Fungal structures in AD CNS

One of the most direct approaches to detect fungal infection in the CNS is the visualisation of fungi in tissue sections. It is well established that the hierarchical pattern of neurofibrillary degeneration and thus the early pathological lessions in AD patients generally starts by modifications in the entorhinal cortex, followed by the hippocampus, association cortex and finally the primary neocortex11. We first analysed fixed sections from different regions of the CNS from one AD patient (AD1) and a control individual (C1) (Supplementary Table I). The regions examined were external frontal cortex (EFC), cerebellar hemisphere (CEH), entorhinal cortex/hippocampus (ERH) and choroid plexus (CP). Notably, fungal cells were detected in the four regions examined from patient AD1 as demonstrated by immunohistochemistry and confocal microscopy using anti-C. glabrata antibodies (Fig. 1). In some instances, fungal cells were clearly visible inside neurons and exhibited an intranuclear location as indicated by counterstaining with the DNA stain, 4'6-diamidino-2-phenylindole (DAPI). The size of the fungal bodies was variable; in some instances, the size was 1–2 μm, whereas the diameter of other fungal bodies was greater (approximately 5–10 μm). In other instances, smaller fungal bodies of 0.4–1 μm were evident depending on the field analysed. The 0.4–1 and the 1–2 μm-sized bodies are similar to those previously reported for some intracellular yeast cells30,31,32. These intracellular forms are known as endomycosomes29,33. Endothelial cells in the CP may also contain fungal bodies. No fungal cells or fungal material were apparent in the different CNS regions from the control (C1) individual (Fig. 1). Curiously, immunostaining of tau protein with specific antibodies localised tau not only in the cytoplasm, but also in the nucleus in both AD1 and C1 sections. This finding is consistent with the observation that nuclear pores are damaged in elderly people, particularly with neurodegenerative diseases and cytoplasmic proteins can relocate to the nucleus34,35. Nuclear tau protein staining was very strong in neurons where intranuclear fungal bodies were detected.

Figure 1 Immunohistochemistry analysis of tissue sections from different regions of the CNS using anti-C. glabrata antibodies. CNS sections from patient AD1 and control individual C1 were obtained from fixed tissue and immunohistochemistry analysis by confocal microscopy was carried out as detailed in Materials and Methods. EFC: external frontal cortex; CEH: cerebellar hippocampus; ERH: entorhinal cortex/hippocampus; CP: choroid plexus. DAPI appears in blue, anti-C. glabrata is shown in green and Tau T100 in red. The different panels in the figure are indicated. Scale bar: 5 μm. Full size image

Wider fields illustrating the presence of additional fungal bodies and a more general view of the fungal infection are shown in Supplementary Figures 1, 2 and 3. Nuclear (DAPI) staining (blue) and double immunofluorescence staining to detect fungal structures (green) and tau protein (red) was carried out and only the merged panel is shown for space restrictions. Several fungal morphologies could be observed in the EFC, with sizes ranging from 0.4–1 and 5–10 μm. The sizes of the fungal bodies found in the CEH were approximately 1–2 μm. Strikingly, two different fields of the ERH revealed amylaceous bodies (corpora amylacea), which were prominently stained in the border of these rounded structures. However, immunostaining was not evenly distributed in all the border zones, indicating that the fungal material is not distributed homogeneously. Collectively, these observations demonstrate the presence of fungal material in different CNS regions examined, but only in the AD patient (AD1). The fungal structures could be detected outside and inside nuclei and, in some instances, fungal cells were positive for DAPI, indicating that they contain nucleic acids (see upper-right panel in Supplementary Figure 1).

Detection of fungal cells and hyphae using different anti-fungal antibodies

It must be kept in mind that the immunoreactivity observed with anti-C. glabrata antibodies does not necessarily mean that this yeast is present. Because the antibodies employed are rabbit polyclonal, they can crossreact with a number of proteins from other fungi. The spectrum of proteins recognised by the different anti-fungal antibodies employed in this work vary and depend on the fungal species present in each case. However, these anti-fungal antibodies do not crossreact with cellular proteins from control individuals. To further assess whether fungal cells were present in patient AD1, we carried out immunohistochemistry analysis using rabbit polyclonal antibodies raised against other fungi. Four additional antibodies (raised against C. famata, C. albicans. P. betae and S. racemosum) detected fungal components (green) in the tissue sections analysed (Fig. 2), as demonstrated by double staining with an anti-neurofilament antibody (red). As indicated previously, the size of the fungal cells detected with the antibodies varied. The additional antibodies also detected long fibrilar structures clearly resembling fungal hyphae, with sizes ranging from 0.1 μm to 1–2 μm. The variety of sizes and morphologies observed in these sections using different anti-fungal antibodies is consistent with the notion that several fungal species were present in the three CNS regions examined. Analyses of tissue sections from C1 using these antibodies failed to reveal any fungal material (results not shown). The possibility that these antibodies recognise human proteins present only in CNS samples from patient AD1 and form structures that resemble different fungal morphologies is unlikely. To further test the presence of fungal proteins in patient AD1, we extracted proteins from different CNS regions of AD1 and C1 and performed western blotting with anti-C. albicans antibodies (Supplementary Figure 4). No specific protein bands were detected by this technique, most probably due to the fact that the amount of fungal proteins is extremely low. To unequivocally identify fungal proteins in brain tissue from AD patients, proteomic methodologies are required as we have previously reported28. Using this approach, we have detected several fungal proteins that are present in CNS samples from AD patients, but not in control individuals.

