Journal of Neurochemistry

Tetrahydrobiopterin (BH4) is an essential cofactor of tyrosine hydroxylase (TH). We found that BH4 and dopa administration in infancy restored the loss of TH protein caused by BH4 deficiency and that the effects of these treatments are attenuated in young adulthood. These findings indicate that the post-natal augmentation of TH protein requires catecholamines and suggest that there is a critical period for the dopaminergic development.

The α2-Antiplasmin (α2AP) protein regulates cellular functions independently or dependently of plasmin, and is highly expressed in the hippocampus. However, the role of α2AP was unclear. Here, we first report that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.

We report here that nanomolar Aβ can induce neuronal loss in culture by activating microglia to release MFG-E8 (Milk-fat globule-EGF factor 8 protein), which binds and opsonizes neurons for phagocytosis by microglia, thereby killing the neurons by phagoptosis. The essential role of MFG-E8 in Aβ-induced phagoptosis, suggests this bridging protein as a potential therapeutic target for Alzheimer's and other neurological diseases involving inflammatory neuronal loss.

We show that the mood-stabilizing drugs, valproic acid (VPA) and Li⁺, could up-regulate FGF-1B promoter through inhibition of HDACs and GSK-3. We further demonstrate that treatment of VPA increases histone acetylation around the regulatory region (18-bp cis-element) of FGF-1B promoter. Importantly, VPA could induce more neuronal differentiation in FGF-1B positive neural stem/progenitor cells than FGF-1B negative cells.

To examine the role of microglia in remyelination, mice were treated with minocycline after cuprizone-induced demyelination. Minocycline treatment reduced the number of oligodendrocytes and CNTF expression in the remyelination phase. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone-induced demyelination.

Read the full article ‘Calcium/calmodulin-dependent serine protein kinase (CASK) is a new intracellular modulator of P2X3 receptors’ on doi: 10.1111/jnc.12272

Energy failure and oxidative stress have been implicated in the pathogenesis of ischemia and cell death. Through the application of the glycolytic inhibitor iodoacetate and the glutathione chelator diethylmaleate, we report a potential link between cytosolic phospholipase A₂ (cPLA₂) activation and energy failure/oxidative stress-induced astrocyte damage involving reactive oxygen species (ROS), signaling through the kinases PKC-α, Src, Raf, and ERK and concurrent elevation of endogenous chelatable zinc.

Control of Visceral Function by Distinct Neurons in the Brainstem

Neurons in the Barrington's nucleus control bladder and other visceral functions through projections to sympathetic and parasympathetic preganglionic neurons in the spinal cord. We found that these two groups of output neurons have distinct membrane properties and are controlled by different excitatory and inhibitory synaptic inputs. This knowledge improves our understanding of the circuitry and central mechanisms regulating visceral functions.

Botulinum neurotoxin is an excellent tool for causing prolonged blockade of secretion offering the possibility of targeted modulation of neuroendocrine functions. We now show the potential of our new protein stapling technique for combinatorial and on-demand assembly of the botulinum enzyme with growth factors and neuropeptides to retarget the botulinum activity into desired cell populations for biomedical applications.

We wished to understand how presynaptic NMDA receptors (preNMDARs) are activated in cerebellar molecular layer interneurons (MLIs). We find that they can detect glutamate by mediating a calcium entry into the presynaptic termini, a signal that is further amplified by their ability to recruit ryanodine receptor. PreNMDARs can convert the glutamate released by volumic transmission into large presynaptic calcium transients.

Synaptotagmin-1, an abundant protein attached to synaptic vesicles, is necessary for the expression of the vesicular Ca²⁺/H⁺ antiport. This low-affinity antiport was previously described using isolated mammalian brain vesicles and its function was analysed in rapid cholinergic transmission. Therefore, in addition to its previously described functions, we here show that synaptotagmin-1 is involved in a rapid vesicular Ca²⁺ sequestration.

Thiazolidinediones (TZDs) decrease tau-Thr²³¹ phosphorylation via the inhibition of CDK5 enzymatic activity, which is mediated by the enhanced proteasomal degradation of p35 and a PPARγ-independent signaling pathway. This molecular mechanism implies a role for TZD drugs in the prevention and treatment of Alzheimer's disease.

