Coordinated activation of AMP‐activated protein kinase,extracellular signal‐regulated kinase,and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.

     

Amyloid beta1–42 (Aβ42) up‐regulates the expression of sortilin via the p75NTR/RhoA signaling pathway

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co‐receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6‐month‐old swedish‐amyloid precursor protein/PS1dE9 transgenic mice. Aβ₄₂ enhanced the protein and mRNA expression levels of sortilin in a dose‐ and time‐dependent manner in SH‐SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75^NTR (P75ECD‐FC), or the antibody against the extracellular domain of p75^NTR, blocked the up‐regulation of sortilin induced by Amyloid‐β protein (Aβ), suggesting that Aβ₄₂ increased the expression level of sortilin and mRNA in SH‐SY5Y via the p75^NTR receptor. Inhibition of ROCK, but not Jun N‐terminal kinase, suppressed constitutive and Aβ₄₂‐induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aβ₄₂ oligomer increases sortilin gene and protein expression through p75^NTR and RhoA signaling pathways, suggesting a potential physiological interaction of Aβ₄₂ and sortilin in Alzheimer's disease.

     

CaMKIIβ is localized in dendritic spines as both drebrin‐dependent and drebrin‐independent pools

Drebrin is a major F‐actin binding protein in dendritic spines that is critically involved in the regulation of dendritic spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel drebrin‐binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca²⁺/calmodulin‐dependent protein kinase II (CaMKIIβ) as a drebrin‐binding protein using a yeast two‐hybrid system, and investigated the drebrin–CaMKIIβ relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIβ in dendrites during the resting state, suggesting that drebrin is involved in the accumulation of CaMKIIβ in dendritic spines. Fluorescence recovery after photobleaching analysis showed that drebrin knockdown increased the stable fraction of CaMKIIβ, indicating the presence of drebrin‐independent, more stable CaMKIIβ. NMDA receptor activation also increased the stable fraction in parallel with drebrin exodus from dendritic spines. These findings suggest that CaMKIIβ can be classified into distinct pools: CaMKIIβ associated with drebrin, CaMKIIβ associated with post‐synaptic density (PSD), and CaMKIIβ free from PSD and drebrin. CaMKIIβ appears to be anchored to a protein complex composed of drebrin‐binding F‐actin during the resting state. NMDA receptor activation releases CaMKIIβ from drebrin resulting in CaMKIIβ association with PSD.

     

Nidogen‐1 is a common target of microRNAs MiR‐192/215 in the pathogenesis of Hirschsprung's disease

Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR‐192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR‐192/215 family in the pathogenesis of HSCR. Quantitative real‐time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual‐luciferase reporter assays, and the functional effects of miR‐192/215 on human 293T and SH‐SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR‐192/215 was significantly down‐regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH‐SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR‐192/215 by dual‐luciferase reporter gene assay and its expression was inversely correlated with that of miR‐192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR‐192/215 inhibitor. The down‐regulation of miR‐192/215 may contribute to HSCR development by targeting NID1.

     

Nuclear diacylglycerol lipase‐α in rat brain cortical neurons: evidence of 2‐arachidonoylglycerol production in concert with phospholipase C‐β activity

In this report, we describe the localization of diacylglycerol lipase‐α (DAGLα) in nuclei from adult cortical neurons, as assessed by double‐immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double‐labeling assays using the anti‐DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα‐signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC‐35‐ and NeuN‐signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C‐β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC‐35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2‐arachidonoylglycerol (2‐AG) by liquid chromatography and mass spectrometry (LC‐MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2‐AG production, and its PLCβ/DAGLα‐dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2‐AG locally produced within the neuronal nucleus.

     

Dyrk1A phosphorylates parkin at Ser‐131 and negatively regulates its ubiquitin E3 ligase activity

Mutations of parkin are associated with the occurrence of autosomal recessive familial Parkinson's disease (PD). Parkin acts an E3 ubiquitin ligase, which ubiquitinates target proteins and subsequently regulates either their steady‐state levels through the ubiquitin–proteasome system or biochemical properties. In this study, we identify a novel regulatory mechanism of parkin by searching for new regulatory factors. After screening human fetal brain using a yeast two hybrid assay, we found dual‐specificity tyrosine‐(Y)‐phosphorylation‐regulated kinase 1A (Dyrk1A) as a novel binding partner of parkin. We also observed that parkin interacts and co‐localizes with Dyrk1A in mammalian cells. In addition, Dyrk1A directly phosphorylated parkin at Ser‐131, causing the inhibition of its E3 ubiquitin ligase activity. Moreover, Dyrk1A‐mediated phosphorylation reduced the binding affinity of parkin to its ubiquitin‐conjugating E2 enzyme and substrate, which could be the underlying inhibitory mechanism of parkin activity. Furthermore, Dyrk1A‐mediated phosphorylation inhibited the neuroprotective action of parkin against 6‐hydroxydopamine toxicity in dopaminergic SH‐SY5Y cells. These findings suggest that Dyrk1A acts as a novel functional modulator of parkin. Parkin phosphorylation by Dyrk1A suppresses its E3 ubiquitin ligase activity potentially contributing to the pathogenesis of PD under PD‐inducing pathological conditions.

