Amphiregulin normalizes altered circuit connectivity for social dominance of the CRTC3 knockout mouse.

Social hierarchy has a profound impact on social behavior, reward processing, and mental health. Moreover, lower social rank can lead to chronic stress and often more serious problems such as bullying victims of abuse, suicide, or attack to society. However, its underlying mechanisms, particularly their association with glial factors, are largely unknown. In this study, we report that astrocyte-derived amphiregulin plays a critical role in the determination of hierarchical ranks. We found that astrocytes-secreted amphiregulin is directly regulated by cAMP response element-binding (CREB)-regulated transcription coactivator 3 (CRTC3) and CREB. Mice with systemic and astrocyte-specific CRTC3 deficiency exhibited a lower social rank with reduced functional connectivity between the prefrontal cortex, a major social hierarchy center, and the parietal cortex. However, this effect was reversed by astrocyte-specific induction of amphiregulin expression, and the epidermal growth factor domain was critical for this action of amphiregulin. These results provide evidence of the involvement of novel glial factors in the regulation of social dominance and may shed light on the clinical application of amphiregulin in the treatment of various psychiatric disorders.

Tau acetylation at K280 regulates tau phosphorylation.

PURPOSE/AIM OF THE STUDY: Accumulation of hyperphosphorylated tau is a key pathological finding of Alzheimer's disease. Recently, acetylation of tau is emerging as another key pathogenic modification, especially regarding the acetylation of tau at K280 of the hexapeptide 275VQIINK280, a critical sequence in driving tau aggregation. However, the relationship between these two key post-translational modifications is not well known. In this study, effect of acetylation of tau at K280 on tau phosphorylation profile was investigated. MATERIALS AND METHODS: The human neuroblastoma cell line, SH-SY5Y, was transfected with p300 acetyltransferase and tau to induce acetylation of tau. Phosphorylation profile after acetylation was evaluated on western blot. K280A-mutant tau was transfected to investigate the effect of acetylation of tau at K280 on tau phosphorylation profile. RESULTS: Overexpression of p300 acetyltransferase in tau-transfected SH-SY5Y human neuroblastoma cells increased acetylation of tau. Meanwhile, tau and its phosphorylation also increased at various sites such as S199/202, S202/T205, T231, and S422, but not at S396. However, blocking acetylation only at K280 with K280A-mutant tau reversed the increased phosphorylation of tau at S202/T205, T231, and S422, but not at S199/202 or S396. CONCLUSION: Here we identified tau phosphorylation profile in the context of p300-induced acetylation and K280A-mutant tau, demonstrating that tau acetylation affects phosphorylation differently by residues and that acetylation at K280 is a determinant of phosphorylation at some residues in the context of pathologic acetyltransferase activity. Yet, our results suggest there is a complex interplay yet to be explored between tau acetylation with tau phosphorylation.

Inhibition of REV-ERBs stimulates microglial amyloid-beta clearance and reduces amyloid plaque deposition in the 5XFAD mouse model of Alzheimer's disease.

A promising new therapeutic target for the treatment of Alzheimer's disease (AD) is the circadian system. Although patients with AD are known to have abnormal circadian rhythms and suffer sleep disturbances, the role of the molecular clock in regulating amyloid-beta (Aß) pathology is still poorly understood. Here, we explored how the circadian repressors REV-ERBα and ß affected Aß clearance in mouse microglia. We discovered that, at Circadian time 4 (CT4), microglia expressed higher levels of the master clock protein BMAL1 and more rapidly phagocytosed fibrillary Aß1-42 (fAß1-42 ) than at CT12. BMAL1 directly drives transcription of REV-ERB proteins, which are implicated in microglial activation. Interestingly, pharmacological inhibition of REV-ERBs with the small molecule antagonist SR8278 or genetic knockdown of REV-ERBs-accelerated microglial uptake of fAß1-42 and increased transcription of BMAL1. SR8278 also promoted microglia polarization toward a phagocytic M2-like phenotype with increased P2Y12 receptor expression. Finally, constitutive deletion of Rev-erbα in the 5XFAD model of AD decreased amyloid plaque number and size and prevented plaque-associated increases in disease-associated microglia markers including TREM2, CD45, and Clec7a. Altogether, our work suggests a novel strategy for controlling Aß clearance and neuroinflammation by targeting REV-ERBs and provides new insights into the role of REV-ERBs in AD.

