Function of Actin Regulatory Proteins in Dendritic Spine and Synapse Formation in Hippocampal Neurons

1. Function of Actin Regulatory Proteins in Dendritic Spine and Synapse Formation in Hippocampal Neurons Caroline Nebhan Dr. Donna Webb Lab BSCI 296 Spring 2009

2. Our lab is interested in synapse formation. Synapses are communicative junctures that form between the axon and dendrite of neurons. -Composed of a pre- and post-synaptic terminal -Post-synaptic terminal consists of actin rich dendritic spine - small extensions from the dendrite that form connections with axonic partnersOur lab is interested in synapse formation. Synapses are communicative junctures that form between the axon and dendrite of neurons. -Composed of a pre- and post-synaptic terminal -Post-synaptic terminal consists of actin rich dendritic spine - small extensions from the dendrite that form connections with axonic partners

3. Development of Dendritic Spines -Dendritic spines are small, actin-rich site that integrate excitatory information. -They constitute the post-synaptic half of excitatory synapses. -It is widely believed that the plasticity of dendritic spines in mature organisms contributes to learning and memory. - Changes in spine number and shape of spines are associated with neurological disorders, such as mental retardation, epilepsy, schizophrenia, and Alzheimer’s disease. -Again, spines are very actin rich -Rho family GTPases are critical regulators of actin-Dendritic spines are small, actin-rich site that integrate excitatory information. -They constitute the post-synaptic half of excitatory synapses. -It is widely believed that the plasticity of dendritic spines in mature organisms contributes to learning and memory. - Changes in spine number and shape of spines are associated with neurological disorders, such as mental retardation, epilepsy, schizophrenia, and Alzheimer’s disease. -Again, spines are very actin rich -Rho family GTPases are critical regulators of actin

4. -A critical group of actin regulators are Rho family GTPases. -Family members include Rho, Rac, and Cdc42 -Molecular switching mechanism Rac- also mediates spines and synaspe formation (previous)- local regualtion of rac activity, spine and synapse formation Rho- same, also plays a role-A critical group of actin regulators are Rho family GTPases. -Family members include Rho, Rac, and Cdc42 -Molecular switching mechanism Rac- also mediates spines and synaspe formation (previous)- local regualtion of rac activity, spine and synapse formation Rho- same, also plays a role

5. PH- Pleckstrin homolgy domain; GTPase Binding Domain; Poly Proline domain; two Verpolin and a cofilin domain; acidic domain. The acidic domain is the binding site of the Arp2/3 complex; the verprolin domain is the site of binding of globular actin. This is how globular actin and the Arp2/3 complex, which mediates actin branching, are brought into contact with each other. Cdc42 is an upstream activator of N-WASP We have demonstrated with Cdc42 RNAi that knockdown of Cdc42 results in a significant decrease in dendritic spines and synapses. Initiates formation new actin filaments a rare X-linked recessive disease characterized by eczema, thrombocytopenia (low platelet count), immune deficiency, and bloody diarrhea WAS generally becomes symptomatic in children. Due to its mode of inheritance, the overwhelming majority are male. It is characterized by bruising caused by thrombocytopenia (low platelet counts), small platelet size on blood film, eczema, recurrent infections, and a propensity for autoimmune disorders and malignancies (mainly lymphoma and leukemia). In Wiskott-Aldrich syndrome, the platelets are small and do not function properly. They are removed by the spleen, which leads to low platelet counts. Splenomegaly is not an uncommon finding. Also, patients develop a type of itchy rash called eczema. Autoimmune disorders are also found in patients with WAS.PH- Pleckstrin homolgy domain; GTPase Binding Domain; Poly Proline domain; two Verpolin and a cofilin domain; acidic domain. The acidic domain is the binding site of the Arp2/3 complex; the verprolin domain is the site of binding of globular actin. This is how globular actin and the Arp2/3 complex, which mediates actin branching, are brought into contact with each other. Cdc42 is an upstream activator of N-WASP We have demonstrated with Cdc42 RNAi that knockdown of Cdc42 results in a significant decrease in dendritic spines and synapses. Initiates formation new actin filaments a rare X-linked recessive disease characterized by eczema, thrombocytopenia (low platelet count), immune deficiency, and bloody diarrhea WAS generally becomes symptomatic in children. Due to its mode of inheritance, the overwhelming majority are male. It is characterized by bruising caused by thrombocytopenia (low platelet counts), small platelet size on blood film, eczema, recurrent infections, and a propensity for autoimmune disorders and malignancies (mainly lymphoma and leukemia). In Wiskott-Aldrich syndrome, the platelets are small and do not function properly. They are removed by the spleen, which leads to low platelet counts. Splenomegaly is not an uncommon finding. Also, patients develop a type of itchy rash called eczema. Autoimmune disorders are also found in patients with WAS.

