Parkinson's Disease: A Comprehensive Analysis of Fungi and Bacteria in Brain Tissue.

Parkinson's disease (PD) is characterized by motor disorders and the destruction of dopaminergic neurons in the substantia nigra pars compacta. In addition to motor disability, many patients with PD present a spectrum of clinical symptoms, including cognitive decline, psychiatric alterations, loss of smell and bladder dysfunction, among others. Neuroinflammation is one of the most salient features of PD, but the nature of the trigger remains unknown. A plausible mechanism to explain inflammation and the range of clinical symptoms in these patients is the presence of systemic microbial infection. Accordingly, the present study provides extensive evidence for the existence of mixed microbial infections in the central nervous system (CNS) of patients with PD. Assessment of CNS sections by immunohistochemistry using specific antibodies revealed the presence of both fungi and bacteria. Moreover, different regions of the CNS were positive for a variety of microbial morphologies, suggesting infection by a number of microorganisms. Identification of specific fungal and bacterial species in different CNS regions from six PD patients was accomplished using nested PCR analysis and next-generation sequencing, providing compelling evidence of polymicrobial infections in the CNS of PD. Most of the fungal species identified belong to the genera Botrytis, Candida, Fusarium and Malassezia. Some relevant bacterial genera were Streptococcus and Pseudomonas, with most bacterial species belonging to the phyla Actinobacteria and Proteobacteria. Interestingly, we noted similarities and differences between the microbiota present in the CNS of patients with PD and that in other neurodegenerative diseases. Overall, our observations lend strong support to the concept that mixed microbial infections contribute to or are a risk factor for the neuropathology of PD. Importantly, these results provide the basis for effective treatments of this disease using already approved and safe antimicrobial therapeutics.

Brain Microbiota in Huntington's Disease Patients.

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.

Searching for Bacteria in Neural Tissue From Amyotrophic Lateral Sclerosis.

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.

Human and Microbial Proteins From Corpora Amylacea of Alzheimer's Disease.

Corpora amylacea (CA) are spherical bodies mainly composed of polyglucans and, to a lesser extent, proteins. They are abundant in brains from patients with neurodegenerative diseases, particularly Alzheimer's disease. Although CA were discovered many years ago, their precise origin and function remain obscure. CA from the insular cortex of two Alzheimer's patients were purified and the protein composition was assessed by proteomic analysis. A number of microbial proteins were identified and fungal DNA was detected by nested PCR.A wide variety of human proteins form part of CA. In addition, we unequivocally demonstrated several fungal and bacterial proteins in purified CA. In addition to a variety of human proteins, CA also contain fungal and bacterial polypeptides.In conclusion, this paper suggests that the function of CA is to scavenge cellular debris provoked by microbial infections.

Infection of Fungi and Bacteria in Brain Tissue From Elderly Persons and Patients With Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia in elderly people. The etiology of this disease remains a matter of intensive research in many laboratories. We have advanced the idea that disseminated fungal infection contributes to the etiology of AD. Thus, we have demonstrated that fungal proteins and DNA are present in nervous tissue from AD patients. More recently, we have reported that bacterial infections can accompany these mycoses, suggesting that polymicrobial infections exist in AD brains. In the present study, we have examined fungal and bacterial infection in brain tissue from AD patients and control subjects by immunohistochemistry. In addition, we have documented the fungal and bacterial species in brain regions from AD patients and control subjects by next-generation sequencing (NGS). Our results from the analysis of ten AD patients reveal a variety of fungal and bacterial species, although some were more prominent than others. The fungal genera more prevalent in AD patients were Alternaria, Botrytis, Candida, and Malassezia. We also compared these genera with those found in elderly and younger subjects. One of the most prominent genera in control subjects was Fusarium. Principal component analysis clearly indicated that fungi from frontal cortex samples of AD brains clustered together and differed from those of equivalent control subjects. Regarding bacterial infection, the phylum Proteobacteria was the most prominent in both AD patients and controls, followed by Firmicutes, Actinobacteria, and Bacteroides. At the family level, Burkholderiaceae and Staphylococcaceae exhibited higher percentages in AD brains than in control brains. These findings could be of interest to guide targeted antimicrobial therapy for AD patients. Moreover, the variety of microbial species in each patient may constitute a basis for a better understanding of the evolution and severity of clinical symptoms in each patient.

Multiple sclerosis and mixed microbial infections. Direct identification of fungi and bacteria in nervous tissue.

