The Role of the Myb-related Transcription Factor Adf-1 in Dendritic Plasticity Open Access

Timmerman, Christina Kimberly (2013)

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Across multiple animal models, there is a much-appreciated role of protein-synthesis in long-term memory formation. This protein-synthesis dependent form of neuronal plasticity requires the coordinated actions of transcription of new gene products and also local translation at relevant synaptic sites. Although, it is unsurprising that mutations in any of these processes affect long-term behavioral adaptations such as memory formation. We investigate a Drosophila long-term memory mutant (nalyot)

and its role in neuronal plasticity. The nalyot mutant is a hypomorphic allele of the Myb-related transcription factor Adf-1. Previous studies have shown that these mutants exhibit mild presynaptic defects. Since a variety of transcription factors are known to influence both pre- and post- synaptic plasticity, we investigated if Adf-1 was involved in post-synaptic (dendritic) plasticity.Dendrites represent sites of signal integration and as such its morphology is intimately associated with how information is processed in any given neuronal circuit. In order to observe Adf-1's role in dendritic morphology in vivo, we turned to a system previously described by our laboratory that reproducibly labels a subset of motor neurons (RP2) in the Drosophila larval ventral nerve cord. Following 3D reconstructions of these RP2 neurons, we find that perturbations in Adf-1 expression result in dramatic loss of dendritic complexity and also lead to severe defects in motor behavior. Furthermore, we find that Adf-1 exerts these effects downstream of neuronal activity and CaMKII signaling. Finally, in order to identify neuronal targets of Adf-1 transcription, we performed ChIP-sequencing from larval brain tissue. We discover that Adf-1 transcriptionally regulates the neural cell-adhesion molecule FasII (NCAM homolog) and the local translation component Staufen in the process of dendritic plasticity.

Table of Contents

Table Of Contents

I. Chapter 1: Introduction to Memory Formation And Processes of Synaptic Plasticity

1. The Identification of Drosophila memory mutants pg. 2-4

a. Phases of Memory Formation pg. 3

b. Mushroom Body in Learning & Memory pg. 4

2. An in vivo system to investigate synaptic plasticity pg. 4-6

3. Drosophila memory mutant: nalyot pg. 6-8

a. Adf-1 Protein Structure pg. 7

b. Adf-1 in Plasticity pg. 7-8

4. CaMKII pg. 8-11

a. CaMKII's Role in Dendrites pg. 9-10

b. CaMKII in Drosophila learning and memory: pg. 10-11

5. Cell Adhesion Molecules in Memory pg. 12-15

a. FasII/ NCAM in memory pg. 13-14

b. Integrins pg. 14-15

6. Local translation pg. 15-19

a. Staufen pg. 17-19

7. Purpose pg. 19

8. Figure and Figure Legends pg. 20-23

a. Figure 1: Distinct Phases of Learning & Memory pg. 20- 21

b. Figure 2: Drosophila Adult Brain Structure pg. 22-23

c. Figure 3: Adf-1 Protein and Gene Structure pg. 24-25

II. Chapter 2: The Drosophila transcription factor Adf-1 (nalyot) regulates dendrite growth by controlling FasII and Staufen expression downstream of CaMKII and neural activity

