Searching for Bacteria in Neural Tissue From Amyotrophic Lateral Sclerosis

Abstract

Despite great efforts in the investigation, the exact etiology of amyotrophic lateral sclerosis (ALS) is a matter of intensive research. We recently advanced the idea that ALS might be caused by fungal infection. Indeed, fungal yeast and hyphal structures can be directly visualized in neural tissue of ALS patients, and a number of fungal species have been identified in the central nervous system (CNS). In the present work, we tested the possibility that bacterial infections can accompany these mycoses. Our findings establish the presence of bacterial DNA in different regions of the CNS from all ALS patients examined. Specifically, we used PCR and next generation sequencing (NGS) to precisely determine the bacterial species present in ALS tissue. Consistent with these findings, immunohistochemistry analysis of CNS sections using specific anti-bacterial antibodies identified prokaryotic cells in neural tissue. Finally, we assayed for the repeat expansion of the hexanucleotide repeat GGGGCC in C9orf72, which is considered the most common genetic cause of ALS in patients, using DNA extracted from ALS CNS tissue. We failed to find this repeated sequence in any of the eleven patients analyzed. Our results indicate that bacterial DNA and prokaryotic cells are present in CNS tissue, leading to the concept that both fungal and bacterial infections coexist in patients with ALS. These observations lay the groundwork for the use of appropriate therapies to eradicate the polymicrobial infections in ALS.

Keywords: amyotrophic lateral sclerosis; neurodegenerative disease; next generation sequencing; polymicrobial infection; repeat expansion C9orf72.

Figure 1

Nested PCR analysis of bacterial DNA extracted from patients with ALS. PCR analysis was carried out as described in section Materials and Methods. (A) Schematic representation of bacterial rRNA genes (16S and 23S) and the intergenic sequence (IGS) region, including the location of the primers employed for the different reactions. (B) Agarose gel electrophoresis of the DNA fragments amplified by nested PCR; analysis of three CNS regions (MC, MD, and SC) from 11 patients amplifying the IGS region Primers 1406(F) – 559(R) and 1492 (F) –242 (R) were used in the first and second round PCR, respectively, C-, control PCR without DNA; CE, control of DNA extraction without DNA; MC, motor cortex; MD, medulla; SC, spinal cord; SC1, SC2, and SC3, three samples from different regions of the spinal cord.

Figure 2

Distribution of bacteria phyla and orders obtained by NGS of DNA from eleven ALS patients. Computational analyses of the sequences obtained on the Illumina platform using Qiime classified the data into bacteria phyla and orders. (A) Panels show the results of bacteria phyla obtained from three CNS regions. (B) Panels show the results of bacteria orders obtained from three CNS regions. SC1, SC2 and SC3, three samples from different regions of the spinal cord.

Figure 3

Principal component analysis of bacteria of CNS regions from ALS patients and AD patients. (A) Three-dimensional (3D) principal component analysis (PCA) between different CNS regions from 11 ALS patients. (B) 3D PCA between ALS and AD patients. MC, motor cortex; MD, medulla; SC, spinal cord, SC1, SC2, and SC3, three samples from different regions of the spinal cord.

Figure 4

Three- and two-dimensional principal component analysis of bacteria of CNS regions from ALS patients and AD patients. Comparison of three separate CNS regions between AD and ALS patients. (A) Left panel: three-dimensional (3D) principal component analysis (PCA) between MC regions of ten ALS patients (plots in red) and 10 AD patients (plots in blue). Right panel: 2D PCA. (B) Left panel: 3D PCA between MD regions of eleven ALS patients (plots in blue) and ten AD patients (plots in red). Right panel: 2D PCA. (C) Left panel: 3D PCA between SC regions of 11 ALS patients (plots in blue) and 10 AD patients (plots in red). Right panel: 2D PCA. The UniFrac method was used to calculate this parameter. MC, motor cortex; MD, medulla; SC, spinal cord.

Figure 5

Three- and two-dimensional principal component analysis of bacteria of CNS regions from ALS patients and control samples. (A) Left panel: three-dimensional (3D) principal component analysis (PCA) between MC regions of 10 ALS patients (plots in blue) and 9 control samples (plots in red). Right panel: 2D PCA. (B) Left panel: 3D principal component analysis between MD regions from 11 ALS patients (plots in blue) and 9 control samples (plots in red). Right panel: 2D PCA. (C) Left panel: 3D PCA between SC regions from 11 ALS patients (plots in blue) and 9 control samples (plots in red). Right panel: 2D PCA. The UniFrac method was used to calculate this parameter. MC, motor cortex; MD, medulla; SC, spinal cord.

Figure 6

Immunohistochemistry to detect peptidoglycan in brain tissue from ALS patients. Double immunostaining and confocal microscopy were carried out as indicated in section Materials and Methods. CNS sections were immunostained with a mouse monoclonal anti-peptidoglycan antibody (green) (1:20 dilution) and a rabbit polyclonal anti-C. albicans antibody (red) (1:500 dilution). DAPI staining of nuclei appears in blue. Scale bar: 5 μm. (A–C) ALS2; (D–F) ALS3; (G–I) ALS4; (J,K) ALS5; (L–N) ALS6; (O) ALS1; (P) ALS7; (Q,R) ALS8; (S) ALS9; (T–V) ALS10; and (W,X) ALS11. (A,D,G,J,L,Q,S,T,W) MC section; (B,E,H,K,M,O,U,X) MD section; and (C,F,I,N,P,R,V) SC section.

Figure 7

Immunohistochemistry to detect C. pneumoniae antigens in brain tissue from ALS patients. Double immunostaining and confocal microscopy were carried out as indicated in section Materials and Methods. CNS sections were immunostained with a rabbit polyclonal C. pneumoniae antibody (green) (1:20 dilution) and a rat polyclonal anti-T. viride antibody (red) (1:20 dilution). DAPI staining of nuclei appears in blue. Scale bar: 5 μm. (A,B) ALS2; (C,D) ALS3; (E) ALS5; (F–H) ALS6; (I,J) ALS1; (K) ALS7; (L,M) ALS8; (N) ALS9; (O) ALS10; and (P) ALS11. (A,C,F,I,K,L,P) MC section; (G,N,O) MD section; and (B,D,E,H,J,M) SC section.

Figure 8

Bacterial proteins in corpora amylacea from ALS patients. Double immunostaining and confocal microscopy were carried out as indicated in section Materials and Methods. CNS sections were immunostained with a mouse monoclonal anti-peptidoglycan antibody (green) (1:20 dilution) and a rabbit polyclonal anti-C. albicans antibody (red) (1:500 dilution). DAPI staining of nuclei appears in blue. Scale bar: 25 μm. (A,B) ALS2; (C) ALS3; (D,E), ALS4; (F) ALS5; (G,H) ALS7; (I,J) ALS8; (K) ALS9; (L–N) ALS10; and (O) ALS11. (A,D,F,L) MC section; (G,I,K,M) MD section; and (B,C,E,H,J,N,O) SC section.