Actin capping protein in dendritic spine development Open Access

Fan, Yanjie (2013)

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Dendritic spines are the tiny membrane protrusions from dendritic processes and they serve as the postsynaptic platform for most excitatory synapses in the mammalian brain. The development of dendritic spines is a key step in forming synaptic contacts and establishing the neuronal circuitry. Actin is a major structural component of dendritic spines and plays a crucial role in spine development. However, the detailed mechanisms that regulate the actin structure and dynamics in spines remain to be elucidated. Our study has identified that the actin capping protein (CP), a regulator of actin filament elongation, plays an essential role in spine development and synapse formation. We found that CP expression in hippocampus was elevated at and after the stage of extensive synapse formation. Moreover, CP was found to gradually localize to dendritic protrusions during development and become highly accumulated in mature dendritic spines. These observations suggest a potential role of CP in spine formation. Utilizing a loss-of function approach, we found CP knockdown in cultured hippocampal neurons resulted in a marked decline in spine density and a concomitant increase of filopodia-like thin dendritic protrusions. The spines which were able to form in CP knockdown cells exhibited an altered morphology, highlighted by multiple thin filopodia-like protrusions emerging from the spine head. Functionally, CP knockdown reduced the number of synapses as evidenced by a reduction in the density of paired pre- and postsynaptic markers. Electrophysiological studies also showed that the frequency of miniature excitatory postsynaptic currents was markedly reduced in CP knockdown neurons. These findings indicate that CP is indispensible for the spine morphogenesis and synaptic formation- likely involved in the conversion of dendritic filopodia to spines. Further experiments suggest that CP knockdown resulted in the instability of dendritic filopodia, as well as a reduction in the clustering of postsynaptic scaffolding proteins in dendritic protrusions. Based on these results, we propose a model in which CP gradually accumulates in dendritic protrusions during synaptic development to stabilize them and facilitate the recruitment of postsynaptic scaffolding proteins. This process enables the morphological differentiation and postsynaptic receptor clustering that are needed for the formation of functional synapses.

Table of Contents

Chapter 1 Introduction 1

1.1 Synapses 2

1.2 Postsynaptic compartment - dendritic spines 6

1.2.1 Size and shape of dendritic spines 6

1.2.2 Postsynaptic receptors 7

1.2.3 Postsynaptic density of dendritic spines 9

1.2.4 Organelles in the dendritic spines 13

1.2.5 Morphology and function correlation of dendritic spines 15

1.3 Cytoskeleton in dendritic spines 19

1.3.1 Actin cytoskeleton in dendritic spines 19

1.3.2 Regulators of actin cytoskeleton in dendritic spines 23

1.3.3 Actin cytoskeleton in synaptic plasticity 26

1.3.3 Microtubule in dendritic spines 29

1.4 Capping protein and its regulation 33

1.4.1 Structure, isoforms and localization of capping protein 33

1.4.2 Regulation of the CP-actin binding 35

1.4.3 CP in cellular processes 38

1.4.4 CP function in the nervous system 39

1.5 Mechanisms of dendritic spine development 41

1.5.1 Three models of dendritic spine formation 41

1.5.2 Extrinsic and intrinsic factors regulating dendritic spine formation 44

1.5.3 Regulation of actin cytoskeleton during dendritic spine formation 46

Chapter 2. Capping protein in the formation of dendritic spines 58

2.1 Summary 59

2.2 Introduction 60

2.3 Results 62

2.3.1 CP expression increases during hippocampal development 62

2.3.2 CP gradually accumulates in dendritic spines during development 63

2.3.3 CP knockdown impairs spine morphogenesis 64

2.3.4 Abnormal spine morphology in CP knockdown is largely actin-dependent 66

2.3.5 CP knockdown affects pre- and postsynaptic specialization and synaptic transmission 67

2.3.6 CP is involved in the conversion of filopodia to dendritic spines 68

2.3.7 Dendritic protrusions during the filopodia-spine conversion are unstable after CP knockdown 69

2.3.8 Dendritic protrusions during the filopodia-spine conversion have reduced PSD clustering after CP knockdown 70

2.4 Discussion 71

2.4.1 Abnormal spine morphology after CP knockdown 71

2.4.2 Alteration of synaptic specialization and efficacy after CP knockdown 72

2.4.3 CP in the conversion of filopodia to dendritic spines 74

2.4.4 CP and actin cytoskeleton in dendritic spine formation 77

2.4.5 CP and microtubules in dendritic spines 81

2.4.6 Possible regulators of CP in dendritic protrusions 82

2.4.7 CP and neurological disorders 83

2.5 Materials and Methods 84

2.6 Figures and Legends 88

Chapter 3 Conclusion and Future Directions 100

3.1 Conclusion 101

3.2 Future directions 103

3.3 Figures and Legends 107

References 108

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