pg. 26-74

1. Introduction pg. 27-30

2. Results pg. 31

a. Adf-1 is expressed in motor neurons in the Drosophila

larval nerve cord pg. 31-32

b. Inhibition of Adf-1 in the RP2 motor neuron leads to

severely reduced dendrite growth and excitability pg. 32-34

c. Adf-1 controls dendrite growth downstream of

CaMKII signaling pg. 35-36

d. Adf-1 regulates activity-dependent developmental

plasticity of dendrites in RP2 motor neurons pg. 37-38

e. Adf-1 inhibition in motor neurons impairs

sensory-motor transmission pg. 38-39

f. Developmental perturbation of Adf-1 in motor

neurons alters locomotor behavior pg. 39-41

g. Potential neuronal targets of Adf-1 identified

through genome-wide ChIP-Seq analysis pg. 42-43

h. Adf-1 controls FasII and Staufen expression to

regulate dendrite growth pg. 43-45

3. Figures and Figure Legends

a. Figure 1: Adf-1 is expressed in larval motor neurons pg. 46-47

b. Figure 2: Normal dendrite growth requires Adf-1 pg. 48-49

c. Figure 3: Adf-1 functions downstream to CaMKII

signaling and neural activity to regulate dendrite

growth in RP2 motor neurons pg. 50-51

d. Figure 4: Adf-1-mediated dendrite phenotypes

affect sensori-motor transmission in larvae pg. 52-53

e. Figure 5: Behavioral consequences of Adf-1 inhibition

in motor neurons pg. 54-55

f. Figure 6: Adf-1 transcriptional targets in the brain

identified by ChIP-Seq analysis pg. 56-57

g. Figure 7: Adf-1 regulates RP2 dendrite growth by

controlling FasII and Staufen expression pg. 58-59

h. Figure 8: Regulation of dendrite plasticity in

Drosophila by the transcription factor Adf-1 pg. 60-61

i. Figure S1 Adf-1 staining in the adult brain, expression

of an Adf-1 enhancer trap line in the larval brain and schematic of transposon insertions in the Adf-1

gene pg. 62-63

j. Figure S2: Adf-1 inhibition reduced dendrite growth in

RP2 motor neurons pg. 64-65

k. Figure S3: Adf-1 functions downstream of CaMKII

signaling and neural activity to control dendrite

growth pg. 66-67

l. Figure S3: Motor phenotypes resulting from Adf-1

inhibition are predominantly developmental in

origin pg. 68-69

m. Figure S4: Analysis of Adf-1 binding sites from brain

and Kc cell ChIP-Seq experiments pg. 70-71

n. Figure S 5:Adf-1 controls FasII and Staufen expression

to regulate dendrite growth pg.72-73

III. Chapter 3: Materials and Methods

1. Drosophila stocks, rearing and genetics pg. 75

2. Antibody generation, immunohistochemistry and western

blotting pg. 76-77

3. Microscopy and 3D reconstructions pg. 78

4. Electrophysiology pg. 75- 76

5. Behavior pg. 77

6. ChIP-Seq analysis & determination of molecular networks pg. 78-79

7. RU-486 Preparation and Feeding pg. 79

8. RNA extraction pg. 79

9. cDNA synthesis/ qRT-PCR pg. 80

IV. Chapter 4: Concluding Remarks and Future Directions pg. 82-90

1. Discussion pg. 82-87

2. Adf-1's Role in the Mushroom Body pg. 87-88

3. Additional Targets of Adf-1 pg. 88

4. Ethanol Sensitivity & Memory Formation pg. 89

5. Future work on Adf-1 phosphorylation by CaMKII pg. 89-90

6. Summary pg. 90

7. Figures and Figure Legends pg. 91-94

a. Figure 1: Perturbation of Adf-1 expression in the

Mushroom Body pg. 91-92

b. Figure 2: Adf-1's role in Actin5c transcription pg. 93-94

V. Appendix

1. Behavioral and electrophysiological outcomes of tissue-specific Smn knockdown in Drosophila melanogaster pg. 95-131

a. Abstract pg. 96-97

b. Introduction pg. 98-100

c. Results pg. 101-111

1. Smn perturbation in neurons or muscle

causes behavioral deficits pg. 101- 103

2. Knockdown of Smn in glutamatergic

neurons, but not cholinergic neurons

leads to behavioral deficits pg. 103-104

3. Neuronal perturbation of Smn does not

alter baseline and high-frequency synaptic

transmission pg. 105-107

4. Smn perturbation in muscle affects quantal

size pg. 108-110

5. Pre-synaptic knockdown of Smn abolishes

long-term homeostatic compensation at the

NMJ pg. 110-112

d. Discussion pg. 113-118

2. Materials and Methods pg. 119-121

3. Figures and Figure Legends pg. 122-133

a. Figure 1 pg. 122-123

b. Figure 2 pg. 124-125

c. Figure 3 pg. 126-127

d. Figure 4 pg. 128-129

e. Figure 5 pg. 130-131

f. Figure 6 pg. 132- 133

VI. References: pg. 134-170

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