Targeting tau in Alzheimer's disease: from mechanisms to clinical therapy.

ABSTRACT: Alzheimer's disease is the most prevalent neurodegenerative disease affecting older adults. Primary features of Alzheimer's disease include extracellular aggregation of amyloid-ß plaques and the accumulation of neurofibrillary tangles, formed by tau protein, in the cells. While there are amyloid-ß-targeting therapies for the treatment of Alzheimer's disease, these therapies are costly and exhibit potential negative side effects. Mounting evidence suggests significant involvement of tau protein in Alzheimer's disease-related neurodegeneration. As an important microtubule-associated protein, tau plays an important role in maintaining the stability of neuronal microtubules and promoting axonal growth. In fact, clinical studies have shown that abnormal phosphorylation of tau protein occurs before accumulation of amyloid-ß in the brain. Various therapeutic strategies targeting tau protein have begun to emerge, and are considered possible methods to prevent and treat Alzheimer's disease. Specifically, abnormalities in post-translational modifications of the tau protein, including aberrant phosphorylation, ubiquitination, small ubiquitin-like modifier (SUMO)ylation, acetylation, and truncation, contribute to its microtubule dissociation, misfolding, and subcellular missorting. This causes mitochondrial damage, synaptic impairments, gliosis, and neuroinflammation, eventually leading to neurodegeneration and cognitive deficits. This review summarizes the recent findings on the underlying mechanisms of tau protein in the onset and progression of Alzheimer's disease and discusses tau-targeted treatment of Alzheimer's disease.

Quercetagitrin Inhibits Tau Accumulation and Reverses Neuroinflammation and Cognitive Deficits in P301S-Tau Transgenic Mice.

Intracellular tau accumulation is a hallmark pathology of Alzheimer's disease (AD) and other tauopathies. Tau protein, in the hyperphosphorylated form, is the component of paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) in AD. Blocking tau aggregation and/or phosphorylation is currently a promising strategy for AD treatment. Here, we elucidate that quercetagitrin, a natural compound derived from African marigold (Tagetes erecta), could inhibit tau aggregation and reduce tau phosphorylation at multiple disease-related sites in vitro. Moreover, the in vivo effect of quercetagitrin was assessed in P301S-tau transgenic via oral administration. The compound treatment restored the cognitive deficits and neuron loss in the mice. The formation of NFTs and tau phosphorylations in the hippocampus and cortex of the mice was also prevented by the compound. Moreover, quercetagitrin feeding displayed neuroinflammation protection through the inhibition of NF-κB activation in the mice. Together, our data reveal that quercetagitrin possesses the potential to further develop as a therapeutic medicine for AD and other tauopathies.

HDAC7 Activates IKK/NF-κB Signaling to Regulate Astrocyte-Mediated Inflammation.

Class IIa histone deacetylases (HDAC) have been shown to drive innate immune cell-mediated inflammation in the peripheral system, but their roles in cerebral inflammatory responses remain largely unknown. Here, we elucidate that HDAC7 is selectively elevated in lipopolysaccharide (LPS)-challenged astrocytes both in vivo and in vitro. We identify that HDAC7 binds to the inhibitory kappa B kinase (IKK) to promote IKKα and IKKß deacetylation and subsequent activation, leading to the activation of nuclear factor κB (NF-κB). Astrocyte-specific overexpression of HDAC7 results in NF-κB activation, pro-inflammatory gene upregulation and anxiety-like behaviors in mice, while downregulating HDAC7 reserves LPS-induced NF-κB activation and inflammatory responses. Furthermore, pharmacological inhibition of HDAC7 by a class IIa HDAC inhibitor attenuates LPS-induced NF-κB activation, inflammatory responses and anxiety-like behaviors both in vivo and in vitro. Together, our data reveal a novel mechanism of HDAC7 in astrocyte-mediated inflammation and suggest that targeting HDAC7 could be a potential therapeutic strategy for the treatment of anxiety and other inflammation-related diseases.

Periphery Biomarkers for Objective Diagnosis of Cognitive Decline in Type 2 Diabetes Patients.

