Deletion of MyD88 in astrocytes prevents ß-amyloid-induced neuropathology in mice.

As the understanding of immune responses in Alzheimer's disease (AD) is in its early phases, there remains an urgency to identify the cellular and molecular processes driving chronic inflammation. In AD, a subpopulation of astrocytes acquires a neurotoxic phenotype which prompts them to lose typical physiological features. While the underlying molecular mechanisms are still unknown, evidence suggests that myeloid differentiation primary response 88 (MyD88) adaptor protein may play a role in coordinating these cells' immune responses in AD. Herein, we combined studies in human postmortem samples with a conditional genetic knockout mouse model to investigate the link between MyD88 and astrocytes in AD. In silico analyses of bulk and cell-specific transcriptomic data from human postmortem brains demonstrated an upregulation of MyD88 expression in astrocytes in AD versus non-AD individuals. Proteomic studies revealed an increase in glial fibrillary acidic protein in multiple brain regions of AD subjects. These studies also showed that although overall MyD88 steady-state levels were unaffected by AD, this protein was enriched in astrocytes near amyloid plaques and neurofibrillary tangles. Functional studies in mice indicated that the deletion of astrocytic MyD88 protected animals from the acute synaptic toxicity and cognitive impairment caused by the intracerebroventricular administration of ß-amyloid (Aß). Lastly, unbiased proteomic analysis revealed that loss of astrocytic MyD88 resulted in altered astrocyte reactivity, lower levels of immune-related proteins, and higher expression of synaptic-related proteins in response to Aß. Our studies provide evidence of the pivotal role played by MyD88 in the regulation of astrocytes response to AD.

Manganese-induced α-synuclein overexpression promotes the accumulation of dysfunctional synaptic vesicles and hippocampal synaptotoxicity by suppressing Rab26-dependent autophagy in presynaptic neurons.

Manganese (Mn) overexposure induces learning and memory impairments in mice by disrupting the functions of synapses and synaptic vesicles (SVs) in the hippocampus, which is associated with α-synuclein (α-Syn) overexpression. Rab26-dependent autophagy is a key signaling step required for impaired SV clearance; however, it is unclear whether Mn-induced α-Syn overexpression is linked to dysregulated Rab26-dependent autophagy in presynaptic neurons. In this study, we developed manganism models in male C57BL/6 mice and hippocampal primary neurons to observe the associations between Mn-induced α-Syn overexpression and impaired SV accumulation. The results of the in vivo experiments showed that 100 and 200 µmol/kg Mn exposure significantly impaired memory and synaptic plasticity in the mice, which was related to the accumulation of impaired SVs in the hippocampus. Consistent with the in vivo outcomes, the level of in vitro injured SVs in the 50 and 100 µmol/L Mn-exposed neuron group were higher than that in the control group. Moreover, 100 µmol/L Mn suppressed the initiation of Rab26-dependent autophagy at the synapse. Then, we transfected neurons with LV-α-Syn short hairpin RNA (shRNA) and exposed the neurons to Mn for an additional 24 h. Surprisingly, the area of colocalization between Rab26 and Atg16L1 and the expression level of LC3II-positive SVs were both higher in Mn-exposed LV-α-Syn shRNA-transfected neurons than those in Mn-treated normal or Mn-treated LV-scrambled shRNA-transfected neurons. Thus, Mn-induced α-Syn overexpression was responsible for the dysregulation of Rab26-dependent autophagy, thereby promoting the accumulation of injured SVs, and causing synaptotoxicity and cognitive and memory deficits in mice.

Melatonin pretreatment alleviates the long-term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor-mediated Wnt signaling modulation.

