zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.

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.

New approaches for solving old problems in neuronal protein trafficking.

Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision.

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.

Local and Use-Dependent Effects of ß-Amyloid Oligomers on NMDA Receptor Function Revealed by Optical Quantal Analysis.

Beta amyloid (Aß) triggers the elimination of excitatory synaptic connections in the CNS, an early manifestation of Alzheimer's disease. Oligomeric assemblies of Aß peptide associate with excitatory synapses resulting in synapse elimination through a process that requires NMDA-type glutamate receptor activation. Whether Aß affects synaptic NMDA receptor (NMDAR) function directly and acts locally at synapses to which it has bound and whether synaptic activity influences Aß synaptic binding and synaptotoxicity have remained fundamental questions. Here, we used subcellular Ca2+ imaging in rat hippocampal neurons to visualize NMDAR function at individual synapses before and after Aß application. Aß triggered a robust impairment of NMDAR Ca2+ entry at most, but not all, synapses. NMDAR function was more severely impaired at highly active synapses and synapses with bound Aß, but activity was not required for Aß synapse binding. Blocking NMDARs during Aß exposure prevented Aß-mediated impairment. Finally, Aß impaired NMDAR Ca2+ entry at doses much lower than those required for NMDAR internalization, revealing a novel, potent mode of NMDAR regulation by Aß. SIGNIFICANCE STATEMENT: Amyloid ß (Aß) is strongly implicated in Alzheimer's disease. Aß triggers the elimination of excitatory synapses through a mechanism that requires NMDA receptors (NMDARs). However, little is known about how or whether Aß influences synaptic NMDAR function. We used an imaging-based assay to investigate the relationship among Aß binding, activity, and NMDAR function at individual synapses. Aß triggered a robust impairment of NMDAR Ca2+ entry at most, but not all, synapses. NMDAR function was more severely impaired at highly active synapses and synapses with bound Aß. Blocking NMDARs during Aß exposure prevented Aß-mediated impairment. Together, our experiments reveal a novel use-dependent, potent, and local mode of Aß-mediated NMDAR impairment.

Sustained inflammation and differential expression of interferons type I and III in PVM-infected interferon-gamma (IFNγ) gene-deleted mice.

Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ(-/-) mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNß, IFNλ2/3 and IFN-stimulated 2-5' oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ(-/-) mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include over-production of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ(-/-) mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.

Defective eosinophil hematopoiesis ex vivo in inbred Rocky Mountain White (IRW) mice.

We examine the proliferation and differentiation of bone marrow (BM) progenitors from inbred Rocky Mountain White (IRW) mice, a strain used primarily for retrovirus infection studies. In contrast to findings with BALB/c and C57BL/6 strains, IRW BM cells cannot proliferate or generate pure eosinophil cultures ex vivo in response to a defined cytokine regimen. Analysis of IRW BM at baseline was unremarkable, including 0.08 ± 0.03% Lin(-)Sca-1(+)c-kit(+) (LSK) hematopoietic stem cells and 5.2 ± 0.3% eosinophils; the percentage of eosinophil progenitors (EoPs; Lin(-)Sca-1(-)c-kit(+)CD34(+)IL-5Rα(+)) was similar in all three mouse strains. Transcripts encoding GM-CSFRα and the IL-3/IL-5/GM-CSF common ß chain were detected at equivalent levels in IRW and BALB/c BM, whereas expression of transcripts encoding IL-5Rα, IL-3Rα, and GATA-2 was diminished in IRW BM compared with BALB/c. Expression of membrane-bound IL-5Rα and intracellular STAT5 proteins was also diminished in IRW BM cells. Diminished expression of transcripts encoding IL-5Rα and GATA-2 and immunoreactive STAT5 in IRW BM persisted after 4 days in culture, along with diminished expression of GATA-1. Western blot revealed that cells from IRW BM overexpress nonsignaling soluble IL-5Rα protein. Interestingly, OVA sensitization and challenge resulted in BM and airway eosinophilia in IRW mice; however, the responses were significantly blunted. These results suggest that IRW mice have diminished capacity to generate eosinophils in culture and in vivo, likely as a result of diminished signaling via IL-5Rα.