Introduction: Type 2 diabetes mellitus (T2DM) is an independent risk factor of Alzheimer's disease (AD), and populations with mild cognitive impairment (MCI) have high incidence to suffer from AD. Therefore, discerning who may be more vulnerable to MCI, among the increasing T2DM populations, is important for early intervention and eventually decreasing the prevalence rate of AD. This study was to explore whether the change of plasma ß-amyloid (Aß) could be a biomarker to distinguish MCI (T2DM-MCI) from non-MCI (T2DM-nMCI) in T2DM patients. Methods: Eight hundred fifty-two T2DM patients collected from five medical centers were assigned randomly to training and validation cohorts. Plasma Aß, platelet glycogen synthase kinase-3ß (GSK-3ß), apolipoprotein E (ApoE) genotypes, and olfactory and cognitive functions were measured by ELISA, dot blot, RT-PCR, Connecticut Chemosensory Clinical Research Center (CCCRC) olfactory test based on the diluted butanol, and Minimum Mental State Examination (MMSE) test, respectively, and multivariate logistic regression analyses were applied. Results: Elevation of plasma Aß1-42/Aß1-40 is an independent risk factor of MCI in T2DM patients. Although using Aß1-42/Aß1-40 alone only reached an AUC of 0.631 for MCI diagnosis, addition of the elevated Aß1-42/Aß1-40 to our previous model (i.e., activated platelet GSK-3ß, ApoE ε4 genotype, olfactory decline, and aging) significantly increased the discriminating efficiency of T2DM-MCI from T2DM-nMCI, with an AUC of 0.846 (95% CI: 0.794-0.897) to 0.869 (95% CI: 0.822-0.916) in the training cohort and an AUC of 0.848 (95% CI: 0.815-0.882) to 0.867 (95% CI: 0.835-0.899) in the validation cohort, respectively. Conclusion: A combination of the elevated plasma Aß1-42/Aß1-40 with activated platelet GSK-3ß, ApoE ε4 genotype, olfactory decline, and aging could efficiently diagnose MCI in T2DM patients. Further longitudinal studies may consummate the model for early prediction of AD.

Medial septum tau accumulation induces spatial memory deficit via disrupting medial septum-hippocampus cholinergic pathway.

Tau accumulation and cholinergic impairment are characteristic pathologies in Alzheimer's disease (AD). However, the causal role of tau accumulation in cholinergic lesion is elusive. Here, we observed an aberrant tau accumulation in the medial septum (MS) of 3xTg and 5xFAD mice, especially in their cholinergic neurons. Overexpressing hTau in mouse MS (MShTau ) for 6 months but not 3 months induced spatial memory impairment without changing object recognition and anxiety-like behavior, indicating a specific and time-dependent effect of MS-hTau accumulation on spatial cognitive functions. With increasing hTau accumulation, the MShTau mice showed a time-dependent cholinergic neuron loss with reduced cholinergic projections to the hippocampus. Intraperitoneal administration of donepezil, a cholinesterase inhibitor, for 1 month ameliorated the MS-hTau-induced spatial memory deficits with preservation of MS-hippocampal cholinergic pathway and removal of tau load; and the beneficial effects of donepezil was more prominent at low dose. Proteomics revealed that MS-hTau accumulation deregulated multiple signaling pathways with numerous differentially expressed proteins (DEPs). Among them, the vacuolar protein sorting-associated protein 37D (VP37D), an autophagy-related protein, was significantly reduced in MShTau mice; the reduction of VP37D was restored by donepezil, and the effect was more significant at low dose than high dose. These novel evidences reveal a causal role of tau accumulation in linking MS cholinergic lesion to hippocampus-dependent spatial cognitive damages as seen in the AD patients, and the new tau-removal and autophagy-promoting effects of donepezil may extend its application beyond simple symptom amelioration to potential disease modification.

A novel small-molecule PROTAC selectively promotes tau clearance to improve cognitive functions in Alzheimer-like models.

Intracellular accumulation of tau is a hallmark pathology in Alzheimer disease (AD) and the related tauopathies, thus targeting tau could be promising for drug development. Proteolysis Targeting Chimera (PROTAC) is a novel drug discovery strategy for selective protein degradation from within cells. Methods: A novel small-molecule PROTAC, named as C004019 with a molecular mass of 1,035.29 dalton, was designed to simultaneously recruite tau and E3-ligase (Vhl) and thus to selectively enhance ubiquitination and proteolysis of tau proteins. Western blotting, immunofluoresence and immunohistochemical staining were employed to verify the effects of C004019 in cell models (HEK293 and SH-SY5Y) and mouse models (hTau-transgenic and 3xTg-AD), respectively. The cognitive capacity of the mice was assessed by a suite of behavior experiments. Electrophysiology and Golgi staining were used to evaluate the synaptic plasticity. Results: C004019 induced a robust tau clearance via promoting its ubiquitination-proteasome-dependent proteolysis in HEK293 cells with stable or transient overexpression of human tau (hTau), and in SH-SY5Y that constitutively overexpress hTau. Furthermore, intracerebral ventricular infusion of C004019 induced a robust tau clearance in vivo. Most importantly, both single-dose and multiple-doses (once per 6 days for a total 5 times) subcutaneous administration of C004019 remarkably decreased tau levels in the brains of wild-type, hTau-transgenic and 3xTg-AD mice with improvement of synaptic and cognitive functions. Conclusions: The PROTAC (C004019) created in the current study can selectively and efficiently promote tau clearance both in vitro and in vivo, which provides a promising drug candidate for AD and the related tauopathies.