Sevoflurane (Sev) is one of the most widely used pediatric anesthetics. The major concern of neonatal repeated application of Sev is its potential long-term impairment of cognition and learning/memory, for which there still lacks effective treatment. At the cellular level, Sev exerts toxic effects in multiple aspects, making it difficult for effective interference. Melatonin is a pineal hormone regulated by and feedbacks to biological rhythm at physiological condition. Recent studies have revealed significant neuroprotective effects of exogenous melatonin or its agonists under various pathological conditions. Whether melatonin could prevent the long-term toxicity of Sev remains elusive. Here, we report that neonatal repeated Sev exposure up-regulated MT1 receptor in hippocampal neurons and oligodendrocytes. Pretreatment with melatonin significantly alleviated Sev-induced synaptic deficiency, dysmyelination, and long-term learning impairment. Both MT1-shRNA and MT1 knockout effectively blocked the protective effects of melatonin on synaptic development, myelination, and behavior performance. Interestingly, long-lasting suppression of Wnt signaling, instead of cAMP/PKA signaling, was observed in hippocampal neurons and oligodendrocytes after neonatal Sev exposure. Pharmacologically activating Wnt signaling rescued both the long-term synaptic deficits and dysmyelination induced by Sev. Further analysis showed that MT1 receptor co-expressed well with ß-catenin and Axin2 and bound to ß-catenin by its C-terminal. Melatonin pretreatment effectively rescued Sev-induced Wnt suppression. Wnt signaling inhibitor XAV939 significantly compromised the protective effects of melatonin. Taken together, our data demonstrated a beneficial effect of melatonin pretreatment on the long-term synaptic impairment and dysmyelination induced by neonatal Sev exposure, and a novel MT1 receptor-mediated interaction between melatonin and canonical Wnt signaling, indicating that melatonin may be clinically applied for improving the safety of pediatric Sev anesthesia.

Non-cell autonomous astrocyte-mediated neuronal toxicity in prion diseases.

Under normal conditions, astrocytes perform a number of important physiological functions centered around neuronal support and synapse maintenance. In neurodegenerative diseases including Alzheimer's, Parkinson's and prion diseases, astrocytes acquire reactive phenotypes, which are sustained throughout the disease progression. It is not known whether in the reactive states associated with prion diseases, astrocytes lose their ability to perform physiological functions and whether the reactive states are neurotoxic or, on the contrary, neuroprotective. The current work addresses these questions by testing the effects of reactive astrocytes isolated from prion-infected C57BL/6J mice on primary neuronal cultures. We found that astrocytes isolated at the clinical stage of the disease exhibited reactive, pro-inflammatory phenotype, which also showed downregulation of genes involved in neurogenic and synaptogenic functions. In astrocyte-neuron co-cultures, astrocytes from prion-infected animals impaired neuronal growth, dendritic spine development and synapse maturation. Toward examining the role of factors secreted by reactive astrocytes, astrocyte-conditioned media was found to have detrimental effects on neuronal viability and synaptogenic functions via impairing synapse integrity, and by reducing spine size and density. Reactive microglia isolated from prion-infected animals were found to induce phenotypic changes in primary astrocytes reminiscent to those observed in prion-infected mice. In particular, astrocytes cultured with reactive microglia-conditioned media displayed hypertrophic morphology and a downregulation of genes involved in neurogenic and synaptogenic functions. In summary, the current study provided experimental support toward the non-cell autonomous mechanisms behind neurotoxicity in prion diseases and demonstrated that the astrocyte reactive phenotype associated with prion diseases is synaptotoxic.

Actin-microtubule crosslinker Pod-1 tunes PAR-1 signaling to control synaptic development and tau-mediated synaptic toxicity.

Partitioning-defective 1 (PAR-1), a conserved cell polarity regulator, plays an important role in synaptic development, and its mutation affects the formation of synaptic boutons and localization of postsynaptic density protein Discs large (Dlg) at the neuromuscular junction (NMJ) in Drosophila. Drosophila PAR-1 and its human homolog, Microtubule affinity-regulating kinases (MARK), are also known to be implicated in Alzheimer's disease (AD) by controlling tau-mediated Aß toxicity. However, the molecular mechanisms of PAR-1 function remain incompletely understood. Here we identified Pod-1, an actin-microtubule crosslinker, which functionally and physically interacts with PAR-1 in Drosophila. Pod-1 prominently co-localizes with PAR-1 in the postsynaptic region and regulates PAR-1 activity at the NMJ. Synaptic defects, including the reduction of boutons and delocalization of Dlg caused by PAR-1 overexpression, were rescued by Pod-1 knockdown. Conversely, the reduction of synaptic boutons in PAR-1 overexpressed NMJ was synergistically enhanced by the overexpression of Pod-1. Furthermore, Pod-1 increases the PAR-1 dependent S262 phosphorylation of tau, which is known to contribute to tau-mediated Aß toxicity. In line with the change of tau phosphorylation, Pod-1 knockdown rescued tau-mediated synaptic toxicity at the NMJ. Our results suggest that Pod-1 may act as a modulator of PAR-1 in synaptic development and tau-mediated toxicity.

Involvement of homodomain interacting protein kinase 2-c-Jun N-terminal kinase/c-Jun cascade in the long-term synaptic toxicity and cognition impairment induced by neonatal Sevoflurane exposure.

