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 β-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 β-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 β-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.

Argyrophylic grain disease (AGD) is a neurodegenerative condition that has been classified among the sporadic tauopathies. Entities in this group present intracellular aggregates of hyperphosphorylated tau, giving rise to characteristic neuronal and glial inclusions. In different tauopathies, the proportion of several tau isoforms present in the aggregates shows specific patterns. AGD has been tentatively classified in the 4R group (predominance of 4R tau isoforms) together with progressive supranuclear palsy and corticobasal degeneration. Pick's disease is included in the 3R group (predominance of 3R isoforms), whereas tau pathology of Alzheimer's disease represents and intermediate group (3 or 4 repeats [3R plus 4R, respectively] isoforms). In this work, we have analyzed tau present in aggregates isolated from brain samples of patients with argyrophylic grain disease. Our results indicate that the main tau isoform present in aggregates obtained from patients with AGD is a hyperphosphorylated isoform containing exons 2 and 10 but lacking exon 3.

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.

The dendritic spines on pyramidal cells represent the main postsynaptic elements of cortical excitatory synapses and they are fundamental structures in memory, learning and cognition. In the present study, we used intracellular injections of Lucifer yellow in fixed tissue to analyse over 19 500 dendritic spines that were completely reconstructed in three dimensions along the length of the basal dendrites of pyramidal neurons in the parahippocampal cortex and CA1 of patients with Alzheimer's disease. Following intracellular injection, sections were immunostained for anti-Lucifer yellow and with tau monoclonal antibodies AT8 and PHF-1, which recognize tau phosphorylated at Ser202/Thr205 and at Ser396/404, respectively. We observed that the diffuse accumulation of phospho-tau in a putative pre-tangle state did not induce changes in the dendrites of pyramidal neurons, whereas the presence of tau aggregates forming intraneuronal neurofibrillary tangles was associated with progressive alteration of dendritic spines (loss of dendritic spines and changes in their morphology) and dendrite atrophy, depending on the degree of tangle development. Thus, the presence of phospho-tau in neurons does not necessarily mean that they suffer severe and irreversible effects as thought previously but rather, the characteristic cognitive impairment in Alzheimer's disease is likely to depend on the relative number of neurons that have well developed tangles.

Immunohistochemical staining of tissues is a powerful tool used to delineate the presence or absence of an antigen. During the last 30 years, antigen visualization in human brain tissue has been significantly limited by the masking effect of fixatives. In the present study, we have used a new method for antigen retrieval in formalin-fixed human brain tissue and examined the effectiveness of this protocol to reveal masked antigens in tissues with both short and long formalin fixation times. This new method, which is based on the use of citraconic acid, has not been previously utilized in brain tissue although it has been employed in various other tissues such as tonsil, ovary, skin, lymph node, stomach, breast, colon, lung and thymus. Thus, we reported here a novel method to carry out immunohistochemical studies in free-floating human brain sections. Since fixation of brain tissue specimens in formaldehyde is a commonly method used in brain banks, this new antigen retrieval method could facilitate immunohistochemical studies of brains with prolonged formalin fixation times.

Seipin is a widely expressed protein but with highest levels found in the brain and testes. Seipin function is not yet completely understood, therefore the aim of this study was to evaluate the expression of BSCL2 transcripts in the central nervous system (CNS) of humans and investigate the effect of their overexpression on a neuron model and their relationship with oxidative stress protection, as well as shed light on the pathogenic mechanisms of Celia's Encephalopathy. We analyzed the expression of BSCL2 transcripts using real-time RT-PCR in samples across the brain regions of subjects who underwent necropsy and from a case with Celia's Encephalopathy. The transcript encoding the long seipin isoform (BSCL2-203, 462 aa) is expressed primarily in the brain and its expression is inversely correlated with age in the temporal lobe, amygdala, and hypothalamus. Strong positive correlations were found between BSCL2 expression and some genes encoding protective enzymes against oxidative stress including SOD1 and SOD2, as well as peroxisome proliferator-activated receptor gamma (PPARG) in the amygdala. These results were experimentally corroborated by overexpressing BSCL2 transcripts in SH-SY5Y cells with lentiviral transduction and assessing their effects on neuron differentiated cells. Confocal microscopy studies showed that both seipin and PEX16 are closely expressed in the hypothalami of healthy human brains, and PEX16 was absent in the same region of the PELD case. We hypothesize that seipin has specific CNS functions and may play a role in peroxisome biogenesis.