Tau-induced upregulation of C/EBPß-TRPC1-SOCE signaling aggravates tauopathies: A vicious cycle in Alzheimer neurodegeneration.

Intracellular accumulating of the hyperphosphorylated tau plays a pivotal role in neurodegeneration of Alzheimer disease (AD), but the mechanisms underlying the gradually aggravated tau hyperphosphorylation remain elusive. Here, we show that increasing intracellular tau could upregulate mRNA and protein levels of TRPC1 (transient receptor potential channel 1) with an activated store-operated calcium entry (SOCE), an increased intraneuronal steady-state [Ca2+ ]i , an enhanced endoplasmic reticulum (ER) stress, an imbalanced protein kinases and phosphatase, and an aggravated tauopathy. Furthermore, overexpressing TRPC1 induced ER stress, kinases-phosphatase imbalance, tau hyperphosphorylation and cognitive deficits in cultured neurons and mice, while pharmacological inhibiting or knockout TRPC1 attenuated the hTau-induced deregulations in SOCE, ER homeostasis, kinases-phosphatase balance, and tau phosphorylation level with improved synaptic and cognitive functions. Finally, an increased CCAAT-enhancer-binding protein (C/EBPß) activity was observed in hTau-overexpressing cells and the hippocampus of the AD patients, while downregulating C/EBPß by siRNA abolished the hTau-induced TRPC1 upregulation. These data reveal that increasing intracellular tau can upregulate C/EBPß-TRPC1-SOCE signaling and thus disrupt phosphorylating system, which together aggravates tau pathologies leading to a chronic neurodegeneration.

Peripheral inflammation promotes brain tau transmission via disrupting blood-brain barrier.

Abnormal aggregation of pathological tau protein is a neuropathological feature of Alzheimer's disease (AD). In the AD patients, the abnormal tau accumulation first appeared in entorhinal cortex (EC) and then propagated to the hippocampus with microglia activation and inflammation, but the mechanism is elusive. Here, we studied the role and mechanisms underlying periphery inflammation on brain tau transmission. By intraperitoneal injection of lipopolysaccharide (LPS) with brain medial entorhinal cortex (MEC)-specific overexpressing P301L human tau (P301L-hTau), we found that both acute and chronic administration of LPS remarkably promoted P301L-hTau transmission from MEC to the hippocampal subsets. Interestingly, the chronic LPS-induced P301L-hTau transmission was still apparent after blocking microglia activation. Further studies demonstrated that LPS disrupted the integrity of blood-brain barrier (BBB) and simultaneous intraperitoneal administration of glucocorticoid (GC) attenuated LPS-promoted P301L-hTau transmission. These data together suggest that a non-microglia-dependent BBB disruption contributes to peripheral LPS-promoted brain P301L-hTau transmission, therefore, maintaining the integrity of BBB can be a novel strategy for preventing pathological tau propagation in AD and other tauopathies.

Tau acetylates and stabilizes ß-catenin thereby promoting cell survival.

Overexpressing Tau counteracts apoptosis and increases dephosphorylated ß-catenin levels, but the underlying mechanisms are elusive. Here, we show that Tau can directly and robustly acetylate ß-catenin at K49 in a concentration-, time-, and pH-dependent manner. ß-catenin K49 acetylation inhibits its phosphorylation and its ubiquitination-associated proteolysis, thus increasing ß-catenin protein levels. K49 acetylation further promotes nuclear translocation and the transcriptional activity of ß-catenin, and increases the expression of survival-promoting genes (bcl2 and survivin), counteracting apoptosis. Mutation of Tau's acetyltransferase domain or co-expressing non-acetylatable ß-catenin-K49R prevents increased ß-catenin signaling and abolishes the anti-apoptotic function of Tau. Our data reveal that Tau preserves ß-catenin by acetylating K49, and upregulated ß-catenin/survival signaling in turn mediates the anti-apoptotic effect of Tau.

Tau inhibits PKA by nuclear proteasome-dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation.

Intraneuronal accumulation of wild-type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full-length wild-type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse- and memory-associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP-response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ-20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau-induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau-induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ-20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.

Co-Expression of Three Wild-Type 3R-Tau Isoforms Induces Memory Deficit via Oxidation-Related DNA Damage and Cell Death: A Promising Model for Tauopathies.