Figure 2 Immunohistochemistry analysis of CNS sections from patient AD1 using different anti-fungal antibodies. Immunohistochemistry analysis of different CNS sections from patient AD1 was carried out as indicated in Fig. 1. EFC: external frontal cortex; CEH: cerebellar hippocampus; ERH: entorhinal cortex/hippocampus. DAPI appears in blue, anti-C. famata, anti-C. albicans, anti-P. betae and anti-S. racemosum are shown in green and human neurofilament in red. The different panels in the figure are indicated. Scale bar: 10 μm. Full size image

In accord with the earlier finding, some fungal cells and hyphae stained positive for DAPI (blue), suggesting that they contain nucleic acids (Fig. 2). In EFC sections stained with anti-C. famata and anti-S. racemosum antibodies, DAPI staining was clearly located inside hyphae. Indeed, DAPI positivity could be seen in the vast majority of cells and hyphae after longer exposure times, but under these conditions neuron nuclei were overexposed. To overcome this limitation, the blue DAPI staining was converted to magenta and the antifungal antibody staining was in green. Under these conditions, positive DAPI staining was observed in all the morphologies found, i.e. yeast-shaped cells and hyphae (Fig. 3) and this was also the case when the anti-C. glabrata antibodies were employed (Fig. 1).

Figure 3 DAPI staining of nuclei of the different fungal morphologies. DAPI staining is shown in magenta to visualize more clearly the fungal nuclei. Immunoreactivity with the fungal antibodies indicated is shown in green. Panels A–G and S: anti-C. glabrata as primary antibody. Panels H–J, T, U, W: anti-C. famata as primary antibody. Panels: K–M, V, X: anti-C. albicans as primary antibody. Panels N, O, Y, Z: anti-S. racemosum. as primary antibody. Panels P-R: anti- P. betae as primary antibody. Panels K, L, S, T, U, Y: EFC of patient AD1. Panels A, H, M–Q, X: CEH of patient AD1. Panels I, J, R, V, W, Z: ERH of patient AD1. Panels B, C, D: CP of patient AD1. Panel E: ERH of patient AD3. Panel F: ERH of patient AD4. Panel G: ERH of patient AD9. Scale bar: 5 μm is the same for all panels shown in the figure. Full size image

Because most of the above results were obtained from only one AD patient and one control individual, it was of interest to examine CNS sections from additional AD patients and controls. To this end, we analysed ERH tissue sections from a further ten AD patients and ten controls by double immunostaining with anti-fungal (green) and anti-tubulin (red) antibodies. Notably, fungal infection was evident in all AD patients studied (Fig. 4), whereas no fungal cells were detected in tissue sections from control individuals (Supplementary Figure 5). The morphology of the fungal structures detected in the additional AD patients was similar to that described for AD1, although not all structures were found in all patients. Moreover, some of these structures were very striking; for example, conidial structures were observed in patients AD2 (anti-C. glabrata) and AD8 (anti-C. albicans) and hyphae formation was observed from a fungal cell in patient AD6 (anti-C. albicans). The small hyphae and yeast cells found in patient AD5 (anti-Phoma staining) was very striking. The existence of different fungal morphologies reinforces the idea that several species can be present, supporting the concept of mixed fungal infections. In conclusion, fungal cells and/or hyphae were found in all AD patients analysed although the morphological characteristics may be different for each patient, thus implying that the fungal species present in each patient may also differ.

