Caracterización de un modelo murino de taupatía tau-P301S Alteraciones en el sistema nervioso periférico y central

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-01-2020Esta tesis tiene embargado el acceso al texto completo hasta el 17-07-2021Los ratones transgénicos que incluyen el gen MAPT humano con algunas de las mutaciones características de la FTDP-17 (demencia frontotemporal con parkinsonismo ligada al cromosoma 17) son frecuentemente empleados como modelo no solo de esta taupatía sino también de otras como la enfermedad de Alzheimer (AD). Aquellos modelos que incluyen mutaciones en el residuo 301 (prolina) están entre los más utilizados por su buena aproximación histopatológica a los efectos de la disfunción asociada a tau en pacientes de AD; en la presente tesis nos centramos en un modelo tau-P301S denominado PS19. Teniendo en cuenta la relación entre tau y la neurogénesis adulta hipocampal, y la ausencia de literatura al respecto en modelos tau-P301S, el estudio de este proceso centró una de las partes de nuestro trabajo. Además de una caracterización de la neurodegeneración en el sistema nervioso central (CNS) de este ratón, quisimos también estudiar el sistema nervioso periférico (PNS), mucho menos tratado en la bibliografía y especialmente interesante a la luz del fuerte fenotipo motor que nuestro modelo P301S desarrolla. La última parte fue dedicada al modelo doble Akp2/P301S, que combina el transgén tau-P301S con una haploinsuficiencia para la fosfatasa alcalina no dependiente de tejido (TNAP), que tiene actividad fosfatasa sobre tau. Teniendo en cuenta que la proteína tau desfosforilada por esta exoenzima puede desencadenar la apoptosis de neuronas vecinas, nuestro objetivo fue determinar si una menor carga de TNAP podría conllevar un amortiguamiento de este efecto neurotóxico y, por lo tanto, una ralentización o interferencia en la progresión de los daños neurodegenerativos. En nuestro modelo P301S observamos una reducción drástica de la supervivencia (hasta una mediana de 11,7 meses) debida a un fenotipo de parálisis motora que suele debutar a partir de los 9 meses. A esta edad, el ratón presenta una sobreexpresión y una hiperfosforilación de la proteína tau transgénica tanto en el hipocampo como en el nervio ciático, atrofia hipocampal acompañada de muerte celular aumentada, gliosis reactiva, y degeneración axonal en el nervio ciático acompañada de los problemas motores evidenciados en la prueba del rotarod; los ratones también mostraron hiperactividad, y no se encontraron diferencias con respecto a los controles a nivel de memoria y navegación espacial dependientes del hipocampo. Se detectó una neurogénesis adulta aumentada, sin diferencias con respecto a los controles en la morfología dendrítica, que podría actuar como mecanismo compensatorio que justificara el mantenimiento de la función hipocampal a pesar de la neurodegeneración observada; el inicio de la expresión del transgén fue observado en etapas tardías de la neurogénesis. El ratón Akp2/P301S mostró los mismos marcadores de neurodegeneración en el CNS que el modelo P301S, así como la neurogénesis aumentada, pero esto no sucedió a nivel del PNS: se observó una recuperación a valores del grupo control de la degeneración axonal en el nervio ciático, así como del rendimiento motor. Estos resultados refuerzan la hipótesis de la ralentización en el avance de la patología, y deberían tenerse en cuenta para explorar la posible utilidad terapéutica de inhibidores de la TNAP.Transgenic mice harbouring human MAPT gene with characteristic FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17) mutations are frequently used as models not only of this tauopathy but also of other tauopathies as Alzheimer’s disease (AD). Those models including mutations in residue 301 (proline) are amongst the most employed ones due to their histopathological similarity to the effects associated with taurelated dysfunction in AD patients; in this thesis we will focus on a P301S-tau model named PS19. Given the connection between tau and adult hippocampal neurogenesis, and the absence of literature on that subject in P301S-tau models, the study of this process was one of the topics of our work. Besides a characterization of the neurodegeneration of the central nervous system (CNS) in this mouse, we also aimed at the peripheral nervous system (PNS), less frequently addressed in the bibliography and especially interesting in light of the strong motor phenotype that our P301S model develops. The last part of this work was dedicated to the model Akp2/P301S, which combines P301S-tau transgene with a haploinsufficiency for the tissue-nonspecific alkaline phosphatase (TNAP), which has phosphatase activity on tau. Taking into account that tau protein, once dephosphorylated by this exoenzyme, can induce apoptosis in neighbouring neurons, our goal was to determine if decreased levels of TNAP could lower this neurotoxic effect, thus interfering with or slowing down the progression of the neurodegenerative damage. We observed in our P301S model a dramatic decrease in survival time (with a median of 11.7 months), which stemmed from a motor-paralysis phenotype that usually made its debut around 9 months of age. At this point, the mouse showed an overexpression and a hyperphosphorylation of the transgenic tau protein, both in the hippocampus and in the sciatic nerve, hippocampal atrophy with increased cell death, reactive gliosis, and axonal degeneration in the sciatic nerve along with motor issues that were patent on the rotarod test; mice also displayed hyperactivity, and no differences with control mice were found regarding hippocampus-dependent memory and spatial navigation. An increased adult neurogenesis was found (with no differences with control mice in respect of dendritic morphology) which could be acting as a compensatory mechanism for neuronal damage and explain the hippocampal-function endurance despite the observed neurodegeneration; the beginning of the transgene expression was found in late stages of this process. Akp2/P301S mice showed the same signs of neurodegeneration in the CNS as the P301S model, as well as an increased adult neurogenesis, but differences were found in the PNS: we observed a recovery, returning to control levels, of axonal degeneration of the sciatic nerve and motor performance. These results reinforce the hypothesis of a deceleration in the progression of the pathology and encourage further exploration of the therapeutic value of TNAP inhibitors

