A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer's disease.

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer's patients' brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3ß, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer's disease and other tauopathies.

PARK2 enhancement is able to compensate mitophagy alterations found in sporadic Alzheimer's disease.

Mitochondrial anomalies have been previously reported in patients' brain and peripheral tissue, suggesting their relevance in sporadic Alzheimer's disease (AD). The present work evaluates mitochondrial function and recycling in human fibroblasts and brain biopsies. Functional studies using patients' skin fibroblasts showed slower mitochondrial membrane potential recovery after a mitochondrial insult together with alterations in lysosomes and autophagy, accompanied by an increase of oxidized and ubiquitinated proteins. Impairment in mitophagy has been proven in these cells due to diminished PARK2 and insufficient vesicle induction, accumulating depolarized mitochondria and PINK1. Augmented Δ1 PINK1 fragment levels suggest an inhibitory effect over PARK2 translocation to the mitochondria, causing the accumulation of activated PINK1. Moreover, the overexpression of PARK2 diminished ubiquitinated proteins accumulation, improves its targeting to mitochondria and potentiates autophagic vesicle synthesis. This allows the reversion of mitophagy failure reflected in the recovery of membrane potential and the decrease of PINK1 and mitochondria accumulation. Sporadic AD fibroblasts exhibited alterations similar to what it could be found in patients' hippocampal samples at early stages of the disease, where there was an accumulation of PINK1 and Δ1 PINK1 together with abnormally increased mitochondrial content. Our findings indicate that mitophagy alterations can be considered a new hallmark of sporadic AD and validate the use of fibroblasts for modelling this pathology.

Patient-derived olfactory mucosa for study of the non-neuronal contribution to amyotrophic lateral sclerosis pathology.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease which currently has no cure. Research using rodent ALS models transgenic for mutant superoxide dismutase 1 (SOD1) has implicated that glial-neuronal interactions play a major role in the destruction of motor neurons, but the generality of this mechanism is not clear as SOD1 mutations only account for less than 2% of all ALS cases. Recently, this hypothesis was backed up by observation of similar effects using astrocytes derived from post-mortem spinal cord tissue of ALS patients which did not carry SOD1 mutations. However, such necropsy samples may not be easy to obtain and may not always yield viable cell cultures. Here, we have analysed olfactory mucosa (OM) cells, which can be easily isolated from living ALS patients. Disease-specific changes observed when ALS OM cells were co-cultured with human spinal cord neurons included decreased neuronal viability, aberrant neuronal morphology and altered glial inflammatory responses. Our results show the potential of OM cells as new cell models for ALS.

Generation of functional neurons from adult human mucosal olfactory ensheathing glia by direct lineage conversion.

A recent approach to promote central nervous system (CNS) regeneration after injury or disease is direct conversion of somatic cells to neurons. This is achieved by transduction of viral vectors that express neurogenic transcription factors. In this work we propose adult human mucosal olfactory ensheathing glia (hmOEG) as a candidate for direct reprogramming to neurons due to its accessibility and to its well-characterized neuroregenerative capacity. After induction of hmOEG with the single neurogenic transcription factor NEUROD1, the cells under study exhibited morphological and immunolabeling neuronal features, fired action potentials and expressed glutamatergic and GABAergic markers. In addition, after engraftment of transduced hmOEG cells in the mouse hippocampus, these cells showed specific neuronal labeling. Thereby, if we add to the neuroregenerative capacity of hmOEG cultures the conversion to neurons of a fraction of their population through reprogramming techniques, the engraftment of hmOEG and hmOEG-induced neurons could be a procedure to enhance neural repair after central nervous system injury.

Intron retention as a productive mechanism in human MAPT: RNA species generated by retention of intron 3.

