Ka Wan Li

Differential Transport and Local Translation of Cytoskeletal, Injury-Response, and Neurodegeneration Protein mRNAs in Axons

Recent studies have begun to focus on the signals that regulate axonal protein synthesis and the functional significance of localized protein synthesis. However, identification of proteins that are synthesized in mammalian axons has been mainly based on predictions. Here, we used axons purified from cultures of injury-conditioned adult dorsal root ganglion (DRG) neurons and proteomics methodology to identify axonally synthesized proteins. Reverse transcription (RT)-PCR from axonal preparations was used to confirm that the mRNA for each identified protein extended into the DRG axons. Proteins and the encoding mRNAs for the cytoskeletal proteins β-actin, peripherin, vimentin, γ-tropomyosin 3, and cofilin 1 were present in the axonal preparations. In addition to the cytoskeletal elements, several heat shock proteins (HSP27, HSP60, HSP70, grp75, αB crystallin), resident endoplasmic reticulum (ER) proteins (calreticulin, grp78/BiP, ERp29), proteins associated with neurodegenerative diseases (ubiquitin C-terminal hydrolase L1, rat ortholog of human DJ-1/Park7, γ-synuclein, superoxide dismutase 1), anti-oxidant proteins (peroxiredoxins 1 and 6), and metabolic proteins (e.g., phosphoglycerate kinase 1 (PGK 1), α enolase, aldolase C/Zebrin II) were included among the axonally synthesized proteins. Detection of the mRNAs encoding each of the axonally synthesized proteins identified by mass spectrometry in the axonal compartment indicates that the DRG axons have the potential to synthesize a complex population of proteins. Local treatment of the DRG axons with NGF or BDNF increased levels of cytoskeletal mRNAs into the axonal compartment by twofold to fivefold but had no effect on levels of the other axonal mRNAs studied. Neurotrophins selectively increased transport of β-actin, peripherin, and vimentin mRNAs from the cell body into the axons rather than changing transcription or mRNA survival in the axonal compartment.

CKAMP44: A Brain-Specific Protein Attenuating Short-Term Synaptic Plasticity in the Dentate Gyrus

Dancing with AMPARs A type of transmembrane receptor for glutamate, known as AMPAR, mediates most of the fast excitatory transmission in the mammalian central nervous system. Their function is regulated by the composition of their subunits, posttranslational modifications, and protein-protein interactions. Recently, several proteins that interact with AMPARs have been identified that affect their subcellular localization, synaptic stabilization, and kinetics. Using proteomic analysis, immunohistochemistry, and electrophysiology, von Engelhardt et al. (p. 1518, published online 25 February; see the Perspective by Farrant and Cull-Candy) identified a protein, CKAMP44, which modulates postsynaptic AMPA receptor gating, deactivation, and desentization. A synaptic protein that regulates postsynaptic AMPA receptor responses has been cloned and functionally characterized. CKAMP44, identified here by a proteomic approach, is a brain-specific type I transmembrane protein that associates with AMPA receptors in synaptic spines. CKAMP44 expressed in Xenopus oocytes reduced GluA1- and A2-mediated steady-state currents, but did not affect kainate- or N-methyl- d-aspartate (NMDA) receptor–mediated currents. Mouse hippocampal CA1 pyramidal neurons expressed CKAMP44 at low abundance, and overexpression of CKAMP44 led to stronger and faster AMPA receptor desensitization, slower recovery from desensitization, and a reduction in the paired-pulse ratio of AMPA currents. By contrast, dentate gyrus granule cells exhibited strong CKAMP44 expression, and CKAMP44 knockout increased the paired-pulse ratio of AMPA currents in lateral and medial perforant path–granule cell synapses. CKAMP44 thus modulates short-term plasticity at specific excitatory synapses.

Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking

Associative learning processes have an important role in the initiation and persistence of heroin-seeking. Here we show in a rat self-administration model that reexposure to cues previously associated with heroin results in downregulation of AMPA receptor subunit GluR2 and concomitant upregulation of clathrin-coat assembly protein AP2m1 in synaptic membranes of the medial prefrontal cortex (mPFC). Reduced AMPA receptor expression in synaptic membranes was associated with a decreased AMPA/NMDA current ratio and increased rectification index in mPFC pyramidal neurons. Systemic or ventral (but not dorsal) mPFC injections of a peptide inhibiting GluR2 endocytosis attenuated both the rectification index and cue-induced relapse to heroin-seeking, without affecting sucrose-seeking. We conclude that GluR2 receptor endocytosis and the resulting synaptic depression in ventral mPFC are crucial for cue-induced relapse to heroin-seeking. As reexposure to conditioned stimuli is a major cause for heroin relapse, inhibition of GluR2 endocytosis may provide a new target for the treatment of heroin addiction.

