Neddylation is required for presynaptic clustering of mGlu7 and maturation of presynaptic terminals

Neddylation is a posttranslational modification in which NEDD8 is conjugated to a target substrate by cellular processes similar to those involved in ubiquitination. Recent studies have identified PSD-95 and cofilin as substrates for neddylation in the brain and have shown that neddylation modulates the maturation and stability of dendritic spines in developing neurons. However, the precise substrates and functional consequences of neddylation at presynaptic terminals remain elusive. Here, we provide evidence that the mGlu7 receptor is a target of neddylation in heterologous cells and rat primary cultured neurons. We found that mGlu7 neddylation is reduced by agonist treatment and is required for the clustering of mGlu7 in the presynaptic active zone. In addition, we observed that neddylation is not required for the endocytosis of mGlu7, but it facilitates the ubiquitination of mGlu7 and stabilizes mGlu7 protein expression. Finally, we demonstrate that neddylation is necessary for the maturation of excitatory presynaptic terminals, providing a key role for neddylation in synaptic function.

Endogenously expressed Ranbp2 (Nup358) is not at the axon initial segment

Ranbp2 (also known as Nup358) is a member of the nucleoporin family that comprises the nuclear pore complex. Ranbp2 localizes at the nuclear membrane and was recently reported at the axon initial segment (AIS). However, we show the anti-Ranbp2 antibody used in previous studies is not specific for Ranbp2. We mapped the antibody binding site to the amino acid sequence KPLQG that is present in both Ranbp2 and Neurofascin, a well-known AIS protein. After silencing Neurofascin expression in neurons, the AIS was not stained by the antibody. Surprisingly, an exogenously expressed N-terminal fragment of Ranbp2 localizes at the AIS. We show this fragment interacts with stable microtubules. Finally, using CRISPR-Cas9 in primary cultured neurons, we inserted an HA-epitope tag at N-terminal, C-terminal, or internal sites of the endogenously expressed Ranbp2. No matter the location of the HA-epitope, endogenous Ranbp2 was found at the nuclear membrane but not the AIS. These results show that endogenously expressed Ranbp2 is not found at axon initial segments.

Targeting of δ-catenin to postsynaptic sites through interaction with the Shank3 N-terminus

Background Neurodevelopmental disorders such as autism spectrum disorder (ASD) may be caused by alterations in genes encoding proteins that are involved in synapse formation and function. This includes scaffold proteins such as Shank3, and synaptic adhesion proteins such as Neurexins or Neuroligins. An important question is whether the products of individual risk genes cooperate functionally (exemplified in the interaction of Neurexin with Neuroligin isoforms). This might suggest a common pathway in pathogenesis. For the SHANK3 gene, heterozygous loss of function, as well as missense mutations have been observed in ASD cases. Several missense mutations affect the N-terminal part of Shank3 which contains the highly conserved Shank/ProSAP N-terminal (SPN) and Ankyrin repeat (Ank) domains. The role of these domains and the relevance of these mutations for synaptic function of Shank3 are widely unknown. Methods We used purification from a synaptic protein fraction, as well as a variety of biochemical and cell biological approaches to identify proteins which associate with the Shank3 N-terminus at postsynaptic sites. Results We report here that δ-catenin, which is encoded by CTNND2, an autism candidate gene, directly interacts with the Ank domain of Shank3 at postsynaptic sites through its Armadillo-repeat domain. The interaction is not affected by well-known posttranslational modifications of δ-catenin, i.e. by phosphorylation or palmitoylation. However, an ASD-associated mutation in the SPN domain of Shank3, L68P, significantly increases the interaction of Shank3 with δ-catenin. By analysis of postsynaptic fractions from mice, we show that the lack of SPN-Ank containing, large isoforms of Shank3 results in the loss of postsynaptic δ-catenin. Further, expression of Shank3 variants containing the N-terminal domains in primary cultured neurons significantly increased the presence of coexpressed δ-catenin at postsynaptic sites. Limitations Work in model organisms such as mice, and in primary cultured neurons may not reproduce faithfully the situation in human brain neurons. Work in primary cultured neurons was also hampered by lack of a specific antibody for endogenous δ-catenin. Conclusions Our data show that the interaction between Shank3 N-terminus and δ-catenin is required for the postsynaptic targeting of δ-catenin. Failure of proper targeting of δ-catenin to postsynaptic sites may contribute to the pathogenesis of autism spectrum disorder.

