VPS35 depletion does not impair presynaptic structure and function

Abstract

The endosomal system is proposed as a mediator of synaptic vesicle recycling, but the molecular recycling mechanism remains largely unknown. Retromer is a key protein complex which mediates endosomal recycling in eukaryotic cells, including neurons. Retromer is important for brain function and mutations in retromer genes are linked to neurodegenerative diseases. In this study, we aimed to determine the role of retromer in presynaptic structure and function. We assessed the role of retromer by knocking down VPS35, the core subunit of retromer, in primary hippocampal mouse neurons. VPS35 depletion led to retromer dysfunction, measured as a decrease in GluA1 at the plasma membrane, and bypassed morphological defects previously described in chronic retromer depletion models. We found that retromer is localized at the mammalian presynaptic terminal. However, VPS35 depletion did not alter the presynaptic ultrastructure, synaptic vesicle release or retrieval. Hence, we conclude that retromer is present in the presynaptic terminal but it is not essential for the synaptic vesicle cycle. Nonetheless, the presynaptic localization of VPS35 suggests that retromer-dependent endosome sorting could take place for other presynaptic cargo.

Retromer in Synaptic Function and Pathology

“…They occur in association with endosomes in the neuronal cell body and in processes (Wen et al, 2011 ; Bhalla et al, 2012 ; Mikhaylova et al, 2016 ). At synapses they are found in both the pre- and postsynaptic compartments (Jakobsson et al, 2011 ; Bhalla et al, 2012 ; Choy et al, 2014 ; Inoshita et al, 2017 ; Vazquez-Sanchez et al, 2018 ). Notably, in postsynaptic dendrites, retromer-bearing endosomes are associated with Golgi satellites, which mediate anterograde protein traffic from the ER as well as retrograde traffic from the plasma membrane (Mikhaylova et al, 2016 ).…”

“…These data thus corroborate the involvement of endosomes in synaptic vesicle recycling in Drosophila larvae, and provide the first evidence for a functional role of retromer in the synaptic vesicle cycle. In the second study, the effects of knocking down VPS35 in mouse hippocampal neurons was investigated (Vazquez-Sanchez et al, 2018 ). To circumvent developmental defects the knockdown was performed when synaptogenesis was essentially completed.…”

“…Notably, ultrastructural analysis did not reveal any difference in the number of synaptic vesicles between control and VPS35-depleted nerve terminals (Figure 1D ). Nor did the knockdown affect synaptic exo- or endocytosis, which were monitored with a pH-sensitive reporter (Vazquez-Sanchez et al, 2018 ).…”

“…(D) Knockdown of retromer in mouse hippocampal neurons does not alter synaptic vesicles in nerve terminals. Quantitative comparison showing that the number of synaptic vesicles is similar in nerve terminals from control neurons and from neurons treated with either of three different shRNAs for VPS35 (reproduced from Vazquez-Sanchez et al, 2018 with permission: http://creativecommons.org/licenses/by/4.0/ ).…”

Retromer subunit, VPS29, regulates synaptic transmission and is required for endolysosomal function in the aging brain

“…In future work, it will be important to further dissect retromer-dependent synaptic mechanisms, including in the mammalian context. Retromer components are present at synaptic terminals in mouse primary neuronal culture (Munsie et al, 2015;Vazquez-Sanchez et al, 2018), and in cortical synaptosome preparations (Tsika et al, 2014). However, functional studies to date have largely focused on the post-synaptic membrane, where evidence supports retromer requirements for trafficking of glutamatergic and adrenergic neurotransmitter receptors (Choy et al, 2014;Tian et al, 2015).…”