BACKGROUND: Tau is a microtubule-binding protein encoded by the MAPT gene. Tau is essential for several physiological functions and associated with pathological processes, including Alzheimer's disease (AD). Six tau isoforms are typically described in the central nervous system, but current research paints a more diverse landscape and a more nuanced balance between isoforms. Recent work has described tau isoforms generated by intron 11 and intron 12 retention. This work adds to that evidence, proving the existence of MAPT transcripts retaining intron 3. Our aim is to demonstrate the existence of mature MAPT RNA species that retain intron 3 in human brain samples and to study its correlation with Alzheimer's disease across different regions. METHODS: Initial evidence of intron-3-retaining MAPT species come from in silico analysis of RNA-seq databases. We further demonstrate the existence of these mature RNA species in a human neuroepithelioma cell line and human brain samples by quantitative PCR. We also use digital droplet PCR to demonstrate the existence of RNA species that retain either intron 3, intron 12 or both introns. FINDINGS: Intron-3-retaining species are even more prominently present that intron-12-retaining ones. We show the presence of MAPT transcripts that retain both introns 3 and 12. These intron-retaining species are diminished in brain samples of patients with Alzheimer's disease with respect to individuals without dementia. Conversely, relative abundance of intron-3- or intron-12-retaining MAPT species with respect to double-retaining species as well as their percentage of expression with respect to total MAPT are increased in patients with Alzheimer's disease, especially in hippocampal samples. Among these TIR-MAPT species, TIR3+12 double truncation allows better classification potential of Alzheimer's disease samples. Moreover, we find a significant increase in intron-3- or intron-12-retaining species and its relative abundance with respect to double-retaining MAPT species in cerebellum in contrast to frontal lateral cortex and hippocampus in individuals with no signs of dementia. INTERPRETATION: Intron retention constitutes a potential mechanism to generate Tau isoforms whose mature RNA expression levels correlate with Alzheimer's pathology showing its potential as a biomarker associated to the disease. FUNDING: This research was funded by the Spanish Ministry of Science, Innovation and Universities: PGC2018-096177-B-I00 (J.A.); Spanish Ministry of Science and Innovation (MCIN): PID2020-113204GB-I00 (F.H.) and PID2021-123859OB-100 from MCIN/AEI/10.13039/501100011033/FEDER, UE (J.A.). It was also supported by CSIC through an intramural grant (201920E104) (J.A.) and the Centre for Networked Biomedical Research on Neurodegenerative Diseases (J.A.). The Centro de Biología Molecular Severo Ochoa (CBMSO) is a Severo Ochoa Center of Excellence (MICIN, award CEX2021-001154-S).

Changes in tau phosphorylation in hibernating rodents.

Tau is a cytoskeletal protein present mainly in the neurons of vertebrates. By comparing the sequence of tau molecule among different vertebrates, it was found that the variability of the N-terminal sequence in tau protein is higher than that of the C-terminal region. The N-terminal region is involved mainly in the binding of tau to cellular membranes, whereas the C-terminal region of the tau molecule contains the microtubule-binding sites. We have compared the sequence of Syrian hamster tau with the sequences of other hibernating and nonhibernating rodents and investigated how differences in the N-terminal region of tau could affect the phosphorylation level and tau binding to cell membranes. We also describe a change, in tau phosphorylation, on a casein kinase 1 (ck1)-dependent site that is found only in hibernating rodents. This ck1 site seems to play an important role in the regulation of tau binding to membranes.

Alzheimer's Disease and Green Tea: Epigallocatechin-3-Gallate as a Modulator of Inflammation and Oxidative Stress.

Alzheimer's disease (AD) is the most common cause of dementia, characterised by a marked decline of both memory and cognition, along with pathophysiological hallmarks including amyloid beta peptide (Aß) accumulation, tau protein hyperphosphorylation, neuronal loss and inflammation in the brain. Additionally, oxidative stress caused by an imbalance between free radicals and antioxidants is considered one of the main risk factors for AD, since it can result in protein, lipid and nucleic acid damage and exacerbate Aß and tau pathology. To date, there is a lack of successful pharmacological approaches to cure or even ameliorate the terrible impact of this disease. Due to this, dietary compounds with antioxidative and anti-inflammatory properties acquire special relevance as potential therapeutic agents. In this context, green tea, and its main bioactive compound, epigallocatechin-3-gallate (EGCG), have been targeted as a plausible option for the modulation of AD. Specifically, EGCG acts as an antioxidant by regulating inflammatory processes involved in neurodegeneration such as ferroptosis and microglia-induced cytotoxicity and by inducing signalling pathways related to neuronal survival. Furthermore, it reduces tau hyperphosphorylation and aggregation and promotes the non-amyloidogenic route of APP processing, thus preventing the formation of Aß and its subsequent accumulation. Taken together, these results suggest that EGCG may be a suitable candidate in the search for potential therapeutic compounds for neurodegenerative disorders involving inflammation and oxidative stress, including Alzheimer's disease.

What's in a Gene? The Outstanding Diversity of MAPT.

