Alberto Gómez-Ramos

Extracellular tau is toxic to neuronal cells

The degeneration of neurons in disorders such as Alzheimers disease has an immediate consequence, the release of intracellular proteins into the extracellular space. One of these proteins, tau, has proven to be toxic when added to cultured neuronal cells. This toxicity varies according to the degree of protein aggregation. The addition of tau to cultured neuroblastoma cells provoked an increase in the levels of intracellular calcium, which is followed by cell death. We suggest that this phenomenon may be mediated by the interaction of tau with muscarinic receptors, which promotes the liberation of calcium from intracellular stores.

Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells.

Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.

Tau overexpression results in its secretion via membrane vesicles.

Background: Tau protein, the main component of neurofibrillary tangles, could be found in the extracellular space upon neuronal death or, as it has recently been suggested, could be secreted from cells through membrane vesicles. Objective: The purpose of this communication is to confirm that upon neuronal death, tau protein can be found, indeed, in the extracellular space and to analyze if tau could be secreted outside the cell in an alternative way. Methods: We have tested not only the extracellular release of tau, but also the toxicity of this extracellular tau. To do these studies, we have used neuronal cell cultures and tau-overexpressing non-neuronal cells. Membrane vesicles were isolated from culture medium from tau-overexpressing non-neuronal cells. Results: Our results indicate that extracellular tau, arising after neuron death, could be a toxic agent for neighboring neurons. On the other hand, we have found that an overexpression of tau protein could result in its secretion through membrane vesicles. However, the presence of this secreted tau does not result in cell death. Conclusion: We conclude that extracellular tau could arise by two different ways, by cell death or by secretion through membrane vesicles.

Characteristics and consequences of muscarinic receptor activation by tau protein

It was recently suggested that tau protein released as a result of neuronal death is toxic to neighbouring cells, an effect that is mediated through the activation of muscarinic M1 and/or M3 receptors. Nevertheless, why tau protein and not other native muscarinic agonists, like ACh, can induce this neurotoxicity remains unknown. To clarify this issue, we analysed the different responses and properties of muscarinic receptors in response to stimulation by tau or ACh. The results revealed that the tau protein has an affinity for muscarinic receptors of around one order of magnitude higher than that of ACh. Furthermore, while the repeated stimulation with ACh induces desensitization of the muscarinic receptors, reiterate stimulation with tau failed to produce this phenomenon. Finally, we found the tau protein to be very stable in the extracellular milieu. These studies provide valuable information to help understand tau toxicity on neural cells bearing M1 or M3 muscarinic receptors and its contribution to neurodegenerative progression in tauopathies.

Tau in neurodegenerative diseases: Tau phosphorylation and assembly

The possible link between tau phosphorylation and tau assembly in these neurodegenerative diseases known as tauopathies is described. Additionally, this link is supported by anin vitro experiment showing the higher capacity of phosphotau to assemble in some specific conditions; and, by a recently reported experiment using a tau transgenic mouse model.

Additional mechanisms conferring genetic susceptibility to Alzheimer’s disease

Familial Alzheimer’s disease (AD), mostly associated with early onset, is caused by mutations in three genes (APP, PSEN1, and PSEN2) involved in the production of the amyloid β peptide. In contrast, the molecular mechanisms that trigger the most common late onset sporadic AD remain largely unknown. With the implementation of an increasing number of case-control studies and the upcoming of large-scale genome-wide association studies there is a mounting list of genetic risk factors associated with common genetic variants that have been associated with sporadic AD. Besides apolipoprotein E, that presents a strong association with the disease (OR∼4), the rest of these genes have moderate or low degrees of association, with OR ranging from 0.88 to 1.23. Taking together, these genes may account only for a fraction of the attributable AD risk and therefore, rare variants and epistastic gene interactions should be taken into account in order to get the full picture of the genetic risks associated with AD. Here, we review recent whole-exome studies looking for rare variants, somatic brain mutations with a strong association to the disease, and several studies dealing with epistasis as additional mechanisms conferring genetic susceptibility to AD. Altogether, recent evidence underlines the importance of defining molecular and genetic pathways, and networks rather than the contribution of specific genes.

