Validation of Suspected Somatic Single Nucleotide Variations in the Brain of Alzheimer's Disease Patients.

Next-generation sequencing techniques and genome-wide association study analyses have provided a huge amount of data, thereby enabling the identification of DNA variations and mutations related to disease pathogenesis. New techniques and software tools have been developed to improve the accuracy and reliability of this identification. Most of these tools have been designed to discover and validate single nucleotide variants (SNVs). However, in addition to germ-line mutations, human tissues bear genomic mosaicism, which implies that somatic events are present only in low percentages of cells within a given tissue, thereby hindering the validation of these variations using standard genetic tools. Here we propose a new method to validate some of these somatic mutations. We combine a recently developed software with a method that cuts DNA by using restriction enzymes at the sites of the variation. The non-cleaved molecules, which bear the SNV, can then be amplified and sequenced using Sanger's technique. This procedure, which allows the detection of alternative alleles present in as few as 10% of cells, could be of value for the identification and validation of low frequency somatic events in a variety of tissues and diseases.

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 ß 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.

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.

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 Alzheimer's 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.

Similarities and differences between exome sequences found in a variety of tissues from the same individual.

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.

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.

Tissue-nonspecific alkaline phosphatase promotes the neurotoxicity effect of extracellular tau.

There is solid evidence indicating that hyperphosphorylated tau protein, the main component of intracellular neurofibrillary tangles present in the brain of Alzheimer disease patients, plays a key role in progression of this disease. However, it has been recently reported that extracellular unmodified tau protein may also induce a neurotoxic effect on hippocampal neurons by activation of M1 and M3 muscarinic receptors. In the present work we show an essential component that links both effects, which is tissue-nonspecific alkaline phosphatase (TNAP). This enzyme is abundant in the central nervous system and is mainly required to keep control of extracellular levels of phosphorylated compounds. TNAP dephosphorylates the hyperphosphorylated tau protein once it is released upon neuronal death. Only the dephosphorylated tau protein behaves as an agonist of muscarinic M1 and M3 receptors, provoking a robust and sustained intracellular calcium increase finally triggering neuronal death. Interestingly, activation of muscarinic receptors by dephosphorylated tau increases the expression of TNAP in SH-SY5Y neuroblastoma cells. An increase in TNAP activity together with increases in protein and transcript levels were detected in Alzheimer disease patients when they were compared with healthy controls.

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.

Role of polyglycine repeats in the regulation of glycogen synthase kinase activity.

Glycogen synthase kinase (GSK3) activity present in one cell is the consequence of the sum of the activities of two different proteins called GSK3alpha and GSK3beta. These isoenzymes are coded by two different genes and share an almost identical sequence at their catalytic domain, but differ in the sequence of putative regulatory regions. In this review, we propose that glycine repeats present only in GSK3alpha may result in the different cleavage of both isoenzymes by the protease calpain, a cleavage that modifies GSK3 activity.

Extracellular tau is toxic to neuronal cells.

The degeneration of neurons in disorders such as Alzheimer's 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.

Inhibition of GSK3 dependent tau phosphorylation by metals.

One of the main pathological characteristics of Alzheimer's disease is the presence in the brain of the patients of an aberrant structure, the paired helical filaments, composed of hyperphosphorylated tau. The level of tau phosphorylation has been correlated with the capacity for tau aggregation. Thus, the mechanism for tau phosphorylation could be important to clarify those pathological features in Alzheimer's disease. Tau protein could be modified by different kinases, being GSK3 the one that could modify more sites of that protein. GSK3 activity could be modulate by the presence of metals like magnesium that can be required for the proper function of the kinase, whereas, metals like manganesum or lithium inhibit the activity of the kinase. Many works have been done to study the inhibition of GSK3 by lithium, a specific inhibitor of that kinase. More recently, it has been indicated that sodium tungstate could also inhibit GSK3 through a different mechanism. In this review, we discuss the effect of these two metals, lithium and tungstate, on GSK3 (or tau I kinase) activity.

Sodium tungstate decreases the phosphorylation of tau through GSK3 inactivation.

Tungstate treatment increases the phosphorylation of glycogen synthase kinase-3beta (GSK3beta) at serine 9, which triggers its inactivation both in cultured neural cells and in vivo. GSK3 phosphorylation is dependent on the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) induced by tungstate. As a consequence of GSK3 inactivation, the phosphorylation of several GSK3-dependent sites of the microtubule-associated protein tau decreases. Tungstate reduces tau phosphorylation only in primed sequences, namely, those prephosphorylated by other kinases before GSK3beta modification, which are serines 198, 199, or 202 and threonine 231. The phosphorylation at these sites is involved in reduction of the interaction of tau with microtubules that occurs in Alzheimer's disease.

