Given a disruption of membrane internalization and an increa

Given a disruption of membrane internalization and an increase in synaptic glutamate receptors in CPG2 knockdown neurons, one might expect an increase in dendritic spine size as membrane continues to be inserted into the spines during receptor exocytosis. However, we observed the opposite phenotype, with dendritic spines on CPG2 knockdown neurons nearly twenty percent smaller than in control neurons. According to our model of CPG2 function, the reduction in spine size may result from an increase in membrane retention within internalized clathrin-coated vesicles, as the clathrin coat cannot be removed and the membrane cannot be recovered from these vesicles. However, since the nutlin 3 is critical for both spine morphology and endocytosis Engqvist-Goldstein and Drubin 2003, Rao and Craig 2000, it is possible that CPG2 regulates the spine cytoskeleton and that its knockdown affects both spine size and endocytosis in parallel. A large proportion of AMPA receptors are inserted into the plasma membrane at extrasynaptic sites, possibly between spines (Passafaro et al., 2001). While normally there is a tight correlation between spine size and synaptic AMPA receptor numbers (Kasai et al., 2003), in CPG2 knockdown neurons, the total amount of synaptic glutamate receptors increases despite smaller spines, suggesting that CPG2 knockdown may disrupt the spine size/receptor number relationship. cpg2 was isolated in a screen for seizure-induced genes and is regulated by physiological activity Nedivi et al. 1993, Nedivi et al. 1996. In general, screens for activity-regulated genes have isolated a number of synaptic proteins that are the components of the basic transmission machinery, indicating that the synaptic rearrangements that occur during plasticity likely involve an augmentation of normal synaptic processes (Nedivi, 1999). Since CPG2 is important for normal synaptic function and for the activity-induced internalization of glutamate receptors that may underlie LTD, an increase in cpg2 expression may belie an increase in synapse formation during synaptic plasticity. However, following LTP, there is an increase in the number of clathrin-coated pits and vesicles in dendritic spines, which may reflect an enhancement of postsynaptic protein cycling during plasticity (Toni et al., 2001). The critical role of CPG2 in activity-dependent glutamate receptor endocytosis may explain the increased need for cpg2 expression during periods of synaptic plasticity. As the product of an activity-regulated transcript that is expressed solely in brain regions with significant synaptic plasticity mechanisms, CPG2 may underlie an adaptation of the clathrin-mediated endocytosis pathway that enables the capacity for postsynaptic plasticity in excitatory synapses.
Experimental Procedures
Acknowledgements We thank Dr. Carlos Lois for the lentivirus vectors, advice on lentivirus design, and use of his equipment; Dr. Michael Ehlers for the clathrin-GFP construct; Dr. Joshua Sanes for the anti-Syne-1 antibody; Dr. Gabor Nyiri for lowicryl-embedded hippocampal sections; Dr. Ulrich Putz for the cpg15-shRNA lentivirus; Wei-Chung Lee and Elizabeth Lester for assistance with the in situ hybridizations; and Shifali Arora for spine size quantifications and subcloning assistance. We would also like to thank Dr. Troy Littleton, Dr. Michele Jacobs, Dr. Tadahiro Fujino, and Wei-Chung Lee for critical comments on the manuscript. This work was supported by NEI (E.N.), NCRR (T.L.H.), and NIDDK (T.L.H.).
Introduction Methotrexate (MTX), as an antagonist of folate, blocks the synthesis of DNA and results in the cell cycle arrest in G1 and/or S phases (Mazur et al., 2009; Nihal et al., 2014). MTX is widely administrated as an anticancer agent as well as the treatment of several autoimmune disorders (Huang et al., 2011; Abdel-Raheem and Khedr, 2014; Favalli et al., 2014; Kivity et al., 2014; Hafez et al., 2015). Over the long term, the administration of MTX results in various organ toxicities (Abo-Haded et al., 2017; Asci et al., 2017). Hepatotoxicity is one of the most frequently reported side effects of MTX (Ali et al., 2014). Low to high doses of MTX may lead to disorders such as liver cirrhosis or fibrosis (Mhatre and Marar, 2016; Mukherjee et al., 2013).