Gustavo Basurto-Islas

Cleavage and conformational changes of tau protein follow phosphorylation during Alzheimer’s disease

Phosphorylation, cleavage and conformational changes in tau protein all play pivotal roles during Alzheimer’s disease (AD). In an effort to determine the chronological sequence of these changes, in this study, using confocal microscopy, we compared phosphorylation at several sites (Ser199/202/396/404/422‐Thr205 and the second repeat domain), cleavage of tau (D421) and the canonical conformational Alz‐50 epitope. While all of these posttranslational modifications are found in neurofibrillary tangles (NFTs) at all stages of the disease, we found significantly higher numbers of phospho‐tau positive NFTs when compared with cleaved tau (P = 0.006 in Braak III; P = 0.002 in Braak IV; P = 0.012 in Braak V) or compared with the Alz‐50 epitope (P < 0.05). Consistent with these findings, in a double transgenic mice model (Tet/GSK‐3β/VLW) overexpressing the enzyme glycogen synthase kinase‐3β (GSK‐3β) and tau with a triple FTDP‐17 mutation (VLW) with AD‐like neurodegeneration, phosphorylation at sites Ser199/202‐Thr205 was greater than truncated tau. Taken together, these data strongly support the notion that the conformational changes and truncation of tau occur after the phosphorylation of tau. We propose two probable pathways for the pathological processing of tau protein during AD, either phosphorylation and cleavage of tau followed by the Alz‐50 conformational change or phosphorylation followed by the conformational change and cleavage as the last step.

Accumulation of Aspartic Acid421- and Glutamic Acid391-Cleaved Tau in Neurofibrillary Tangles Correlates With Progression in Alzheimer Disease

Truncations of tau protein at aspartic acid421 (D421) and glutamic acid391 (E391) residues are associated with neurofibrillary tangles (NFTs) in the brains of Alzheimer disease (AD) patients. Using immunohistochemistry with antibodies to D421- and E391-truncated tau (Tau-C3 and MN423, respectively), we correlated the presence of NFTs composed of these truncated tau proteins with clinical and neuropathologic parameters in 17 AD and 23 non-AD control brains. The densities of NFTs composed of D421- or E391-truncated tau correlated with clinical dementia index and Braak staging in AD. Glutamic acid391 tau truncation was prominent in the entorhinal cortex, whereas D421 truncation was prominent in the subiculum, suggesting that NFTs composed of either D421- or E391-truncated tau may be formed mutually exclusively in these areas. Both truncations were associated with the prevalence of the apolipoprotein E ϵ4 allele. By double labeling, intact tau in NFTs was commonly associated with D421-cleaved tau but not with E391-truncated tau; D421-cleaved tau was never associated with E391-truncated tau. These results indicate that tau is not randomly proteolyzed at different domains, and that proteolysis occurs sequentially from the C-terminus to inner regions of tau in AD progression. Identification of NFTs composed of tau at different stages of truncation may facilitate assessment of neurofibrillary pathology in AD.

Truncation of Tau Protein and its Pathological Significance in Alzheimer's Disease

Abnormal posttranslational modifications of tau protein lead it to aggregate into paired helical filaments in Alzheimers disease (AD). The mechanisms involved in the early pathological processing of tau and the induction of a polymeric state seem to progress through a sequential pattern of changes mainly involving abnormal phosphorylation, conformational changes and truncation. While proteolytic cleavage of tau protein during the progression of AD has not been comprehensively analyzed, tau is a substrate for several intracellular proteases. Furthermore, abnormal regulation of proteolytic events, including those associated with apoptosis, may generate truncated tau subproducts which in turn may be toxic to neurons per se and capable of polymerization at a faster rate. Accumulation of tau fibrils has long been controversial, with much debate concerning the true toxicity of polymerized tau. The development of different transgenic mice overexpressing tau protein, the generation of cell models expressing tau, and the in vitro polymerization paradigms have significantly enhanced our understanding of the biophysics and pathological properties of tau polymers in AD, as well as in other tau pathologies. This review will discuss the pathological role of truncated tau protein in the context of toxicity and neurofibrillary tangle formation and maturation and its significance in clinical dementia.