Characterization of Alzheimer paired helical filaments by electron microscopy.

We show how electron microscopy can be used to answer several critical issues in neurodegenerative disorders that course with the formation of aberrant filamentous structures. Thus, electron microscopy is a useful technique to study in vitro assembly of pathogenic proteins, to map the regions involved in filament formation, as well as to detect by immunoelectron microscopy which proteins bind to the filaments. Furthermore, electron microscopy is the main technique used to discover if an animal model develops fibrillar pathology and if those filaments are similar to those found in human patients. This review focuses on Alzheimer's disease and related tauopathies, although similar studies have been done with other neurodegenerative disorders as, for example, Huntington's disease.

Tau phosphorylation and assembly.

Neurofibrillary tangles, one of the aberrant structures found in the brain of Alzheimer's disease patients are mainly composed of tau in hyperphosphorylated form. Thus, a possible relation between phosphorylation and assembly of tau proteins has been analysed. By doing in vitro studies we have observed that in certain conditions, where compounds from oxidative stress are present, the capacity of tau for self assembly increases upon phosphorylation.

Expression of an altered form of tau in Sf9 insect cells results in the assembly of polymers resembling Alzheimer's paired helical filaments.

Tau is the main component of the paired helical filaments (PHFs), aberrant structures that develop in the brain of Alzheimer's disease (AD) patients and other tauopathies like frontotemporal dementia and parkinsonism associated to chromosome 17 (FTDP-17). Previous work has shown that tau overexpression in Sf9 insect cells results in the formation of long cytoplasmatic extensions as a consequence of microtubule stabilization and bundling. Throughout this work, we have taken studies in this system further by overexpression of an altered form of tau characteristic of FTDP-17, which includes three mutations (G272V, P301L and R406W) and biochemically behaves as a hyperphosphorylated form of the protein, with the aim of developing an in vitro model which would favour the formation of tau aggregates. Our results indicate that filaments resembling PHFs assemble when Sf9 cells overexpress FTDP-17 tau. The amount of these polymers is reduced in lithium treated cells which suggests that phosphorylation of FTDP-17 tau by GSK3 induces a conformational change favouring the formation of fibrillar polymers.

Assembly in vitro of tau protein and its implications in Alzheimer's disease.

Tau is a microtubule associated protein that is also the main component of the aberrant filaments that form aberrant structures like the neuropil threads or the neurofibrillary tangles, found in the brain of Alzheimer's disease patients. The assembly of tau aberrant filaments could be reproduced in vitro by using a high concentration of tau protein or, at lower protein concentrations, by adding some compounds like polyanions, fatty acids (and derivates), and others. In this mini-review a descriptive analysis of the different conditions needed for in vitro tau polymerization are summarized.

Effect of the lipid peroxidation product acrolein on tau phosphorylation in neural cells.

A hallmark of several neurodegenerative disorders, including Alzheimer's disease and tauopathies, is the hyperphosphorylation of the microtubule-associated protein tau. Tau phosphorylation by proline-directed and non-proline-directed protein kinases has been tested using antibodies PHF1 and 12E8, respectively. The effect of the lipid peroxidation product acrolein on these modes of phosphorylation has been assayed. We have found that acrolein, a peroxidation product from arachidonic acid, increases the phosphorylation of tau at the site recognized by PHF-1 both in human neuroblastoma cells and in primary cultures of mouse embryo cortical neurons. Whereas the basal phosphorylation of tau protein at the PHF1 site seems to be largely mediated by glycogen synthase kinase-3 (which is also activated in response to Abeta peptide), the acrolein-induced tau hyperphosphorylation at the same site is also due to p38 stress-activated kinase. These results support the view that oxidative stress and subsequent formation of lipid peroxidation products may contribute to tau protein phosphorylation in Alzheimer's disease and tauopathies.

Formation of aberrant phosphotau fibrillar polymers in neural cultured cells.

Here we show, for the first time, the in vitro formation of filamentous aggregates of phosphorylated tau protein in SH-SY5Y human neuroblastoma cells. The formation of such aberrant aggregates, similar to those occurring in vivo in Alzheimer's disease and other tauopathies, requires okadaic acid, a phosphatase inhibitor, to increase the level of phosphorylated tau, and hydroxynonenal, a product of oxidative stress that selectively adducts and modifies phosphorylated tau. Our findings suggest that both phosphorylation and oxidative modification are required for tau filament formation. Importantly, the in vitro formation of intracellular tau aggregates could be used as a model of tau polymerization and facilitate the development of novel therapeutic approaches.