RÉSUMÉ
AIMS: Mutations in the MAPT gene encoding tau protein can cause autosomal dominant neurodegenerative tauopathies including frontotemporal dementia (often with Parkinsonism). In Alzheimer's disease, the most common tauopathy, synapse loss is the strongest pathological correlate of cognitive decline. Recently, Positron Emission Tomography (PET) imaging with synaptic tracers revealed clinically relevant loss of synapses in primary tauopathies; however, the molecular mechanisms leading to synapse degeneration in primary tauopathies remain largely unknown. In this study, we examined post-mortem brain tissue from people who died with frontotemporal dementia with tau pathology (FTDtau) caused by the MAPT intronic exon 10 + 16 mutation, which increases splice variants containing exon 10 resulting in higher levels of tau with four microtubule-binding domains. METHODS: We used RNA sequencing and histopathology to examine temporal cortex and visual cortex, to look for molecular phenotypes compared to age, sex and RNA integrity matched participants who died without neurological disease (n = 12 FTDtau10 + 16 and 13 controls). RESULTS: Bulk tissue RNA sequencing reveals substantial downregulation of gene expression associated with synaptic function. Upregulated biological pathways in human MAPT 10 + 16 brain included those involved in transcriptional regulation, DNA damage response and neuroinflammation. Histopathology confirmed increased pathological tau accumulation in FTDtau10 + 16 cortex as well as a loss of presynaptic protein staining and region-specific increased colocalization of phospho-tau with synapses in temporal cortex. CONCLUSIONS: Our data indicate that synaptic pathology likely contributes to pathogenesis in FTDtau10 + 16 caused by the MAPT 10 + 16 mutation.
Sujet(s)
Démence frontotemporale , Mutation , Synapses , Protéines tau , Humains , Protéines tau/génétique , Protéines tau/métabolisme , Démence frontotemporale/génétique , Démence frontotemporale/anatomopathologie , Mâle , Femelle , Synapses/anatomopathologie , Synapses/métabolisme , Sujet âgé , Adulte d'âge moyen , Expression des gènes/génétique , Encéphale/anatomopathologie , Encéphale/métabolisme , Tauopathies/génétique , Tauopathies/anatomopathologie , Tauopathies/métabolismeRÉSUMÉ
Mutations in the MAPT gene encoding tau protein can cause autosomal dominant neurodegenerative tauopathies including frontotemporal dementia (often with Parkinsonism). In Alzheimer's disease, the most common tauopathy, synapse loss is the strongest pathological correlate of cognitive decline. Recently, PET imaging with synaptic tracers revealed clinically relevant loss of synapses in primary tauopathies; however, the molecular mechanisms leading to synapse degeneration in primary tauopathies remain largely unknown. In this study, we examined post-mortem brain tissue from people who died with frontotemporal dementia with tau pathology (FTDtau) caused by the MAPT intronic exon 10+16 mutation, which increases splice variants containing exon 10 resulting in higher levels of tau with four microtubule binding domains. We used RNA sequencing and histopathology to examine temporal cortex and visual cortex, to look for molecular phenotypes compared to age, sex, and RNA integrity matched participants who died without neurological disease (n=12 per group). Bulk tissue RNA sequencing reveals substantial downregulation of gene expression associated with synaptic function. Upregulated biological pathways in human MAPT 10+16 brain included those involved in transcriptional regulation, DNA damage response, and neuroinflammation. Histopathology confirmed increased pathological tau accumulation in FTDtau cortex as well as a loss of presynaptic protein staining, and region-specific increased colocalization of phospho-tau with synapses in temporal cortex. Our data indicate that synaptic pathology likely contributes to pathogenesis in FTDtau caused by the MAPT 10+16 mutation.
RÉSUMÉ
We applied a novel application of FLIM-FRET to in situ measurement and quantification of protein interactions to explore isoform specific differences in Aß-ApoE interaction and ApoE tertiary conformation in senile plaques in human Alzheimer brain. ApoE3 interacts more closely with Aß than ApoE4, but a greater proportion of Aß molecules within plaques are decorated with ApoE4 than ApoE3, lending strong support to the hypothesis that isoform specific differences in ApoE are linked with Aß deposition. We found an increased number of ApoE N-terminal fragments in ApoE4 plaques, consistent with the observation that ApoE4 is more easily cleaved than ApoE3. In addition, we measured a small but significant isoform specific difference in ApoE domain interaction. Based on our in situ data, supported by traditional biochemical data, we propose a pathway by which isoform specific conformational differences increase the level of cleavage at the hinge region of ApoE4, leading to a loss of ApoE function to mediate clearance of Aß and thereby increase the risk of AD for carriers of the APOEε4 allele.