     

Functional analysis and purification of a Pen‐2 fusion protein for γ‐secretase structural studies

The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.

     

Positive allosteric modulation of alpha‐7 nicotinic receptors promotes cell death by inducing Ca2+ release from the endoplasmic reticulum

Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7‐nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca²⁺ signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca²⁺ signaling and cell viability. We used human SH‐SY5Y neuroblastoma cells overexpressing α7‐nAChRs (α7‐SH) and their control (C‐SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca²⁺ with Fura‐2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch‐clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7‐SH but not of C‐SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca²⁺ concentration; (iii) released Ca²⁺ from the ER by a Ca²⁺‐induced Ca²⁺ release mechanism only in α7‐SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7‐nAChRs, ryanodine receptors, or IP₃ receptors. In conclusion, positive allosteric modulation of α7‐nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca²⁺, overloading of intracellular Ca²⁺, and neuronal cell death.

     

Postnatal ethanol exposure alters levels of 2‐arachidonylglycerol‐metabolizing enzymes and pharmacological inhibition of monoacylglycerol lipase does not cause neurodegeneration in neonatal mice

The consumption of ethanol by pregnant women may cause neurological abnormalities, affecting learning and memory processes in children, and are collectively described as fetal alcohol spectrum disorders. However, the molecular mechanisms underlying these changes are still poorly understood. In our previous studies, we found that ethanol treatment of postnatal day 7 (P7) mice significantly enhances the anandamide levels but not the 2‐arachidonylglycerol (2‐AG) levels and induces widespread neurodegeneration, but the reason for the lack of significant effects of ethanol on the 2‐AG level is unknown. In this study, we examined developmental changes in diacylglycerol lipase‐α, β (DAGL‐α and β) and monoacylglycerol lipase (MAGL). We found that the levels of these proteins were significantly higher in adult brains compared to those detected early in brain development. Next, we examined the influence of P7 ethanol treatment on these enzymes, finding that it differentially altered the DAGL‐α protein and mRNA levels but consistently enhanced those of the DAGL‐β. Interestingly, the ethanol treatment enhanced MAGL protein and mRNA levels. Inhibition of MAGL with KML29 failed to induce neurodegeneration in P7 mice. Collectively, these findings suggest that ethanol significantly activates DAGL‐β and MAGL in the neonatal brain, resulting in no net change in 2‐AG levels.

     

The metabolic impact of β‐hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity

Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β‐hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β‐hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β‐hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β‐hydroxybutyrate was present in these neurons. In addition, the NMDA receptor‐induced calcium responses in the neurons were diminished in the presence of β‐hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β‐hydroxybutyrate augmented transmitter release induced by the K_ATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of K_ATP channels in the effects of ketone bodies on transmitter release.

     

Alpha‐ketoglutarate dehydrogenase complex‐dependent succinylation of proteins in neurons and neuronal cell lines

Reversible post‐translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remain unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α‐ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans‐succinylase that mediates succinylation in an α‐ketoglutarate‐dependent manner. Inhibition of KGDHC reduced succinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl‐CoA suggests that the catalysis owing to the E2k succinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC.

     

Targeting cyclin D3/CDK6 activity for treatment of Parkinson's disease

At present, treatment for Parkinson's disease (PD) is only symptomatic; therefore, it is important to identify new targets tackling the molecular causes of the disease. We previously found that lymphoblasts from sporadic PD patients display increased activity of the cyclin D3/CDK6/pRb pathway and higher proliferation than control cells. These features were considered systemic manifestations of the disease, as aberrant activation of the cell cycle is involved in neuronal apoptosis. The main goal of this work was to elucidate whether the inhibition of cyclin D3/CDK6‐associated kinase activity could be useful in PD treatment. For this purpose, we investigated the effects of two histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic (SAHA) acid and sodium butyrate (NaB), and the m‐TOR inhibitor rapamycin on cell viability and cyclin D3/CDK6 activity. Moreover, the potential neuroprotective action of these drugs was evaluated in 6‐hydroxy‐dopamine (6‐OHDA) treated dopaminergic SH‐SY5Y cells and primary rat mesencephalic cultures. Here, we report that both compounds normalized the proliferative activity of PD lymphoblasts and reduced the 6‐OHDA‐induced cell death in neuronal cells by preventing the over‐activation of the cyclin D3/CDK6/pRb cascade. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it is plausible that they may serve as novel therapeutic drugs for PD.

