Evidence for fungal infection in cerebrospinal fluid and brain tissue from patients with amyotrophic lateral sclerosis

Abstract

Among neurogenerative diseases, amyotrophic lateral sclerosis (ALS) is a fatal illness characterized by a progressive motor neuron dysfunction in the motor cortex, brainstem and spinal cord. ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown. In the present work, we have explored the possibility of fungal infection in cerebrospinal fluid (CSF) and in brain tissue from ALS patients. Fungal antigens, as well as DNA from several fungi, were detected in CSF from ALS patients. Additionally, examination of brain sections from the frontal cortex of ALS patients revealed the existence of immunopositive fungal antigens comprising punctate bodies in the cytoplasm of some neurons. Fungal DNA was also detected in brain tissue using PCR analysis, uncovering the presence of several fungal species. Finally, proteomic analyses of brain tissue demonstrated the occurrence of several fungal peptides. Collectively, our observations provide compelling evidence of fungal infection in the ALS patients analyzed, suggesting that this infection may play a part in the etiology of the disease or may constitute a risk factor for these patients.

Keywords: Amyotrophic lateral sclerosis; brain histological analysis; fungal PCR; fungal infection; proteomic analysis..

Figure 1

Analysis of C. albicans and enolase antigens in CSF by slot-blot. 20 μl CSF samples were diluted with 180 μl TBS and were blotted onto a nitrocellulose membrane, which was incubated with the rabbit antiserum against C. albicans or enolase or recombinant MBP-enolase (primary antibody) as indicated and afterwards incubated with a rabbit anti rat IgG (secondary antibody). Positive control: control + 200 ng yeast protein or purified MBP-enolase. Negative control: control - corresponds to TBS alone.

Figure 2

PCR analysis of DNA obtained from CSF. A) Schematic representation of fungal rRNA genes and the ITS1 sequence. Location of the primers employed for the PCR: primers 1 employed in the first PCR; primers 2A employed in the second PCR; primers 2B employed in the second PCR and previously described as panfungal primers. B) PCR was carried out as described from DNA samples obtained from CSF of ALS patients or controls. The primers employed were primers 1 for the first round PCR and primers 2A for the second round. After PCR, the samples were separated on agarose gels and stained with ethidium bromide. DNA size markers are shown on the left. Fungal species detected after sequencing each product is shown on the right. C) The samples obtained after the first PCR (primers 1) were amplified using primers 2B. PCR products were separated on agarose gels, extracted and sequenced. Fungal species detected are depicted on the right. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers.

Figure 3

Immunohistochemistry analysis of brain sections from the frontal cortex of ALS patients. Brain sections (frontal cortex) from ALS patients 9, 10 and 11 were observed with a confocal laser scanning microscope. Sections were obtained from fixed tissue and immunohistochemistry analyses were carried out. Sections were incubated with anti-C. glabrata antibodies (1:500 dilution), followed by incubation with secondary antibody donkey anti-rabbit IgG conjugated to Alexa 555 (1:500). Subsequently, sections were incubated with DAPI (1 μg/ml). The different panels in the figure are indicated. Scale bar: 10 μm.

Figure 4

Immunohistochemistry analysis of brain sections from the frontal cortex of an ALS patient and a control. Brain sections (frontal cortex) from ALS patient 11 and control 6 were observed with a confocal laser scanning microscope. Sections were obtained from fixed tissue and immunohistochemistry analyses were carried out. Double immunofluorescence assay using anti-tubulin and anti-C. glabrata antibodies was carried out as detailed in Materials and Methods. DAPI appears in blue and anti-C. glabrata is shown in green. Human tubulin appears in red. The different panels in the figure are indicated.

Figure 5

PCR analysis of different brain regions from three ALS patients. PCR was carried out as described. A) PCR analysis of different brain regions and controls using primers 1 and 2a. Table shows fungal species detected. B) PCR analysis of different brain regions and controls using primers 1 and 2b. Table shows fungal species detected. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers. FC: Frontal cortex. C: Cerebellum. O: Occipital cortex. WM: White matter. GM: Grey matter.

Figure 6

Functional grouping of human proteins specifically present in the frontal cortex from ALS patients. Human proteins which are common in the three ALS patients that are not present in control brain grouped by: a) cellular component; b) molecular function; c) protein class.