In the present study, we show that the microtubule plus-end binding protein CLASP2 is an important regulator of neuronal polarity and synaptic function. We found endogenous CLASP2 protein expression steadily increased during neuronal development and was present in both axonal and somato-dendritic compartments (). CLASP2 downregulation in primary neurons impairs axon elongation and dendritic branching which was rescued by human CLASP2. Conversely, overexpression of CLASP2 induced the growth of multiple axons, enhanced dendritic branching and ultimately to functional alterations in synaptic structure and function (–).
CLASP2 was initially identified as a binding partner of the cytoplasmic linker protein of 170 kDa (CLIP-170) in motile fibroblasts and as an important regulator in cell polarity by stabilizing microtubules at their plus ends (Akhmanova et al., 2001
; Galjart, 2005
). The asymmetric distribution of CLASP in regulating cellular polarity was shown to be mediated by PI3K signaling through GSK3β. The spatially activated PI3K signaling is conveyed downstream through localized inhibition of GSK3β activity that enhances CLASP2 binding to microtubules (Akhmanova et al., 2001
; Wittmann and Waterman-Storer, 2005
; Kumar et al., 2009
; Watanabe et al., 2009
; Al-Bassam et al., 2010
). Interestingly, these two spatially coupled kinases have been shown to control axon growth via
regulation of the +TIP adenomatous polyposis coli (APC) (Zhou et al., 2004
). Recent studies also show that CLASP2 is enriched in growth cones and supports axon regeneration by stabilizing the growing ends of axonal microtubules downstream of GSK3β (Hur et al., 2011
). In line with other +TIPs such as APC (Zhou et al., 2004
) and CLIPs (Neukirchen and Bradke, 2011
), our data show that overexpression of CLASP2 in primary hippocampal neurons promotes axon elongation and induces growth of multiple axons per neuron (). Also, we show that PI3K-driven axon elongation depends on microtubule dynamics of CLASP2 ().
In addition to increasing axon outgrowth during neuronal polarization, CLASP2 overexpression induced dendritic development. Overexpression of CLASP2 significantly increased dendritic length and the total number of MAP2-positive processes at 5 DIV with extensive dendrite outgrowth in both primary and secondary dendritic branching (). These results strongly suggest that CLASP2 plays an active role in regulating the initial stabilization of both axons and dendrites; however the cellular and molecular mechanisms underlying their specific targeting to the growing plus-ends of neurites remain elusive. The differential regulation of +TIP interactions with microtubule ends and the composition of the microtubule cytoskeleton can influence whether a process becomes an axon or a dendrite either as a result of specific motor-based loading or regional control of post-translational modifications (Akhmanova and Steinmetz, 2008
). For instance, axon-specific microtubules are uniformly oriented with plus-ends pointing outward that efficiently recruit kinesin-1 motor-driven transport, whereas the microtubule orientations of mixed polarity in dendrites facilitate dynein motor entry (Kapitein and Hoogenraad, 2011
). Axonal and dendritic microtubule arrays also differ in their patterns of post-translational modification. For example, acetylated and detyrosinated microtubules are enriched in axons and selectively recruit kinesin-1 motor proteins (Witte and Bradke, 2008
; Konishi and Setou, 2009
). Since CLASP2 is regulated by the PI3K-GSK3β signaling pathway, it is possible that phosphorylation is a molecular switch capable of inducing rapid changes in the microtubule dynamic between axons and dendrites.
In non-neuronal cells, there is a substantial Golgi-associated pool of CLASPs and previous results indicate that CLASP2 promotes microtubule nucleation from the trans-Golgi network leading to asymmetry of microtubule arrays in polarized cells (Efimov et al., 2007
). In addition, Golgi-derived CLASP-dependent microtubules are crucial for establishing continuity and proper morphology of the Golgi complex which is essential for cell polarization and motility (Miller et al., 2009
). Microtubules nucleated at the Golgi are preferentially oriented towards the leading edge and direct polarized trafficking in motile cells similar to the microtubule-dependent trafficking pathway during axon specification (Hoogenraad et al., 2001
; Witte et al., 2008
; Miller et al., 2009
). In neurons, the Golgi apparatus has also been implicated in neuronal polarity where the position of the Golgi and the adjoined centrosome correlates with newly emerging axons (Zmuda and Rivas, 1998
; de Anda et al., 2005
; de Anda et al., 2010
). Specialized Golgi outposts that populate exclusively along dendrites have been shown to regulate extension and retraction of dendritic branching (Horton et al., 2005
; Ye et al., 2007
; Hanus and Ehlers, 2008
). Here, we found that CLASP2 colocalizes with the Golgi marker GM130 in primary neurons and that increasing CLASP2 expression leads to increased Golgi condensation (). We also found CLASP2 overexpression led to increased neuritic branching only in neurons with stacked as opposed to ribbon Golgi morphology (), suggesting that CLASP2 mediates specific changes in the Golgi that stabilize the cytoskeleton to promote branching.