Tau protein is a microtubule-associated protein encoded by the MAPT gene that carries out a myriad of physiological functions and has been linked to certain pathologies collectively termed tauopathies, including Alzheimer's disease, frontotemporal dementia, Huntington's disease, progressive supranuclear palsy, etc. Alternative splicing is a physiological process by which cells generate several transcripts from one single gene and may in turn give rise to different proteins from the same gene. MAPT transcripts have been proven to be subjected to alternative splicing, generating six main isoforms in the central nervous system. Research throughout the years has demonstrated that the splicing landscape of the MAPT gene is far more complex than that, including at least exon skipping events, the use of 3' and 5' alternative splice sites and, as has been recently discovered, also intron retention. In addition, MAPT alternative splicing has been showed to be regulated spatially and developmentally, further evidencing the complexity of the gene's splicing regulation. It is unclear what would drive the need for the existence of so many isoforms encoded by the same gene, but a wide range of functions have been ascribed to these Tau isoforms, both in physiology and pathology. In this review we offer a comprehensive up-to-date exploration of the mechanisms leading to the outstanding diversity of isoforms expressed from the MAPT gene and the functions in which such isoforms are involved, including their potential role in the onset and development of tauopathies such as Alzheimer's disease.

Specific Peptide from the Novel W-Tau Isoform Inhibits Tau and Amyloid ß Peptide Aggregation In Vitro.

W-Tau, a new tau human-specific splicing isoform generated by intron retention, has been recently described. This isoform contains an 18-residue unique sequence corresponding to the translation of the retained region of intron 12. In this work, we have described that such 18-amino-acid peptide from the retained intron 12 can inhibit tau and ß amyloid peptides aggregation under in vitro conditions. This inhibitory function is also present in smaller fragments of the 18-residue peptide.

Mouse and Human Tau Expression in Different Brain Areas.

Background: An increase in tau protein is believed to be necessary for tau aggregation. However, whether this is due to increased expression of the endogenous tau promoter or protein accumulation due to proteostasis failure remains uncertain. Objective: To analyze the expression of GFP protein under endogenous tau promoter across different ages and within different brain areas. Methods: We have measured direct expression of Mapt gene promotor by western blot and immunofluorescence, by means of a commercial tau knock-out mice generated by integrating GFP-encoding cDNA into exon 1 of the Mapt gene. Besides, we have analyzed the MAPT gene expression in human samples. Results: Mapt expression is similar in the cortex, hippocampus, and cerebellum in mice and in human samples although some differences exist between dentate gyrus and CA1 hippocampal areas in mice. Besides, we have analyzed the murine Mapt gene expression during aging (at 2, 6, 12, and 18 moths) and no differences in endogenous tau promoter expression were observed. Conclusion: Our results suggest that Mapt promoter activity is similar in the brain areas studied and, therefore, tau accumulation due to aging is likely due to proteostasis failure rather than occurring at the transcriptional level.

p38 Inhibition Decreases Tau Toxicity in Microglia and Improves Their Phagocytic Function.

Alzheimer's disease (AD) and other tauopathies are histopathologically characterized by tau aggregation, along with a chronic inflammatory response driven by microglia. Over the past few years, the role of microglia in AD has been studied mainly in relation to amyloid-ß (Aß) pathology. Consequently, there is a substantial knowledge gap concerning the molecular mechanisms involved in tau-mediated toxicity and neuroinflammation, thus hindering the development of therapeutic strategies. We previously demonstrated that extracellular soluble tau triggers p38 MAPK activation in microglia. Given the activation of this signaling pathway in AD and its involvement in neuroinflammation processes, here we evaluated the effect of p38 inhibition on primary microglia cultures subjected to tau treatment. Our data showed that the toxic effect driven by tau in microglia was diminished through p38 inhibition. Furthermore, p38 blockade enhanced microglia-mediated tau phagocytosis, as reflected by an increase in the number of lysosomes. In conclusion, these results contribute to our understanding of the functions of p38 in the central nervous system (CNS) beyond tau phosphorylation in neurons and provide further insights into the potential of p38 inhibition as a therapeutic strategy to halt neuroinflammation in tauopathies.

Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration.

Olfactory ensheathing glia (OEG) cells are known to facilitate repair following axotomy of adult neurons, although the molecular mechanisms involved are not fully understood. We previously identified plasminogen activator inhibitor-1 (PAI-1), proteinase-activated receptor-1 (PAR-1), and thrombomodulin (TM) as candidates to regulate rat OEG-dependent axonal regeneration. In this study, we have validated the involvement of these proteins in promoting axonal regeneration by immortalized human OEGs. We studied the effect of silencing these proteins in OEGs on their capacity to promote the regeneration of severed adult retinal ganglion cells (RGCs) axons. Our results support the role of glial PAI-1 as a downstream effector of PAR-1 in promoting axon regeneration. In contrast, we found that TM inhibits OEG induced-axonal regeneration. We also assessed the signaling pathways downstream of PAR-1 that might modulate PAI-1 expression, observing that specifically inhibiting Gα(i), Rho kinase, or PLC and PKC downregulated the expression of PAI-1 in OEGs, with a concomitant reduction in OEG-dependent axon regeneration in adult RGCs. Our findings support an important role for the thrombin system in regulating adult axonal regeneration by OEGs.