Sujet(s)
Maladie d'Alzheimer/métabolisme , Précurseur de la protéine bêta-amyloïde/métabolisme , Apolipoprotéines E/métabolisme , Encéphale/anatomopathologie , Plaque amyloïde/anatomopathologie , Sujet âgé , Sujet âgé de 80 ans ou plus , Maladie d'Alzheimer/anatomopathologie , Apolipoprotéine E3/métabolisme , Apolipoprotéine E4/métabolisme , Apolipoprotéines E/composition chimique , Femelle , Humains , Mâle , Liaison aux protéines , Isoformes de protéines/composition chimique , Isoformes de protéines/métabolisme , Structure tertiaire des protéinesRÉSUMÉ
Tau protein is present in six different splice forms in the human brain and interacts with microtubules via either 3 or 4 microtubule binding repeats. An increased ratio of 3 repeat to 4 repeat isoforms is associated with neurodegeneration in inherited forms of frontotemporal dementia. Tau over-expression diminishes axonal transport in several systems, but differential effects of 3 repeat and 4 repeat isoforms have not been studied. We examined the effects of tau on mitochondrial transport and found that both 3 repeat and 4 repeat tau change normal mitochondrial distribution within the cell body and reduce mitochondrial localization to axons; 4 repeat tau has a greater effect than 3 repeat tau. Further, we observed that the 3 repeat and 4 repeat tau cause different alterations in retrograde and anterograde transport dynamics with 3 repeat tau having a slightly stronger effect on axon transport dynamics. Our results indicate that tau-induced changes in axonal transport may be an underlying theme in neurodegenerative diseases associated with isoform specific changes in tau's interaction with microtubules.
Sujet(s)
Transport axonal/physiologie , Mitochondries/physiologie , Neurones/physiologie , Tauopathies/physiopathologie , Protéines tau/génétique , Protéines tau/métabolisme , Animaux , Lignée cellulaire tumorale , Cortex cérébral/cytologie , Gliome , Protéines à fluorescence verte/génétique , Humains , Souris , Techniques d'analyse microfluidique , Neurones/cytologie , Transport des protéines/physiologie , Séquences répétées d'acides nucléiques/physiologie , Tauopathies/génétique , Tauopathies/métabolisme , TransfectionRÉSUMÉ
Neurodegenerative tauopathies are marked by their common pathologic feature of aggregates formed of hyperphosphorylated tau protein, which are associated with synapse and neuronal loss. Changes in tau conformation result in both loss of normal function and gain of fibrillogenicity that leads to aggregation. Here, we discuss the pathophysiology of tau and emerging evidence of how changes in this protein might ultimately lead to neuronal death. In particular, based on recent evidence, we propose that a non-apoptotic caspase-associated form of death is occurring in tauopathy.
Sujet(s)
Dégénérescence nerveuse/anatomopathologie , Dégénérescence nerveuse/physiopathologie , Tauopathies/physiopathologie , Protéines tau/métabolisme , Animaux , Humains , Dégénérescence nerveuse/génétique , Tauopathies/génétique , Tauopathies/anatomopathologie , Protéines tau/génétiqueRÉSUMÉ
Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.
Sujet(s)
Arachidonate 5-lipoxygenase/métabolisme , Protéines de transport/métabolisme , Leucotriènes/biosynthèse , Protéines membranaires/métabolisme , Complexes multiprotéiques/analyse , Enveloppe nucléaire/métabolisme , Protéines d'activation de la 5-lipoxygénase , Animaux , Arthrite/enzymologie , Arthrite/métabolisme , Protéines membranaires/analyse , Souris , Cellules myéloïdes/composition chimique , Cellules myéloïdes/métabolisme , Granulocytes neutrophiles/composition chimique , Granulocytes neutrophiles/métabolisme , Enveloppe nucléaire/composition chimiqueRÉSUMÉ
The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.