Multiple sclerosis (MS) is the prototypical inflammatory disease of the central nervous system (CNS), leading to multifocal demyelination and neurodegeneration. The etiology of this incurable disease is unknown and remains a matter of intensive research. The possibility that microbial infections, such as viruses or bacteria, can trigger an autoimmune reaction in CNS tissue has been suggested. However, the recent demonstration that bacteria are present in CNS tissue points to a direct involvement of microbial infections in the etiology of MS. In the present study, we provide the first evidence of fungal infection in CNS tissue of MS patients, and demonstrate that fungal DNA from different species can be detected in the CNS. We used, nested PCR assays together with next-generation sequencing to identify the fungal species in the nervous tissue of 10 patients with MS. Strikingly, Trichosporon mucoides was found in the majority of MS patients, and particularly high levels of this fungus were found in two patients. Importantly, T. mucoides was not detected in the CNS of control subjects. We were also able to visualize fungal structures in CNS tissue sections by immunohistochemistry using specific antifungal antibodies, which also revealed the accumulation of a number of microbial cells in microfoci. Again, microbial structures were not observed in CNS sections from controls. In addition to fungi, neural tissue from MS patients was also positive for bacteria. In conclusion, our present observations point to the novel concept that MS could be caused by polymicrobial infections. Thus, mycosis of the CNS may be accompanied by opportunistic bacterial infection, promoting neuroinflammation and directly causing focal lesions, followed by demyelination and axonal injury.

Fungal infection in neural tissue of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease and the main cause of motor neuron pathology. The etiology of the disease remains unknown, and no effective therapy exists to halt the disease or improve the quality of life. Here, we provide compelling evidence for the existence of fungal infection in ALS. Immunohistochemistry analysis using a battery of antifungal antibodies revealed fungal structures such as yeast and hyphae in the motor cortex, the medulla and the spinal cord, in eleven patients with ALS. Some fungal structures were localized intracellularly and even intranuclearly, indicating that this infection is not the result of post-mortem colonization. By contrast, this burden of fungal infection cannot be observed in several CNS areas of control subjects. PCR analysis and next generation sequencing of DNA extracted from frozen neural tissue identified a variety of fungal genera including Candida, Malassezia, Fusarium, Botrytis, Trichoderma and Cryptococcus. Overall, our present observations provide strong evidence for mixed fungal infections in ALS patients. The exact mixed infection varies from patient to patient consistent with the different evolution and severity of symptoms in each ALS patient. These novel findings provide a logical explanation for the neuropathological observations of this disease, such as neuroinflammation and elevated chitinase levels, and could help to implement appropriate therapies.

Polymicrobial Infections In Brain Tissue From Alzheimer's Disease Patients.

Several studies have advanced the idea that the etiology of Alzheimer's disease (AD) could be microbial in origin. In the present study, we tested the possibility that polymicrobial infections exist in tissue from the entorhinal cortex/hippocampus region of patients with AD using immunohistochemistry (confocal laser scanning microscopy) and highly sensitive (nested) PCR. We found no evidence for expression of early (ICP0) or late (ICP5) proteins of herpes simplex virus type 1 (HSV-1) in brain sections. A polyclonal antibody against Borrelia detected structures that appeared not related to spirochetes, but rather to fungi. These structures were not found with a monoclonal antibody. Also, Borrelia DNA was undetectable by nested PCR in the ten patients analyzed. By contrast, two independent Chlamydophila antibodies revealed several structures that resembled fungal cells and hyphae, and prokaryotic cells, but most probably were unrelated to Chlamydophila spp. Finally, several structures that could belong to fungi or prokaryotes were detected using peptidoglycan and Clostridium antibodies, and PCR analysis revealed the presence of several bacteria in frozen brain tissue from AD patients. Thus, our results show that polymicrobial infections consisting of fungi and bacteria can be revealed in brain tissue from AD patients.

Identification of Fungal Species in Brain Tissue from Alzheimer's Disease by Next-Generation Sequencing.

The possibility that patients diagnosed with Alzheimer's disease (AD) have disseminated fungal infection has been recently advanced by the demonstration of fungal proteins and DNA in nervous tissue from AD patients. In the present study, next-generation sequencing (NGS) was used to identify fungal species present in the central nervous system (CNS) of AD patients. Initially, DNA was extracted from frozen tissue from four different CNS regions of one AD patient and the fungi in each region were identified by NGS. Notably, whereas a great variety of species were identified using the Illumina platform, Botrytis cinerea and Cryptococcus curvatus were common to all four CNS regions analyzed. Further analysis of entorhinal/cortex hippocampus samples from an additional eight AD patients revealed a variety of fungal species, although some were more prominent than others. Five genera were common to all nine patients: Alternaria, Botrytis, Candida, Cladosporium, and Malassezia. These observations could be used to guide targeted antifungal therapy for AD patients. Moreover, the differences found between the fungal species in each patient may constitute a basis to understand the evolution and severity of clinical symptoms in AD.