6. Experimental Goals What is the role of N-WASP in dendritic spine and synapse formation? What effect do the different domains of N-WASP have in spine morphology? What upstream regulators and downstream effectors of N-WASP play a role in the development of dendritic spines and synapses Cdc42 Arp2/3 What is the role of VASP in dendritic spine and synapse formation? We want to explore the role of N-WASP in neurons by looking at its localization and function. We also want to understand how the different domains of N-WASP play a role in its function. We are also interested in the upstream regulators and downstream effectors of N-WASP Expresion correlates with synapse formation, expressed highly in brain We want to explore the role of N-WASP in neurons by looking at its localization and function. We also want to understand how the different domains of N-WASP play a role in its function. We are also interested in the upstream regulators and downstream effectors of N-WASP Expresion correlates with synapse formation, expressed highly in brain

7. N-WASP Localizes to Synapses In our lab, we harvest rat hippocampal neurons for use as our model system. A. N-WASP antibody SV2 stains synaptic vesicle protein 2, a pre-synaptic marker PSD-95 stains the post-synaptic density; a post-synaptic marker High-resolution overlays show that N-WASP localizes directly next to SV2 and directly with PSD-95. This suggests that N-WASP indeed localizes to the post-synaptic half of a synapse. B. We have transfected neurons with GFP-tagged N-WASP, then stain with SV2 and PSD-95, respectively. Here, we see similar localization of N-WASP to synapses as demonstrated nwith endogenous N-WASP. In our lab, we harvest rat hippocampal neurons for use as our model system. A. N-WASP antibody SV2 stains synaptic vesicle protein 2, a pre-synaptic marker PSD-95 stains the post-synaptic density; a post-synaptic marker High-resolution overlays show that N-WASP localizes directly next to SV2 and directly with PSD-95. This suggests that N-WASP indeed localizes to the post-synaptic half of a synapse. B. We have transfected neurons with GFP-tagged N-WASP, then stain with SV2 and PSD-95, respectively. Here, we see similar localization of N-WASP to synapses as demonstrated nwith endogenous N-WASP.

8. Inhibition of N-WASP by Wiskostatin Decreases the Number of Spines and Synapses To investigate the specific role of N-WASP, we treated neurons with an N-WASP specific inhibitor, Wiskostatin. We saw a dose dependent decrease in the number of dendritic spines and synapses. In these images, SV2 staining is shown in green, while we have stained for actin with phalloidin (a toxin that binds actin) in red. Because dendritic spines are very actin-rich, we can use phalloidin to visualize them. To investigate the specific role of N-WASP, we treated neurons with an N-WASP specific inhibitor, Wiskostatin. We saw a dose dependent decrease in the number of dendritic spines and synapses. In these images, SV2 staining is shown in green, while we have stained for actin with phalloidin (a toxin that binds actin) in red. Because dendritic spines are very actin-rich, we can use phalloidin to visualize them.

9. N-WASP Activity is Required for the Formation of Excitatory but not Inhibitory Synapses We noticed that the decrease in synapses was concentrated to the dendritic shafts, and did not see much change in synaspe number around the cell body. This hinted that N-WASP may only be affecting only excitatory synapses, which are more concentrated on the shaft and in dendritic spines. To explore this, we treated neurons with 5 um Wiskostatin, then stained two separate treatments One with GAD6, which selectively stains inhibitory synapses One with PSD-95, which selectively stains excitatory synapses Our results show no significant change in inhibitory synapse number after treatment with the N-WASP specific inhibitor, but we show a significant decrease in excitatory synapses after treatment with 5uM Wiskostatin. We noticed that the decrease in synapses was concentrated to the dendritic shafts, and did not see much change in synaspe number around the cell body. This hinted that N-WASP may only be affecting only excitatory synapses, which are more concentrated on the shaft and in dendritic spines. To explore this, we treated neurons with 5 um Wiskostatin, then stained two separate treatments One with GAD6, which selectively stains inhibitory synapses One with PSD-95, which selectively stains excitatory synapses Our results show no significant change in inhibitory synapse number after treatment with the N-WASP specific inhibitor, but we show a significant decrease in excitatory synapses after treatment with 5uM Wiskostatin.

