MAPT/Tau accumulation represses autophagy flux by disrupting IST1-regulated ESCRT-III complex formation: a vicious cycle in Alzheimer neurodegeneration.

Macroautophagy/autophagy deficit induces intracellular MAPT/tau accumulation, the hallmark pathology in Alzheimer disease (AD) and other tauopathies; however, the reverse role of MAPT accumulation in autophagy and neurodegeneration is not clear. Here, we found that overexpression of human wild-type full-length MAPT, which models MAPT pathologies as seen in sporadic AD patients, induced autophagy deficits via repression of autophagosome-lysosome fusion leading to significantly increased LC3 (microtubule-associated protein 1 light chain 3)-II and SQSTM1/p62 (sequestosome 1) protein levels with autophagosome accumulation. At the molecular level, intracellular MAPT aggregation inhibited expression of IST1 (IST1 factor associated with ESCRT-III), a positive modulator for the formation of ESCRT (the Endosomal Sorting Complex Required for Transport) complex that is required for autophagosome-lysosome fusion. Upregulating IST1 in human MAPT transgenic mice attenuated autophagy deficit with reduced MAPT aggregation and ameliorated synaptic plasticity and cognitive functions, while downregulating IST1 per se induced autophagy deficit with impaired synapse and cognitive function in naïve mice. IST1 can facilitate association of CHMP2B (charged multivesicular body protein 2B) and CHMP4B/SNF7-2 to form ESCRT-III complex, while lack of IST1 impeded the complex formation. Finally, we demonstrate that MAPT accumulation suppresses IST1 transcription with the mechanisms involving the ANP32A-regulated mask of histone acetylation. Our findings suggest that the AD-like MAPT accumulation can repress autophagosome-lysosome fusion by deregulating ANP32A-INHAT-IST1-ESCRT-III pathway, which also reveals a vicious cycle of MAPT accumulation and autophagy deficit in the chronic course of AD neurodegeneration.Abbreviations: AAV: adeno-associated virus; Aß: ß-amyloid; aCSF: artificial cerebrospinal fluid; AD: Alzheimer disease; ANP32A: acidic nuclear phosphoprotein 32 family member A; ATG: autophagy related; AVs: autophagic vacuoles; CEBPB: CCAAT enhancer binding protein beta; CHMP: charged multivesicular body protein; DMEM: Dulbecco's modified eagle's medium; EBSS: Earle's balanced salt solution; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; fEPSPs: field excitatory postsynaptic potentials; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3B: glycogen synthase kinase 3 beta; HAT: histone acetyl transferase; HDAC: histone deacetylase; INHAT: inhibitor of histone acetyl transferase; IST1: IST1 factor associated with ESCRT-III; LAMP2: lysosomal associated membrane protein 2; LTP: long-term potentiation; MAP1LC3: microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MVB: multivesicular bodies; MWM: Morris water maze; PBS: phosphate-buffered saline solution; RAB7: member RAS oncogene family; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SQSTM1/p62: sequestosome 1.

Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression.

Intracellular tau accumulation forming neurofibrillary tangles is hallmark pathology of Alzheimer's disease (AD), but how tau accumulation induces synapse impairment is elusive. By overexpressing human full-length wild-type tau (termed hTau) to mimic tau abnormality as seen in the brain of sporadic AD patients, we find that hTau accumulation activates JAK2 to phosphorylate STAT1 (signal transducer and activator of transcription 1) at Tyr701 leading to STAT1 dimerization, nuclear translocation, and its activation. STAT1 activation suppresses expression of N-methyl-D-aspartate receptors (NMDARs) through direct binding to the specific GAS element of GluN1, GluN2A, and GluN2B promoters, while knockdown of STAT1 by AAV-Cre in STAT1flox/flox mice or expressing dominant negative Y701F-STAT1 efficiently rescues hTau-induced suppression of NMDAR expression with amelioration of synaptic functions and memory performance. These findings indicate that hTau accumulation impairs synaptic plasticity through JAK2/STAT1-induced suppression of NMDAR expression, revealing a novel mechanism for hTau-associated synapse and memory deficits.

Human wild-type full-length tau accumulation disrupts mitochondrial dynamics and the functions via increasing mitofusins.