Causes versus effects: the increasing complexities of Alzheimer's disease pathogenesis

Amyloid plaques and neurofibrillary tangles are the hallmarks of Alzheimer’s disease and have been the focus of disease etiology and pathogenesis. However, in the larger picture of a complex disease, the precise etiology of the lesions per se, as well as the clinical disease, remain to be defined. In this regard, to date no single process has been identified as a useful target and treatment efforts have shown no meaningful progress. Therefore, alternative ideas that may lead to new and effective treatment options are much needed.

Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau

Background: In Alzheimer brain, I2PP2A is translocated from the neuronal nucleus to the cytoplasm and promotes abnormal hyperphosphorylation of Tau. Results: Inactivation of nuclear localization signal (NLS) causes retention of I2PP2A in the cell cytoplasm, where it promotes Tau hyperphosphorylation by affecting PP2A signaling. Conclusion: Retention of I2PP2A in cell cytoplasm results in Tau hyperphosphorylation. Significance: The study provides potential tools for investigating Tau-based therapeutics. Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I2PP2A). In AD brain, I2PP2A is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I2PP2A containing a conserved basic motif, 179RKR181, which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I2PP2A in which the NLS was inactivated by 179RKR181 → AAA along with 168KR169 → AA mutations. In this model, the mutant NLS (mNLS)-I2PP2A (I2PP2AAA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I2PP2A activated CAMKII and GSK-3β, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I2PP2A with PP2A and GSK-3β but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I2PP2A-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology.

Conformational changes and cleavage; are these responsible for the tau aggregation in Alzheimer’s disease?

In the past, post-translational modifications of tau protein, such as phosphorylation, cleavage and conformational changes, have long been implicated in the pathogenesis of Alzheimer’s disease. Unfortunately, the accurate role and relationship between these pathological modifications during tau aggregation remains under extensive study. We had proposed a chronological model of tau pathological processing during Alzheimer´s disease, in which phosphorylation and cleavage could lead to conformational changes causing aggregation and therefore, cell toxicity. We discuss this issue and review in vitro and in situ evidence that supports the relevance of tau modifications that cause its pathological conformations and toxic aggregation. Thus, we offer a brief discussion regarding conformational change and cleavage as future clinical targets.

Animal Models of the Sporadic Form of Alzheimer's Disease: Focus on the Disease and Not Just the Lesions

Alzheimers disease is multifactorial and involves several different mechanisms. The sporadic form of the disease accounts for over 99% of the cases. As of yet, there is no practical and widely available animal model of the sporadic form of the disease. In the Alzheimers disease brain, the lysosomal enzyme asparaginyl endopeptidase is activated and translocated from the neuronal lysosomes to the cytoplasm, probably due to brain acidosis caused by ischemic changes associated with age-associated microinfarcts. The activated asparaginyl endopeptidase cleaves inhibitor-2 of protein phosphatase-2A, I2(PP2A), into I(2NTF) and I(2CTF) which translocate to the neuronal cytoplasm and inhibit the protein phosphatase activity and consequently the abnormal hyperphosphorylation of tau. Employing adeno-associated virus serotype 1 (AAV1) vector containing I(2NTF-CTF) and transduction of the brains of newborn rat pups with this virus, an animal model has been generated. The AAV1-I(2NTF-CTF) rats show neurodegeneration and cognitive impairment at 4 months and abnormal hyperphosphorylation and aggregation of tau and intraneuronal accumulation of amyloid-β at 13 months. The AAV1-I(2NTF-CTF) rats not only offer a disease-relevant model of the sporadic form of Alzheimers disease but also represent a practical and widely available animal model. This short perspective on the need to focus on and develop the disease-relevant models of the sporadic form of Alzheimers disease very much reflects the thinking of Inge Grundke-Iqbal who passed away on September 22, 2012 and to whom this article is dedicated.