Sujet(s)
Algorithmes , Artéfacts , Transfert d'énergie par résonance de fluorescence/méthodes , Amélioration d'image/méthodes , Interprétation d'images assistée par ordinateur/méthodes , Microscopie de fluorescence/méthodes , Technique de soustraction , Animaux , Souris , Souris transgéniques , Reproductibilité des résultats , Sensibilité et spécificitéRÉSUMÉ
BACKGROUND AND PURPOSE: In focal ischemic cortex, cerebral blood flow autoregulation is impaired, and perfusion passively follows blood pressure variations. Although it is generally agreed that profound hypotension is harmful in acute stroke, the hemodynamic and metabolic impact of increased blood pressure on the ischemic core and penumbra are less well understood. We, therefore, tested whether pharmacologically induced hypertension improves cerebral blood flow and metabolism and tissue outcome in acute stroke using optical imaging with high spatiotemporal resolution. METHODS: Cerebral blood flow, oxyhemoglobin, and cerebral metabolic rate of oxygen were measured noninvasively using simultaneous multispectral reflectance imaging and laser speckle flowmetry during distal middle cerebral artery occlusion in mice. Hypertension was induced by phenylephrine infusion starting 10 or 60 minutes after ischemia to raise blood pressure by 30% for the duration of ischemia; control groups received saline infusion. RESULTS: Mild induced hypertension rapidly increased cerebral blood flow, oxyhemoglobin, and cerebral metabolic rate of oxygen in both the core and penumbra and prevented the expansion of cerebral blood flow deficit during 1 hour distal middle cerebral artery occlusion. Induced hypertension also diminished the deleterious effects of periinfarct depolarizations on cerebral blood flow, oxyhemoglobin, and cerebral metabolic rate of oxygen without altering their frequency. Consistent with this, mild induced hypertension reduced infarct volume by 48% without exacerbating tissue swelling when measured 2 days after 1 hour transient distal middle cerebral artery occlusion. CONCLUSIONS: Our data suggest that mild induced hypertension increases collateral cerebral blood flow and oxygenation and improves cerebral metabolic rate of oxygen in the core and penumbra, supporting its use as bridging therapy in acute ischemic stroke until arterial recanalization is achieved.
Sujet(s)
Encéphalopathie ischémique/traitement médicamenteux , Circulation cérébrovasculaire/effets des médicaments et des substances chimiques , Hypertension artérielle/induit chimiquement , Consommation d'oxygène , Accident vasculaire cérébral/traitement médicamenteux , Vasoconstricteurs/pharmacologie , Animaux , Encéphalopathie ischémique/métabolisme , Encéphalopathie ischémique/physiopathologie , Modèles animaux de maladie humaine , Métabolisme énergétique/effets des médicaments et des substances chimiques , Métabolisme énergétique/physiologie , Homéostasie/effets des médicaments et des substances chimiques , Homéostasie/physiologie , Hypertension artérielle/physiopathologie , Infarctus du territoire de l'artère cérébrale moyenne/traitement médicamenteux , Infarctus du territoire de l'artère cérébrale moyenne/métabolisme , Infarctus du territoire de l'artère cérébrale moyenne/physiopathologie , Fluxmétrie laser Doppler , Souris , Oxyhémoglobines/effets des médicaments et des substances chimiques , Oxyhémoglobines/métabolisme , Phényléphrine/pharmacologie , Phényléphrine/usage thérapeutique , Accident vasculaire cérébral/métabolisme , Accident vasculaire cérébral/physiopathologie , Résultat thérapeutique , Vasoconstricteurs/usage thérapeutiqueRÉSUMÉ
Accumulation of neurofibrillary tangles (NFTs) in Alzheimer's disease correlates with neuronal loss and cognitive decline, but the precise relationship between NFTs and neuronal death and downstream mechanisms of cell death remain unclear. Caspase cleaved products accumulate in tangles, implying that tangles may contribute to apoptotic neuronal death. To test this hypothesis, we developed methods using multiphoton imaging to detect both neurofibrillary pathology and caspase activation in the living mouse brain. We examined rTg4510 mice, a reversible mouse model of tauopathy that develops tangles and neuronal loss. Only a small percentage of imaged neurons were caspase activity positive, but the vast majority of the cells with active caspases contained NFTs. We next tested the hypothesis that caspase activation led to acute, apoptotic neuronal death. Caspase positive cell bodies did not degenerate over hours of imaging, despite the presence of activated executioner caspases. Suppression of the transgene, which stops ongoing death, did not suppress caspase activity. Finally, histochemical assessments revealed evidence of caspase-cleaved tau, but no TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) positive or apoptotic nuclei. With the novel technique of observing NFTs and caspase activation in the living brain, we demonstrate that aggregated tau in neurons can be associated with caspase activation, but that caspase activation is not sufficient to cause acute neuronal death in this model.