     

Analysis of the amyloid precursor protein role in neuritogenesis reveals a biphasic SH‐SY5Y neuronal cell differentiation model

The existence of an intrinsic programme controlling neuritogenesis and activated during early neuronal differentiation and regeneration stages is well established. However, the identity and role of each molecular player and event, as well as how such a programme is modified by environmental signals, remain a focus of research. The amyloid precursor protein (APP) is a neuromodulator of the developing and mature nervous system, although in a highly complex manner which is far from clear. To study APP‐induced neuritogenesis, the retinoic acid (RA)‐induced SH‐SY5Y cell differentiation model was first minutely characterized in terms of RA dose, morphological outputs and relevant biochemical markers. The findings reported here unveiled two differentiation phases for the 10 μM RA dose: 1–4 (4 days excluded) and 4–8 days, clearly defined by fold increases in the ratio between APP and acetylated Tubulin. Moreover, we describe, for the first time, a unique peak of secreted APP (sAPP)/APP ratio in the first phase. Subsequent APP and sAPP modulations confirmed that a high sAPP/APP ratio potentiates the elongation of smaller processes at the earlier neuritogenic phase. This sAPP/APP ratio drops in the second phase, as holoAPP levels increase to assist the maintenance of the longer neurites, potentially via their stabilization.

     

Lipid peroxidation is essential for α‐synuclein‐induced cell death

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.

     

Phenolic compounds prevent the oligomerization of α‐synuclein and reduce synaptic toxicity

Lewy bodies, mainly composed of α‐synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low‐order αS oligomers are neurotoxic and critical species in the pathogenesis of α‐synucleinopathies. To develop disease modifying therapies for α‐synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo‐induced cross‐linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N‐terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long‐term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers.

     

MicroRNA‐27a‐3p suppression of peroxisome proliferator‐activated receptor‐γ contributes to cognitive impairments resulting from sevoflurane treatment

Sevoflurane is the most widely used anaesthetic administered by inhalation. Exposure to sevoflurane in neonatal mice can induce learning deficits and abnormal social behaviours. MicroRNA (miR)‐27a‐3p, a short, non‐coding RNA that functions as a tumour suppressor, is up‐regulated after inhalation of anaesthetic, and peroxisome proliferator‐activated receptor γ (PPAR‐γ) is one of its target genes. The objective of this study was to investigate how the miR‐27a‐3p–PPAR‐γ interaction affects sevoflurane‐induced neurotoxicity. A luciferase reporter assay was employed to identify the interaction between miR‐27a‐3p and PPAR‐γ. Primary hippocampal neuron cultures prepared from embryonic day 0 C57BL/6 mice were treated with miR‐27a‐3p inhibitor or a PPAR‐γ agonist to determine the effect of miR‐27a‐3p and PPAR‐γ on sevoflurane‐induced cellular damage. Cellular damage was assessed by a flow cytometry assay to detect apoptotic cells, immunofluorescence to detect reactive oxygen species, western blotting to detect NADPH oxidase 1/4 and ELISA to measure inflammatory cytokine levels. In vivo experiments were performed using a sevoflurane‐induced anaesthetic mouse model to analyse the effects of miR‐27a‐3p on neurotoxicity by measuring the number of apoptotic neurons using the Terminal‐deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) method and learning and memory function by employing the Morris water maze test. Our results revealed that PPAR‐γ expression was down‐regulated by miR‐27a‐3p following sevoflurane treatment in hippocampal neurons. Down‐regulation of miR‐27a‐3p expression decreased sevoflurane‐induced hippocampal neuron apoptosis by decreasing inflammation and oxidative stress‐related protein expression through the up‐regulation of PPAR‐γ. In vivo tests further confirmed that inhibition of miR‐27a‐3p expression attenuated sevoflurane‐induced neuronal apoptosis and learning and memory impairment. Our findings suggest that down‐regulation of miR‐27a‐3p expression ameliorated sevoflurane‐induced neurotoxicity and learning and memory impairment through the PPAR‐γ signalling pathway. MicroRNA‐27a‐3p may, therefore, be a potential therapeutic target for preventing or treating sevoflurane‐induced neurotoxicity.

     

LPS antagonism of TGF‐β signaling results in prolonged survival and activation of rat primary microglia

Accumulating evidence indicates that activated microglia contribute to the neuropathology involved in many neurodegenerative diseases and after traumatic injury to the CNS. The cytokine transforming growth factor‐beta 1 (TGF‐β1), a potent deactivator of microglia, should have the potential to reduce microglial‐mediated neurodegeneration. It is therefore perplexing that high levels of TGF‐β1 are found in conditions where microglia are chronically activated. We hypothesized that TGF‐β1 signaling is suppressed in activated microglia. We therefore activated primary rat microglia with lipopolysaccharide (LPS) and determined the expression of proteins important to TGF‐β1 signaling. We found that LPS treatment decreased the expression of the TGF‐β receptors, TβR1 and TβR2, and reduced protein levels of Smad2, a key mediator of TGF‐β signaling. LPS treatment also antagonized the ability of TGF‐β to suppress expression of pro‐inflammatory cytokines and to induce microglial cell death. LPS treatment similarly inhibited the ability of the TGF‐β related cytokine, Activin‐A, to down‐regulate expression of pro‐inflammatory cytokines and to induce microglial cell death. Together, these data suggest that microglial activators may oppose the actions of TGF‐β1, ensuring continued microglial activation and survival that eventually may contribute to the neurodegeneration prevalent in chronic neuroinflammatory conditions.