Microtubules act as the main cytoskeletal tracks for transport of materials to and from the synapse (Conde and Caceres, 2009
; Hoogenraad and Bradke, 2009
; Goellner and Aberle, 2012
). However, little is known about the role of the cytoskeletal machinery, specifically +TIPs on synapse formation and function in mammals. Our results indicate that CLASP2 has a functional role in spontaneous neurotransmission (). The shRNA knockdown of CLASP2 reduced miniature event frequency in excitatory synapses, while overexpression of CLASP2 caused an increase in both miniature event frequency and amplitude specifically in excitatory synapses, suggesting that CLASP2 regulates both presynaptic neurotransmitter release machinery and postsynaptic receptor trafficking. The current changes in spontaneous neurotransmission could be due to CLASP2 effects on axon and dendritic outgrowth, exclusively targeting excitatory neurons which have been shown to vary greatly in their somatic, dendritic and axonal morphologies compared to inhibitory neurons (Markram et al., 2004
). Interestingly, we found that CLASP2 induced ultrastructural changes in asymmetric excitatory synapses that support the presynaptic changes we observed electrophysiologically (). Quantitative electron microscopy revealed a selective increase in presynaptic terminal circumference that may be a consequence of increased vesicle fusion with the plasma membrane. In line with the ultrastructural data, the size and number of synapses as assessed by synapsin-PSD95 colocalization was significantly enhanced in neurons overexpressing CLASP2.
We surveyed several synaptic proteins to determine whether CLASP2 has selective effects on the composition of the synapse. We found that CLASP2 overexpression caused pronounced increases in pre- (synapsin, synaptobrevin, and SNAP25) but not post- (PSD95, GluR1) synaptic protein expression (). Recent studies demonstrate that while the presynaptic complex which gives rise to either action potential-evoked or spontaneous neurotransmitter release utilizes the same molecular machinery, they rely on distinct molecular interactions of the same components for normal function (Ramirez and Kavalali, 2011
). Structure-function analyses of neuronal SNARE proteins (syntaxin, synaptobrevin and SNAP25) revealed key differences between molecular interactions that give rise to spontaneous versus evoked fusion (Ramirez and Kavalali, 2011
). For example, loss of SNAP25 or synaptobrevin largely abolishes calcium-dependent evoked release but leaves spontaneous fusion release intact suggesting a role for alternate SNAREs in mediating spontaneous release (Schoch et al., 2001
; Washbourne et al., 2002
; Bronk et al., 2007
). The CLASP2-mediated increase in synapsin synaptobrevin, and SNAP25 may provide possible mechanistic detail of the functional increase in evoked mEPSCs as alterations in expression levels of these proteins have been shown to cause calcium-dependent evoked neurotransmitter release (Lu et al., 1992
; Rosahl et al., 1995
; Schoch et al., 2001
; Washbourne et al., 2002
; Bronk et al., 2007
At the postsynaptic membrane, we found that CLASP2 plays an important role in glutamate receptor trafficking, including an increase in surface levels of GluA1 in CLASP2 overexpressing neurons. This raises the possibility that CLASP2 functions as a factor controlling the delivery of synaptic components to synaptic terminals (). Previously, +TIPs have been shown to play important roles in positioning neurotransmitter receptors and ion channels. APC and end-binding protein1 (EB1) have been shown to be involved in assembly and stabilization of α3 nicotinic acetylcholine receptor at the postsynaptic side of cholinergic synapses by anchoring microtubules (Temburni et al., 2004
). EB1 was also shown to target voltage-gated potassium (Kv1) channels to axons (Gu et al., 2006
). In principal, localized remodeling of the cytoskeleton through +TIPs could achieve the spatial remodeling required for synapse-specific and activity-dependent synaptic plasticity. It is possible that binding of CLASP2 to microtubules or to other end-binding proteins is important for the phenotypes we observed, but further studies will be necessary to investigate this possibility.
In summary, our results suggest that the control of microtubule organization by CLASP2 may represent a mechanism for regulated growth cone motility and synaptic growth and plasticity. We find that increased CLASP2 expression leads to accelerated and enhanced neuron and synaptic development. Although the cellular and molecular mechanism remains to be determined, CLASP2 has a number of specific binding domains that allow for protein interactions at the Golgi, cytoskeleton and the growing tips of neurites. The enhanced neuronal development may therefore arise due to altered Golgi function, increased trafficking along microtubules or increased vesicle fusion due to stabilization of the cytoskeleton. The specific CLASP2 domains responsible for these functional changes will be important to address in the future.