Sujet(s)
Apoptose/physiologie , Caspases/métabolisme , Enchevêtrements neurofibrillaires/enzymologie , Neurones/enzymologie , Animaux , Caspases/analyse , Mort cellulaire/physiologie , Activation enzymatique/physiologie , Humains , Souris , Souris transgéniques , Microscopie de fluorescence multiphotonique , Enchevêtrements neurofibrillaires/composition chimique , Enchevêtrements neurofibrillaires/anatomopathologie , Neurones/composition chimique , Neurones/anatomopathologie , Protéines tau/métabolisme , Protéines tau/physiologieRÉSUMÉ
Dendritic spines, the site of most excitatory synapses in the brain, are lost in Alzheimer's disease and in related mouse models, undoubtedly contributing to cognitive dysfunction. We hypothesized that spine loss results from plaque-associated alterations of spine stability, causing an imbalance in spine formation and elimination. To investigate effects of plaques on spine stability in vivo, we observed cortical neurons using multiphoton microscopy in a mouse model of amyloid pathology before and after extensive plaque deposition. We also observed age-matched nontransgenic mice to study normal effects of aging on spine plasticity. We found that spine density and structural plasticity are maintained during normal aging. Tg2576 mice had normal spine density and plasticity before plaques appeared, but after amyloid pathology is established, severe disruptions were observed. In control animals, spine formation and elimination were equivalent over 1 hour of observation ( approximately 5% of observed spines), resulting in stable spine density. However, in aged Tg2576 mice spine elimination increased, specifically in the immediate vicinity of plaques. Spine formation was unchanged, resulting in spine loss. These data show a small population of rapidly changing spines in adult and even elderly mouse cortex; further, in the vicinity of amyloid plaques, spine stability is markedly impaired leading to loss of synaptic structural integrity.
Sujet(s)
Maladie d'Alzheimer/anatomopathologie , Cortex cérébral/anatomopathologie , Épines dendritiques/anatomopathologie , Plaque amyloïde/anatomopathologie , Précurseur de la protéine bêta-amyloïde/génétique , Animaux , Modèles animaux de maladie humaine , Humains , Souris , Souris transgéniques , Neurones/anatomopathologieRÉSUMÉ
The Tg2576 transgenic mouse model of human cerebral amyloid angiopathy is characterized by age-dependent cerebrovascular deposition of amyloid-beta (Abeta) starting from 9 months of age and progressively worsening to involve most pial arterioles by 18 months; soluble Abeta levels are elevated long before vascular deposition takes place in this model. It has been suggested that elevated soluble Abeta levels alone are sufficient to impair cerebral blood flow (CBF) regulation thereby contributing to the early progression of Alzheimer's disease. Using laser speckle flowmetry through an intact skull, we studied the impact of elevated soluble Abeta levels and vascular Abeta deposition on a wide range of CBF responses to evaluate vasodilation and vasoconstriction in young or aged Tg2576 mice. Nineteen-month-old Tg2576 with severe vascular Abeta deposits showed an attenuated hyperaemic response during hypercapnia and whisker stimulation compared to wild-type littermates. The anticipated increase in CBF due to isoflurane anaesthesia was also suppressed, as were the typical hypoperfusion responses during cortical spreading depression and alpha-chloralose anaesthesia. The responses of 8-month-old Tg2576 with elevated soluble Abeta levels, but without vascular Abeta deposition, did not differ from age-matched controls. In conclusion, our data suggest that vascular Abeta deposition is associated with impaired vasodilator as well as vasoconstrictor responses to a wide range of stimuli. These responses do not differ from controls when studied non-invasively prior to vascular Abeta deposition, thus challenging the view that elevated soluble Abeta levels are sufficient to cause cerebrovascular dysfunction.
