Activity-Dependent Control of Slow Synaptic Vesicle Endocytosis by Cyclin-Dependent Kinase 5

Abstract

The stimulated dephosphorylation of the dephosphin group of endocytic proteins by calcineurin and their subsequent rephosphorylation by cyclin-dependent kinase 5 (cdk5) is required for synaptic vesicle (SV) retrieval in central nerve terminals. However, the specific endocytic pathway(s) controlled by these enzymes is unknown. To address this issue, we combined functional and morphological assays of endocytosis in primary neuronal cultures with pharmacological and molecular ablation of calcineurin and cdk5 activity. During strong stimulation, inhibition of calcineurin or cdk5 blocked uptake of the activity-dependent membrane marker FM1-43, but not the more hydrophilic FM2-10. However, FM2-10 uptake-measured poststimulation was sensitive to cdk5 and calcineurin inhibition, indicating that a slow form of endocytosis persists after termination of stimulation. In parallel EM studies, inhibition of cdk5 during strong stimulation greatly reduced horseradish peroxidase labeling of plasma membrane-derived nerve terminal endosomes, but not SVs. Furthermore, during mild stimulation, FM1-43 uptake was unaffected by cdk5 inhibition and the SV membrane was exclusively retrieved via a single SV route, suggesting that recruitment of the endosomal route of membrane retrieval is activity dependent. Thus, we propose that the calcineurin/cdk5-dependent phosphorylation cycle of the dephosphins specifically controls a slow endocytic pathway that proceeds via endosomal intermediates and is activated by strong physiological stimulation in central nerve terminals.

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

“…FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals [3][4][5][6][7][8] . They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes.…”

“…As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation 1,2 . It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrindependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals [3][4][5][6][7][8] . They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes.…”

Cdk5 Modulates Cocaine Reward, Motivation, and Striatal Neuron Excitability

“…Cdk5 is activated after association with either of the homologous cofactors p35 (Lew et al, 1994;Tsai et al, 1994) or p39 (Cai et al, 1997). This kinase regulates numerous aspects of neuronal function, including cAMP and Ca 2ϩ signal transduction cascades (for review, see Bibb, 2003), presynaptic machinery (Tan et al, 2003;Tomizawa et al, 2003;Evans and Cousin, 2007), and synaptic plasticity (Fischer et al, 2005;Centonze et al, 2006;. In the striatum, Cdk5 controls dopamine neurotransmission through the regulation of the protein phosphatase-1 inhibitor, DARPP-32 (Bibb et al, 1999), as well as presynaptic components of dopamine synthesis and release (Chergui et al, 2004;Kansy et al, 2004;Moy and Tsai, 2004).…”

Bulk endocytosis at neuronal synapses

“…As mentioned above, dynamin I is only dephosphorylated by calcineurin during neuronal activity that triggers ADBE [40]. Furthermore, inhibition of the protein kinases responsible for the rephosphorylation of dynamin I after stimulation, cyclin-dependent kinase 5 [28] and glycogen synthase kinase 3 [44], specifically blocks ADBE but not CME. Another important consequence of dynamin dephosphorylation is its interaction with the synaptic protein, syndapin I [45,46].…”

“…Thus, the dephosphorylation function of calcineurin is linked to the activity dependency of ADBE. Indeed, pharmacological disruption of calcineurin function results in inhibition of bulk endocytosis in cultured neurons [28].…”