Somatic Signature of Brain-Specific Single Nucleotide Variations in Sporadic Alzheimer's Disease

BACKGROUND Although genome-wide association studies have shown that genetic factors increase the risk of suffering late-onset, sporadic Alzheimers disease (SAD), the molecular mechanisms responsible remain largely unknown. OBJECTIVE The aim of the study was to investigate the presence of somatic, brain-specific single nucleotide variations (SNV) in the hippocampus of SAD samples. METHODS By using bioinformatic tools, we compared whole exome sequences in paired blood and hippocampal genomic DNAs from 17 SAD patients and from 2 controls and 2 vascular dementia patients. RESULTS We found a remarkable number of SNVs in SAD brains (~575 per patient) that were not detected in blood. Loci with hippocampus-specific (hs)-SNVs were common to several patients, with 38 genes being present in 6 or more patients out of the 17. While some of these SNVs were in genes previously related to SAD (e.g., CSMD1, LRP2), most hs-SNVs occurred in loci previously unrelated to SAD. The most frequent genes with hs-SNVs were associated with neurotransmission, DNA metabolism, neuronal transport, and muscular function. Interestingly, 19 recurrent hs-SNVs were common to 3 SAD patients. Repetitive loci or hs-SNVs were underrepresented in the hippocampus of control or vascular dementia donors, or in the cerebellum of SAD patients. CONCLUSION Our data suggest that adult blood and brain have different DNA genomic variations, and that somatic genetic mosaicism and brain-specific genome reshaping may contribute to SAD pathogenesis and cognitive differences between individuals.

AD genetic risk factors and tau spreading.

Development of tau pathology is associated with progressive neuronal loss and cognitive decline. In the brains of Alzheimers disease (AD) patients, tau pathology propagates according to an anatomically defined pattern with relatively uniform distribution, and contributes to cognitive decline in age-associated tauopathy (Braak and Braak, 1991; Saito et al., 2004). Recently, it has been revealed that tau, which is an intracellular protein, can appear in the extracellular space, likely due to an exocytosis mechanism. Such extracellular tau could then be internalized into neighboring cells in at least two different ways depending on its aggregation state. In the case of soluble monomeric or small oligomeric tau protein, the endocytosis appears to be clathrin dependent (reviewed in Rubinsztein, 2006). In contrast, larger aggregates of tau could bind heparin in the extracellular matrix and be internalized through macropinocytosis (Holmes et al., 2014). As a result of exocytosis and endocytosis, the spreading of tau can occur in various neurodegenerative diseases (tauopathies) including AD. In this opinion article we have focused on the endocytosis mechanism. Several genetic risk factors have been associated with a higher probability of developing sporadic Alzheimers disease (SAD). The Alzheimer Association (http://www.alzforum.org/) has ranked the top six risk genes, shown in Table ​Table1,1, based on genome-wide association studies (GWAS). Table 1 The top six AD risk genes that interact with tau.

Distinct X-chromosome SNVs from some sporadic AD samples.

Sporadic Alzheimer disease (SAD) is the most prevalent neurodegenerative disorder. With the development of new generation DNA sequencing technologies, additional genetic risk factors have been described. Here we used various methods to process DNA sequencing data in order to gain further insight into this important disease. We have sequenced the exomes of brain samples from SAD patients and non-demented controls. Using either method, we found a higher number of single nucleotide variants (SNVs), from SAD patients, in genes present at the X chromosome. Using the most stringent method, we validated these variants by Sanger sequencing. Two of these gene variants, were found in loci related to the ubiquitin pathway (UBE2NL and ATXN3L), previously do not described as genetic risk factors for SAD.

Variations in brain DNA

It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain) of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain.