Aging did not influence IGF1R gene expression in the male CP (Fig. 2A & Supplementary Fig. 1C), whereas, in females, both the absolute (P < 0.01) and relative (P < 0.001) expression indicate a significant reduction in IGF1R gene transcription (Fig. 2B & Supplementary Fig. 1D). The IGF peptides have a short lifespan unless they are bound by specific binding proteins that transport them in circulation and deliver them to specific tissues. IGFBPs are found throughout the body in various fluids and tissues [46, 47]. IGFBP has binding affinities for IGF-I and IGF-II comparable to the ligands for IGF-IR. Most IGFBPs inhibit IGF-induced cell growth by binding to IGFs and acting as a time-release mechanism. Here, we analyzed the transcription pattern of IGFBP4 and IGFBP7. Quantitative PCR gene expression analysis showed that IGFBP4 and IGFBP7 expression in CP is gender-specific; in males, the gene expression remains unchanged (Supplementary Fig. 1c), and in females (Supplementary Fig. 1D), both IGFBP4 (P < 0.001) and IGFBP7 (P < 0.0001) expression significantly reduces as age progresses. Gene expression study using QIAcuity dPCR (Fig. 2B) confirms the age-induced reduction (P < 0.0001) in the IGFBP transcription.
Figure 3: Longevity factor klotho expression was significantly altered in the aged choroid plexus of both genders: Age-related transcriptional level reduction was observed in the CP of A Males and B Females. A quantitative analysis of Klotho protein was conducted using Jess capillary separation in the CP of C males and E females. Expression was normalized using β actin. Klotho protein expression was significantly reduced in aged CP of D males and F females. H Klotho (Red) -IsolectinB4 (Green) co-labeling confirmed the absence of Klotho from Choroid vasculature. I Pectin (Green)was used to mark the epithelial cells, and the basolateral sides of the choroid epithelial cells are rich in Klotho (Red) protein. G The mean pixel intensity of IHC images confirmed the protein level reduction of klotho in aged male CP. Aging also reduced klotho staining in the ependymal cells in the ventricles (marked with white arrows). Scalebar-100µm. * P < 0.05**P < 0.01, *** P < 0.001, **** P < 0.0001
Longevity factor Klotho is reduced in the aged choroid plexus.
α-Klotho is a glycosylated transmembrane protein that has been extensively studied as an anti-aging protein. Klotho is expressed highly in the choroid plexus, and its precise functions are largely unknown. We examined the gender and age-specific expression pattern of klotho in the CP. Klotho showed a reduced copy number (Fig. 3A&B) in the aged CPs of both males (P < 0.01) and females (P < 0.001), indicating an age-related reduction in Klotho gene expression. The reduction (Supplementary Fig. 1B) was evident in females (P < 0.001) from middle age onwards.
We extended our investigation to examine whether the changes in the mRNA level are reflected at the protein level. First, we used Western blot analysis to examine membrane-bound Klotho protein expression in the aged CP of both genders (Fig. 3C& 3E). Klotho protein expression revealed an age-related reduction (Fig. 3D& 3F) in the CP of males (P < 0.05) and females (P < 0.05). Next, we used IHC analysis to identify age-related changes in klotho protein expression patterns in the choroid epithelium and vasculature. Co-localization of Klotho with plectin indicates that Klotho protein expression is mainly concentrated in the choroid epithelial cells (Fig. 3I & Supplementary Fig. 4B). The apical part of epithelium lacks klotho expression, whereas the basolateral side is rich in klotho protein. Klotho is not co-labeled with isolectin B4, indicating the absence of Klotho protein in the choroid vasculature (Fig. 3H & Supplementary Fig. 4A). IHC analyses clearly indicate an age-dependent reduction (P < 0.0001) in klotho protein expression in CP of males (P < 0.0001) and females (P < 0.001) (Fig. 3G & Supplementary Fig. 4C). Reduced klotho protein expression was also observed in the ventricular lining of the old mice.
Figure 4: Water channel protein AQP1 expression is reduced in the aged choroid plexus. Aging significantly reduces the water channel protein AQP1 gene expression in the CP of males and B females. D Aqp1-Isolectin B4 co-labeling confirmed the absence of Aqp-1 protein expression in the adult and old CP vasculature. E AQP1- Plectin co-labeling study indicates Aqp1 localization in the apical part of the choroid epithelial cells. C The mean pixel intensity of IHC images showed a reduction in AQP1 Protein expression in the aged CP of males and females. Scalebar-100µm **P < 0.01, *** P < 0.001, **** P < 0.0001
Water channel protein AQP1 expression is altered in the aged choroid plexus.