The CLAVATA3/ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences.

It is believed that CLAVATA3 (CLV3) encodes a peptide ligand that interacts with the CLV1/CLV2 receptor complex to limit the number of stem cells in the shoot apical meristem of Arabidopsis thaliana; however, the exact composition of the functional CLV3 product remains a mystery. A recent study on CLV3 shows that the CLV3/ESR (CLE) motif, together with the adjacent C-terminal sequence, is sufficient to execute CLV3 function when fused behind an N-terminal sequence of ERECTA. Here we show that most of the sequences flanking the CLE motif of CLV3 can be deleted without affecting CLV3 function. Using a liquid culture assay, we demonstrate that CLV3p, a synthetic peptide corresponding to the CLE motif of CLV3, is able to restrict the size of the shoot apical meristem in clv3 seedlings but not in clv1 seedlings. In accordance with this decrease in meristem size, application of CLV3p to in vitro-grown clv3 seedlings restricts the expression of the stem cell-promoting transcription factor WUSCHEL. Thus, we propose that the CLE motif is the functional region of CLV3 and that this region acts independently of its adjacent sequences.

Rat liver peroxisomes after fibrate treatment: A survey using quantitative mass spectrometry

Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal β-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor α-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibrate-specific induction scheme showing the variability of peroxisome proliferator-activated receptorα-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22–C26) but rather metabolize preferentially long chain fatty acids (C16–18).

Lasting synaptic changes underlie attention deficits caused by nicotine exposure during adolescence

Tobacco smoking and nicotine exposure during adolescence interfere with prefrontal cortex (PFC) development and lead to cognitive impairments in later life. The molecular and cellular underpinnings of these consequences remain elusive. We found that adolescent nicotine exposure induced lasting attentional disturbances and reduced mGluR2 protein and function on presynaptic terminals of PFC glutamatergic synapses. Restoring mGluR2 activity in vivo by local infusion of a group II mGluR agonist in adult rats that received nicotine as adolescents rescued attentional disturbances.

CRNF, a Molluscan Neurotrophic Factor That Interacts with the p75 Neurotrophin Receptor

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

Extracellular matrix plasticity and GABAergic inhibition of prefrontal cortex pyramidal cells facilitates relapse to heroin seeking

Successful treatment of drug addiction is hampered by high relapse rates during periods of abstinence. Neuroadaptation in the medial prefrontal cortex (mPFC) is thought to have a crucial role in vulnerability to relapse to drug seeking, but the molecular and cellular mechanisms remain largely unknown. To identify protein changes that contribute to relapse susceptibility, we investigated synaptic membrane fractions from the mPFC of rats that underwent 21 days of forced abstinence following heroin self-administration. Quantitative proteomics revealed that long-term abstinence from heroin self-administration was associated with reduced levels of extracellular matrix (ECM) proteins. After extinction of heroin self-administration, downregulation of ECM proteins was also present in the mPFC, as well as nucleus accumbens (NAc), and these adaptations were partially restored following cue-induced reinstatement of heroin seeking. In the mPFC, these ECM proteins are condensed in the perineuronal nets that exclusively surround GABAergic interneurons, indicating that ECM adaptation might alter the activity of GABAergic interneurons. In support of this, we observed an increase in the inhibitory GABAergic synaptic inputs received by the mPFC pyramidal cells after the re-exposure to heroin-conditioned cues. Recovering levels of ECM constituents by metalloproteinase inhibitor treatment (FN-439; i.c.v.) prior to a reinstatement test attenuated subsequent heroin seeking, suggesting that the reduced synaptic ECM levels during heroin abstinence enhanced sensitivity to respond to heroin-conditioned cues. We provide evidence for a novel neuroadaptive mechanism, in which heroin self-administration-induced adaptation of the ECM increased relapse vulnerability, potentially by augmenting the responsivity of mPFC GABAergic interneurons to heroin-associated stimuli.

Proteomics, Ultrastructure, and Physiology of Hippocampal Synapses in a Fragile X Syndrome Mouse Model Reveal Presynaptic Phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.

Proteomics of the Injured Rat Sciatic Nerve Reveals Protein Expression Dynamics During Regeneration

Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the ∼1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, α-crystallin B, α-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.