Alzheimer's disease (AD) is characterized by a progressive loss of neurons and cognitive functions. Therefore, early diagnosis of AD is critical. The development of practical and non-invasive diagnostic tests for AD remains, however, an unmet need. In the present proof-of-concept study we investigated tear fluid as a novel source of disease-specific protein and microRNA-based biomarkers for AD development using samples from patients with mild cognitive impairment (MCI) and AD. Tear protein content was evaluated via liquid chromatography-mass spectrometry and microRNA content was profiled using a genome-wide high-throughput PCR-based platform. These complementary approaches identified enrichment of specific proteins and microRNAs in tear fluid of AD patients. In particular, we identified elongation initiation factor 4E (eIF4E) as a unique protein present only in AD samples. Total microRNA abundance was found to be higher in tears from AD patients. Among individual microRNAs, microRNA-200b-5p was identified as a potential biomarker for AD with elevated levels present in AD tear fluid samples compared to controls. Our study suggests that tears may be a useful novel source of biomarkers for AD and that the identification and verification of biomarkers within tears may allow for the development of a non-invasive and cost-effective diagnostic test for AD.

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele.

Components of the innate immune complement system have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS); however, a comprehensive examination of complement expression in this disease has not been performed. This study therefore aimed to determine the expression of complement components (C1qB, C4, factor B, C3/C3b, C5 and CD88) and regulators (CD55 and CD59a) in the lumbar spinal cord of hSOD1(G93A) mice during defined disease stages.

Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) represent two distinct clinical entities belonging to a wider group, generically named as conformational disorders that share common pathophysiologic mechanisms. It is well-established that the APOE ε4 allele and homozygosity at polymorphic codon 129 in the PRNP gene are the major genetic risk factors for AD and human prion diseases, respectively. However, the roles of PRNP in AD, and APOE in CJD are controversial. In this work, we investigated for the first time, APOE and PRNP genotypes simultaneously in 474 AD and 175 sporadic CJD (sCJD) patients compared to a common control population of 335 subjects. Differences in genotype distribution between patients and control subjects were studied by logistic regression analysis using age and gender as covariates. The effect size of risk association and synergy factors were calculated using the logistic odds ratio estimates. Our data confirmed that the presence of APOE ε4 allele is associated with a higher risk of developing AD, while homozygosity at PRNP gene constitutes a risk for sCJD. Opposite, we found no association for PRNP with AD, nor for APOE with sCJD. Interestingly, when AD and sCJD patients were stratified according to their respective main risk genes (APOE for AD, and PRNP for sCJD), we found statistically significant associations for the other gene in those strata at higher previous risk. Synergy factor analysis showed a synergistic age-dependent interaction between APOE and PRNP in both AD (SF = 3.59, p = 0.027), and sCJD (SF = 7.26, p = 0.005). We propose that this statistical epistasis can partially explain divergent data from different association studies. Moreover, these results suggest that the genetic interaction between APOE and PRNP may have a biological correlate that is indicative of shared neurodegenerative pathways involved in AD and sCJD.

Mutations in the gene encoding fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), but the mechanisms by which these mutants trigger neurodegeneration remain unknown. Endoplasmic reticulum (ER) stress is increasingly recognized as an important and early pathway to motor neuron death in ALS. FUS is normally located in the nucleus but in ALS, FUS redistributes to the cytoplasm and forms inclusions. In this study, we investigated whether FUS induces ER stress in a motor neuron like cell line (NSC-34). We demonstrate that ER stress is triggered in cells expressing mutant FUS, and this is closely associated with redistribution of mutant FUS to the cytoplasm. Mutant FUS also colocalized with protein disulfide-isomerase (PDI), an important ER chaperone, in NSC-34 cells and PDI was colocalized with FUS inclusions in human ALS lumbar spinal cords, in both sporadic ALS and mutant FUS-linked familial ALS tissues. These findings implicate ER stress in the pathophysiology of FUS, and provide evidence for common pathogenic pathways in ALS linked to the ER.