A primary function of choroid epithelium is the production of CSF. Aquaporins (AQPs) are a family of small transmembrane proteins that facilitate water transport across plasma membranes during CSF production. AQP1 is the primary water channel expressed in the choroid plexus epithelium. We examined the age-dependent transcriptional changes in the male and female CP. AQP1 gene expression (Supplementary Fig. 2A& 2B) is significantly lowered in the aged CP of both males (P < 0.001) and females (P < 0.0001). The reduction was evident from middle age onwards. The ddPCR analysis further confirmed the low copy number in the aged CP (Fig. 4A&B), indicating a significantly reduced concentration of AQP1 transcripts in males (P < 0.01) and females (P < 0.001). The protein expression analysis using IHC indicates that, like AQP1 mRNA transcription, AQP1 Protein expression also showed an age-dependent reduction (P < 0.0001) in the CP epithelium of aged males and females (Fig. 4E & Supplementary Fig. 5C). IHC analysis indicates that the AQP1 protein is absent in the vasculature (Fig. 4D & Supplementary Fig. 5A) and present only in the ventricular-facing surface of the choroid plexus epithelium (Fig. 4E & Supplementary Fig. 5B).
Figure: 5. Tight junction proteins are significantly altered in the aged choroid plexus. A Tight junctional protein gene expression showed a differential expression in the aged male CP CLDN-1 mRNA transcription was unchanged along with reduced expression of CLDN-2, CLDN-5, OCLDN, and JAM2. B In females, CLDN-1, CLDN-2, CLDN-5, OCLDN, and JAM2 transcription was significantly reduced. C BCSF barrier permeability was assessed by injecting dextran average mol wt 40000 intracardially. In adult CP, fluorescence is mainly observed in the choroid vasculature and basolateral side of the epithelium, indicating that the tight junction prevents the diffusion of FITC dextran to the ventricles (Fig. 5c). In aged mice, fluorescence can be observed in and out of the apical side of endothelial cells, indicating that dextran 40000 was able to cross the tight junction. **P < 0.01, *** P < 0.001, **** P < 0.0001
Aging-altered tight junction protein expression compromises tight junction integrity.
The distribution of tight junctions in the choroid plexus differs from other brain regions. Choroid vasculature is fenestrated and lacks tight junctions [48]. The epithelial cells are connected by tight junctions in the apical region and maintain BCSF integrity [49]. Claudins are one of the most crucial tight junction proteins in the choroid epithelium. In males, real-time PCR analysis showed significant downregulation (P < 0.05) of CLDN1 gene expression in the middle-aged CP, and the gene expression came back to normal in the aged CP (Supplementary Fig. 2C). In female CP, CLDN1 m RNA transcription showed a trend toward decrease but was not statistically significant (Supplementary Fig. 2D). QIAcuity ddPCR analysis showed a consistent copy number of CLDN1 transcripts in the adult and aged male CP (Fig. 5A). However, the copy number in females was significantly (P < 0.001) reduced in aged CP (Fig. 5B). CLDN2 and CLDN5 mRNA expression were significantly (P < 0.0001) reduced in the aged CP of females (Supplementary Fig. 2D). ddPCR analysis further confirmed the reduction in CLDN2 and CLDN5 gene copy numbers (Fig. 5B). In males, CLDN2 (P < 0.0001) and CLDN5(P < 0.001) gene expression were significantly downregulated in the aged CP (Supplementary Fig. 2C). QIAcuity ddPCR further confirmed the same pattern of reduction in CLDN2 and CLDN5 gene expression (Fig. 5A). The mRNA expression of JAM2 is significantly downregulated in the aged CP of both males (P < 0.001) and females (P < 0.0001) (Fig. 5A & 5B, Supplementary Fig. 2C &2D). DdPCR confirms the reduction in JAM2 mRNA gene expression. To study whether the altered tight junction transcription affects the BCSF barrier integrity, we injected FITC-Dextran (MW 40000) intravenously. Fluorescent imaging showed that barrier permeability was intact in adult CP, and fluorescence was restricted within the basolateral side (Fig. 5C). However, dextran leakage can be observed from the apical membrane in the aged CP, indicating that BCSF integrity is compromised.
Figure 6: Transthyretin gene expression was significantly downregulated in the aged CP of males and females. A Thyroid hormone distributor protein TTR gene expression in the aged CP of males and B females. TTR transcription was significantly downregulated in the aged CP of both genders. *** P < 0.001.
Aging altered Transport protein Transthyretin gene expression
Transthyretin (TTR) is the major protein synthesized by the choroid epithelial cells and is expressed at remarkably high levels. Functionally, TTR binds and distributes thyroid hormones (THs) in the blood and cerebrospinal fluid [50]. Here, we examined the age and gender-specific changes in the TTR gene transcription. Relative gene expression showed that aging significantly accelerated the downregulation of TTR mRNA (Supplementary Fig. 6E &6F) in the CP of males (P < 0.001) and females(P < 0.001). The ddPCR further confirmed the reduced TTR expression (Fig. 6A &6B) in aged mice.