Small-molecule drug screening identifies drug Ro 31-8220 that reduces toxic phosphorylated tau in Drosophila melanogaster.

The intraneuronal aggregates of hyperphosphorylated and misfolded tau (neurofibrillary tangles, NFTs) cause a stereotypical spatiotemporal Alzheimer's disease (AD) progression that correlates with the severity of the associated cognitive decline. Kinase activity contributes to the balance between neuron survival and cell death. Hyperactivation of kinases including the conventional protein kinase C (PKC) is a defective molecular event accompanying associative memory loss, tau phosphorylation, and progression of AD or related neurodegenerative diseases. Here, we investigated the ability of small therapeutic compounds (a custom library) to improve tau-induced rough-eye phenotype in a Drosophila melanogaster model of frontotemporal dementia. We also assessed the tau phosphorylation in vivo and selected hit compounds. Among the potential hits, we investigated Ro 31-8220, described earlier as a potent PKCα inhibitor. Ro 31-8220 robustly improved the rough-eye phenotype, reduced phosphorylated tau species in vitro and in vivo, reversed tau-induced memory impairment, and improved the fly motor functions. In a human neuroblastoma cell line, Ro 31-8220 reduced the PKC activity and the tau phosphorylation pattern, but we also have to acknowledge the compound's wide range of biological activity. Nevertheless, Ro 31-8220 is a novel therapeutic mitigator of tau-induced neurotoxocity.

Orexin Impairs the Phagocytosis and Degradation of Amyloid-ß Fibrils by Microglial Cells.

BACKGROUND: Intracranial accumulation of amyloid-ß (Aß) is a characteristic finding of Alzheimer's disease (AD). It is thought to be the result of Aß overproduction by neurons and impaired clearance by several systems, including degradation by microglia. Sleep disturbance is now considered a risk factor for AD, but studies focusing on how sleep modulates microglial handling of Aß have been scarce. OBJECTIVE: To determine whether phagocytosis and degradation of extracellular Aß fibrils by BV2 microglial cells were impaired by treatment with orexin-A/B, a major modulator of the sleep-wake cycle, which may mimic sleep deprivation conditions. METHODS: BV2 cells were treated with orexin and Aß for various durations and phagocytic and autophagic processes for degradation of extracellular Aß were examined. RESULTS: After treatment with orexin, the formation of actin filaments around Aß fibrils, which is needed for phagocytosis, was impaired, and phagocytosis regulating molecules such as PI3K, Akt, and p38-MAPK were downregulated in BV2 cells. Orexin also suppressed autophagic flux, through disruption of the autophagosome-lysosome fusion process, resulting in impaired Aß degradation in BV2 cells. CONCLUSIONS: Our results demonstrate that orexin can hinder clearance of Aß through the suppression of phagocytosis and autophagic flux in microglia. This is a novel mechanism linking AD and sleep, and suggests that attenuated microglial function, due to sleep deprivation, may increase Aß accumulation in the brain.

Regulation of amyloid precursor protein processing by its KFERQ motif.

Understanding of trafficking, processing, and degradation mechanisms of amyloid precursor protein (APP) is important because APP can be processed to produce ß-amyloid (Aß), a key pathogenic molecule in Alzheimer's disease (AD). Here, we found that APP contains KFERQ motif at its C-terminus, a consensus sequence for chaperone-mediated autophagy (CMA) or microautophagy which are another types of autophagy for degradation of pathogenic molecules in neurodegenerative diseases. Deletion of KFERQ in APP increased C-terminal fragments (CTFs) and secreted N-terminal fragments of APP and kept it away from lysosomes. KFERQ deletion did not abolish the interaction of APP or its cleaved products with heat shock cognate protein 70 (Hsc70), a protein necessary for CMA or microautophagy. These findings suggest that KFERQ motif is important for normal processing and degradation of APP to preclude the accumulation of APP-CTFs although it may not be important for CMA or microautophagy. [BMB Reports 2016; 49(6): 337-342].