A controversy exists as to whether de novo-generated neuronal tetraploidy (dnNT) occurs in Alzheimer's disease. In addition, the presence of age-associated dnNT in the normal brain remains unexplored. Here we show that age-associated dnNT occurs in both superficial and deep layers of the cerebral cortex of adult mice, a process that is blocked in the absence of E2F1, a known regulator of cell cycle progression. This blockage correlates with improved cognition despite compromised neurogenesis in the adult hippocampus was confirmed in mice lacking the E2f1 gene. We also show that the human cerebral cortex contains tetraploid neurons. In normal humans, age-associated dnNT specifically occurs in the entorhinal cortex whereas, in Alzheimer, dnNT also affects association cortices prior to neurofibrillary tangle formation. Alzheimer-associated dnNT is likely potentiated by altered amyloid precursor protein (APP) processing as it is enhanced in the cerebral cortex of young APPswe/PS1deltaE9 mice, long before the first amyloid plaques are visible in their brains. In contrast to age-associated dnNT, enhanced dnNT in APPswe/PS1deltaE9 mice mostly affects the superficial cortical layers. The correlation of dnNT with reduced cognition in mice and its spatiotemporal course, preceding and recapitulating Alzheimer-associated neuropathology, makes this process a potential target for intervention in 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.

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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons in the brain and spinal cord. ALS and frontotemporal dementia (FTD) are overlapping diseases with shared pathological features. Affected neurons of people with ALS and FTD typically contain ubiquitin-immunoreactive inclusions, of which TDP-43 (Tar DNA-binding protein of 43 kDa) is a major component. However, what triggers the formation of these abnormal TDP-43 inclusions is unclear. Previously, we identified CCNF mutations in cohorts of familial and sporadic cases of ALS and FTD. CCNF encodes cyclin F, the substrate-binding component of a multiprotein E3 ubiquitin ligase complex that ubiquitylates and subsequently directs a set of protein substrates for proteasomal degradation. Here, we explored the relationship between cyclin F and TDP-43.

Amyotrophic lateral sclerosis (ALS)- and frontotemporal dementia (FTD)-linked mutations in CCNF have been shown to cause dysregulation to protein homeostasis. CCNF encodes for cyclin F, which is part of the cyclin F-E3 ligase complex SCFcyclinF known to ubiquitylate substrates for proteasomal degradation. In this study, we identified a function of cyclin F to regulate substrate solubility and show how cyclin F mechanistically underlies ALS and FTD disease pathogenesis. We demonstrated that ALS and FTD-associated protein sequestosome-1/p62 (p62) was a canonical substrate of cyclin F which was ubiquitylated by the SCFcyclinF complex. We found that SCFcyclin F ubiquitylated p62 at lysine(K)281, and that K281 regulated the propensity of p62 to aggregate. Further, cyclin F expression promoted the aggregation of p62 into the insoluble fraction, which corresponded to an increased number of p62 foci. Notably, ALS and FTD-linked mutant cyclin F p.S621G aberrantly ubiquitylated p62, dysregulated p62 solubility in neuronal-like cells, patient-derived fibroblasts and induced pluripotent stem cells and dysregulated p62 foci formation. Consistently, motor neurons from patient spinal cord tissue exhibited increased p62 ubiquitylation. We suggest that the p.S621G mutation impairs the functions of cyclin F to promote p62 foci formation and shift p62 into the insoluble fraction, which may be associated to aberrant mutant cyclin F-mediated ubiquitylation of p62. Given that p62 dysregulation is common across the ALS and FTD spectrum, our study provides insights into p62 regulation and demonstrates that ALS and FTD-linked cyclin F mutant p.S621G can drive p62 pathogenesis associated with ALS and FTD.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.

DNA is the most stable nucleic acid and most important store of genetic information. DNA sequences are conserved in virtually all the cells of a multicellular organism. To analyze the sequences of various individuals with distinct pathological disorders, DNA is routinely isolated from blood, independently of the tissue that is the target of the disease. This approach has proven useful for the identification of familial diseases where mutations are present in parental germinal cells. With the capacity to compare DNA sequences from distinct tissues or cells, present technology can be used to study whether DNA sequences in tissues are invariant. Here we explored the presence of specific SNVs (Single Nucleotide Variations) in various tissues of the same individual. We tested for the presence of tissue-specific exonic SNVs, taking blood exome as a control. We analyzed the chromosomal location of these SNVs. The number of SNVs per chromosome was found not to depend on chromosome length, but mainly on the number of protein-coding genes per chromosome. Although similar but not identical patterns of chromosomal distribution of tissue-specific SNVs were found, clear differences were detected. This observation supports the notion that each tissue has a specific SNV exome signature.

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.

During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases.