Brain Microbiota in Huntington's Disease Patients

Abstract

One of the most important challenges facing medical science is to better understand the cause of neuronal pathology in neurodegenerative diseases. Such is the case for Huntington's disease (HD), a genetic disorder primarily caused by a triplet expansion in the Huntingtin gene (HTT). Although aberrant HTT is expressed from embryogenesis, it remains puzzling as to why the onset of disease symptoms manifest only after several decades of life. In the present study, we investigated the possibility of microbial infection in brain tissue from patients with HD, reasoning that perhaps mutated HTT could be deleterious for immune cells and neural tissue, and could facilitate microbial colonization. Using immunohistochemistry approaches, we observed a variety of fungal structures in the striatum and frontal cortex of seven HD patients. Some of these fungi were found in close proximity to the nucleus, or even as intranuclear inclusions. Identification of the fungal species was accomplished by next-generation sequencing (NGS). Interestingly, some genera, such as Ramularia, appeared unique to HD patients, and have not been previously described in other neurodegenerative diseases. Several bacterial species were also identified both by PCR and NGS. Notably, a curved and filamentous structure that immunoreacts with anti-bacterial antibodies was characteristic of HD brains and has not been previously observed in brain tissue from neurodegenerative patients. Prevalent bacterial genera included Pseudomonas, Acinetobacter, and Burkholderia. Collectively, our results represent the first attempt to identify the brain microbiota in HD. Our observations suggest that microbial colonization may be a risk factor for HD and might explain why the onset of the disease appears after several decades of life. Importantly, they may open a new field of investigation and could help in the design of new therapeutic strategies for this devastating disorder.

Keywords: Huntington’s disease; endomycosomes; fungal infection; neurodegenerative diseases; next generation sequencing; polymicrobial infections.

FIGURE 1

Immunohistochemistry of striatum sections from Huntington’s disease patients using a battery of antifungal antibodies. The striatum CNS region of four HD patients (HD4–HD7) was processed for immunohistochemistry as described in section “Materials and Methods.” Paraffin sections were immunostained with rabbit polyclonal antibodies against C. albicans, C. glabrata, and P. notatum (green). Nuclei were stained with DAPI (blue). Scale bar: 5 μm.

FIGURE 2

Immunohistochemistry of brain sections from seven Huntington’s disease patients using anti-C. albicans and anti-S. racemosum antibodies. Two CNS regions (striatum, ST and frontal cortex, FC) from seven HD patients (HD1–HD7) were processed for immunohistochemistry analysis. Paraffin sections were immunostained with rabbit polyclonal antibodies against C. albicans (A) or S. racemosum (B) (green). Nuclei were stained with DAPI (blue). Scale bar: 5 μm.

FIGURE 3

Fungal structures in the frontal cortex region from one Huntington’s disease patient (HD5) analyzed by immunohistochemistry. Immunohistochemistry analysis was carried out using polyclonal antibody against C. albicans (green). Nuclei were stained with DAPI (blue). Scale bar: 5 μm.

FIGURE 4

Orthogonal projection and 3D analysis of intracellular microbial structures. Immunohistochemistry was carried out as described in section “Materials and Methods.” Orthogonal projections (A,B) and different stacks from a 3D image (C,D) (see also Supplementary Movies 1, 2) from the striatum of HD6 patient. Panels (A,C) show the same cell that appear in panels (B,D), respectively. Samples were immunostained with an anti-C. albicans antibody (green). Nuclei were stained with DAPI (blue). All scale bars: 5 μm.

FIGURE 5

Analysis of corpora amylacea in Huntington’s disease brain sections by immunohistochemistry. Striatum sections from HD patients were incubated with rabbit polyclonal antibodies against C. albicans (A), P. notatum (B–D), P. betae (E,F), chitin (G–I) and enolase (J–L) appear in green. (B,G,K) HD4; (A,C,E): HD5; (D,H,K): HD6; (F,I,L): HD7. Nuclei were stained with DAPI (blue). Scale bar: 20 μm.

FIGURE 6

Identification of fungal phylum order and genus by next-generation sequencing. DNA was extracted from HD brain tissue for next-generation sequencing. Computational analyses of the sequences using QIIME classified the data into fungal phyla, order and genera. (A) Striatum tissue from seven HD brains. (B) Frontal cortex tissue from seven HD brains. Asc: Ascomycota, Bas: Basidiomycota, Chy: Chytridiomycota.

FIGURE 7

Principal component analysis of fungi identified by next-generation sequencing. 3D principal component analysis scatter plots of HD and Parkinson’s disease (PD) patients. Panel (A) shows the distribution between striatum (plots in blue) and frontal cortex (plots in red) regions of seven HD patients. Panel (B) shows the distribution between HD patients (plots in red) and PD patients (plots in blue). The UniFrac method was used to calculate this parameter. ST, striatum; FC, frontal cortex; MC, motor cortex; MS, mesencephalon.

FIGURE 8

Prokaryotic structures in Huntington’s disease brain sections using an anti-C. pneumoniae antibody. Different sections from HD patients were incubated with a rabbit polyclonal antibody against C. pneumoniae (green). DAPI staining is shown in blue. (A,C,E,F,H,J,K) striatum region; (B,D,G,I,L) frontal cortex region; (A,B) HD1; (C,D) HD2; (E) HD3; (F,G) HD4; (H,I) HD5; (J) HD6; (K,L) HD7. Scale bar: 5 μm.

FIGURE 9

Distribution of bacterial phyla and classes in CNS samples from Huntington’s disease patients. Computational analyses of the sequences obtained using next-generation sequencing were carried out with QIIME. Bacterial phyla and classes obtained with this program are shown. Panel (A) shows bacterial phyla and classes detected in striatum of HD patients. Panel (B) shows bacterial phyla and classes detected from frontel cortex of PD patients.

FIGURE 10

Principal component analysis of bacterial identified by next-generation sequencing. 3D principal component analysis scatter plots of HD and Parkinson’s disease patients. Panel (A) shows the distribution between striatum (plots in blue) and frontal cortex (plots in red) regions of seven HD patients. Panel (B) shows the distribution between HD patients (plots in red) and Parkinson’s disease patients (plots in blue). The UniFrac method was used to calculate this parameter. ST, striatum; FC, frontal cortex; MC, motor cortex; MS, mesencephalon.