Neural stem cells (NSCs) produce synaptamide (N-docosahexaenoylethanolamine) from docosahexaenoic acid (DHA). Synaptamide potently induces neuronal differentiation of NSCs through PKA-CREB signaling. The neurogenic capacity of NSCs is linked to the endogenous synaptamide level which depends on synaptamide stability and DHA availability in NSCs. Dietary omega-3 fatty acid intake that influences endogenous DHA and synaptamide levels may have significant impact on neurodevelopment.

Sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions. Regarding stroke, there is no direct evidence. We have demonstrated that citicoline increases SIRT1 protein levels in brain concomitantly to neuroprotection. Citicoline fails to reduce infarct volume in Sirt1-/- mice. Our findings suggest that therapeutic strategies acting on SIRT1 may be useful in the treatment of stroke.

ATP-gated P2X3 receptors are important transducers of painful stimuli. This study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in interacting and controlling P2X3 receptor expression and function in sensory ganglion neurons. As CASK-mediated control of P2X3 receptors was dependent on the receptor activation state, CASK might be an intracellular gateway to regulate purinergic nociceptive signaling.

We proposed the following mechanism for LPS preconditioning-induced retinal prevention against ischemia: LPS preconditioning-induced retinal prevention against ischemic damages is mediated by toll-like receptor (TLR4) signaling through microglia. This TLR4 priming by LPS preconditioning dominantly activates TRIF-IRF3 (TIR-domain-containing adapter-inducing interferon-β/interferon regulatory factor 3) pathway and suppresses MyD88 - NF-κB pathway. Thus, detailed study of these findings may provide novel idea for discovery of new drugs against ischemic disorders.

Knockdown of glycogen branching enzyme in neurons led to accumulation of an insoluble form of glycogen called polyglucosan, to apoptosis and to activation of glycogen synthase. These effects were reversed by glycogen synthase inhibition through starvation and rapamycin treatments, suggesting a potential therapeutic value of glycogen synthase inhibition for treating glycogen storage disorders.

Adult offspring of isolated rats exhibited an increased brain level of allopregnanolone, increased basal activity of the HPA axis, reduction of hypothalamic CRF and CRFR1 in the pituitary. There was also attenuation of corticosterone response by acute stress and up-regulation of GR expression in the hippocampus. In conclusion, a stressful experience before mating influence neuroendocrine signalling in the next generation.

ABCA1 actively eliminates 24-OHC in the presence of HDL and protects neuronal cells from apoptosis. High cholesterol turnover is essential for neural functions and 24(S)-hydroxycholesterol (24-OHC) is the major cholesterol metabolite in the brain. However, 24-OHC is toxic to neuronal cells. We propose that two ABC proteins, ABCA1 and ABCG1, are involved in active elimination of 24-OHC in the presence of HDL as a lipid acceptor and protect neuronal cells from apoptosis.

We have shown that ethanol promotes TLR4 activation through its interactions with the lipid rafts microdomains. Here, we report that ethanol triggers TLR4/TLR2 recruitment into microglia lipid rafts/caveolae, promoting TLR4 and TLR2 interactions and signaling and leading to the production of proinflammatory cytokines, which cause ROS generation and neuronal apoptosis. These findings might provide a mechanism for alcohol-triggered neuroinflammation and brain damage.

Pro-inflammatory cytokines such as TNF-α and IL-1β increase kidney-type mitochondrial glutaminase in the cytosol of neurons and TNF-α may further release glutaminase to the extracellular space. Consequently, intracellular and extracellular glutamate levels increase in neurons during inflammation. The excess levels of glutamate in the extracellular space may induce neuronal injury and death. Therefore, neuronal glutaminase is a potential component of neurotoxicity during inflammation.

This study describes the binding site for 2-arachidonyl glycerol (2-AG) on β₂ subunit-containing GABA_A receptors. From the fact that the important amino acid residues in the fourth transmembrane segment (M4) span > 18 Å, we conclude that the hydrophobic tail of the bound 2-AG molecule must be near linear and that the site mainly locates to the inner leaflet of the bilayer but stretches far into the membrane. An allosteric modulator acting at this drug binding site would be specific for β₂ subunit-containing GABA_A receptors and an antagonist would block endocannabinoid action on GABA_A receptors.