Sevoflurane is one of the most widely used anesthetics with recent concerns rising about its pediatric application. The synaptic toxicity and mechanisms underlying its long-term cognition impairment remain unclear. In this study, we investigated the expression and roles of homeodomain interacting protein kinase 2 (HIPK2), a stress activating kinase involved in neuronal survival and synaptic plasticity, and its downstream c-Jun N-terminal kinase (JNK)/c-Jun signaling in the long-term toxicity of neonatal Sevoflurane exposure. Our data showed that neonatal Sevoflurane exposure results in impairment of memory, enhancement of anxiety, less number of excitatory synapses and lower levels of synaptic proteins in the hippocampus of adult rats without significant changes of hippocampal neuron numbers. Up-regulation of HIPK2 and JNK/c-Jun was observed in hippocampal granular neurons shortly after Sevoflurane exposure and persisted to adult. 5-((6-Oxo-5-(6-(piperazin-1-yl)pyridin-3-yl)-1,6-dihydropyridin-3-yl)methylene)thiazolidine-2,4-dione trifluoroacetate, antagonist of HIPK2, could significantly rescue the cognition impairment, decrease in long-term potentiation, reduction in spine density and activation of JNK/c-Jun induced by Sevoflurane. JNK antagonist SP600125 partially restored synapse development and cognitive function without affecting the expression of HIPK2. These data, in together, revealed a novel role of HIPK2-JNK/c-Jun signaling in the long-term synaptic toxicity and cognition impairment of neonatal Sevoflurane exposure, indicating HIPK2-JNK/c-Jun cascade as a potential target for reducing the synaptic toxicity of Sevoflurane. Cover Image for this issue: doi: 10.1111/jnc.14757.

Hsp60 Protects against Amyloid ß Oligomer Synaptic Toxicity via Modification of Toxic Oligomer Conformation.

Alzheimer's disease (AD) is the leading cause of dementia worldwide. While the etiology of AD remains uncertain, neurotoxic effects of amyloid beta oligomers (Aßo) on synaptic function, a well-established early event in AD, is an attractive area for the development of novel strategies to modify or cease the disease's progression. In this work, we tested the protective action of the mitochondrial chaperone Hsp60 against Aßo neurotoxicity, by determining the direct effect of Hsp60 in changing Aßo toxic conformations and thus reducing their dysfunctional synaptic binding and consequent suppression of long-term potentiation. Our data suggest that Hsp60 has a direct impact on Aßo, resulting in a reduction of cytotoxicity and rescue of Aßo-driven synaptic damage, thus proposing Hsp60 as an attractive therapeutic target candidate.

Using Molecular Tweezers to Remodel Abnormal Protein Self-Assembly and Inhibit the Toxicity of Amyloidogenic Proteins.

Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly, which act by binding selectively to lysine and arginine residues. Through this unique mechanism of action, MTs inhibit formation of toxic oligomers and aggregates. Their efficacy and safety have been demonstrated in vitro, in cell culture, and in animal models. Here, we discuss the application of MTs in diverse in vitro and in vivo systems, the experimental details, the scope of their use, and the limitations of the approach. We also consider methods for administration of MTs in animal models to measure efficacy, pharmacokinetic, and pharmacodynamic parameters in proteinopathies.

Role of microtubule-associated protein tau phosphorylation in Alzheimer's disease.

As a major microtubule-associated protein, tau plays an important role in promoting microtubule assembly and stabilizing microtubules. In Alzheimer's disease (AD) and other tauopathies, the abnormally hyperphosphorylated tau proteins are aggregated into paired helical filaments and accumulated in the neurons with the form of neurofibrillary tangles. An imbalanced regulation in protein kinases and protein phosphatases is the direct cause of tau hyperphosphorylation. Among various kinases and phosphatases, glycogen synthase kinase-3ß (GSK-3ß) and protein phosphatase 2A (PP2A) are the most implicated. Accumulation of the hyperphosphorylated tau induces synaptic toxicity and cognitive impairments. Here, we review the upstream factors or pathways that can regulate GSK-3ß or PP2A activity mainly based on our recent findings. We will also discuss the mechanisms that may underlie tau-induced synaptic toxicity.