The Neurotrophic and Anti-Inflammatory Effect of β-asarone on the DA-Induced the Pathology of Minimal Hepatic Encephalopathy

Background: The mechanism underlying the impaired cognitive function and memory loss in Minimal hepatic encephalopathy (MHE) remains unclear. Dopamine (DA) is reported to be associated with dementia. Methods: In this study, we investigated mechanism underlying DA-induced MHE pathology by immunoblotting, ELISA, FM4-64 and fluorescence staining. Results: We observed that MHE brains showed the increased content of DA, after administration of anti-DA antibody, and cognitive loss in MHE rats was recovered to the normal level, indicating the involvement of DA in the pathogenesis of MHE. Moreover, DA (10 μM) treatment obviously induced the decrease in the production of GDNF/NGF and the increase in TNFα levels in primary cultured neurons, which were blocked by addition of β-asarone (βASA). We also demonstrated that DA stimulated the activation of ASK1/JNK1 pathway. and the addition of anti-TNFα antibody reversed the inactivation of Notch signaling, the downregulation of neurotrophins and synaptic loss.Conclusions: Overall, we suggested that DA stimulated abundant production and secretion of neuronal TNFα, which elicited progressive loss of neurotrophic factors, leading to cognitive disorder of MHE.

The Neurotrophic and Anti-Inflammatory Effect of β-asarone on the DA-Induced the Pathology of Minimal Hepatic Encephalopathy

Background: The mechanism underlying the impaired cognitive function and memory loss in Minimal hepatic encephalopathy (MHE) remains unclear. Dopamine (DA) is reported to be associated with dementia. Methods: In this study, we investigated mechanism underlying DA-induced MHE pathology by immunoblotting, ELISA, FM4-64 and fluorescence staining. Results: We observed that MHE brains showed the increased content of DA, after administration of anti-DA antibody, and cognitive loss in MHE rats was recovered to the normal level, indicating the involvement of DA in the pathogenesis of MHE. Moreover, DA (10 μM) treatment obviously induced the decrease in the production of GDNF/NGF and the increase in TNFα levels in primary cultured neurons, which were blocked by addition of β-asarone (βASA). We also demonstrated that DA stimulated the activation of ASK1/JNK1 pathway. and the addition of anti-TNFα antibody reversed the inactivation of Notch signaling, the downregulation of neurotrophins and synaptic loss.Conclusions: Overall, we suggested that DA stimulated abundant production and secretion of neuronal TNFα, which elicited progressive loss of neurotrophic factors, leading to cognitive disorder of MHE.

α-synuclein strains that cause distinct pathologies differentially inhibit proteasome

Abnormal α-synuclein aggregation has been implicated in several diseases and is known to spread in a prion-like manner. There is a relationship between protein aggregate structure (strain) and clinical phenotype in prion diseases, however, whether differences in the strains of α-synuclein aggregates account for the different pathologies remained unclear. Here, we generated two types of α-synuclein fibrils from identical monomer and investigated their seeding and propagation ability in mice and primary-cultured neurons. One α-synuclein fibril induced marked accumulation of phosphorylated α-synuclein and ubiquitinated protein aggregates, while the other did not, indicating the formation of α-synuclein two strains. Notably, the former α-synuclein strain inhibited proteasome activity and co-precipitated with 26S proteasome complex. Further examination indicated that structural differences in the C-terminal region of α-synuclein strains lead to different effects on proteasome activity. These results provide a possible molecular mechanism to account for the different pathologies induced by different α-synuclein strains.