Prevention of senescence progression in reversibly immortalized human ensheathing glia permits their survival after deimmortalization.

Reversible immortalization holds great potential for primary tissue expansion to develop cell-based therapies as well as for basic research. Human olfactory ensheathing glia (hOEG) are promising candidates for treating spinal cord injury and for studying extrinsic neuroregenerative mechanisms. We used lentivectors with Cre/loxP technology to achieve reversible gene transfer of BMI1, SV40 large T antigen (TAg), a short hairpin RNA against p53 (shp53), and the catalytic subunit of telomerase (TERT) in primary cultures of hOEG from human donor cadaver olfactory bulbs. Several combinations of these genes were able to immortalize hOEG, conserving their antigenic markers and neuroregenerative properties but only those transduced by BMI1/TERT did not accumulate karyotypic alterations or increase senescence marker levels. Strikingly, these were also the only cells which continued to proliferate after transgene removal by Cre recombinase delivery, whereas hOEG immortalized by shp53 or TAg in combination with TERT entered into growth arrest and died. These data support the idea that immortalization and halting senescent changes are separate processes; hOEG immortalized by BMI1/TERT can revert back to their former primary cell replicative state when deimmortalized, whereas those transduced by the other combinations depend on the presence of these transgenes to maintain their aberrant proliferative state.

Corrigendum to "Slower Dynamics and Aged Mitochondria in Sporadic Alzheimer's Disease".

[This corrects the article DOI: 10.1155/2017/9302761.].

Reversibly immortalized human olfactory ensheathing glia from an elderly donor maintain neuroregenerative capacity.

A continuous normal function of olfactory ensheathing glia (OEG) is to promote axonal regeneration from the olfactory neuroepithelium to the brain, and their neuroregenerative potential in other CNS sites such as the injured spinal cord has been studied for over a decade. However, human OEG are difficult to obtain in large amounts directly from tissues, and the derived primary cultures have a limited duplication capacity. Thus, although auto-transplantation may be an obvious option for initial proof-of-concept trials, alternatives must be explored to obtain large quantities of homogeneous, pre-characterized OEG for wide-scale therapeutic use. We have cultured primary human OEG derived from olfactory bulbs (OB) obtained by necropsy and successfully extended the replicative lifespan of these cells using lentivectors encoding Bmi-1 and TERT transgenes flanked by loxP sites. In contrast to the primary cells which could only be expanded for a limited number of passages (approximately 12), adult human OEG immortalized Bmi-1/TERT divided indefinitely in culture. Clonal lines were isolated and the floxed transgenes could be excised by lentivector-mediated Cre recombinase delivery. Primary, immortalized, and deimmortalized human OEG all expressed typical markers of this cell type and importantly, were all able to promote axonal regeneration of adult rat retinal ganglion neurons (RGN) in co-culture assays.

The IDH-TAU-EGFR triad defines the neovascular landscape of diffuse gliomas.

Gliomas that express the mutated isoforms of isocitrate dehydrogenase 1/2 (IDH1/2) have better prognosis than wild-type (wt) IDH1/2 gliomas. However, how these mutant (mut) proteins affect the tumor microenvironment is still a pending question. Here, we describe that the transcription of microtubule-associated protein TAU (MAPT), a gene that has been classically associated with neurodegenerative diseases, is epigenetically controlled by the balance between wt and mut IDH1/2 in mouse and human gliomas. In IDH1/2 mut tumors, we found high expression of TAU that decreased with tumor progression. Furthermore, MAPT was almost absent from tumors with epidermal growth factor receptor (EGFR) mutations, whereas its trancription negatively correlated with overall survival in gliomas carrying wt or amplified (amp) EGFR We demonstrated that the overexpression of TAU, through the stabilization of microtubules, impaired the mesenchymal/pericyte-like transformation of glioma cells by blocking EGFR, nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and the transcriptional coactivator with PDZ-binding motif (TAZ). Our data also showed that mut EGFR induced a constitutive activation of this pathway, which was no longer sensitive to TAU. By inhibiting the transdifferentiation capacity of EGFRamp/wt tumor cells, TAU protein inhibited angiogenesis and favored vascular normalization, decreasing glioma aggressiveness and increasing their sensitivity to chemotherapy.

Glioma regression in vitro and in vivo by a suicide combined treatment.