Role of Microtubule-associated Protein Tau Phosphorylation in Alzheimer's Disease / 华中科技大学学报（医学）（英德文版）

As a major microtubule-associated protein,tau plays an important role in promoting microtubule assembly and stabilizing microtubules.In Alzheimer's disease (AD) and other tauopathies,the abnormally hyperphosphorylated tau proteins are aggregated into paired helical filaments and accumulated in the neurons with the form of neurofibrillary tangles.An imbalanced regulation in protein kinases and protein phosphatases is the direct cause of tau hyperphosphorylation.Among various kinases and phosphatases,glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) are the most implicated.Accumulation of the hyperphosphorylated tau induces synaptic toxicity and cognitive impairments.Here,we review the upstream factors or pathways that can regulate GSK-3β or PP2A activity mainly based on our recent findings.We will also discuss the mechanisms that may underlie tau-induced synaptic toxicity.

Alzheimer's Aß interacts with cellular prion protein inducing neuronal membrane damage and synaptotoxicity.

A major feature of Alzheimer's disease is the accumulation of ß-amyloid (Aß) peptide in the brain. Recent studies have indicated that Aß oligomers (Aßo) can interact with the cellular prion protein (PrPc). Therefore, this interaction might be driving some of Aß toxic effects in the synaptic region. In the present study, we report that Aßo binds to PrPc in the neuronal membrane playing a role on toxic effects induced by Aß. Phospholipase C-enzymatic cleavage of PrPc from the plasma membrane attenuated the association of Aßo to the neurons. Furthermore, an anti-PrP antibody (6D11) decreased the association of Aßo to hippocampal neurons with a concomitant reduction in Aßo and PrPc co-localization. Interestingly, this antibody blocked the increase in membrane conductance and intracellular calcium induced by Aßo. Thus, the data indicate that PrPc plays a role on the membrane perforations produced by Aßo, the increase in calcium ions and the release of synaptic vesicles that subsequently leads to synaptic failure. Future studies blocking Aßo interaction with PrPc could be important for the discovery of new therapeutic strategies for Alzheimer's disease.

Mechanism of synapse redox stress in Okadaic acid (ICV) induced memory impairment: Role of NMDA receptor.

The N-methyl-D-aspartate (NMDA) receptor is a subtype of ionotropic glutamate receptor that is involved in synaptic mechanisms of learning and memory, and mediates excitotoxic neuronal injury. In this study, we tested the hypothesis that NMDA receptor subunit gene expression is altered in cortex and hippocampus of OKA induced memory impairment. Therefore in the present study, we checked the effect of OKA (ICV) on NMDA receptor regulation and synapse function. The memory function anomalies and synaptosomal calcium ion (Ca(2+)) level were increased in OKA treated rats brain; which was further protected by MK801 (0.05mg/kg. i.p) treatment daily for 13days. To elucidate the involvement of NMDA receptor, we estimated NR1, NR2A and NR2B (subunits) expression in rat brain. Results showed that expression of NR1 and NR2B were significantly increased, but expression of NR2A had no significant change in OKA treated rat brain. We also observed decrease in synapsin-1 mRNA and protein expression which indicates synapse dysfunction. In addition, we detected an increase in MDA and nitrite levels and a decrease in GSH level in synapse preparation which indicates synapse altered redox stress. Moreover, neuronal loss was also confirmed by nissl staining in periventricular cortex and hippocampus. Altered level of oxidative stress markers along with neuronal loss confirmed neurotoxicity. Further, MK801 treatment restored the level of NR1, NR2B and synapsin-1 expression, and protected from neuronal loss and synapse redox stress. In conclusion, Okadaic acid (OKA) induced expression of NR1 and NR2B deteriorates synapse function in rat brain which was confirmed by the neuroprotective effect of MK801.

Inhibition of amyloid beta-induced synaptotoxicity by a pentapeptide derived from the glycine zipper region of the neurotoxic peptide.

A major characteristic of Alzheimer's disease is the presence of amyloid beta (Aß) oligomers and aggregates in the brain. Aß oligomers interact with the neuronal membrane inducing perforations, causing an influx of calcium ions and increasing the release of synaptic vesicles that leads to a delayed synaptic failure by vesicle depletion. Here, we identified a neuroprotective pentapeptide anti-Aß compound having the sequence of the glycine zipper region of the C-terminal of Aß (G33LMVG37). Docking and Förster resonance energy transfer experiments showed that G33LMVG37 interacts with Aß at the C-terminal region, which is important for Aß association and insertion into the lipid membrane. Furthermore, this pentapeptide interfered with Aß aggregation, association, and perforation of the plasma membrane. The synaptotoxicity induced by Aß after acute and chronic applications were abolished by G33LMVG37. These results provide a novel rationale for drug development against Alzheimer's disease.