10. Knockdown of N-WASP by RNAi We generated a RNAi construct to knock down expression of endogenous NWASP to further explore the role of NWASP in dendritic spine and synapse development. We tested the RNAi construct in non-neuronal cells before we transfected the neurons with this construct. This construct specifically knocked down NWASP and not a related protein WAVE1. We also tested a Scrambled RNAi construct to use as a control, which did not affect NWASP expression. We generated a RNAi construct to knock down expression of endogenous NWASP to further explore the role of NWASP in dendritic spine and synapse development. We tested the RNAi construct in non-neuronal cells before we transfected the neurons with this construct. This construct specifically knocked down NWASP and not a related protein WAVE1. We also tested a Scrambled RNAi construct to use as a control, which did not affect NWASP expression.

11. Knockdown of N-WASP Expression Affects Spines and Synapses When neurons were transfected with the NWASP-RNAi, a 2-3 fold decrease in spines and synapse density was observed, compared to control cells expressing the empty vector or the scrambled RNAi peptide. To confirm that the loss of spine and synapses was specific to loss of NWASP, rescue experiment was performed, in which we co-transfected bovine NWASP with rat-NWASP RNAi. The rat N-WASP RNAi target sequence does not significantly affect expression of bovine N-WASP due to several nucleotide mismatches. Bovine NWASP completely rescued rat RNAi mediated reduction of spines and synapses. These results suggested that NWASP plays a critical role in spine and synapse formation. When neurons were transfected with the NWASP-RNAi, a 2-3 fold decrease in spines and synapse density was observed, compared to control cells expressing the empty vector or the scrambled RNAi peptide. To confirm that the loss of spine and synapses was specific to loss of NWASP, rescue experiment was performed, in which we co-transfected bovine NWASP with rat-NWASP RNAi. The rat N-WASP RNAi target sequence does not significantly affect expression of bovine N-WASP due to several nucleotide mismatches. Bovine NWASP completely rescued rat RNAi mediated reduction of spines and synapses. These results suggested that NWASP plays a critical role in spine and synapse formation.

12. Next, we wanted to further understand N-WASP by exploring the functions of various domains. We used truncated forms of N-WASP, N-WASP deletion constructs, to do this. Here you see GFP transfected neurons; this panel shows neurons transfected with GFP-tagged N-WASP We used three different truncated forms of N-WASP. The deltaWH1 construct lacked the N-terminus of the protein, which includes the pleckstrin homology domain. The deltaWA construct lacked the c-terminus of the protein, which includes verprolin domain where monomeric actin binds, as well as the acidic domain, where the Arp2/3 complex binds. An interesting phenotype that we noticed in neurons transfected with deltaWA was a high number of dendritic protrusions, but a low number of dendritic spines. Again, the WA region interacts with the Arp2/3 complex. Next, we wanted to further understand N-WASP by exploring the functions of various domains. We used truncated forms of N-WASP, N-WASP deletion constructs, to do this. Here you see GFP transfected neurons; this panel shows neurons transfected with GFP-tagged N-WASP We used three different truncated forms of N-WASP. The deltaWH1 construct lacked the N-terminus of the protein, which includes the pleckstrin homology domain. The deltaWA construct lacked the c-terminus of the protein, which includes verprolin domain where monomeric actin binds, as well as the acidic domain, where the Arp2/3 complex binds. An interesting phenotype that we noticed in neurons transfected with deltaWA was a high number of dendritic protrusions, but a low number of dendritic spines. Again, the WA region interacts with the Arp2/3 complex.

13. The Arp2/3 Complex Creates Branched Actin Networks -Arp2/3 complex is responsible for creating branched actin networks. -Nucleation of new actin filaments at 70 degree angles from the mother filament -VCA region seems to be important, so we wanted to look at the Arp2/3 complex. -For this reason, we think it may play a role in spine head enlargement. -Arp2/3 complex is responsible for creating branched actin networks. -Nucleation of new actin filaments at 70 degree angles from the mother filament -VCA region seems to be important, so we wanted to look at the Arp2/3 complex. -For this reason, we think it may play a role in spine head enlargement.

14. Arp3 Localizes to Spines and Excitatory Synapses First, we used an antibody for endogenous Arp3 We also used GFP-tagged Arp3 to demonstrate that Arp3 localizes to spines and synapses - close apposition verses directly on top of each otherFirst, we used an antibody for endogenous Arp3 We also used GFP-tagged Arp3 to demonstrate that Arp3 localizes to spines and synapses - close apposition verses directly on top of each other

15. Knockdown of Arp3 Expression Using RNAi To explore this, we created an Arp3 RNAi that we transfected into neurons. Here is the Western blot, again in HEK cells. 81% Knockdown. To explore this, we created an Arp3 RNAi that we transfected into neurons. Here is the Western blot, again in HEK cells. 81% Knockdown.