Intracellular accumulation of tau protein is hallmark of sporadic Alzheimer's disease (AD), however, the cellular mechanism whereby tau accumulation causes neurodegeneration is poorly understood. Here we report that overexpression of human wild-type full-length tau (termed htau) disrupted mitochondrial dynamics by enhancing fusion and induced their perinuclear accumulation in HEK293 cells and rat primary hippocampal neurons. The htau accumulation at later stage inhibited mitochondrial functions shown by the decreased ATP level, the ratio of ATP/ADP and complex I activity. Simultaneously, the cell viability was decreased with retraction of the cellular/neuronal processes. Further studies demonstrated that htau accumulation increased fusion proteins, including OPA1 and mitofusins (Mfn1, Mfn2) and reduced the ubiquitination of Mfn2. Downregulation of the mitofusins by shRNA to ~45% or ~52% of the control levels attenuated the htau-enhanced mitochondrial fusion and restored the functions, while downregulation of OPA1 to ~50% of the control level did not show rescue effects. Finally, abnormal mitochondrial accumulation and dysfunction were also observed in the brains of htau transgenic mice. Taken together, our data demonstrate that htau accumulation decreases cell viability and causes degeneration via enhancing mitofusin-associated mitochondrial fusion, which provides new insights into the molecular mechanisms underlying tauopathies.

Tau accumulation impairs mitophagy via increasing mitochondrial membrane potential and reducing mitochondrial Parkin.

Intracellular accumulation of wild type tau is a hallmark of sporadic Alzheimer's disease (AD). However, the molecular mechanisms underlying tau toxicity is not fully understood. Here, we detected mitophagy deficits evidenced by the increased levels of mitophagy markers, including COX IV, TOMM20, and the ratio of mtDNA to genomic DNA indexed as mt-Atp6/Rpl13, in the AD brains and in the human wild type full-length tau (htau) transgenic mice. More interestingly, the mitophagy deficit was only shown in the AD patients who had an increased total tau level. Further studies demonstrated that overexpression of htau induced mitophagy deficits in HEK293 cells, the primary hippocampal neurons and in the brains of C57 mice. Upon overexpression of htau, the mitochondrial membrane potential was increased and the levels of PTEN-induced kinase 1 (PINK1) and Parkin decreased in the mitochondrial fraction, while upregulation of Parkin attenuated the htau-induced mitophagy deficits. Finally, we detected a dose-dependent allocation of tau proteins into the mitochondrial outer membrane fraction along with its cytoplasmic accumulation. These data suggest that intracellular accumulation of htau induces mitophagy deficits by direct inserting into the mitochondrial membrane and thus increasing the membrane potential, which impairs the mitochondrial residence of PINK1/Parkin. Our findings reveal a novel mechanism underlying the htau-induced neuronal toxicities in AD and other tauopathies.

Silencing [Formula: see text] Rescues Tau Pathologies and Memory Deficits through Rescuing PP2A and Inhibiting GSK-3ß Signaling in Human Tau Transgenic Mice.

Increase of inhibitor-2 of protein phosphatase-2A [Formula: see text] is associated with protein phosphatase-2A (PP2A) inhibition and tau hyperphosphorylation in Alzheimer's disease (AD). Down-regulating [Formula: see text] attenuated amyloidogenesis and improved the cognitive functions in transgenic mice expressing amyloid precursor protein (tg2576). Here, we found that silencing [Formula: see text] by hippocampal infusion of [Formula: see text] down-regulated [Formula: see text] (~45%) with reduction of tau phosphorylation/accumulation, improvement of memory deficits, and dendritic plasticity in 12-month-old human tau transgenic mice. Silencing [Formula: see text] not only restored PP2A activity but also inhibited glycogen synthase kinase-3ß (GSK-3ß) with a significant activation of protein kinase A (PKA) and Akt. In HEK293/tau and N2a/tau cells, silencing [Formula: see text] by [Formula: see text] also significantly reduced tau hyperphosphorylation with restoration of PP2A activity and inhibition of GSK-3ß, demonstrated by the decreased GSK-3ß total protein and mRNA levels, and the increased inhibitory phosphorylation of GSK-3ß at serine-9. Furthermore, activation of PKA but not Akt mediated the inhibition of GSK-3ß by [Formula: see text] silencing. We conclude that targeting [Formula: see text] can improve tau pathologies and memory deficits in human tau transgenic mice, and activation of PKA contributes to GSK-3ß inhibition induced by silencing [Formula: see text]in vitro, suggesting that [Formula: see text] is a promising multiple target of AD.

Silencing PP2A inhibitor by lenti-shRNA interference ameliorates neuropathologies and memory deficits in tg2576 mice.

