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Dr. Alaa El-Husseini

Last Research Projects in the Lab:

1. Control of synaptic balance
2. Assembly of proteins at nascent neuronal contacts
3. Protein sorting and trafficking
4. Regulation of protein sorting and function by palmitoylation

1. Control of synaptic balance

Excitation and inhibition in the CNS are mediated mainly by the neurotransmitters glutamate and γ-amino butyric acid (GABA), respectively. Neurons take exquisite care in outfitting each synapse type with characteristic structural and neurochemical features. For example, most excitatory synapses are formed through contact between glutamate-releasing axonal terminals and postsynaptic dendritic spines containing glutamate receptors. Conversely, inhibitory synapses are formed on the dendritic shaft where GABA receptors are found apposed to terminals positive for GABA biosynthetic enzymes. The number of excitatory versus inhibitory contacts that a single neuron receives dictates neuronal excitability and function. Thus, precise control systems must be established in each neuron to maintain appropriate numbers of excitatory and inhibitory synapses. However, factors that trigger the transformation of initial sites of contact to either excitatory or inhibitory synapses and ultimately modulate synaptic balance have only recently been discovered. Recent investigations by our lab indicates that assembly of scaffolding proteins such as the postsynaptic density protein PSD-95 and adhesion molecules such as neuroligins is critical for establishing synaptic balance.

This newly discovered mechanism has important implications in neurodevelopmental psychiatric disorders such as autism and some forms of mental retardation in which an imbalance in excitatory/inhibitory (E/I) synaptic ratio is thought to occur. In particular, it has been proposed that enhanced excitability associated with autism underlies the expression of abnormal social behavior characteristic of this disorder. On going work in the lab is focused on elucidating the role of these proteins, as well as other related molecules, in controlling synapse number, type and morphology and understanding how this process is potentially disrupted in brain disorders.

Neuroligins and PSD-95 modulate excitatory and inhibitory synapse development. (A) An example of the effects of a member of the neuroligin (NLG) family, NLG2 (green), on synapse formation. Expression of NLG2 in hippocampal neurons increases the number of excitatory (VGLUT-positive; red) and inhibitory (VGAT-positive; blue) presynaptic contacts. (B) Endogenous NLG2 (red) is normally localized at inhibitory synaptic contacts (VGAT-positive; blue). Overexpression of PSD-95 shifts NLG2 from inhibitory to excitatory (PSD-95-positive) synapses (colocalization of NLG2 and PSD-95 appears in orange).

Proteins implicated in the control of excitatory to inhibitory synaptic ratio. (A) Cell adhesion molecules such as neuroligin-1 (NLG1) and NLG2 induce excitatory and inhibitory synapses. NLG1 is enriched at excitatory sites whereas NLG2 is concentrated at inhibitory sites. Neuroligins associate with PSD-95 which is exclusively localized at excitatory sites. Interaction with PSD-95 enhances NLG1 accumulation at excitatory synapses. Other unidentified scaffolding proteins sequester NLG2 at inhibitory synapses. The relative levels of these proteins ensure appropriate excitatory to inhibitory (E/I) synaptic ratio received by individual neurons. (B) Altered expression of neuroiligins or PSD-95 manipulates the E/I ratio. An example showing that enhanced levels of PSD-95 redistributes NLG2 from inhibitory to excitatory synapses. This results in an enhancement of excitatory presynaptic terminals and a reduction in the number of inhibitory contacts, thus shifting the E/I synaptic ratio toward higher overall excitation.

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2. Assembly of proteins at nascent neuronal contacts

PSD-95 is a major component of the PSD at glutamatergic synapses. Recent findings suggest that PSD-95 plays important role in structurally and functionally organizing the PSD by coupling signal transduction proteins close to synaptic receptors using its multiple protein-protein interaction sites. Hence, we have initiated studies to determine the importance of synaptic targeting of PSD-95 in assembly of proteins at the PSD and its role in synapse development. We have demonstrated that synaptic clustering of PSD-95 drives the assembly of specific postsynaptic proteins including GKAP, Shank, neuroligins and the glutamate receptor subunit GluR1 at the synapse. Future work will look at the trafficking of other proteins to nascent contact sites, and the protein-protein interactions that regulate their assembly.

Recruitment of clusters of synaptic proteins at early sites of contact between axons and dendrites.(A) Shows an accumulation of a synaptophysin cluster at a contact site between dendritic filopodia of a cell transfected with a membrane targeted GFP and an axon from a neuron transfected with synaptophysin tagged with DsRed (SYN DsRed). (B) Time lapse images showing accumulation of SYN DsRed at a site apposed to an existing PSD-95 GFP cluster, occurred over a time (t) period of 20 minutes.