Calpain-mediated cleavage of DARPP-32 in Alzheimer's disease.

Toxicity induced by aberrant protein aggregates in Alzheimer's disease (AD) causes synaptic disconnection and concomitant progressive neurodegeneration that eventually impair cognitive function. cAMP-response element-binding protein (CREB) is a transcription factor involved in the molecular switch that converts short-term to long-term memory. Although disturbances in CREB function have been suggested to cause memory deficits in both AD and AD animal models, the mechanism of CREB dysfunction is still unclear. Here, we show that the dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32), a key inhibitor of protein phosphate-1 (PP-1) that regulates CREB phosphorylation, is cleaved by activated calpain in both AD brains and neuronal cells treated with amyloid-ß or okadaic acid, a protein phosphatase-2A inhibitor that induces tau hyperphosphorylation and neuronal death. We found that DARPP-32 is mainly cleaved at Thr(153) by calpain and that this cleavage of DARPP-32 reduces CREB phosphorylation via loss of its inhibitory function on PP1. Our results suggest a novel mechanism of DARPP-32-CREB signalling dysregulation in AD.

Disease-Associated Mutations of TREM2 Alter the Processing of N-Linked Oligosaccharides in the Golgi Apparatus.

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune-modulatory receptor involved in phagocytosis and inflammation. Mutations of Q33X, Y38C and T66M cause Nasu-Hakola disease (NHD) which is characterized by early onset of dementia and bone cysts. A recent, genome-wide association study also revealed that single nucleotide polymorphism of TREM2, such as R47H, increased the risk of Alzheimer's disease (AD) similar to ApoE4. However, how these mutations affect the trafficking of TREM2, which may affect the normal functions of TREM2, was not known. In this study, we show that TREM2 with NHD mutations are impaired in the glycosylation with complex oligosaccharides in the Golgi apparatus, in the trafficking to plasma membrane and further processing by γ-secretase. Although R47H mutation in AD affected the glycosylation and normal trafficking of TREM2 less, the detailed pattern of glycosylated TREM2 differs from that of the wild type, thus suggesting that precise regulation of TREM2 glycosylation is impaired when arginine at 47 is mutated to histidine. Our results suggest that the impaired glycosylation and trafficking of TREM2 from endoplasmic reticulum/Golgi to plasma membrane by mutations may inhibit its normal functions in the plasma membrane, which may contribute to the disease.

Vancomycin blocks autophagy and induces interleukin-1ß release in macrophages.

Systemic inflammatory response syndrome (SIRS) is a serious condition that can cause organ failure as an exaggerated immunoresponse to the infection or other causes. Recently, autophagy was reported as a key process that regulates inflammatory responses in macrophages. Vancomycin is one of the most commonly prescribed antibiotics for sepsis treatment or following surgery. However, there are no studies on how vancomycin affects autophagy or inflammation. Here, we treated macrophage cell lines with vancomycin and lipopolysaccharides and found that vancomycin blocks autophagy and increases inflammatory responses. This finding suggests that vancomycin should be more cautiously administered in order to prevent unwanted SIRS during sepsis.

zVLL-CHO at low concentrations acts as a calpain inhibitor to protect neurons against okadaic acid-induced neurodegeneration.

There is evidence that ß-secretase and amyloid precursor protein ß-C-terminal fragments (APP-CTF) are involved in the pathogenesis of Alzheimer's disease (AD). Previously, we have reported that N-benzyloxycarbonyl-Val-Leu-leucinal (zVLL-CHO) reduced APP ß-CTF accumulation in axonal swellings of degenerating neurons. Here, in an effort to discover more effective neuroprotective agents, we examined the effects of the ß-secretase inhibitors, H-KTEEISEVN-stat-VAEF-OH (VAEF) and H-EVNstatineVAEF-NH2 (GL-189) as well as zVLL-CHO on OA (okadaic acid)-induced neurodegeneration. Unexpectedly, we found that pretreatment with zVLL-CHO (1 µM) protected neurons after OA treatment, whereas both VAEF and GL-189 lacked neuroprotective effects. Interestingly, 1 µM zVLL-CHO did not inhibit ß-secretase. We previously reported that calpain is activated by OA treatment and calpain inhibitors protect against OA-induced neurodegeneration. The data presented here show that pretreatment with 1 µM zVLL-CHO decreased the levels of calpain-cleaved α-spectrin with a concomitant decrease in LDH release and an increase in average dendritic branch length compared to neurons treated with OA alone. These findings suggest that zVLL-CHO protects against OA-induced neurodegeneration via calpain inactivation.