Spreading depression (SD), the likely cause of migraine, occurs with increased TNF-α and oxidative stress (OS), which we show is specific to microglia and astrocytes. We then show TNF-α and SD itself increase susceptibility to subsequent SD. IGF-1 decreases TNF-α and microglial OS from SD. These findings support IGF-1 and microglial OS as potential therapeutic targets against SD and perhaps migraine.

The article describes the functionality of an extramitochondrial oxidative phosphorylation (OXPHOS) in peripheral (sciatic) nerve. Isolated myelin vesicles express functional respiratory complexes and produce ATP, consuming oxygen. This ability is impaired when myelin is isolated from dys/demyelinated (CMT1A) sciatic nerve, although the OXPHOS proteins are present. This suggests that myelin structure is fundamental for the activity of the respiratory complexes.

We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3′UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3′UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43′s 3′UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth.

The vertical alignment between the channel-lining transmembrane segment M2 and segment M3 in Cys-loop ligand-gated ion channels is a topic of controversy. While the vertical alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel (GLIC) and eukaryotic Caenorhabditis elegans glutamate-gated chloride channel α (GluClα) are in agreement, the Torpedo nAChR model based on electronmicroscopy differs. We determined the register between M2 and M3 in muscle nAChR by disulfide crosslinking. Our results are best reflected if modeled on GluClα, but not if modeled on the current Torpedo nAChR model. Overall, this indicates that the vertical alignment in nAChR is conserved between prokaryotes and eukaryotes.

Histaminergic neurons in the tuberomammillary nucleus (TMN) play an important role in the regulation of sleep-wakefulness. In this study, we found that norepinephrine acts on presynaptic α_2A adrenoceptors to inhibit GABAergic transmission onto TMN neurons. This α_2A adrenoceptor-mediated presynaptic modulation may affect the excitability of TMN neurons and thus contribute to the behavioral state-dependent consolidation of wakefulness.

Inflammation is a characteristic of Alzheimer's disease. The P2Y₂ protein receptor has been found to be upregulated under inflammatory conditions. Activation of the receptor by UTP promotes interactions with α_vβ_3/5 integrin and signals G₁₂-dependent Rho activation leading to cofilin phosphorylation involved in neurite extension. This study indicates that up-regulation of P2Y₂Rs in mouse primary cortical neurons under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.

In this study, we describe a method for enriching alpha-synuclein (α-syn) from a variety of biological samples, from cultured cells to brain tissues. Enrichment of α-syn was achieved by heating samples, further facilitating the identification of specific post-translational modifications by immunoblot, or mass spectrometry-based techniques. This approach will contribute to the clarification of the role of α-syn PTMs in Parkinson's disease.

WNTs are lipoglycoproteins, which affect microglia, the macrophages of the CNS, in a pro-inflammatory manner. Here, we show for the first time that WNTs in co-stimulation with lipopolysaccharides, however, reduce the lipopolysaccharide-induced pro-inflammatory effects e g on cyclooxygenase expression. Based on our findings we suggest that WNTs act as homeostatic regulators acting pro- or anti-inflammatory in a context-dependent manner.

Presynaptic Zn²⁺ release and post-synaptic accumulation is implicated in neuronal physiology and pathology; however, direct detection of Zn²⁺ translocation can be masked by intracellular buffering. This study exploited intracellular dialysis by whole-cell clamp to deplete intracellular Zn²⁺ buffers and promote detection of Zn²⁺ translocation. The approach described here should help understand Zn²⁺ homeostasis by assisting identification of Zn²⁺ source(s) contributing to Zn²⁺ signalling.

Inflammation and iron accumulation are present in a variety of neurodegenerative diseases including Alzheimer's and Parkinson's disease. We analyzed the effects of the inflammatory cytokines TNF-α and Il-6 and of LPS on total cell iron content and on the expression and abundance of the iron transporters DMT1 and FPN1 in neurons, astrocytes, and microglia. Inflammatory stimuli increased expression of DMT1 in neurons, astrocytes, and microglia, induced the expression of hepcidin in astrocytes and microglia but not in neurons. Incubation with hepcidin transiently decreased the expression of FPN1 in the three cell types. The net result of these changes was increased iron accumulation in neurons and microglia but not in astrocytes. The findings establish a causal association between inflammation and iron accumulation in brain cells, presumably by changes in DMT1 and FPN1 expression and mediated in part by hepcidin.