Fungal Enolase, ß-Tubulin, and Chitin Are Detected in Brain Tissue from Alzheimer's Disease Patients.

Recent findings provide evidence that fungal structures can be detected in brain tissue from Alzheimer's disease (AD) patients using rabbit polyclonal antibodies raised against whole fungal cells. In the present work, we have developed and tested specific antibodies that recognize the fungal proteins, enolase and ß-tubulin, and an antibody that recognizes the fungal polysaccharide chitin. Consistent with our previous studies, a number of rounded yeast-like and hyphal structures were detected using these antibodies in brain sections from AD patients. Some of these structures were intracellular and, strikingly, some were found to be located inside nuclei from neurons, whereas other fungal structures were detected extracellularly. Corporya amylacea from AD patients also contained enolase and ß-tubulin as revealed by these selective antibodies, but were devoid of fungal chitin. Importantly, brain sections from control subjects were usually negative for staining with the three antibodies. However, a few fungal structures can be observed in some control individuals. Collectively, these findings indicate the presence of two fungal proteins, enolase and ß-tubulin, and the polysaccharide chitin, in CNS tissue from AD patients. These findings are consistent with our hypothesis that AD is caused by disseminated fungal infection.

Corpora Amylacea of Brain Tissue from Neurodegenerative Diseases Are Stained with Specific Antifungal Antibodies.

The origin and potential function of corpora amylacea (CA) remains largely unknown. Low numbers of CA are detected in the aging brain of normal individuals but they are abundant in the central nervous system of patients with neurodegenerative diseases. In the present study, we show that CA from patients diagnosed with Alzheimer's disease (AD) contain fungal proteins as detected by immunohistochemistry analyses. Accordingly, CA were labeled with different anti-fungal antibodies at the external surface, whereas the central portion composed of calcium salts contain less proteins. Detection of fungal proteins was achieved using a number of antibodies raised against different fungal species, which indicated cross-reactivity between the fungal proteins present in CA and the antibodies employed. Importantly, these antibodies do not immunoreact with cellular proteins. Additionally, CNS samples from patients diagnosed with amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) also contained CA that were immunoreactive with a range of antifungal antibodies. However, CA were less abundant in ALS or PD patients as compared to CNS samples from AD. By contrast, CA from brain tissue of control subjects were almost devoid of fungal immunoreactivity. These observations are consistent with the concept that CA associate with fungal infections and may contribute to the elucidation of the origin of CA.

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Multiple Sclerosis.

Several neurochemical, neuropathological, and experimental data suggest a possible role of oxidative stress in the ethiopathogenesis of multiple sclerosis(MS). Heme-oxygenases(HMOX) are an important defensive mechanism against oxidative stress, and HMOX1 is overexpressed in the brain and spinal cord of MS patients and in experimental autoimmune encephalomyelitis(EAE). We analyzed whether common polymorphisms affecting the HMOX1 and HMOX2 genes are related with the risk to develop MS. We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations(CNVs) of these genes in 292 subjects MS and 533 healthy controls, using TaqMan assays. The frequencies of HMOX2 rs1051308AA genotype and HMOX2 rs1051308A and HMOX1 rs2071746A alleles were higher in MS patients than in controls, although only that of the SNP HMOX2 rs1051308 in men remained as significant after correction for multiple comparisons. None of the studied polymorphisms was related to the age at disease onset or with the MS phenotype. The present study suggests a weak association between HMOX2 rs1051308 polymorphism and the risk to develop MS in Spanish Caucasian men and a trend towards association between the HMOX1 rs2071746A and MS risk.

Different Brain Regions are Infected with Fungi in Alzheimer's Disease.

The possibility that Alzheimer's disease (AD) has a microbial aetiology has been proposed by several researchers. Here, we provide evidence that tissue from the central nervous system (CNS) of AD patients contain fungal cells and hyphae. Fungal material can be detected both intra- and extracellularly using specific antibodies against several fungi. Different brain regions including external frontal cortex, cerebellar hemisphere, entorhinal cortex/hippocampus and choroid plexus contain fungal material, which is absent in brain tissue from control individuals. Analysis of brain sections from ten additional AD patients reveals that all are infected with fungi. Fungal infection is also observed in blood vessels, which may explain the vascular pathology frequently detected in AD patients. Sequencing of fungal DNA extracted from frozen CNS samples identifies several fungal species. Collectively, our findings provide compelling evidence for the existence of fungal infection in the CNS from AD patients, but not in control individuals.

Cerebrospinal Fluid from Alzheimer's Disease Patients Contains Fungal Proteins and DNA.

The identification of biomarkers for Alzheimer's disease is important for patient management and to assess the effectiveness of clinical intervention. Cerebrospinal fluid (CSF) biomarkers constitute a powerful tool for diagnosis and monitoring disease progression. We have analyzed the presence of fungal proteins and DNA in CSF from AD patients. Our findings reveal that fungal proteins can be detected in CSF with different anti-fungal antibodies using a slot-blot assay. Additionally, amplification of fungal DNA by PCR followed by sequencing distinguished several fungal species. The possibility that these fungal macromolecules could represent AD biomarkers is discussed.

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

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.

Direct visualization of fungal infection in brains from patients with Alzheimer's disease.

Recently, we have reported the presence of fungal infections in patients with Alzheimer's disease (AD). Accordingly, fungal proteins and DNA were found in brain samples, demonstrating the existence of infection in the central nervous system. In the present work, we raised antibodies to specific fungal species and performed immunohistochemistry to directly visualize fungal components inside neurons from AD patients. Mice infected with Candida glabrata were initially used to assess whether yeast can be internalized in mammalian tissues. Using polyclonal rabbit antibodies against C. glabrata, rounded immunopositive cells could be detected in the cytoplasm of cells from liver, spleen, and brain samples in infected, but not uninfected, mice. Immunohistochemical analyses of tissue from the frontal cortex of AD patients revealed the presence of fungal material in a small percentage (~10%) of cells, suggesting the presence of infection. Importantly, this immunopositive material was absent in control samples. Confocal microscopy indicated that this fungal material had an intracellular localization. The specific morphology of this material varied between patients; in some instances, disseminated material was localized to the cytoplasm, whereas small punctate bodies were detected in other patients. Interestingly, fungal material could be revealed using different anti-fungal antibodies, suggesting multiple infections. In summary, fungal infection can only be observed using specific anti-fungal antibodies and only a small percentage of cells contain fungi. Our findings provide an explanation for the hitherto elusive detection of fungi in AD brains, and are consistent with the idea that fungal cells are internalized inside neurons.

NQO1 gene rs1800566 variant is not associated with risk for multiple sclerosis.

BACKGROUND: A possible role of oxidative stress in the pathogenesis of multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis has been suggested. The detoxification enzyme NAD(P)H dehydrogenase, quinone 1 (NQO1) has been found up-regulated in MS lesions. A previous report described an association between the SNP rs1800566 in the NQO1 gene and the risk for MS in the Greek population. The aim of this study was to replicate a possible influence of the. SNP rs1800566 in the NQO1 gene in the risk for MS in the Spanish Caucasian population. METHODS: We analyzed allelic and genotypic frequency of NQO1 rs1800566 in 290 patients with MS and 310 healthy controls, using TaqMan Assays. RESULTS: NQO1 rs1800566 allelic and genotypic frequencies did not differ significantly between MS patients and controls, and were unrelated with age of onset of MS, gender, and clinical type of MS. CONCLUSIONS: Our results indicate that NQO1 rs1800566 does not have an effect on MS disease risk.

Fungal infection in patients with Alzheimer's disease.

Alzheimer's disease is a progressive neurodegenerative disorder that leads to dementia mainly among the elderly. This disease is characterized by the presence in the brain of amyloid plaques and neurofibrillary tangles that provoke neuronal cell death, vascular dysfunction, and inflammatory processes. In the present work, we have analyzed the existence of fungal infection in Alzheimer's disease patients. A proteomic analysis provides compelling evidence for the existence of fungal proteins in brain samples from Alzheimer's disease patients. Furthermore, PCR analysis reveals a variety of fungal species in these samples, dependent on the patient and the tissue tested. DNA sequencing demonstrated that several fungal species can be found in brain samples. Together, these results show that fungal macromolecules can be detected in brain from Alzheimer's disease patients. To our knowledge these findings represent the first evidence that fungal infection is detectable in brain samples from Alzheimer's disease patients. The possibility that this may represent a risk factor or may contribute to the etiological cause of Alzheimer's disease is discussed.

MAPT gene rs1052553 variant is not associated with the risk for multiple sclerosis.

BACKGROUND/OBJECTIVES: Some experimental data suggest a possible role of tau protein in the pathogenesis of multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis. The aim of this study was to investigate a possible influence of the SNP rs1052553 in the MAPT gene in the risk for relapsing bout onset (relapsing-remitting and secondary progressive) MS. METHODS: We analyzed the allelic and genotype frequency of MAPT rs1052553, which has been associated with some neurodegenerative diseases, in 259 patients with relapsing bout onset MS and 291 healthy controls, using TaqMan Assays. RESULTS: MAPT rs1052553 allelic and genotype frequencies did not differ significantly between relapsing bout onset MS patients and controls, and were unrelated with the age of onset of MS or gender. CONCLUSIONS: These results suggest that MAPT rs1052553 polymorphism is not related with the risk for relapsing bout onset MS.