Pamela Arstikaitis

Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation

Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins—68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is the finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl), both with regard to localization and function: Cdc42-palm concentrates in dendritic spines and has a special role in inducing these post-synaptic structures. Furthermore, assessing palmitoylation dynamics in drug-induced activity models identifies rapidly induced changes for Cdc42 as well as for other synaptic palmitoyl proteins, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.

Huntingtin-Interacting Protein HIP14 Is a Palmitoyl Transferase Involved in Palmitoylation and Trafficking of Multiple Neuronal Proteins

In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.

Palmitoylation of huntingtin by HIP14 is essential for its trafficking and function.

Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.

Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity

Palmitoylation, a post‐translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate‐enzyme specificity for these PATs. Using the acyl‐biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC‐3, DHHC‐8, HIP14L (DHHC‐13), and HIP14 (DHHC‐17). Exogenous expression of enzymes and substrates in COS cells reveals that HIP14L and HIP14 modulate huntingtin palmitoylation, DHHC‐8 modulates paralemmin‐1 palmitoylation, and DHHC‐3 shows the least substrate specificity. These in vitro data were validated by lentiviral siRNA‐mediated knockdown of endogenous HIP14 and DHHC‐3 in cultured rat cortical neurons. PATs require the presence of palmitoylated cysteines in order to interact with their substrates. To understand the elements that influence enzyme/ substrate specificity further, we fused the HIP14 ankryin repeat domain to the N terminus of DHHC‐3, which is not a PAT for huntingtin. This modification enabled DHHC‐3 to behave similarly to HIP14 by modulating palmitoylation and trafficking of huntingtin. Taken together, this study indicates that individual PATs have specific substrate preference, determined by regulatory domains outside the DHHC domain of the enzymes.— Huang, K., Sanders, S., Singaraja, R., Orban, P., Cij‐souw, T., Arstikaitis, P., Yanai, A., Hayden, M. R., El‐Husseini, A. Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity. FASEB J. 23, 2605–2615 (2009)

Palmitoylation of ATP-Binding Cassette Transporter A1 Is Essential for Its Trafficking and Function

ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.

Paralemmin-1, a Modulator of Filopodia Induction Is Required for Spine Maturation

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.

Proteins That Promote Filopodia Stability, but Not Number, Lead to More Axonal-Dendritic Contacts

Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation.

Ministrokes in Channelrhodopsin-2 Transgenic Mice Reveal Widespread Deficits in Motor Output Despite Maintenance of Cortical Neuronal Excitability

We evaluated the effects of ministrokes targeted to individual pial arterioles on motor function in Thy-1 line 18 channelrhodopsin-2 (ChR2) transgenic mice within the first hours after ischemia. Using optogenetics, we directly assessed both the excitability and motor output of cortical neurons in a manner independent of behavioral state or training. Occlusion of individual arterioles within the motor cortex led to a ministroke that was verified using laser speckle contrast imaging. Surprisingly, ministrokes targeted to a relatively small region of the forelimb motor map, with an ischemic core of 0.07 ± 0.03 mm2, impaired motor responses evoked from points across widespread areas of motor cortex even 1.5 mm away. Contrasting averaged ChR2-evoked electroencephalographic, spinal (ChR2 evoked potential), and electromyographic responses revealed a mismatch between measures of cortical excitability and motor output within 60 min after stroke. This mismatch suggests that apparently excitable cortical neurons (even >1 mm into peri-infarct areas, away from the infarct core) were impaired in their capacity to generate spinal potentials leading to even more severe deficits in motor output at muscles. We suggest that ischemia, targeted to a subset of motor cortex, leads to relatively small reductions in excitability within motor cortex, and cumulative depression of both descending spinal circuits and motor output in response to the activation of widespread cortical territories even outside of the area directly affected by the ischemia.

Myosin-Va-interacting protein, RILPL2, controls cell shape and neuronal morphogenesis via Rac signaling.