Deficits of protein phosphatase-2A (PP2A) play a crucial role in tau hyperphosphorylation, amyloid overproduction, and synaptic suppression of Alzheimer's disease (AD), in which PP2A is inactivated by the endogenously increased inhibitory protein, namely inhibitor-2 of PP2A (I2(PP2A)). Therefore, in vivo silencing I2(PP2A) may rescue PP2A and mitigate AD neurodegeneration. By infusion of lentivirus-shRNA targeting I2(PP2A) (LV-siI2(PP2A)) into hippocampus and frontal cortex of 11-month-old tg2576 mice, we demonstrated that expression of LV-siI2(PP2A) decreased remarkably the elevated I2(PP2A) in both mRNA and protein levels. Simultaneously, the PP2A activity was restored with the mechanisms involving reduction of the inhibitory binding of I2(PP2A) to PP2A catalytic subunit (PP2AC), repression of the inhibitory Leu309-demethylation and elevation of PP2AC. Silencing I2(PP2A) induced a long-lasting attenuation of amyloidogenesis in tg2576 mice with inhibition of amyloid precursor protein hyperphosphorylation and ß-secretase activity, whereas simultaneous inhibition of PP2A abolished the antiamyloidogenic effects of I2(PP2A) silencing. Finally, silencing I2(PP2A) could improve learning and memory of tg2576 mice with preservation of several memory-associated components. Our data reveal that targeting I2(PP2A) can efficiently rescue Aß toxicities and improve the memory deficits in tg2576 mice, suggesting that I2(PP2A) could be a promising target for potential AD therapies.

Glycogen synthase kinase-3ß regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1.

Protein phosphatase-2A (PP2A) activity is significantly suppressed in Alzheimer's disease. We have reported that glycogen synthase kinase-3ß (GSK-3ß) inhibits PP2A via upregulating the phosphorylation of PP2A catalytic subunit (PP2A(C)). Here we studied the effects of GSK-3ß on the inhibitory demethylation of PP2A at leucine-309 (dmL309-PP2A(C)). We found that GSK-3ß regulates dmL309-PP2A(C) level by regulating PME-1 and PPMT1. Knockdown of PME-1 or PPMT1 eliminated the effects of GSK-3ß on PP2A(C). GSK-3 could negatively regulate PP2A regulatory subunit protein level. We conclude that GSK-3ß can inhibit PP2A by increasing the inhibitory L309-demethylation involving upregulation of PME-1 and inhibition of PPMT1.

Effects of endogenous beta-amyloid overproduction on tau phosphorylation in cell culture.

Alzheimer's disease is characterized by beta-amyloid (Abeta) overproduction and tau hyperphosphorylation. Recent studies have shown that synthetic Abeta promotes tau phosphorylation in vitro. However, whether endogenously overproduced Abeta promotes tau phosphorylation and the underlying mechanisms remain unknown. Here, we used mouse neuroblastoma N2a stably expressing wild-type amyloid precursor protein (APPwt) or the Swedish mutant APP (APPswe) to determine the alterations of phosphorylated tau and the related protein kinases. We found that phosphorylation of tau at paired helical filament (PHF)-1, pSer396 and pThr231 epitopes was significantly increased in cells transfected with APPwt and APPswe, which produced higher levels of Abeta than cells transfected with vector or amyloid precursor-like protein 1. The activity of glycogen synthase kinase-3 (GSK-3) was up-regulated with a concomitant reduction in the inhibitory phosphorylation of GSK-3 at its N-terminal Ser9 residue. In contrast, the activity of cyclin-dependent kinase-5 (CDK-5) and protein kinase C (PKC) was down-regulated. Inhibition of GSK-3 by LiCl, but not inhibition of CDK-5 by roscovitine, arrested Abeta secretion and tau phosphorylation. Inhibition of PKC by GF-109203X activated GSK-3, whereas activation of PKC by phorbol-12,13-dibutyrate inhibited GSK-3. These results suggest that endogenously overproduced Abeta induces increased tau phosphorylation through activation of GSK-3, and that inactivation of PKC is at least one of the mechanisms involved in GSK-3 activation.

Effect of melatonin on calyculin A-induced tau hyperphosphorylation.

We have found in the present study that incubation of neuroblastma N2a with calyculin A, an inhibitor of protein phosphatase-2A (PP-2A) and protein phosphatase-1 (PP-1), reduces cell viability in a dose-dependent manner, and leads to tau hyperphosphorylation at tau-1 (Ser198/199/202) and PHF-1 (Ser396/404) epitopes. In addition to inhibit PP-2A, calyculin A treatment also results in significant activation of glycogen synthase kinase-3 (GSK-3). Calyculin A induces oxidative stress manifested by elevated level of malondialdehyde and decreased activity of superoxide dismutase. When the cells were incubated simultaneously with calyculin A and melatonin (25 microM or 50 microM), the calyculin A-induced decrease in cell viability, tau hyperphosphorylation, PP-2A/GSK-3 imbalance and oxidative stress were attenuated accordingly. These results suggest (i) that calyculin A induces tau hyperphosphorylation not only by inhibition of PP-2A, but also by activation of GSK-3 in N2a cells; (ii) that melatonin efficiently attenuates the calyculin A-induced damages through not only its antioxidant effect but also its modulation to the phosphorylation system.