Evidence of Presynaptic Localization and Function of the c-Jun N-Terminal Kinase

The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity

Role of Glycogen Synthase Kinase-3β in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons

The phosphorylation of Amyloid Precursor Protein (APP) at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (Cdk5) can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 μM) for 30'-45' led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3β activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3β reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3β signaling pathway

Crosstalk between JNK and SUMO Signaling Pathways: deSUMOylation Is Protective against H2O2-Induced Cell Injury

Background: Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown. Methodology/Principal Findings: With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H 2O 2 induced-oxidative stress. Our data show that H 2O 2 modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNK’s action with the D-JNKI1 peptide rescued cells from death. Following H2O2, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H 2O 2 induced death. Importantly, in our model, direct interaction between these proteins can occur. Conclusions/Significance: Taken together our results show that SUMOylation may significantly contribute to modulation o

C-Jun-N-terminal kinase regulates Aβ oligomers production, synapthopathy and cognitive deficits in Alzheimer's disease

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder characterized by Amyloid-B CAP) and tau deposition in the brain. The number of patients suffering AD is estimated at 36 million worldwide, making AD the most common fonn of dementia. There is no efficient therapy for AD, thus efforts to develop new pharmacological strategies to treat AD need to be intensified. Increasing evidence establishes a central role of soluble and oligomeric form of AP peptide in the pathogenesis of AD. AP accumulates in the synaptic compartment and disrupts the synaptic functionality, leading at least to synaptic loss and neurodegeneration. These events strongly correlate with cognitive deficits characteristic of the pathology. The mechanisms promoting AP production as well as the intracellular pathways responsible for synaptic degeneration are not well known. Objective of this study was thus to decipher the signaling pathways involved in A~ oligorners toxicity, focusing on cj Jun N-terminal kinase (JNK). JNK has been extensively studied for its role in stress stimuli and AD. Here we found that JNK participates to the production of AP oiigomers, promoting phosphorylation of APP at Thr668 residue. Moreover we reported a strong activation of the JNK pathway in the synaptic compartment in both in vitro and in vivo models of synaptopathy. This correlates with the reduction of dendritic spines density and a decrease of postsynaptic markers (AMP AR and NMDAR subunits, PSD-95 and drebrin). To confirm the involvement of JNK in synaptic degeneration induced by AP oligomers we phannacologically inhibited JNK action, using the specific cell permeable peptide, D-JNKll. Treatment with D-JNKII reduces the amyloidogenic cleavage of APP, and thus the production of AP oiigomers. Moreover, D-JNKll reverts synaptic degeneration preventing loss of dendritic spines and proteins from the postsynaptic membrane. Finally, the peptide prevents long term potentiation and long term depression alterations and completely reverts cognitive deficits. These results provide essential new infotmation about the molecular changes that ultimately lead to the disruption of synaptic functionality and validate JNK inhibition as a new pharmacological strategy for the treatment of AD.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Combinatorial expression of neurexins and LAR-type phosphotyrosine phosphatase receptors instructs assembly of a cerebellar circuit

Abstract Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits. SAMs are broadly expressed in the brain, suggesting that they may instruct synapse formation and specification via a combinatorial logic. Here, we generate sextuple conditional knockout mice targeting all members of the two major families of presynaptic SAMs, Neurexins and leukocyte common antigen-related-type receptor phospho-tyrosine phosphatases (LAR-PTPRs), which together account for the majority of known trans-synaptic complexes. Using synapses formed by cerebellar Purkinje cells onto deep cerebellar nuclei as a model system, we confirm that Neurexins and LAR-PTPRs themselves are not essential for synapse assembly. The combinatorial deletion of both neurexins and LAR-PTPRs, however, decreases Purkinje-cell synapses on deep cerebellar nuclei, the major output pathway of cerebellar circuits. Consistent with this finding, combined but not separate deletions of neurexins and LAR-PTPRs impair motor behaviors. Thus, Neurexins and LAR-PTPRs are together required for the assembly of a functional cerebellar circuit

Correction: Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

[This corrects the article DOI: 10.1371/journal.pone.0158295.]

Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges

Triple ESyt123 KO does not increase the susceptibility of neurons to stress, induce obvious changes in the ER, or affect calcium dynamics in neurons.

<p>(A) Hippocampal cultures obtained from ESyt123 conditional KO mice and infected with lentiviruses expressing GFP-Cre and GFP-ΔCre. Images show GFP fluorescence merged with brightfield signal to illustrate the high efficiency of infection of the virus for both Cre and ΔCre conditions. (B) Neuronal cell death in response to mild stress (DTT, Tunicamycin, Thapsigargin and Paraquat) was assessed by the MTT assay. The stress conditions induced partial neuronal cell death, which was unchanged in neurons infected with Cre or ΔCre, suggesting that loss of ESyts does not increase susceptibility to toxic agents. Data were normalized to the control ΔCre condition, and are expressed as means ± SEM. Two-way ANOVA, Bonferroni post-hoc test, *p<0.05 vs CTR, **p<0.01 vs CTR, ***p<0.001 vs control, n = 8. (C) Confocal images showing hippocampal neurons transfected with Sec61β-EGFP in green and mCherry signal in red. Insert shows a magnification of the soma to better visualize the ER associated with the nucleus. (D) Quantification of the total neuronal area (mCherry signal) and the fraction of the nuclear-associated ER (Sec61β area normalized to mCherry area) showing no difference between ΔCre and Cre treated neurons. Data are expressed as means ± SEM. Student t-test, p>0.05, n = 18 ΔCre, n = 15 Cre. (E) Representative images of Sec61β-EGFP signal (green) acquired at higher exposure, showing that cytoplasmic ER is present in dendrites as well as in the neck of a subset of spines (see arrowheads) in both ΔCre and Cre treated neurons. The mCherry signal (red) allows visualization of dendrites and dendritic spines. (F) Representative images of GCaMP6M-expressing hippocampal neurons at resting (left panel-absence of Ca2+-signal) or during network activity (right panel-presence of a Ca2+-signal) for ΔCre (top panel) and Cre (low panel) treated neurons. Calcium activity is present in both ΔCre and Cre treated neurons. (G) Quantification of the number of calcium peaks, the integrated peak area, as well as the rise time and decay time kinetics, during 5min recording of neuronal activity. No significant differences has been observed between ΔCre and Cre treated neurons. Data are expressed as means ± SEM. Student t-test, p>0.05, n = 5 pups (total of analyzed neurons = 184) ΔCre, n = 5 pups (total of analyzed neurons = 129) Cre.</p

Loss of ESyts does not affect the level of synaptic and ER markers in the brain.

<p>(A and B) Immunoblots and relative quantifications of major pre-synaptic proteins in WT and constitutive ESyt123 triple KO mice. Data are shown as means ± SEM, Student’s t-test, p>0.05, n = 4. (C and D) Western blot and relative quantification of ER proteins, showing no differences between WT and 123EC KO mice. Data are shown as means ± SEM, Student’s t-test, p>0.05, n = 4.</p