The three isoforms of 3R-tau are predominantly deposited in neurons bearing neurofibrillary tangles in Alzheimer's disease (AD), while only 3R-tau accumulation has been detected in Pick's disease (PiD), suggesting the involvement of 3R-tau in neurodegeneration. However, both the role and the molecular mechanism of 3R-tau in neurodegeneration are elusive. Here, we co-expressed three isoforms of human wild-type 3R-tau in adult mouse hippocampal to mimic the pathologic tau accumulating observed in PiD patients. We found that co-expressing three 3R-tau isoforms induced hyperphosphorylation and accumulation of tau proteins; simultaneously, the mice showed remarkable neuron death with synapse and memory deficits. Further in vitro and in vivo studies demonstrated that co-expressing 3R-tau isoforms caused oxidative stress evidenced by an increased malondialdehyde, and the decreased superoxide dismutase and glutathione peroxidase; the 3R-tau accumulation also induced significant glial activation and DNA double-strand breaks (DSBs). Notably, the toxic effects of 3R-tau accumulation were efficiently reversed by administration of antioxidants Vitamin E (VitE) and Vitamin C (VitC), respectively. These data reveal that intracellular accumulation of 3R-tau isoforms in adult brain induces significant neuron death and memory deficits with the mechanism involving oxidation-mediated DSBs; and the antioxidants VitE and VitC can efficiently attenuate the toxicities of 3R-tau. Given that no significant cell death has been detected in the currently available wild-type tau-accumulating models, co-expressing 3R-tau isoforms could be a promising model for drug development of tauopathies, such as PiD.

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.

New Anti-Inflammatory Aporphine and Lignan Derivatives from the Root Wood of Hernandia nymphaeifolia.

A new aporphine, 3-hydroxyhernandonine (1) and a new lignin, 4'-O-demethyl-7-O-methyldehydropodophyllotoxin (2), have been isolated from the root wood of Hernanadia nymphaeifolia, together with thirteen known compounds (3⁻15). The structures of these compounds were determined through mass spectrometry (MS) and spectroscopic analyses. The known isolate, 2-O-methyl-7-oxolaetine (3), was first isolated from natural sources. Among the isolated compounds, 3-hydroxyhernandonine (1), 4'-O-demethyl-7-O-methyldehydropodophyllotoxin (2), hernandonine (4), oxohernangerine (5), and oxohernagine (6) displayed inhibition (IC50 values ≤5.72 µg/mL) of superoxide anion production by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). In addition, 3-hydroxyhernandonine (1), 4'-O-demethyl-7-O-methyldehydropodophyllotoxin (2), oxohernangerine (5), and oxohernagine (6) suppressed fMLP/CB-induced elastase release with IC50 values ≤5.40 µg/mL.

TRPC1 Null Exacerbates Memory Deficit and Apoptosis Induced by Amyloid-ß.

The transient receptor potential cation (TRPC) channels are widely expressed in nervous system but their functions remain largely unclear. Here, we found that TRPC1 deletion did not affect learning and memory in physiological conditions, while it aggravated learning and memory deficits induced by amyloid-ß (Aß), the major component of the senile plaques observed in the brains of Alzheimer's disease (AD). Further studies demonstrated that TRPC1 deletion did not affect cell apoptosis in physiological condition, but it exacerbated the Aß-induced cell death in mouse hippocampus. Moreover, the level of TRPC1 was decreased in AD cell and mouse models, and upregulation of TRPC1 decreased Aß levels with attenuation of apoptosis in the cells stably overexpressing amyloid-ß protein precursor (AßPP). Finally, the transmembrane domain of TRPC1 could bind to AßPP and thus decreased Aß production. These findings indicate that loss of TRPC1 exacerbates Aß-induced memory deficit and cell apoptosis, though it does not impair cognitive function or induce cell death in physiological conditions.

Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca2+ concentration with a simultaneous increase in the phosphorylation of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca2+/CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca2+/calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca2+/CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2.

Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer's disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments.

Tau accumulation induces synaptic impairment and memory deficit by calcineurin-mediated inactivation of nuclear CaMKIV/CREB signaling.

Intracellular accumulation of wild-type tau is a hallmark of sporadic Alzheimer's disease (AD), but the molecular mechanisms underlying tau-induced synapse impairment and memory deficit are poorly understood. Here we found that overexpression of human wild-type full-length tau (termed hTau) induced memory deficits with impairments of synaptic plasticity. Both in vivo and in vitro data demonstrated that hTau accumulation caused remarkable dephosphorylation of cAMP response element binding protein (CREB) in the nuclear fraction. Simultaneously, the calcium-dependent protein phosphatase calcineurin (CaN) was up-regulated, whereas the calcium/calmodulin-dependent protein kinase IV (CaMKIV) was suppressed. Further studies revealed that CaN activation could dephosphorylate CREB and CaMKIV, and the effect of CaN on CREB dephosphorylation was independent of CaMKIV inhibition. Finally, inhibition of CaN attenuated the hTau-induced CREB dephosphorylation with improved synapse and memory functions. Together, these data indicate that the hTau accumulation impairs synapse and memory by CaN-mediated suppression of nuclear CaMKIV/CREB signaling. Our findings not only reveal new mechanisms underlying the hTau-induced synaptic toxicity, but also provide potential targets for rescuing tauopathies.