Differences Between Human and Murine Tau at the N-terminal End

Human tauopathies, such as Alzheimer’s disease (AD), have been widely studied in transgenic mice overexpressing human tau in the brain. The longest brain isoforms of Tau in mice and humans show 89% amino acid identity; however, the expression of the isoforms of this protein in the adult brain of the two species differs. Tau 3R isoforms are not present in adult mice. In contrast, the adult human brain contains Tau 3R and also Tau 4R isoforms. In addition, the N-terminal sequence of Tau protein in mice and humans differs, a Tau peptide (residues 17–28) being present in the latter but absent in the former. Here we review the main published data on this N-terminal sequence that suggests that human and mouse Tau proteins interact with different endogenous proteins and also show distinct secretion patternsThis study was funded by grants from Spanish Ministry of Economy and Competitiveness (Ministerio de Economía, Industria y Competitividad, Gobierno de España; BFU2016-77885-P), Structural Funds of the European Union from the Comunidad de Madrid [S2017/BMD-3700 (NEUROMETAB-CM)], institutional funding from the Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), and an institutional grant from the Fundación R. Areces. JM-R has a fellowship from the Fundación La Caix

Alteration in the Synaptic and Extrasynaptic Organization of AMPA Receptors in the Hippocampus of P301S Tau Transgenic Mice

Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD

Different modes of synaptic and extrasynaptic NMDA receptor alteration in the hippocampus of P301S tau transgenic mice

N-methyl-d-aspartate receptors (NMDARs) are pivotal players in the synaptic transmission and synaptic plasticity underlying learning and memory. Accordingly, dysfunction of NMDARs has been implicated in the pathophysiology of Alzheimer disease (AD). Here, we used histoblot and sodium dodecylsulphate-digested freeze-fracture replica labelling (SDS-FRL) techniques to investigate the expression and subcellular localisation of GluN1, the obligatory subunit of NMDARs, in the hippocampus of P301S mice. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered at 3 months. Using the SDS-FRL technique, excitatory synapses and extrasynaptic sites on spines of pyramidal cells and interneuron dendrites were analysed throughout all dendritic layers in the CA1 field. Our ultrastructural approach revealed a high density of GluN1 in synaptic sites and a substantially lower density at extrasynaptic sites. Labelling density for GluN1 in excitatory synapses established on spines was significantly reduced in P301S mice, compared with age-matched wild-type mice, in the stratum oriens (so), stratum radiatum (sr) and stratum lacunosum-moleculare (slm). Density for synaptic GluN1 on interneuron dendrites was significantly reduced in P301S mice in the so and sr but unaltered in the slm. Labelling density for GluN1 at extrasynaptic sites showed no significant differences in pyramidal cells, and only increased density in the interneuron dendrites of the sr. This differential alteration of synaptic versus extrasynaptic NMDARs supports the notion that the progressive accumulation of phospho-tau is associated with changes in NMDARs, in the absence of amyloid-β pathology, and may be involved in the mechanisms causing abnormal network activity of the hippocampal circui

TNAP upregulation is a critical factor in Tauopathies and its blockade ameliorates neurotoxicity and increases life-expectancy

