We have used biochemical, electrophysiological, genetic and microscopy tools to elucidate the function of dynamin isoforms in synaptic transmission. Our studies demonstrate a major function for dynamin 3 presynaptically that overlaps and synergizes with that of dynamin 1. However, while absence of dynamin 3 worsens the phenotype produced by the loss of dynamin 1, both at the organismal and synaptic levels, nervous system development is not grossly affected by the lack of both isoforms. Neurons lacking both dynamins develop, differentiate and establish synapses in vitro. Most strikingly, nerve terminals can recycle synaptic vesicles in their absence implying that dynamin 2 alone and/or dynamin-independent mechanisms are sufficient to support basic synaptic function. These results collectively demonstrate that neither dynamin 1 nor 3 are essential for regenerating synaptic vesicles, but rather contribute to the efficiency of this process.
The overlapping function of dynamin 1 and 3 in nerve terminals is supported by their similar localizations and interactions and by the more striking structural and functional defects of the presynapse observed in dynamin 1, 3 DKO neurons relative to dynamin 1 KO neurons. Furthermore, the neonatal lethal phenotype of the DKO far exceeded the severity of the dynamin 1 single KO phenotype (Ferguson et al., 2007
) in spite of the lack of an obvious phenotype in dynamin 3 KO mice. While this genetic interaction could conceivably arise due to multiple mechanisms, our data suggests a synergistic function of dynamin 1 and 3 in synaptic vesicle endocytosis. This interpretation is further supported by the strong enrichment of dynamin 3 at presynaptic terminals of dynamin 1 KO neurons (Ferguson et al, 2007
). Unique functions of dynamin 1 and 3 relative to dynamin 2 likely exist, including differential interactions with other proteins and perhaps phosphorylation-based regulatory mechanisms, but these functions are not essential for the basic mechanism of synaptic vesicle endocytosis.
A selective enrichment of dynamin 3 in dendritic spines was reported previously (Gray et al., 2003
; Lu et al., 2007
). However, we have shown here that the signal produced by the dynamin 3 antibody used in those studies is not abolished in dynamin 3 KO neurons (). In an additional observation relating to evaluation and validation of anti-dynamin antibody specificity in our KO mice, we observed that the prominent presynaptic immunoreactivity recognized by the widely used Hudy 1 antibody (Warnock et al., 1995
), that is typically thought to reflect dynamin 1 (Takei et al., 1995
), was not abolished in dynamin 1 KO neurons (Fig. S1A and B
). Importantly, although our results demonstrate a major function of dynamin 3 presynaptically, they do not speak against a likely function for both dynamin 3 and dynamin 1 in clathrin mediated endocytosis that takes place in other neuronal sub-compartments including post-synaptically but which are beyond the scope of the present study.
While a dramatic accumulation of clathrin coated pits was observed at a subset of DKO synapses, these intermediates converted to synaptic vesicles in the majority of neurons within hours upon silencing of neuronal activity (). Furthermore, endocytic recovery from stimulation-evoked exocytosis was still observed, albeit at a slower rate, in DKO neurons (). Thus, the critical contributions of dynamin 1 and 3 to the recycling of synaptic vesicles do not seem to reflect a unique and specific function of these two dynamins. A simple interpretation of our results is that dynamin 2, which is expressed in neurons at a much lower concentration than the combined concentration of dynamin 1 and dynamin 3, yet at a concentration that is in the same range of that of dynamin 2 in non-neuronal cells (Ferguson et al., 2007
), can support a low rate of synaptic vesicle endocytosis in addition to house-keeping forms of clathrin mediated endocytosis. A contribution of dynamin 2 to synaptic vesicle recycling is supported by the partial ability of this isoform to rescue the dynamin 1 KO phenotype when it is over-expressed (Ferguson et al., 2007
The potential existence of a dynamin-independent pathway for synaptic vesicle reformation also warrants consideration and is supported by reports that a much limited form of synaptic transmission persists after manipulations expected to perturb dynamin function, such as microinjection of GTPγS or peptides (Xu et al, 2008
; Shupliakov et al, 1997
; Sundborger et al, 2011
). Furthermore, studies with dynasore, an inhibitor of dynamin GTPase activity, have demonstrated a complete block of the compensatory synaptic vesicle internalization that follows after triggered exocytosis (Newton et al., 2006
) but synaptic vesicle endocytosis still occurred under conditions of spontaneous release (Chung et al, 2010
). Dynasore only inhibits dynamin’s GTPase activity by ~80%, based on biochemical studies (Macia et al., 2006
; Chung et al, 2010
) thus sparing ~20% activity, an amount that is more than the percent of total dynamin acounted for by dynamin 2 in neurons (). The high efficacy of dynasore in some studies, in spite of its incomplete inhibition of dynamin, suggests a dominant-negative effect of dynasore-bound dynamin or an off-target effect of the drug.
