Mechanisms and Role of Dendritic Membrane Trafficking for Long-Term Potentiation.

Long-term potentiation (LTP) of excitatory synapses is a major form of plasticity for learning and memory in the central nervous system. While the molecular mechanisms of LTP have been debated for decades, there is consensus that LTP induction activates membrane trafficking pathways within dendrites that are essential for synapse growth and strengthening. Current models suggest that key molecules for synaptic potentiation are sequestered within intracellular organelles, which are mobilized by synaptic activity to fuse with the plasma membrane following LTP induction. While the identity of the factors mobilized to the plasma membrane during LTP remain obscure, the field has narrowly focused on AMPA-type glutamate receptors. Here, we review recent literature and present new experimental data from our lab investigating whether AMPA receptors trafficked from intracellular organelles directly contribute to synaptic strengthening during LTP. We propose a modified model where membrane trafficking delivers distinct factors that are required to maintain synapse growth and AMPA receptor incorporation following LTP. Finally, we pose several fundamental questions that may guide further inquiry into the role of membrane trafficking for synaptic plasticity.

L-Type Voltage-Gated Ca2+ Channels Regulate Synaptic-Activity-Triggered Recycling Endosome Fusion in Neuronal Dendrites.

The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs) with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs) regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines.

Neurons face the challenge of regulating the abundance, distribution and repertoire of integral membrane proteins within their immense, architecturally complex dendritic arbors. While the endoplasmic reticulum (ER) supports dendritic translation, most dendrites lack the Golgi apparatus (GA), an essential organelle for conventional secretory trafficking. Thus, whether secretory cargo is locally trafficked in dendrites through a non-canonical pathway remains a fundamental question. Here we define the dendritic trafficking itinerary for key synaptic molecules in rat cortical neurons. Following ER exit, the AMPA-type glutamate receptor GluA1 and neuroligin 1 undergo spatially restricted entry into the dendritic secretory pathway and accumulate in recycling endosomes (REs) located in dendrites and spines before reaching the plasma membrane. Surprisingly, GluA1 surface delivery occurred even when GA function was disrupted. Thus, in addition to their canonical role in protein recycling, REs also mediate forward secretory trafficking in neuronal dendrites and spines through a specialized GA-independent trafficking network.

Optogenetic Control of Synaptic Composition and Function.

The molecular composition of the postsynaptic membrane is sculpted by synaptic activity. During synaptic plasticity at excitatory synapses, numerous structural, signaling, and receptor molecules concentrate at the postsynaptic density (PSD) to regulate synaptic strength. We developed an approach that uses light to tune the abundance of specific molecules in the PSD. We used this approach to investigate the relationship between the number of AMPA-type glutamate receptors in the PSD and synaptic strength. Surprisingly, adding more AMPA receptors to excitatory contacts had little effect on synaptic strength. Instead, we observed increased excitatory input through the apparent addition of new functional sites. Our data support a model where adding AMPA receptors is sufficient to activate synapses that had few receptors to begin with, but that additional remodeling events are required to strengthen established synapses. More broadly, this approach introduces the precise spatiotemporal control of optogenetics to the molecular control of synaptic function.

Computer Simulations Support a Morphological Contribution to BDNF Enhancement of Action Potential Generation.

Brain-derived neurotrophic factor (BDNF) regulates both action potential (AP) generation and neuron morphology. However, whether BDNF-induced changes in neuron morphology directly impact AP generation is unclear. We quantified BDNF's effect on cultured cortical neuron morphological parameters and found that BDNF stimulates dendrite growth and addition of dendrites while increasing both excitatory and inhibitory presynaptic inputs in a spatially restricted manner. To gain insight into how these combined changes in neuron structure and synaptic input impact AP generation, we used the morphological parameters we gathered to generate computational models. Simulations suggest that BDNF-induced neuron morphologies generate more APs under a wide variety of conditions. Synapse and dendrite addition have the greatest impact on AP generation. However, subtle alterations in excitatory/inhibitory synapse ratio and strength have a significant impact on AP generation when synaptic activity is low. Consistent with these simulations, BDNF rapidly enhances spontaneous activity in cortical cultures. We propose that BDNF promotes neuron morphologies that are intrinsically more efficient at translating barrages of synaptic activity into APs, which is a previously unexplored aspect of BDNF's function.

Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation.

Dendritic spines are major sites of excitatory synaptic transmission and changes in their numbers and morphology have been associated with neurodevelopmental and neurodegenerative disorders. Brain-derived Neurotrophic Factor (BDNF) is a secreted growth factor that influences hippocampal, striatal and neocortical pyramidal neuron dendritic spine density. However, the mechanisms by which BDNF regulates dendritic spines and how BDNF interacts with other regulators of spines remain unclear. We propose that one mechanism by which BDNF promotes dendritic spine formation is through an interaction with Wnt signaling. Here, we show that Wnt signaling inhibition in cultured cortical neurons disrupts dendritic spine development, reduces dendritic arbor size and complexity, and blocks BDNF-induced dendritic spine formation and maturation. Additionally, we show that BDNF regulates expression of Wnt2, and that Wnt2 is sufficient to promote cortical dendrite growth and dendritic spine formation. Together, these data suggest that BDNF and Wnt signaling cooperatively regulate dendritic spine formation.

The beta amyloid peptide can act as a modular aggregation domain.

Although there is compelling evidence that the beta amyloid peptide (Abeta) can be centrally involved in Alzheimer's disease, the natural role (if any) of this peptide remains unclear. Here we use green fluorescent protein (GFP) fusions to demonstrate that the Abeta sequence, like prion domains, can act as a modular aggregation domain when terminally appended to a normally soluble protein. We find that a single amino acid substitution (Leu(17) to Pro) in the beta peptide sequence can abolish this cis capacity to induce aggregation. Introduction of this substitution into full-length APP (i.e., a Leu(613)Pro substitution in APP695) alters the processing of APP leading to the accumulation of the C99 C-terminal fragment (CTF). We suggest that in at least some aggregation disease-related proteins the presence of an aggregation domain is not "accidental", but reflects a selected role of these domains in modulating the trafficking or metabolism of the parental protein.

Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.

A non-natural 16-residue "degron" peptide has been reported to convey proteasome-dependent degradation when fused to proteins expressed in yeast (Gilon, T., Chomsky, O., and Kulka, R. (2000) Mol. Cell. Biol. 20, 7214-7219) or when fused to green fluorescent protein (GFP) and expressed in mammalian cells (Bence, N. F., Sampat, R. M., and Kopito, R. R. (2001) Science 292, 1552-1555). We find that expression of the GFP::degron in Caenorhabditis elegans muscle or neurons results in the formation of stable perinuclear deposits. Similar perinuclear deposition of GFP::degron was also observed upon transfection of primary rat hippocampal neurons or mouse Neuro2A cells. The generality of this observation was supported by transfection of HEK 293 cells with both GFP::degron and DsRed(monomer)::degron constructs. GFP::degron expressed in C. elegans is less soluble than unmodified GFP and induces the small chaperone protein HSP-16, which co-localizes and co-immunoprecipitates with GFP::degron deposits. Induction of GFP::degron in C. elegans muscle leads to rapid paralysis, demonstrating the in vivo toxicity of this aggregating variant. This paralysis is suppressed by co-expression of HSP-16, which dramatically alters the subcellular distribution of GFP::degron. Our results suggest that in C. elegans, and perhaps in mammalian cells, the degron peptide is not a specific proteasome-targeting signal but acts instead by altering GFP secondary or tertiary structure, resulting in an aggregation-prone form recognized by the chaperone system. This altered form of GFP can form toxic aggregates if its expression level exceeds the capacity of chaperone-based degradation pathways. GFP::degron may serve as an instructive "generic" aggregating control protein for studies of disease-associated aggregating proteins, such as huntingtin, alpha-synuclein, and the beta-amyloid peptide.

Compensatory regulation among ER chaperones in C. elegans.

We have identified a particularly clear case of compensatory transcriptional regulation among ER chaperones in Caenorhabditis elegans using a GFP reporter transgene that is under the control of the promoter of hsp-4, a C. elegans homolog of GRP-78/BiP. Knockdown by RNA interference of 9 known or predicted ER chaperones induced hsp-4 upregulation via the ire-1/xbp-1 signaling cascade employed in the unfolded protein response. We show that this compensatory regulation is specific for ER chaperones, not dependent on RNA interference, and required for maintaining viability in worms containing a deletion of the hsp-3 gene.