Neuronal morphology plays an essential role in neuronal function. The establishment and maintenance of neuronal morphology is intimately linked to the actin cytoskeleton; however, the molecular mechanisms that regulate changes in neuronal morphology are poorly understood. Here we identify a novel myosin-Va (MyoVa)-interacting protein, RILPL2, which regulates cellular morphology. Overexpression of this protein in young or mature hippocampal neurons results in an increase in the number of spine-like protrusions. By contrast, knockdown of endogenous RILPL2 in neurons by short hairpin RNA (shRNA) interference results in reduced spine-like protrusions, a phenotype rescued by overexpression of an shRNA-insensitive RILPL2 mutant, suggesting a role for RILPL2 in both the establishment and maintenance of dendritic spines. Interestingly, we demonstrate that RILPL2 and the Rho GTPase Rac1 form a complex, and that RILPL2 is able to induce activation of Rac1 and its target, p21-activated kinase (Pak). Notably, both RILPL2-mediated morphological changes and activation of Rac1-Pak signaling were blocked by expression of a truncated tail form of MyoVa or MyoVa shRNA, demonstrating that MyoVa is crucial for proper RILPL2 function. This might represent a novel mechanism linking RILPL2, the motor protein MyoVa and Rac1 with neuronal structure and function.

BARS at the synapse: PICK-1 lipid binding domain regulates targeting, trafficking, and synaptic plasticity.

Editors Note: These short reviews of a recent paper in the Journal, written exclusively by graduate students or postdoctoral fellows, are intended to mimic the journal clubs that exist in your own departments or institutions. For more information on the format and purpose of the Journal Club, please see Review of Jin et al. Targeting of AMPA receptors (AMPARs) to synaptic sites is critical for synaptic plasticity, including long-term potentia-tion (LTP) and long-term depression (LTD) (Barry and Ziff, 2002; Bredt and Nicoll, 2003). In hippocampal pyramidal neurons, AMPA receptors mediate rapid synaptic transmission. Trafficking of AMPARs to synaptic sites occurs by two main mechanisms: (1) glutamate receptor (GluR)2/3 subunits constitutively cycle to and from the synapse, and (2) GluR1/2 subunits are inserted into the synapse in an activity-dependent manner. A number of proteins regulate the trafficking and insertion of AMPA receptors at synaptic sites, including protein-interacting C ki-nase (PICK1). PICK1 is localized to exci-tatory synapses in cultured hippocampal neurons, and it interacts with the C-terminal PSD-95/Discs large/zona occludens-1 (PDZ) domain of GluR2/3 subunits (Xia et al., 1999). Furthermore, PICK1 promotes colocalization and clustering of AMPA receptors at excitatory sites (Xia et al., 1999). Structurally, PICK1 has multiple domains that function as protein-interaction sites, including a PDZ domain located at the N terminus, a central Bin/amphiphysin/Rvs (BAR) domain, and an acidic domain. The recent paper by Jin et al. (2006) in The Journal of Neuro-science demonstrates that the PICK1 BAR domain is important for the targeting of PICK1 and expression of LTD. The authors show that PICK1 binds phospho-inositide lipids via the BAR domain and that lipid binding is critical for the synap-tic targeting of PICK1, trafficking of AMPA receptors to the membrane surface , and induction of LTD. Jin et al. (2006) demonstrate that PICK1 binds lipids in vitro via specific residues present in the BAR domain [Jin et al. Using a Sindbis viral expression system, they infected hippocampal neurons and found that mutations in the BAR domain of PICK1 (PICK1 2K-E, PICK1 3K-E, and PICK1 5K-E) impaired its synaptic targeting , as shown by a reduction in the number and intensity of clusters. Furthermore, the PICK1 2KE mutant did not colocalize at excitatory synapses with PSD-95 [Jin et al.gesting that the lipid-binding domain is important for the synaptic targeting of PICK1. To explore how mutations in the BAR domain would affect synaptic targeting of GluR1 and GluR2, the authors …