Emerging Roles of Neuronal Extracellular Vesicles at the Synapse.

Extracellular vesicles (EVs) are secreted from most, if not all, cell types and are implicated in short- and long-distance signaling throughout the body. EVs are also secreted from neurons and represent an emergent neuronal communication platform. Understanding the functional implications of EV signaling to recipient neurons and glia requires understanding the cell biology involved in EV biogenesis, cargo loading, secretion, uptake, and signal transduction in the recipient cell. Here we review these major questions of EV biology while highlighting recent new insights and examples within the nervous system, such as modulating synaptic function or morphogenesis in recipient neurons.

Sympathetic neurons secrete retrogradely transported TrkA on extracellular vesicles.

Proper wiring of the peripheral nervous system relies on neurotrophic signaling via nerve growth factor (NGF). NGF secreted by target organs (i.e. eye) binds to the TrkA receptor expressed on the distal axons of postganglionic neurons. Upon binding, TrkA is internalized into a signaling endosome and retrogradely trafficked back to the soma and into the dendrites to promote cell survival and postsynaptic maturation, respectively. Much progress has been made in recent years to define the fate of the retrogradely trafficked TrkA signaling endosome, yet it has not been fully characterized. Here we investigate extracellular vesicles (EVs) as a novel route of neurotrophic signaling. Using the mouse superior cervical ganglion (SCG) as a model, we isolate EVs derived from sympathetic cultures and characterize them using immunoblot assays, nanoparticle tracking analysis, and cryo-electron microscopy. Furthermore, using a compartmentalized culture system, we find that TrkA derived from endosomes originating in the distal axon can be detected on EVs secreted from the somatodendritic domain. In addition, inhibition of classic TrkA downstream pathways, specifically in somatodendritic compartments, greatly decreases TrkA packaging into EVs. Our results suggest a novel trafficking route for TrkA: it can travel long distances to the cell body, be packaged into EVs, and be secreted. Secretion of TrkA via EVs appears to be regulated by its own downstream effector cascades, raising intriguing future questions about novel functionalities associated with TrkA+ EVs.

Dynamics and distribution of endosomes and lysosomes in dendrites.

All cells are filled with membrane-bound organelles which are responsible for the synthesis and transport as well as degradation of membrane proteins. The localization of these organelles inside cells is highly regulated. The regulation of organelle positioning has been widely studied in many cell types. In neurons, organelle positioning and its regulation is of particular interest because of the enormous size of neurons and the high spatial heterogeneity of different functional domains, such as axons, proximal and distal portions of dendrites, and synapses. We will discuss new discoveries with regard to the dynamic positioning of endosomes and lysosomes between soma and along dendrites. Just as the "how" of dynamic endosome/lysosome positioning is still being investigated, the "why" is also being explored. An exciting possibility is that synaptic activity influences organelle behaviors. We will discuss what is currently known about the how and the why of endosome/lysosome dynamics in dendrites.

Mice Harboring a Non-Functional CILK1/ICK Allele Fail to Model the Epileptic Phenotype in Patients Carrying Variant CILK1/ICK.

CILK1 (ciliogenesis associated kinase 1)/ICK (intestinal cell kinase) is a highly conserved protein kinase that regulates primary cilia structure and function. CILK1 mutations cause a wide spectrum of human diseases collectively called ciliopathies. While several CILK1 heterozygous variants have been recently linked to juvenile myoclonic epilepsy (JME), it remains unclear whether these mutations cause seizures. Herein, we investigated whether mice harboring either a heterozygous null Cilk1 (Cilk1+/-) mutation or a heterozygous loss-of-function Cilk1 mutation (Cilk1R272Q/+) have epilepsy. We first evaluated the spontaneous seizure phenotype of Cilk1+/- and Cilk1R272Q/+ mice relative to wildtype littermates. We observed no electrographic differences among the three mouse genotypes during prolonged recordings. We also evaluated electrographic and behavioral responses of mice recovering from isoflurane anesthesia, an approach recently used to measure seizure-like activity. Again, we observed no electrographic or behavioral differences in control versus Cilk1+/- and Cilk1R272Q/+ mice upon isoflurane recovery. These results indicate that mice bearing a non-functional copy of Cilk1 fail to produce electrographic patterns resembling those of JME patients with a variant CILK1 copy. Our findings argue against CILK1 haploinsufficiency being the mechanism that links CILK1 variants to JME.

Different Inhibitors of Aß42-Induced Toxicity Have Distinct Metal-Ion Dependency.

Oligomers of amyloid ß-protein (Aß) are thought to be the proximal toxic agents initiating the neuropathologic process in Alzheimer's disease (AD). Therefore, targeting the self-assembly and oligomerization of Aß has been an important strategy for designing AD therapeutics. In parallel, research into the metallobiology of AD has shown that Zn2+ can strongly modulate the aggregation of Aß in vitro and both promote and inhibit the neurotoxicity of Aß, depending on the experimental conditions. Thus, successful inhibitors of Aß self-assembly may have to inhibit the toxicity not only of Aß oligomers themselves but also of Aß-Zn2+ complexes. However, there has been relatively little research investigating the effects of Aß self-assembly and toxicity inhibitors in the presence of Zn2+. Our group has characterized previously a series of Aß42 C-terminal fragments (CTFs), some of which have been shown to inhibit Aß oligomerization and neurotoxicity. Here, we asked whether three CTFs shown to be potent inhibitors of Aß42 toxicity maintained their activity in the presence of Zn2+. Biophysical analysis showed that the CTFs had different effects on oligomer, ß-sheet, and fibril formation by Aß42-Zn2+ complexes. However, cell viability experiments in differentiated PC-12 cells incubated with Aß42-Zn2+ complexes in the absence or presence of these CTFs showed that the CTFs completely lost their inhibitory activity in the presence of Zn2+ even when applied at 10-fold excess relative to Aß42. In light of these results, we tested another inhibitor, the molecular tweezer CLR01, which coincidentally had been shown to have a high affinity for Zn2+, suggesting that it could disrupt both Aß42 oligomerization and Aß42-Zn2+ complexation. Indeed, we found that CLR01 effectively inhibited the toxicity of Aß42-Zn2+ complexes. Moreover, it did so at a lower concentration than needed for inhibiting the toxicity of Aß42 alone. In agreement with these results, CLR01 inhibited ß-sheet and fibril formation in Aß42-Zn2+ complexes. Our data suggest that, for the development of efficient therapeutic agents, inhibitors of Aß self-assembly and toxicity should be examined in the presence of relevant metal ions and that molecular tweezers may be particularly attractive candidates for therapy development.