Figure 4 Immunohistochemistry analysis of entorhinal cortex sections from ten different AD patients. Entorhinal cortex sections from ten different AD patients were incubated with different antibodies (anti-C. glabrata, anti-C albicans and anti-P. betae) and are shown in green; human α-tubulin immunostaining is shown in red. Double immunofluorescence assay and confocal microscopy was carried out as indicated in Fig. 1 and Materials and Methods. DAPI appears in blue. Scale bar: 5 μm. Full size image

Fungal infection of the neurovascular system

The majority of AD patients exhibit pathological lesions of the vascular system in the CNS16,36. Up to 90% of AD patients present various cerebrovascular pathologies, including cerebral amyloid angiopathy, microinfarcts, haemorrhages and microvascular degeneration. Accordingly, deposits of amyloid β in the walls of capillaries, small arterioles and medium-size arteries are evident in most AD patients studied. These amyloid deposits give rise to cerebral amyloid angiopathy. We reasoned that if fungal infection is present in blood vessels, it might induce the formation of amyloid deposits and angiopathy. To assess fungal infection in blood vessels from EFC, ERH and CP regions, we carried out confocal immunofluorescence analysis. Fungal cells of different sizes and hyphae were detected inside capillaries and other blood vessels (Fig. 5). Some staining was also evident in the vessel walls, demonstrating that fungal infection can be detected in the neurovascular system. Also, the CP region from patient AD1 (Fig. 5), but not from C1 (Supplementary Figure 6), contained fungal cells and hyphae that immunoreacted with the different anti-fungal antibodies. These results are in good agreement with the notion that several fungal species can infect blood vessels and cause pathological modifications37,38,39. These studies were extended to the analysis of the CP region from three additional AD patients (AD12, 13, 14) using several anti-fungal antibodies., Consistent with the previous result, fungal material was detected in all three cases examined, but not in the control (Supplementary Figure 6). In conclusion, fungal structures can be observed also in the vascular system of AD patients.

Figure 5 Fungal bodies in CNS blood vessels from patient AD1 detected by immunohistochemistry. Double immunofluorescence assay analyzed by confocal microscopy as described in Materials and Methods. Blood vessels from different CNS regions from patient AD1 are shown. Panels A–E: EFC. Panels F and G: ERH. Panels H–L: CP. Panels A and I: anti-C. famata as primary antibody. Panels B, C, J: anti-C. albicans as primary antibody. Panels D, G, K: anti-P. betae as primary antibody. Panels E and L: anti-S. racemosum as primary antibody. Panels F and H: anti-C. glabrata as primary antibody. Anti-fungal antibodies are shown in green. An anti-human neurofilament antibody was used in all panels, except panels F and H, which were stained with anti-Tau T100 antibodies (red.) DAPI appears in blue. Scale bar: 10 μm. Full size image

Identification of fungal species in the different CNS regions

DNA amplification and sequencing is the most precise approach to determine specific fungal species present in CNS tissues. Because the vast majority of DNA from tissue samples is human, we previously developed a sensitive nested PCR assay to amplify fungal DNA present in very minor amounts28,33,40,41. We recently used this tecnhnique successfully to identify several fungal species in brain samples from AD patients40. To identify the fungal species present in the different brain regions of patient AD1, we extracted DNA from frozen tissue and performed PCR using primers that amplify two internal transcribed spacers (ITS-1 and ITS-2) located between ribosomal RNA genes (see scheme, Supplementary Figure 7). Several different PCR assays were carried out since in our experience the use of several primer pairs ensures the amplification of DNA from different fungal species. We first amplified ITS-1 with primers ITS-1 (external) and then three different PCR assays were subsequently performed with internal primer pairs (ITS-1, internal). In addition, ITS-2 was first amplified with primers ITS-2 (external) followed by a second PCR with an ITS-2 (internal) primer pair. DNA amplified by each PCR was separated on agarose gels and we sequenced the extracted fragments. A typical PCR result after amplification of the ITS-1 using DNA extracted from the different CNS regions of patient AD1 and C1 is shown in Supplementary Figure 7. No PCR products were amplified from DNA extracted from the different regions of C1, or from controls for DNA extraction and PCR. These findings reveal that no fungal contamination occurred in the PCR assay or during DNA extraction. Sequencing of each fragment from the four PCR assays using DNA from patient AD1 revealed a number of fungal species as listed in Table 1. Of note, several species could be detected from the same region, supporting the concept of mixed fungal infection. Conversely, no single fungal species was present in all four regions of the CNS examined. Of note, some of the species detected, such as Malasezzia spp., Phoma and S. cerevisiae, have been previously identified in AD brains40. Significantly, the majority of the species listed in Table 1 are described as human pathogens42,43. The possibility that different fungal species or a combination of species serves as a risk factor or represents the cause of AD might explain the diversity observed in the evolution and severity of clinical symptoms in each AD patient.