So far, no new therapeutic agents for Alzheimer's disease (AD) have reached the clinics based on reducing amyloid β-peptide (Aβ) levels through the use of secretase inhibitors or immunotherapy. Here, we highlight emerging areas of amyloid precursor protein (APP) function that may provide new insights into synaptic development, cognition, and gene regulation. Endogenous APP in primary cortical neurons has a key role in maintaining neuronal calcium homeostasis essential for synaptic transmission. Disruption of this homeostatic mechanism predisposes to aging and AD. The intracellular domain of APP (AICD) is also a key epigenetic regulator of various genes, including APP itself, the amyloid-degrading enzyme neprilysin (NEP), and aquaporin-1.

Read the full article ‘Loss of Bace2 in zebrafish affects melanocyte migration and is distinct from Bace1 knock out phenotypes’ on doi: 10.1111/jnc.12198.

Pallidal hyperactivity and hyperdopaminergic states found in a cytokine model for schizophrenia

The indirect pathway of the basal ganglia circuit is implicated in cognition, motor coordination, learning and antipsychotic pharmacology. The activity of globus pallidus neurons in this pathway is markedly modulated by dopaminergic afferents. Perinatal expose to EGF induces pallidal hyperdopaminergic innervation and elevates their firing rates, resulting in increased GABA release. This pallidal dysfunction contributes to the neurobehavioral deficits of this animal model for schizophrenia.

This study showed that a PD toxin MPP⁺ causes selective degeneration of dopaminergic (DA) neurons in a Drosophila primary culture. We also found that MPP⁺-mediated neurodegeneration was rescued by D2 agonists. Furthermore, activation of Drosophila D2 autoreceptors (D2R_auto) prevents MPP⁺ toxicity through suppression of DA neuronal excitability (or excitotoxicity).

Reduced expression of both Reelin and Brain-Derived Neurotrophic factor (BDNF) genes has been associated with schizophrenia in human post-mortem studies. We report a female-specific interaction between these two genes in a specific region of the hippocampus in mice, and this was mediated by estradiol. These data provide a better understanding of the sex-specific pathogenesis of schizophrenia.

We investigate whether dysregulation of serotonergic modulation of glutamatergic transmission in the NAc would have a significant role in the development of cocaine addiction. We find that a single in vivo exposure to cocaine impairs the induction of 5-HT-LTD in the NAc. It provides major advances in our understanding of the functional role of the 5-HT system in the NAc.

In this study, we demonstrated that TRAF5 deficiency leads to inhibited inflammation, BBB disruption and neuronal apoptosis in the ischemic brain probably via the suppression of NF-κB signaling and the activation of Akt/FoxO1 signaling. Neuron-specific TRAF5 transgenic mice showed an opposite phenotype which confirmed the findings.

We have synthesized a ~ 5 kDa Aβ binding peptide (ABP-p-4-5), a derivative of the human pericentriolar material-1 protein that is involved in cell cycle transit and neurogenesis. ABP-p-4-5 selectively binds with high affinity Aβ_1-42 oligomers that are believed to be the toxic drivers of AD (Alzheimer's disease). These observations suggest that ABP-p-4-5 peptide may prove to be an AD therapeutic.

Gene expression profiling revealed that expression of the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in neural stem cells migrating toward tumor cells. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that nNOS is a relevant regulator in controlling tumor tropism of neural stem cells. This finding suggests a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy.

The study demonstrates that isolectin IB4, commonly used as a microglial marker in the brain, binds to the glycosylated ectodomain of Ret tyrosine kinase receptor, the signaling component for the family ligands of the glial cell line-derived neurotrophic factor. Binding was observed in microglia under physiological and ischemic conditions, thus indicating Ret as one of the IB4-reactive glycoconjugate accounting for the IB4 stain in microglia.

Amyloid plaques in Alzheimer's brains consist of mostly Aβ42, and some plaques contain only Aβ42, even though Aβ40 is several-fold more concentrated than Aβ42. Here, we show that a mixture of Aβ42 and Aβ40 forms interlaced fibrils in vitro, seemingly contradicting in vivo observations. We propose some potential mechanisms that may contribute to the preferential deposition of Aβ42 in Alzheimer's.