Studies of dynamin 1 KO neurons revealed that the strong presynaptic endocytic phenotype was predominantly restricted to GABAergic synapses under conditions of spontaneous network activity (Hayashi et al., 2008
). This selective phenotype was proposed to arise from a higher rate of tonic activity at such synapses. However, in dynamin 1, 3 DKO neurons, clathrin coated pits accumulated robustly at both excitatory and inhibitory synapses. We suggest that the combined loss of dynamin 1 and 3 lowers the endocytic capacity to a point where it can no longer keep pace with the spontaneous network activity level even in excitatory neurons. We further suspect that an initial loss of inhibition, due to selective vulnerability of GABAergic interneurons, could disinhibit network activity within the DKO cultures resulting in excitatory neurons that drive themselves to the point of exhaustion. Importantly, there was evidence for a greater sensitivity of parvalbumin-positive GABAergic neurons, as indicated by a stronger and irreversible endocytic phenotype. This observation may reflect a more general vulnerability of this subpopulation of GABAergic interneurons as their high activity levels have been proposed to confer added sensitivity to another genetic perturbation (Garcia-Junco-Clemente et al., 2010
). Furthermore, a spontaneous dynamin 1 missense mutation in mice was recently reported that is permissive for development but which confers seizure susceptibility that could arise from greater sensitivity of GABAergic interneurons to endocytic perturbation (Boumil et al, 2010
Interestingly, in spite of the strong decrease in average EPSC amplitude, the frequency and amplitude of mEPSCs was not markedly affected in DKO cultures (). Perhaps, under conditions where efficiency of recycling is severely impaired, newly formed vesicles are rapidly made available for spontaneous release and even the very low levels of dynamin 2 or dynamin-independent mechanisms may be adequate to replenish vesicles consumed by the more modest rates of spontaneous release. These considerations fit with the previous report that spontaneous transmission was relatively spared following treatment of cultured neurons with dynasore (Chung et al, 2010
The morphology of the endocytic intermediates that accumulate in DKO nerve terminals provides new insight into the mechanisms acting upstream of dynamin in endocytosis and, more generally, in the cell biology of nerve terminals. Like in fibroblasts that lack dynamin (Ferguson et al., 2009
), the ability of clathrin coated pits to mature to a very advanced state with narrow necks argues against essential functions for dynamin earlier in the process. Coated pits of dynamin 1, 3 mutant nerve terminals, however, are quite different from those observed in fibroblasts with no dynamin: 1) They are considerably smaller and highly homogeneous in diameter, consistent with their being direct precursors of synaptic vesicles. Thus, factors other than neuron-specific dynamin isoforms or high dynamin abundance must impose this small curvature. 2) Their narrow necks, while constricted and elongated, are shorter than the narrow long stalks of arrested fibroblastic clathrin coated pits. Considering that actin polymerization was shown to be required for the formation of the long necks in non-neuronal cells (Ferguson et al., 2009
), it is of interest that clathrin mediated endocytosis of synaptic vesicles was reported not to be dependent on actin (Sankaranarayanan et al., 2003
). 3) Clathrin coated pits of DKO nerve terminals typically originate from deep invaginations of the plasma membrane that are often decorated by numerous, sometimes 100’s of such pits. Each such tubular invagination is connected to the outer plasma membrane by a narrow constriction similar to the neck of clathrin coated pits (Fig. S6G and H
). This structural arrangement allows for accommodation of the vast increase in plasma membrane area produced by massive synaptic vesicle exocytosis without expanding the outer surface of the presynaptic terminal.
The great abundance of clathrin coated pits emphasizes the importance of fission as a trigger to uncoating. This is consistent with the report that auxilin, a critical co-factor for clathrin uncoating (Yim et al., 2010
), is most strongly recruited to pits only after fission (Massol et al., 2006
). Accordingly, while immunoreactivity for a variety of major endocytic clathrin coat components analyzed (light chain of clathrin, AP-2, AP180, epsin) was clustered in DKO nerve terminals, auxilin, which is an abundant component of purified brain clathrin coated vesicles (Ahle and Ungewickell, 1990
), was not (). These findings fit with a model in which i) endocytic clathrin coated pits can assemble only at the PI(4,5)P2
rich plasma membrane, ii) fission is coupled to PI(4,5)P2
dephosphorylation, iii) modification of the lipid composition of the membrane triggers uncoating by promoting adaptor dissociation and auxilin/Hsc70 recruitment (Cremona et al, 1999; Massol et al, 2006
; Yim et al, 2010
; Guan et al., 2010
Overall, our findings reveal a remarkable plasticity of nerve terminals. In a subset of dynamin 1,3 DKO neurons, spontaneous network activity resulted in a nearly complete shift from the typical “secretion-ready state” (abundance of synaptic vesicles clustered at active zones) to an “endocytic state” in which the great majority, possibly all, synaptic vesicles were replaced by clathrin coated pits. This shift implies a cross-talk between the cytosol and the membranes because proteins peripherally associated with the cytosolic face of synaptic vesicles, such as synapsin and Rab3, must be replaced, in a reversible fashion, with proteins of the clathrin coats and other endocytic factors. The different lipid environment (phosphoinositides and possibly other phospholipids) that surrounds synaptic vesicle proteins as they cycle between vesicles and the plasma membrane likely plays a role in these changes (Di Paolo and De Camilli, 2006
). However, the enhancement of the phosphorylation state of synapsin at sites 2 and 3 when nerve terminals are in an endocytic state, in spite of a global decrease in activity-dependent parameters, suggests the additional occurrence of feed-back mechanisms between the progression of synaptic vesicle membranes along their exo-endocytic cycle and signaling pathways within nerve terminals. Elucidating the mechanisms underlying this link will be an interesting focus of future work.