Tauopathies are a family of neurodegenerative diseases characterized by the presence of abnormally hyperphosphorylated Tau protein. Several studies have proposed that increased extracellular Tau (eTau) leads to the spread of cerebral tauopathy. However, the molecular mechanisms underlying eTau-induced neurotoxicity remain unclear. Previous in vitro studies reported that the ecto-enzyme tissue-nonspecific alkaline phosphatase (TNAP) dephosphorylate eTau at different sites increasing its neurotoxicity. Here, we confirm TNAP protein upregulation in the brains of Alzheimer's patients and found a similar TNAP increase in Pick's disease patients and P301S mice, a well-characterized mouse model of tauopathies. Interestingly, the conditional overexpression of TNAP causes intracellular Tau hyperphosphorylation and aggregation in cells neighbouring those overexpressing the ectoenzyme. Conversely, the genetic disruption of TNAP reduced the dephosphorylation of eTau and decreased neuronal hyperactivity, brain atrophy, and hippocampal neuronal death in P301S mice. TNAP haploinsufficiency in P301S mice prevents the decreased anxiety-like behaviour, motor deficiency, and increased memory capacity and life expectancy. Similar results were observed by the in vivo pharmacological blunting of TNAP activity. This study provides the first in vivo evidence demonstrating that raised TNAP activity is critical for Tau-induced neurotoxicity and suggest that TNAP blockade may be a novel and efficient therapy to treat tauopathiesThis work was supported by funding from the following: Spanish Ministry of Economy and Competitiveness RTI2018-095753-B-I00 (to M.D.-H.), BFU2016-77885-P (to F.H.) and PGC2018-096177-B-I00 (to J.A.); European Union H2020 program H2020-MSCA-ITN-2017 number 766124 (to M.D-H); European Regional Development Funds from the Comunidad de Madrid S2017/BMD-3700 (NEUROMETAB-CM) (to F.H.); UCM-Santander Central Hispano Bank PR41/17–21,014 (to M.D-H); CIBERNED-ISCIII; and the Fundación R. Areces (to F.H.). A.S-S was hired by RTI2018-095753-B-I00 grant and as postdoctoral researcher by UCM (CT48/19), C.dL. and C.B. were hired by H2020-MSCA-ITN-2017 (grant number 766124), and J M-R had a fellowship from the Fundación La Caixa. This work was supported in part by ERD

Tau-positive nuclear indentations in P301S tauopathy mice

Increased incidence of neuronal nuclear indentations is a well-known feature of the striatum of Huntington's disease (HD) brains and, in Alzheimer's disease (AD), neuronal nuclear indentations have recently been reported to correlate with neurotoxicity caused by improper cytoskeletal/nucleoskeletal coupling. Initial detection of rod-shaped tau immunostaining in nuclei of cortical and striatal neurons of HD brains and in hippocampal neurons of early Braak stage AD led us to coin the term 'tau nuclear rods (TNRs).' Although TNRs traverse nuclear space, they in fact occupy narrow cytoplasmic extensions that fill indentations of the nuclear envelope and we will here refer to this histological hallmark as Tau-immunopositive nuclear indentations (TNIs). We reasoned that TNI formation is likely secondary to tau alterations as TNI detection in HD correlates with an increase in total tau, particularly of the isoforms with four tubulin binding repeats (4R-tau). Here we analyze transgenic mice that overexpress human 4R-tau with a frontotemporal lobar degeneration-tau point mutation (P301S mice) to explore whether tau alteration is sufficient for TNI formation. Immunohistochemistry with various tau antibodies, immunoelectron microscopy and double tau-immunofluorescence/DAPI-nuclear counterstaining confirmed that excess 4R-tau in P301S mice is sufficient for the detection of abundant TNIs that fill nuclear indentations. Interestingly, this does not correlate with an increase in the number of nuclear indentations, thus suggesting that excess total tau or an isoform imbalance in favor of 4R-tau facilitates tau detection inside preexisting nuclear indentations but does not induce formation of the latter. In summary, here we demonstrate that tau alteration is sufficient for TNI detection and our results suggest that the neuropathological finding of TNIs becomes a possible indicator of increased total tau and/or increased 4R/3R-tau ratio in the affected neurons apart from being an efficient way to monitor pathology-associated nuclear indentations

MicroRNA-22 Controls Aberrant Neurogenesis and Changes in Neuronal Morphology After Status Epilepticus