Sites apposed to stationary non-synaptic scaffold clusters are readily transformed to active presynaptic terminals. DIV 5 hippocampal neurons were transfected with PSD-95 GFP and analyzed 24-36 hours post-transfection for changes in the number of active presynaptic terminals by subsequent loading (left panels) and unloading (right panels) of the vital dye FM 4-64.Example of a stationary FM 4-64 negative PSD-95 GFP (closed arrowhead, load 1), which became FM 4-64 positive within 2 hours of the initial load.

Click below to download a movie showing contact formation between a dendritic filopodia and an axon (movie 1), or Movie 2 to see the recruitment of postsynaptic PSD-95 at the site of axonal contact.

Movie1 (Quicktime .mov file)
Movie2 (Quicktime .mov file)

Click here to download the latest version of Quicktime Player

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3. Protein sorting and trafficking

Proper sorting and transport of neurotransmitter receptors and associated proteins is essential for neuronal activity and plasticity. Recent studies have identified several proteins that regulate clustering of neurotransmitter receptors at the synapse. However, it remains unknown what proteins mediate sorting and delivery of receptors from the soma to postsynaptic sites. Molecular motors that regulate cargo trafficking on both actin filaments and microtubules have been implicated in initial transport and delivery to specific subcellular sites. In particular, class V of unconventional myosins is actin-based motors thought to regulate trafficking of organelles and associated proteins in neuronal cells. Our investigations revealed a novel mechanism for the transport of a specific glutamate receptor subunit in neurons mediated by a member of the myosin V family.

Future studies will be directed towards understanding how protein sorting is regulated by motor proteins and how this process regulates synapse function.

Expression of a mutant form of myosin Vb (MyoVb CT) lacking the cargo binding domain results in abnormal accumulation of GluR1, but not NR1 in a perinuclear region in the soma (arrowheads).

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4. Regulation of protein sorting and function by palmitoylation

Modification of proteins with the lipid palmitate regulates targeting to specific vesicular compartments and synaptic membranes. Mounting evidence indicates that this lipid modification modulates diverse aspects of neuronal development and synaptic transmission. In particular, palmitoylation regulates the function of proteins that control neuronal differentiation, axonal pathfinding and filopodia formation. In addition, trafficking of numerous proteins associated with synaptic vesicle release machinery requires protein palmitoylation. Remarkably, reversible palmitoylation of specific scaffolding proteins and signaling molecules dynamically regulates ion channel clustering and synaptic strength.

Neurons possess an elaborate plasma membrane architecture that consists of highly specialized morphological structures, including axons, dendrites and synapses. Modulation of plasma membrane dynamics and composition regulates the development of these unique structures. Ongoing research in the lab is exploring proteins, particularly palmitoylated forms, that modulate the formation and structure of the plasma membrane as well as their participation in synapse formation.

Filopodia inducing motifs (FIMs): Specific palmitoylated protein motifs, characterized by two adjacent cysteines and nearby basic residues, are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and the branching of dendrites and axons in neurons. Such motifs are present at the N-terminus of GAP-43 and the C-terminus of paralemmin, two neuronal proteins implicated in cytoskeletal organization and filopodial outgrowth. Filopodia induction is blocked by mutations of the palmitoylated sites or by treatment with 2-bromopalmitate, an agent that inhibits protein palmitoylation. Moreover, overexpression of a constitutively active form of ARF6, a GTPase that regulates membrane cycling and dendritic branching reversed the effects of the acylated protein motifs. Filopodia induction by the specific palmitoylated motifs was also reduced upon overexpression of a dominant negative form of the GTPase cdc42.

Palmitoylation plays multiple roles in synapse development and function. (A) Palmitoylation is required for paralemmin-induced filopodia, dendritic protrusions implicated in spine formation. Filopodia induction requires ARF6 and cdc-42, two GTPases that regulate bulk membrane cycling and actin dynamics. In growth cones, palmitoylation of cell adhesion molecules such as NCAM140 and DCC regulates axonal pathfinding and synapse formation. Palmitoylation of GAP43 directs it to neurites and regulates growth cone maturation. (B) Palmitoylation of a variety of proteins regulates diverse aspects of synaptic function (some examples are highlighted in red). On the presynaptic side, palmitoylation of proteins such as, GAD65, synaptotagmin I, and the SNARE proteins SNAP-25 and Ykt6 regulate neurotransmitter synthesis, synaptic vesicle fusion and transmitter release. Palmitate attachment modulates voltage sensing and current amplitude of several voltage gated ion channels including subunit of Kv1.1 and 2A-subunit of Ca2+ channel. On the postsynaptic side, numerous G-protein-coupled receptors (GPCRs), G-proteins and signaling proteins such as Fyn (a member of the Src family of non-receptor tyrosine kinases) and the small GTPase Ras are palmitoylated. Palmitoylation regulates multimerization and clustering of PSD-95. This process modulates clustering AMPA-type glutamate receptors. Clustering of GABAA receptor γ2 subunit also requires palmitoylation, whereas palmitoylation of the nicotinic α-bungarotoxin binding receptors (BgtRs) is required for targeting to lipid rafts.

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