Expression of Tau Produces Aberrant Plasma Membrane Blebbing in Glial Cells Through RhoA-ROCK-Dependent F-Actin Remodeling.

Abnormal aggregation of Tau in glial cells has been reported in Alzheimers disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

Pathological Stages of Abnormally Processed Tau Protein During Its Aggregation into Fibrillary Structures in Alzheimer’s Disease

Abnormal aggregation of tau protein within the cytoplasm of susceptible neurons has been considered one of the major hallmarks that define the neuropathology of Alzheimer’s disease (AD) (Iqbal et al., 2010; Pritchard et al., 2011). At early stages of neuronal degeneration tau protein is accumulated in the form of early non-assembled aggregates that may alter the normal functioning of affected neurons (Hoozemans et al., 2009; Luna-Munoz et al., 2007). Nonfibrillar aggregation of tau protein as a pre-tangle state has been reported to occur early in the disease but also observed in nondemented very old individuals (Garcia-Sierra et al., 2000). Some studies have reported that oligomeric species of tau protein represent the toxic structures rather than fibrillary structures (Berger et al., 2007; Maeda et al., 2006), however few studies have analyzed and determined a positive correlation between the load of pre-tangle carrying neurons and the clinical symptoms of AD. Further alterations in neurons may occur when the soluble aggregates of tau become assembled into insoluble polymers referred to as paired helical filaments (PHFs) that may also obstruct the transit and distribution of intracellular components, modify the neuronal morphology and alter the cytoskeleton (Ballatore, Lee & Trojanowski, 2007; Kidd, 2006). These filaments progressively coalesce into neurofibrillary tangles (NFTs) which eventually lead to the neuronal death (Guo & Lee, 2011). It is generally accepted that in AD cases, the density of NFTs is the best correlate with the dementia score (Arriagada, Marzloff & Hyman, 1992; Gomez-Isla et al., 1997).

Upregulation of casein kinase 1 epsilon may be involved in tau pathology in Alzheimer's disease brain

Background: Dysfunction of microtubule-associated protein tau and tau aggregates are prominent features in Alzheimer disease (AD) and other tauopathies, but the mechanisms underlying loss of Tau function-related synaptic and cognitive deficits remain poorly understood. Here we investigated whether loss of tau function in tau knock out (KO) mice can induce synaptic, cognitive and motor deficits and whether these deficits can be treated with supplements in diet. Methods: Young to middle-aged and aged tau KO mice and age-matched C57 wild type control mice were used for the experiment that was raised on PMI 5015 with some 18:n-3 but no long chain n-3. Treatment intervention includes DHA and the combination of DHAwith a -lipoate. Results: Aged (19-20 months old) but not middle-aged (8-9 months old) tau knockout (KO) mice develop severe hippocampal excitatory synaptic marker loss and behavior deficits implying an age-related failure to compensate for loss of tau by other microtubule-associated proteins (MAP1 and MAP2). We also find that in aged tau KO mice, the dietary n-3 fatty acid docosahexaenoic acid (DHA) partially protected, while DHA plus a -lipoate fully protected against synaptic and cognitive deficits by maintaining active MAPs and microtubule stability via inhibition of C-Jun N-terminal kinases (JNKs). Motor deficits and loss of tyrosine hydroxylase (TH) appeared in middle-aged tau KO mice without further decline in aged tau KO mice, the combination of DHA with a -lipoate diet prevented the loss of TH in OKOmice. Our preliminary data also suggested hippocampal loss of the neuronal marker NeuN and rescue by DHA/ALA. Conclusions: Our results imply loss of tau function with aging contributes to synaptic, cognitive and motor deficits that can be treated by safe interventions that rescue MAPs.