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. 2022 Dec 30;24(1):660.
doi: 10.3390/ijms24010660.

Cerebral Folate Metabolism in Post-Mortem Alzheimer's Disease Tissues: A Small Cohort Study

Naila Naz  1 , Syeda F Naqvi  1 , Nadine Hohn  1 , Kiara Whelan  1 , Phoebe Littler  1 , Federico Roncaroli  1   2 , Andrew C Robinson  1   2 , Jaleel A Miyan  1
Affiliations

Cerebral Folate Metabolism in Post-Mortem Alzheimer's Disease Tissues: A Small Cohort Study

Naila Naz et al. Int J Mol Sci. .

Abstract

We investigated the cerebral folate system in post-mortem brains and matched cerebrospinal fluid (CSF) samples from subjects with definite Alzheimer's disease (AD) (n = 21) and neuropathologically normal brains (n = 21) using immunohistochemistry, Western blot and dot blot. In AD the CSF showed a significant decrease in 10-formyl tetrahydrofolate dehydrogenase (FDH), a critical folate binding protein and enzyme in the CSF, as well as in the main folate transporter, folate receptor alpha (FRα) and folate. In tissue, we found a switch in the pathway of folate supply to the cerebral cortex in AD compared to neurologically normal brains. FRα switched from entry through FDH-positive astrocytes in normal, to entry through glial fibrillary acidic protein (GFAP)-positive astrocytes in the AD cortex. Moreover, this switch correlated with an apparent change in metabolic direction to hypermethylation of neurons in AD. Our data suggest that the reduction in FDH in CSF prohibits FRα-folate entry via FDH-positive astrocytes and promotes entry through the GFAP pathway directly to neurons for hypermethylation. This data may explain some of the cognitive decline not attributable to the loss of neurons alone and presents a target for potential treatment.

Keywords: ALDH1L1; Alzheimer’s disease; astrocytes; cerebral folate; cerebrospinal fluid; folate metabolism; methylation; neurons.

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Conflict of interest statement

The authors declare no conflict of interest in this publication.

Figures

Figure 1
Figure 1
Western and dot blot analysis of CSF for FDH, FRα and folate. The images of the relevant bands and dots from different blots for FDH, FRα and folate are shown at top with optical density measurements plotted as mean ±SEM for n = 21 per group. *, ** and *** show significance at p < 0.05, 0.001, and 0.0001, respectively. The mean optical density measurements are shown in the table. Images of the full blot membranes are shown in Supplementary Figure S1.
Figure 2
Figure 2
Immunofluorescence staining of Braak 0-II brain sections for FDH (green). Left panel (a) shows the entire section of cortex shown at low power (5×; scale bar: 2 mm) to orientate the different regions of brain shown in the right panels (b–f) at 200×, scale bar: 50 µm. (b) Cortex near the pia mater, (c) Mid cortex region, (d) White matter region, (e) sub-ventricle zone, (f) ventricular zone. The image is representative of n = 3 brains.
Figure 3
Figure 3
Immunofluorescence staining of Braak 0-II brain for FDH, GFAP, FRα and GFAP. Double immunofluorescence for FDH (green) and GFAP (red) staining of neurologically normal brain in cortical region near the pia mater (a,b) 100×, scale: 100 µm, white matter region (c), sub ventricular zone (d) and ventricular zone (e,f) at 200×, scale: 50 µm. The white arrows indicate FDH+/GFAP+ whereas red arrow indicates FDH+/GFAP−. Double immunofluorescence staining of FDH (green) and S100 (red) (g) to demonstrate FDH+ /S100+ astrocytes 200×, scale: 50 µm. Double immunofluorescence staining for FRα and GFAP (h, 100×; scale bar 100 µm). The figure is representative of n = 3.
Figure 4
Figure 4
Immunofluorescence staining for FDH, FRα and NeuN. Double immunofluorescence staining for FRα (red) and FDH (green) in Braak 0-II (a,b) and Braak V-VI (c–e) brains ((a,c) 100×, scale:100 µm and (b) 400×, 20 µm). Co-localisation (yellow) of FRα with FDH in the FDH-positive astrocytes in normal brains (a,b) shows almost complete separation in Alzheimer brain (c–e). FRα appears as speckled red staining in normal neuronal cell bodies (white arrows in (b)) while in Alzheimer cortex these cells are full of red, FRα (d,e). Double immunofluorescence staining for FRα (red) and NeuN (blue) in normal (f) and AD (i) brains show colocalisation, which is shown in separate channels in (g,h) and (j,k) ((f), 200×, scale: 50 µm ((g,h,j,k), 400×, scale: 20 µm). The figure is representative of neurologically normal, Braak 0-II n = 3 and AD, Braak V-VI n = 4.
Figure 5
Figure 5
Immunofluorescence staining for GFAP, FRα and FDH. Upper panel: Double immunofluorescence staining for FDH (green) and GFAP (red) in normal and AD brains. Co-localisation (yellow) is observed in astrocytes. Lower panel: Double immunofluorescence staining for FRα (red) and GFAP (green) in neurologically normal and AD brains. Co-localisation is observed as bright yellow colour. Magnification 200×, scale bar: 50 µm. The figure is representative of neurologically normal n = 3 and AD brains n = 4.
Figure 6
Figure 6
Immunofluorescence staining for folate, FRα and FDH. Double immunofluorescence staining for FDH (green) and folate (red) in neurologically normal ((a) 100 × 100 µm and (b) 400 × 20 µm) and AD brain ((c) 100 × 100 µm and (d) 400 × 20 µm). Some co-localisation (yellow) of folate and FDH ((e) 400 × 20 µm) is observed. Double immunofluorescence staining for FRα (green) and folate (red) in normal ((f) 100 × 100 µm and (g) 400 × 20 µm) and AD brain ((h) 100 × 100 µm and (i) 400 × 20 µm). The figure is representative of neurologically normal n = 3 and AD n = 4 brains.
Figure 7
Figure 7
Immunofluorescence staining for 5-methyl cytosine (5mc, marker of methylation) and 5-hydroxy methyl cytosine (5hmc, marker of demethylation) in neurologically normal (Braak 0-II) and AD (Braak V-VI) brains. Double immunofluorescence staining for 5-methyl cytosine (red) and 5-hydroxy methyl cytosine (green) in normal cortical cells ((a) at 100×, scale: 100 µm and (b) 400×, scale: 20 µm) show a balance of methylation and demethylation (seen as colocalised, yellow staining). In AD brain ((c) at 100×, scale: 100 µm and (d) 400×, scale: 20 µm) cells show almost exclusive methylation with very little demethylation. Double immunofluorescence staining for 5mc (green) and the neuronal marker NeuN (blue) in normal (e) and AD (h) are also shown in separated channels (f,g,i,j) to show that many cells have colocalised with many more in AD brain, indicating hypermethylation of neurones in AD. (e,h), at 100×, scale: 100 µm. (f–j) at 200×, scale: 50 µm. The figure is representative of neurologically normal n = 3 and AD n = 4 brains.
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