16. Arp3 Knockdown Decreases the Number of Spines and Synapses We transfected the Arp3 RNAi into neurons and saw a significant decrease in spine and synapse number. No significant change was observed with the scrambled RNAi peptide. We transfected the Arp3 RNAi into neurons and saw a significant decrease in spine and synapse number. No significant change was observed with the scrambled RNAi peptide.

17. VASP VASP: Vasodilator-stimulated phosphoprotein Actin regulatory protein A role in the initial protrusion of dendritic spines? VASP is involved in actin bundling. For this reason, we think it may play a role in the a dendrite extending an initial protrusion. VASP is involved in actin bundling. For this reason, we think it may play a role in the a dendrite extending an initial protrusion.

19. VASP expression increases the number of spines and synapses

20. Knockdown of VASP Expression Affects Spines and Synapses

22. Conclusions N-WASP localizes to active, excitatory synapses. Inhibition of N-WASP by wiskostatin or knockdown by siRNA results in a decrease in excitatory synapses and dendritic spines. Elimination of the Arp 2/3 binding region of N-WASP decreases the number of spines and synapses. Knockdown of Arp3 by siRNA decreases the number of spines and synapses. VASP localizes to active synapses. Knockdown of VASP expression is rescued to restore wild type phenotype

23. Further understanding the role of VASP in spine and synapse formation Quantify spine head size Deletion Domains

25. FM4-64 is a styryl dye that is used to visualize active synapses - dye inserts hydrophobic tail into lipid bilayer - dye is easily washed out (because it cannot cross the lipid bilayer) - only membrane involved in active movement/being actively pinocytosed is protected from washing - uptake dependent on depolarization -K+ solution/K+-free solution Neurons expressing GFP-N-WASP were labeled with FM4-64 FM4-64 puncta represent functional, active synapses N-WASP puncta colocalize with active synapses 85% of FM4-64 labeled synapses also contain N-WASP, indicating that N-WASP is present in most active synapses FM4-64 is a styryl dye that is used to visualize active synapses - dye inserts hydrophobic tail into lipid bilayer - dye is easily washed out (because it cannot cross the lipid bilayer) - only membrane involved in active movement/being actively pinocytosed is protected from washing - uptake dependent on depolarization -K+ solution/K+-free solution Neurons expressing GFP-N-WASP were labeled with FM4-64 FM4-64 puncta represent functional, active synapses N-WASP puncta colocalize with active synapses 85% of FM4-64 labeled synapses also contain N-WASP, indicating that N-WASP is present in most active synapses

26. Interestingly, several recently identified gene mutations found in mental retardation patients are involved in Rho family signaling. The Rho family of small GTPases include Rho Rac and Cdc42. The three mutated genes encode oligophrenin1, a Rho GAP, alphaPIX, a GEF for Rac and Cdc42 and PAK3, an effector for Rac and Cdc42. Notice that both PIX and PAK are on our pathway that regulates spine and synapse formation. And more interestingly, many forms of mental retardation are characterized by dendritic spine abnormalities. And the most common forms of abnormalities are either long thin spines or decreased spine density. Interestingly, several recently identified gene mutations found in mental retardation patients are involved in Rho family signaling. The Rho family of small GTPases include Rho Rac and Cdc42. The three mutated genes encode oligophrenin1, a Rho GAP, alphaPIX, a GEF for Rac and Cdc42 and PAK3, an effector for Rac and Cdc42. Notice that both PIX and PAK are on our pathway that regulates spine and synapse formation. And more interestingly, many forms of mental retardation are characterized by dendritic spine abnormalities. And the most common forms of abnormalities are either long thin spines or decreased spine density.

27. We also wanted to explore the role of Cdc42 in regulating spine and synapse formation, as Cdc42 is a known activator of N-WASP We also wanted to explore the role of Cdc42 in regulating spine and synapse formation, as Cdc42 is a known activator of N-WASP

28. The effect of the Cdc42-RNAi was rescued with expression of the WA region of N-WASP Dominant negative Sequesters the GEF, with disallows the activation of Cdc42The effect of the Cdc42-RNAi was rescued with expression of the WA region of N-WASP Dominant negative Sequesters the GEF, with disallows the activation of Cdc42