Luba Kojic

Role of NMDA receptor–dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries

Excitotoxic neuronal damage caused by overactivation of N-methyl-D-aspartate glutamate receptors (NMDARs) is thought to be a principal cause of neuronal loss after stroke and brain trauma. Here we report that activation of sterol regulatory element binding protein-1 (SREBP-1) transcription factor in affected neurons is an essential step in NMDAR-mediated excitotoxic neuronal death in both in vitro and in vivo models of stroke. The NMDAR-mediated activation of SREBP-1 is a result of increased insulin-induced gene-1 (Insig-1) degradation, which can be inhibited with an Insig-1–derived interference peptide (Indip) that we have developed. Using a focal ischemia model of stroke, we show that systemic administration of Indip not only prevents SREBP-1 activation but also substantially reduces neuronal damage and improves behavioral outcome. Our study suggests that agents that reduce SREBP-1 activation such as Indip may represent a new class of neuroprotective therapeutics against stroke.

Subunit-selective palmitoylation regulates the intracellular trafficking of AMPA receptor

The AMPA receptor (AMPAR) subunits GluR1 and GluR2 show different properties in central neurons and affect AMPAR trafficking via distinct mechanisms. This subunit‐specificity is partly achieved by recruiting unique protein modifications on different subunits. Here, we show that palmitoylation of GluR1 and GluR2 subunits also displays subunit‐specific properties and functions. Our findings indicate that GluR1 palmitoylation requires dynamic anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus. In contrast, GluR2 subunits are primarily palmitoylated locally in the ER as immature receptors, and an intact microtubule network is required for their palmitoylation. Interestingly, the majority of palmitoylated GluR2 subunits are not associated with GluR1 subunits. We found that preventing palmitoylation results in loss of mature GluR2, but leaves GluR1 intact, as palmitoylation on GluR2 in the ER prevents their sorting to the lysosome after receptor maturation. Moreover, palmitoylation on GluR1 and GluR2 subunits responds differently to neuronal activity. Blocking neuronal activity by tetrodotoxin increased the pool size of palmitoylated GluR2, but not GluR1. Acute stimulation by NMDA and AMPA also differentially affect AMPAR palmitoylation in a subunit‐specific manner. The present findings thus indicate that AMPAR palmitoylation is a subunit‐specific process that contributes to its regulation and trafficking.

Parainflammation associated with advanced glycation endproduct stimulation of RPE in vitro: Implications for age-related degenerative diseases of the eye

Age related macular degeneration (AMD) is one of the leading causes of blindness in Western society. A hallmark of early stage AMD are drusen, extracellular deposits that accumulate in the outer retina. Advanced glycation endproducts (AGE) accumulate with aging and are linked to several age-related diseases such as Alzheimers disease, osteoarthritis, atherosclerosis and AMD. AGE deposits are found in drusen and in Bruchs membrane of the eye and several studies have suggested its role in promoting oxidative stress, apoptosis and lipofuscin accumulation. Recently, complement activation and chronic inflammation have been implicated in the pathogenesis of AMD. While AGEs have been shown to promote inflammation in other diseases, whether it plays a similar role in AMD is not known. This study investigates the effects of AGE stimulation on pro- and anti-inflammatory pathways in primary culture of human retinal pigment epithelial cells (RPE). Differential gene expression studies revealed a total of 41 up- and 18 down-regulated RPE genes in response to AGE stimulation. These genes fell into three categories as assessed by gene set enrichment analysis (GSEA). The main categories were inflammation (interferon-induced, immune response) and proteasome degradation, followed by caspase signaling. Using suspension array technology, protein levels of secreted cytokines and growth factors were also examined. Anti-inflammatory cytokines including IL10, IL1ra and IL9 were all overexpressed. Pro-inflammatory cytokines including IL4, IL15 and IFN-γ were overexpressed, while other pro-inflammatory cytokines including IL8, MCP1, IP10 were underexpressed after AGE stimulation, suggesting a para-inflammation state of the RPE under these conditions. Levels of mRNA of chemokine, CXCL11, and viperin, RSAD2, were up-regulated and may play a role in driving the inflammatory response via the NF-kB and JAK-STAT pathways. CXCL11 was strongly immunoreactive and associated with drusen in the AMD eye. The pathways and novel genes identified here highlight inflammation as a key response to AGE stimulation in primary culture of human RPE, and identify chemokine CXCL11 as putative novel agent associated with the pathogenesis of AMD.

The regulatory role of long-term depression in juvenile and adult mouse ocular dominance plasticity

The study of experience-dependent ocular dominance (OD) plasticity has greatly contributed to the understanding of visual development. During the critical period, preventing input from one eye results in a significant impairment of vision, and loss of cortical responsivity via the deprived eye. Residual ocular dominance plasticity has recently been observed in adulthood. Accumulating evidence suggests that OD plasticity involves N-methyl-D-aspartate receptor (NMDAR)-dependent long-term depression (LTD). Here we report that the administration of a selective LTD antagonist prevented the ocular dominance shift during the critical period. The NMDAR co-agonist D-serine facilitated adult visual cortical LTD and the OD shift in short-term monocularly deprived (MD) adult mice. When combined with reverse suture, D-serine proved effective in restoring a contralaterally-dominated visual input pattern in long-term MD mice. This work suggests LTD as a key mechanism in both juvenile and adult ocular dominance plasticity, and D-serine as a potential therapeutic in human amblyopic subjects.

Isoform-specific palmitoylation of JNK regulates axonal development

The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1–3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3s distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching.

JNK3 Couples the Neuronal Stress Response to Inhibition of Secretory Trafficking

Palmitoylated JNK3 sequesters the phosphatase Sac1 at the Golgi to limit trafficking of glutamate receptors to the cell surface. JNK3 Golgi Traffic Cop in Neurons Neuronal integrity is maintained by the secretory pathway. The phosphatase Sac1, which metabolizes phosphatidylinositol 4-phosphate (PI4P), inhibits the trafficking of proteins through the secretory pathway by depleting PI4P at the Golgi. Yang et al. found that JNK3 (c-Jun N-terminal kinase 3) was palmitoylated and associated with the Golgi in neurons exposed to NMDA (N-methyl-d-aspartate), a glutamate receptor agonist that can be excitotoxic. Independently of its kinase activity, JNK3 bound to Sac1 and sequestered it at the Golgi, resulting in decreased abundance of PI4P and inhibition of Golgi-to-surface trafficking of AMPA-type GluR1 (glutamate receptor subunit 1). Inhibiting the JNK3-Sac1 interaction with synthetic peptides prevented NMDA-induced reduction of surface GluR1 and synaptic loss. Thus, JNK3 appears to function in the neuronal response to stress to limit trafficking through the secretory pathway. Secretory trafficking through the Golgi complex is critical for neuronal development, function, and stress response. Altered secretion is associated with the pathogenesis of various neurological diseases. We found that c-Jun amino-terminal kinase 3 (JNK3) inhibited secretory trafficking by promoting the depletion of phosphatidylinositol 4-phosphate (PI4P) in the Golgi complex of COS7 cells and primary rat neurons. Exposure of cultured primary rat neurons to excitotoxic concentrations of NMDA (N-methyl-d-aspartate), an agonist of a class of ionotropic glutamate receptors, or overexpression of zD17 (a palmitoyl transferase) resulted in JNK3 palmitoylation and association with the Golgi complex. Analysis of mutant constructs of JNK3 indicated that Golgi association was independent of its kinase activity but depended on its palmitoylation. The association of JNK3 with the Golgi in cultured neurons decreased the secretory trafficking of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 (glutamate receptor subunit 1), a component of ionotropic glutamate receptors found at glutamatergic synapses. Palmitoylated JNK3 bound to the phosphatase Sac1, increasing its abundance at the Golgi and thereby decreasing the abundance of PI4P, a lipid necessary for post-Golgi trafficking. Disrupting the JNK3-Sac1 interaction with two synthetic peptides prevented the loss of surface GluR1 and preserved synaptic integrity in cultured neurons exposed to NMDA. Together, our results suggest that JNK3 participates in an adaptive response to neuronal hyperexcitation by impeding secretory trafficking at the Golgi complex.

A microfluidic based in vitro model of synaptic competition.

Synaptic competition is widely believed to be central to the formation and function of neuronal networks, yet the underlying mechanisms are poorly described. To investigate synaptic competition in vitro, we have developed a novel two input pathway competition model using a 3-compartment microfluidic device. Axons from cultured rat cortical neurons from two different lateral compartments (inputs) innervate a common neuronal population in a separate central compartment. Inhibiting one inputs activity, using the GABAAR agonist muscimol, resulted in increased synapse numbers and axon elongation of the opposing untreated (uninhibited) inputs in the central compartment. Time lapse imaging revealed that uninhibited inputs outgrew and outconnected their inhibited counterparts. This form of competition occurs during a sensitive period ending prior to 21 DIV and is NMDAR and CamKII dependent. Surprisingly, this form of plasticity was dependent on the age of the center compartment neurons but not of the competing inputs.