Sujet(s)
Vieillissement/physiologie , Angiopathie amyloïde cérébrale/physiopathologie , Circulation cérébrovasculaire , Peptides bêta-amyloïdes/métabolisme , Animaux , Angiopathie amyloïde cérébrale/génétique , Angiopathie amyloïde cérébrale/métabolisme , Cortex cérébral/physiopathologie , Modèles animaux de maladie humaine , Hypercapnie/physiopathologie , Hyperhémie/physiopathologie , Fluxmétrie laser Doppler/méthodes , Mâle , Souris , Souris de lignée C57BL , Souris transgéniques , Stimulation physique , Vasoconstriction , Vasodilatation , VibrissesRÉSUMÉ
Normobaric hyperoxia is under investigation as a treatment for acute ischaemic stroke. In experimental models, normobaric hyperoxia reduces cerebral ischaemic injury and improves functional outcome. The mechanisms of neuroprotection are still debated because, (i) inhalation of 100% O2 does not significantly increase total blood O2 content; (ii) it is not known whether normobaric hyperoxia increases O2 delivery to the severely ischaemic cortex because of its short diffusion distance; and (iii) hyperoxia may reduce collateral cerebral blood flow (CBF) to ischaemic penumbra because it can cause vasoconstriction. We addressed these issues using real-time two-dimensional multispectral reflectance imaging and laser speckle flowmetry to simultaneously and non-invasively determine the impact of normobaric hyperoxia on CBF and oxygenation in ischaemic cortex. Ischaemia was induced by distal middle cerebral artery occlusion (dMCAO) in normoxic (30% inhaled O2, arterial pO2 134 +/- 9 mmHg), or hyperoxic mice (100% inhaled O2 starting 15 min after dMCAO, arterial pO2 312 +/- 10 mmHg). Post-ischaemic normobaric hyperoxia caused an immediate and progressive increase in oxyhaemoglobin (oxyHb) concentration, nearly doubling it in ischaemic core within 60 min. In addition, hyperoxia improved CBF so that the area of cortex with < or =20% residual CBF was decreased by 45% 60 min after dMCAO. Furthermore, hyperoxia reduced the frequency of peri-infarct depolarizations (PIDs) by more than 60%, and diminished their deleterious effects on CBF and metabolic load. Consistent with these findings, infarct size was reduced by 45% in the hyperoxia group 2 days after 75 min transient dMCAO. Our data show that normobaric hyperoxia increases tissue O2 delivery, and that novel mechanisms such as CBF augmentation, and suppression of PIDs may afford neuroprotection during hyperoxia.
Sujet(s)
Circulation cérébrovasculaire , Infarctus du territoire de l'artère cérébrale moyenne/thérapie , Consommation d'oxygène , Oxygénothérapie , Animaux , Modèles animaux de maladie humaine , Hémoglobines/métabolisme , Infarctus du territoire de l'artère cérébrale moyenne/anatomopathologie , Infarctus du territoire de l'artère cérébrale moyenne/physiopathologie , Souris , Souris de lignée C57BL , Résultat thérapeutiqueRÉSUMÉ
Ischemic depolarizing events, such as repetitive spontaneous periinfarct spreading depolarizations (PIDs), expand the infarct size after experimental middle cerebral artery (MCA) occlusion. This worsening may result from increased metabolic demand, exacerbating the mismatch between cerebral blood flow (CBF) and metabolism. Here, we present data showing that anoxic depolarization (AD) and PIDs caused vasoconstriction and abruptly reduced CBF in the ischemic cortex in a distal MCA occlusion model in mice. This reduction in CBF during AD increased the area of cortex with 20% or less residual CBF by 140%. With each subsequent PID, this area expanded by an additional 19%. Drugs that are known to inhibit cortical spreading depression (CSD), such as N-methyl-D-aspartate receptor antagonists MK-801 and 7-chlorokynurenic acid, and sigma-1 receptor agonists dextromethorphan and carbetapentane, did not reduce the frequency of PIDs, but did diminish the severity of episodic hypoperfusions, and prevented the expansion of severely hypoperfused cortex, thus improving CBF during 90 mins of acute focal ischemia. In contrast, AMPA receptor antagonist NBQX, which does not inhibit CSD, did not impact the deterioration in CBF. When measured 24 h after distal MCA occlusion, infarct size was reduced by MK-801, but not by NBQX. Our results suggest that AD and PIDs expand the CBF deficit, and by so doing negatively impact lesion development in ischemic mouse brain. Mitigating the vasoconstrictive neurovascular coupling during intense ischemic depolarizations may provide a novel hemodynamic mechanism of neuroprotection by inhibitors of CSD.