Effective relief of neuropathic pain by adeno-associated virus-mediated expression of a small hairpin RNA against GTP cyclohydrolase 1.

BACKGROUND: Recent studies show that transcriptional activation of GTP cyclohydrolase I (GCH1) in dorsal root ganglia (DRG) is significantly involved in the development and persistency of pain symptoms. We thus hypothesize that neuropathic pain may be attenuated by down-regulation of GCH1 expression, and propose a gene silencing system for this purpose. RESULTS: To interrupt GCH1 synthesis, we designed a bidirectional recombinant adeno-associated virus encoding both a small hairpin RNA against GCH1 and a GFP reporter gene (rAAV-shGCH1). After rAAV-shGCH1 was introduced into the sciatic nerve prior to or following pain-inducing surgery, therapeutic efficacy and the underlying mechanisms were subsequently validated in animal models. The GFP expression data indicates that rAAV effectively delivered transgenes to DRG. Subsequently reduced GCH1 expression was evident from immunohistochemistry and western-blotting analysis. Along with the down-regulation of GCH1, the von Frey test correspondingly indicated a sharp decline in pain symptoms upon both pre- and post-treatment with rAAV-shGCH1. Interestingly, GCH1 down-regulation additionally led to decreased microglial activation in the dorsal horn, implying an association between pain attenuation and reduced inflammation. CONCLUSION: Therefore, the data suggests that GCH1 levels can be reduced by introducing rAAV-shGCH1, leading to pain relief. Based on the results, we propose that GCH1 modulation may be developed as a clinically applicable gene therapy strategy to treat neuropathic pain.

Autophagy in coxsackievirus-infected neurons.

Autophagy is a process to engulf aberrant organelles or protein aggregates into double-membrane vesicles for lysosomal breakdown. Autophagy is a protective process against some intracellular bacteria and viruses; however, it is also used for replication by some viruses, such as poliovirus. We recently found that coxsackievirus B4 (CVB4) also induces the autophagy pathway and activates the calpain system for replication in neurons. Notably, the inhibition of autophagy with 3-methyladenine (3MA) reduced calpain activation and virus replication. Calpain inhibitors also reduced autophagosome formation and virus replication. This finding indicates that calpain and the autophagy pathway are closely connected with each other during the infection. Interestingly, we also found that 3MA and calpain inhibitors enhanced the caspase-3 specific cleavage of spectrin during CVB4 infection, suggesting that autophagy inhibition by these drugs triggered apoptosis. Thus, autophagy and apoptosis may balance each other in CVB4-infected neurons. Here, we show that inhibition of caspase with zVAD increased autophagosome formation, further proposing the cross-talk between autophagy and apoptosis in CVB4-infected neurons.

Dynein cleavage and microtubule accumulation in okadaic acid-treated neurons.

Impairment of protein phosphatase 2A (PP2A) activity is implicated in tau hyperphosphorylation and microtubule (MT) instability in Alzheimer's disease (AD). Here, we report that okadaic acid, an effective PP2A inhibitor, suppresses the levels of acetylated and detyrosinated tubulins, but enhances tyrosinated tubulins in rat primary cortical neuron cultures. Immunocytochemistry experiments reveal that MTs accumulate intensely around soma and proximal neurites, implying impairment of MT transport to distal neurites which is mediated by dynein and dynactin. Here, we reveal that they can be cleaved by calpain. Notably, shortening of process length in OA-treated neurons is alleviated when calpain cleavage activity is inhibited. Based on these results, we propose that calpain-mediated dynein cleavage in OA-treated neurons is responsible for the MT transport deficit, and consequently, neurite retraction.