Inhibition of BACE1 protease activity has therapeutic importance for Alzheimer's disease. Analysis of BACE1 and BACE2 knock-out zebrafish revealed that they exhibit distinct phenotypes. bace1 mutants display hypomyelination in the PNS and supernumerary neuromasts while in bace2 mutants the shape and migration of melanocytes is affected. These phenotypes are not further enhanced in the viable double mutants. Our data suggest that blocking BACE1 activity is a safe therapeutic approach.

Read the Editorial Highlight for this article on doi: 10.1111/jnc.12200

We demonstrate distinct subpopulations of midbrain dopaminergic neurons based on their requirement for the transcription factor Pitx3. Calbindin-D28k-positive dopaminergic neurons are preserved in Pitx3 hypomorphic mice and show resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced injury. In contrast, Pitx3-dependent neurons are susceptible to toxic stress, and this vulnerability is augmented upon reduced Pitx3 expression. These data suggest a potential link between Pitx3 and differential vulnerability of dopaminergic populations involved in Parkinson's disease.

Insights into the oligomerization of CRMPs: Crystal structure of human collapsin response mediator protein 5.

Collapsin response mediator proteins (CRMPs) are cytosolic homo- and/or hetero-tetrameric phosphoproteins crucial for neuronal development and repair. We determined the crystal structure of human CRMP-5, revealing a homo-tetramer assembly similar to that of other CRMPs. Solution studies indicate that subunit interactions in CRMP-5 and CRMP-1 homo-tetramers are weaker compared with CRMP-2, and that divalent cations, Ca²⁺ and Mg²⁺, destabilize them further.

We propose that uncoupling NMDA receptors from PSD95 provides neuroprotection from stroke by enhancing Ca²⁺/Calmodulin-dependent neurotrophic signalling. Blocking NMDAR-PSD95 interactions allows enhanced activation of nuclear CaM-kinase IV and prolonged phosphorylation of the CREB transcription factor, resulting in enhanced neurotrophic transcription. These findings demonstrate the importance of maintaining neurotrophic, Ca^2 + -dependent signalling during neuronal injury and provide novel targets for neuroprotection in stroke.

TDP-43 is an important contributor to ALS and FTLD. In this study, we revealed that TDP-43 associates with stalled ribosomes and that its silencing disturbs mRNA stability and cell survival during cellular stress. Our findings suggest that TDP-43 is a stress-response protein that functions in translational control mechanisms and determines cell survival.

Monoclonal antibodies selective for α-synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and transgenic mice expressing the disease-causing A30P mutation.

Aggregation of α-synuclein with formation of toxic oligomers/protofibrils is a central feature in the brain of Parkinson's disease and dementia with Lewy bodies. We developed monoclonal antibodies with high affinity and strong selectivity against such α-synuclein species. The novel antibodies recognize relevant α-synuclein pathology in both transgenic mice (A) and human brain. Apart from advancing our understanding of the disease processes, these antibodies may be valuable both for immunotherapy and as reagents in biomarker assays.

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ) tract within the androgen receptor (AR) gene. Here, we report that genistein, an isoflavone found in soy, disrupts the interaction between AR and ARA70 and promotes the degradation of mutant AR and ameliorates behavioral abnormalities in SBMA transgenic mice.

This study investigated the neuroprotective effect of acute ethanol therapy following ischemic stroke. During early reperfusion, there is a detrimental increase in glycolysis and NADPH oxidase (NOX) activity. We found that ethanol reduced hyperglycolysis and NOX activation during early reperfusion, which correlated with a reduction in infarct volume and improvement in functional outcome, suggesting ethanol's utility as a post-stroke therapy.

The calcium binding protein EFhd2 is a novel amyloid protein that co-aggregates with pathological tau proteins in Alzheimer's disease. EFhd2 is overexpressed in Alzheimer's disease brain and its coiled-coil domain mediates the association with tau proteins. The formation of EFhd2 oligomers may contribute to the aggregation of tau proteins, to either promote neuronal death or survival. This study further demonstrates that EFhd2 is a molecular factor involved in the pathobiology of tauopathies.

Brain injury-induced proteolysis is reduced in a novel calpastatin overexpressing transgenic mouse

Following brain injury, the activation of calpain proteases results in protein cleavage and eventual cell death. Calpain inhibition via calpastatin is an advantageous approach due to its highly specific interaction with calpains. Augmenting calpastatin levels in a novel transgenic mouse resulted in decreased breakdown of selected proteins, thereby identifying a potential therapeutic agent for both brain injury and neurodegenerative disorders.