We present here a suicide therapy against malignant gliomas based on the transfer to tumor cells of a gene encoding a beta-glucosidase, linamarase (lis), which in the presence of the innocuous substrate linamarin (lin) produces cyanide, blocking the mitochondrial respiratory chain. Dog glioma cells carrying the lis gene are thus sensitive to lin (IC(50) of 250 microg/mL at 48 hours) and cell death is accompanied by mitochondrial fission and ATP depletion. The combination of lis/lin with an otherwise nontoxic level of glucose oxidase (GO) enhances the therapeutic potential (IC(50) of 50 microg/mL at 48 hours). GO produces hydrogen peroxide, inducing oxidative damage and increasing cellular stress. We show here the antitumoral effect of the lis/lin/GO therapy in a canine glioma cell line and in a xenograft glioma model in nude mice. The synergic combination causes mitochondrial membrane depolarization and phosphatidylserine externalization and accelerates death by 48 hours. The lethal process is caspase independent; poly(ADP-ribose) polymerase 1 is not implicated; and there is no apoptosis-inducing factor translocation to the nucleus. The combined system induces autophagic cell death that can be rescued by 3-methyladenine and is characterized by the presence of double-membrane vesicles and punctate LC-3 pattern.

Modulating Effect of Diet on Alzheimer's Disease.

As life expectancy is growing, neurodegenerative disorders, such as Alzheimer's disease, are increasing. This disease is characterised by the accumulation of intracellular neurofibrillary tangles formed by hyperphosphorylated tau protein, senile plaques composed of an extracellular deposit of ß-amyloid peptide (Aß), and neuronal loss. This is accompanied by deficient mitochondrial function, increased oxidative stress, altered inflammatory response, and autophagy process impairment. The present study gathers scientific evidence that demonstrates that specific nutrients exert a direct effect on both Aß production and Tau processing and their elimination by autophagy activation. Likewise, certain nutrients can modulate the inflammatory response and the oxidative stress related to the disease. However, the extent to which these effects come with beneficial clinical outcomes remains unclear. Even so, several studies have shown the benefits of the Mediterranean diet on Alzheimer's disease, due to its richness in many of these compounds, to which can be attributed their neuroprotective properties due to the pleiotropic effect they show on the aforementioned processes. These indications highlight the potential role of adequate dietary recommendations for clinical management of both Alzheimer's diagnosed patients and those in risk of developing it, emphasising once again the importance of diet on health.

Mitophagy Failure in APP and Tau Overexpression Model of Alzheimer's Disease.

Mitochondrial alterations and oxidative stress are common features of Alzheimer's disease brain and peripheral tissues. Moreover, mitochondrial recycling process by autophagy has been found altered in the sporadic form of the disease. However, the contribution of the main proteins involved in this pathology such as amyloid-ß protein precursor (AßPP) and tau needs to be achieved. With this aim, human unmodified fibroblasts were transduced with lentivectors encoding APP and Tau and treated with CCCP to study the mitophagy process. Both AßPP and tau separately increased autophagy flux mainly by improving degradation phase. However, in the specific case of mitophagy, labeling of mitochondria by PINK1 and PARK2 to be degraded by autophagy seemed reduced, which correlates with the long-term accumulation of mitochondria. Nevertheless, the combination of tau and AßPP was necessary to cause a mitophagy functional impairment reflected in the accumulation of depolarized mitochondria labeled by PINK1. The overexpression of Tau and APP recapitulates the mitophagy failure previously found in sporadic Alzheimer's disease.

Benefit of Oleuropein Aglycone for Alzheimer's Disease by Promoting Autophagy.

Alzheimer's disease is a proteinopathy characterized by accumulation of hyperphosphorylated Tau and ß-amyloid. Autophagy is a physiological process by which aggregated proteins and damaged organelles are eliminated through lysosomal digestion. Autophagy deficiency has been demonstrated in Alzheimer's patients impairing effective elimination of aggregates and damaged mitochondria, leading to their accumulation, increasing their toxicity and oxidative stress. In the present study, we demonstrated by microarray analysis the downregulation of fundamental autophagy and mitophagy pathways in Alzheimer's patients. The benefits of the Mediterranean diet on Alzheimer's disease and cognitive impairment are well known, attributing this effect to several polyphenols, such as oleuropein aglycone (OLE), present in extra virgin olive oil. OLE is able to induce autophagy, achieving a decrease of aggregated proteins and a reduction of cognitive impairment in vivo. This effect is caused by the modulation of several pathways including the AMPK/mTOR axis and the activation of autophagy gene expression mediated by sirtuins and histone acetylation or EB transcription factor. We propose that supplementation of diet with extra virgin olive oil might have potential benefits for Alzheimer's patients by the induction of autophagy by OLE.