Prolonged seizures (status epilepticus, SE) may drive hippocampal dysfunction and epileptogenesis, at least partly, through an elevation in neurogenesis, dysregulation of migration and aberrant dendritic arborization of newly-formed neurons. MicroRNA-22 was recently found to protect against the development of epileptic foci, but the mechanisms remain incompletely understood. Here, we investigated the contribution of microRNA-22 to SE-induced aberrant adult neurogenesis. SE was induced by intraamygdala microinjection of kainic acid (KA) to model unilateral hippocampal neuropathology in mice. MicroRNA-22 expression was suppressed using specific oligonucleotide inhibitors (antagomir-22) and newly-formed neurons were visualized using the thymidine analog iodo-deoxyuridine (IdU) and a green fluorescent protein (GFP)-expressing retrovirus to visualize the dendritic tree and synaptic spines. Using this approach, we quantified differences in the rate of neurogenesis and migration, the structure of the apical dendritic tree and density and morphology of dendritic spines in newly-formed neurons.SE resulted in an increased rate of hippocampal neurogenesis, including within the undamaged contralateral dentate gyrus (DG). Newly-formed neurons underwent aberrant migration, both within the granule cell layer and into ectopic sites. Inhibition of microRNA-22 exacerbated these changes. The dendritic diameter and the density and average volume of dendritic spines were unaffected by SE, but these parameters were all elevated in mice in which microRNA-22 was suppressed. MicroRNA-22 inhibition also reduced the length and complexity of the dendritic tree, independently of SE. These data indicate that microRNA-22 is an important regulator of morphogenesis of newly-formed neurons in adults and plays a role in supressing aberrant neurogenesis associated with SE

Peripheral nervous system effects in the PS19 tau transgenic mouse model of tauopathy

It is well known that transgenic mice overexpressing human tau protein with P301S mutation driven by the mouse prion protein promoter show clasping and limb retraction, hunched back and paralysis, followed by inability to feed that results in death around 12 months of age. To understand these motor deficits, we have carried out rotarod tests on PS19 line and demonstrated how they worsened during aging. Then, we have analyzed if these phenotypic characteristics correlate with sciatic nerve degeneration. We first demonstrated by western blot and immunohistochemistry that the sciatic nerve expresses the transgenic tau protein; then, electron microscopy studies showed alterations in myelin, mainly a detachment of myelin lamellae at Schmidt-Lanterman clefts. Similar motor deficits and myelin alterations have been previously reported in tau knockout and overexpressing transgenic mice; taking into account that PS19 model is widely used to study tauopathies, we suggest that analyzing the expression of transgenic tau protein and myelin abnormalities in the sciatic nerve should be considered when studying some features as motor performance or survival.Spanish Ministry of Economy and Competitiveness (BFU2016-77885-P), Comunidad de Madrid cofinanced with the Structural Funds of the European Union (S2017/BMD-3700 (NEUROMETAB-CM)), Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), and from an institutional grant from the Fundación R. Arece

Differences Between Human and Murine Tau at the N-terminal End

Human tauopathies, such as Alzheimer’s disease (AD), have been widely studied in transgenic mice overexpressing human tau in the brain. The longest brain isoforms of Tau in mice and humans show 89% amino acid identity; however, the expression of the isoforms of this protein in the adult brain of the two species differs. Tau 3R isoforms are not present in adult mice. In contrast, the adult human brain contains Tau 3R and also Tau 4R isoforms. In addition, the N-terminal sequence of Tau protein in mice and humans differs, a Tau peptide (residues 17–28) being present in the latter but absent in the former. Here we review the main published data on this N-terminal sequence that suggests that human and mouse Tau proteins interact with different endogenous proteins and also show distinct secretion patterns.Spanish Ministry of Economy and Competitiveness (Ministerio de Economía, Industria y Competitividad, Gobierno de España; BFU2016-77885-P), Structural Funds of the European Union from the Comunidad de Madrid [S2017/BMD-3700 (NEUROMETABCM)], institutional funding from the Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), and an institutional grant from the Fundación R. Areces. JM-R has a fellowship from the Fundación La Caixa

Implications of CB2 cannabinoid receptors in the neurodegenerative process associated to tauopathies

Resumen del trabajo presentado al 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Santander del 10 al 13 de septiembre de 2018.[Background] Tauopathies are a group of neurodegenerative diseases characterized by the aggregation of TAU protein for which there is no effective treatment yet. This is the reason why new approaches are now being investigated, such as the endocannabinoid system. In Alzheimer’s disease, it has been shown that the levels of type-2 cannabinoid receptors (CB2) are increased in the glial cells that surround amyloid plaques. Nevertheless, the effects that TAU accumulation has on CB2 expression and its implication in the neurodegenerative process are still unknown.[Results] We have seen an increase in CB2 expression in hippocampal neurons of 12 month-old transgenic mice overexpressing hTAUP301S protein. This alteration is an early event on the pathology, as CB2 levels are also increased in another animal model overexpressing hTAUP301L where there is not neuronal death. Moreover, CB2 expression in these mice can be modulated by dimethyl fumarate (DMF) treatment, an NRF2 inductor with neuroprotective effects. These results were reproduced in vitro when hTAUP301L was overexpressed in HT22 cells. Finally, we found an antioxidant response element on CNR2 promoter and our analysis in vitro point out at the possibility that CB2 expression could be modulated by NRF2.[Conclusions] Overexpression of hTAU protein increases CB2 levels in neurons and can be modulated by DMF treatment.Peer reviewe