INVESTIGATIONS ON THE ROLES OF CLASS 3 SEMAPHORINS IN THE ADULT HIPPOCAMPUS

Ph.DDOCTOR OF PHILOSOPH

Alternative Splicing of P/Q-Type Ca2+ Channels Shapes Presynaptic Plasticity

Alternative splicing of pre-mRNAs is prominent in the mammalian brain, where it is thought to expand proteome diversity. For example, alternative splicing of voltage-gated Ca2+ channel (VGCC) a1 subunits can generate thousands of isoforms with differential properties and expression patterns. However, the impact of this molecular diversity on brain function, particularly on synaptic transmission, which crucially depends on VGCCs, is unclear. Here, we investigate how two major splice isoforms of P/Q-type VGCCs (Cav2.1[EFa/b]) regulate presynaptic plasticity in hippocampal neurons. We find that the efficacy of P/Q-type VGCC isoforms in supporting synaptic transmission is markedly different, with Cav2.1[EFa] promoting synaptic depression and Cav2.1[EFb] synaptic facilitation. Following a reduction in network activity, hippocampal neurons upregulate selectively Cav2.1[EFa], the isoform exhibiting the higher synaptic efficacy, thus effectively supporting presynaptic homeostatic plasticity. Therefore, the balance between VGCC splice variants at the synapse is a key factor in controlling neurotransmitter release and presynaptic plasticity

Characterization of splice variations in the EF-hand of the Cav 2.1 P/Q - Type voltage-gated Calcium channel in the Central Nervous System

Master'sMASTER OF SCIENC

Single cell knockout of NRP1 in neural progenitors results in neurons with impaired dendritic formations is independent of vascular endothelial growth factor (VEGF)-binding of NRP1.

<p>(A) Graphs showing quantification of total dendritic length (A) and branch number (B) of newborn DGCs expressing GFP (CTR) or Cre-recombinase (CRE) at 14 DPI in NRP1 conditional knockout mice (NRP1 Floxed/Floxed) or mice expressing one single copy of Floxed-NRP1 and a knockin (KI) of an altered ligand binding site variant of Nrp1 (NRP1 Floxed/KI). (C) Representative tracings of dendritic morphology of adult-born neurons from the above indicated groups. (Scale bar = 20 µm).</p

Overexpression of constitutively active FAK rescues the effects of roscovitine inhibition of semaphorin-induced dendritic growth.

<p>(A) Representative images of primary hippocampal neurons transfected with constitutively active FAK (S732D). Neurons were treated with AP control (AP), 3A-AP (3A), 3F-AP (3F), AP with roscovitine pre-treatment (AP+R), 3A-AP or 3F-AP with roscovitine pre-treatment (3A+R). Scale bar = 20 µm. (B) Graphs showing total dendritic length of primary hippocampal neurons expressing S732D FAK treated as indicated above. 40–50 neurons from each group were used for analysis. Values represent mean ± s.e.m. (No significant difference by 2-way ANOVA).</p

Overexpression of Cdk5 or FAK rescued dendritic phenotypes of NRP-1 and -2 deficient neurons.

<p>(A) Representative images of adult-born neurons co-infected with retrovirus expressing shNRP1 and mCherry shNRP1 and mCherry-T2A-Cdk5 and shNRP1 and mCherry-T2A-FAK. Scale bar = 20 µm. Quantification of total dendritic length (B, D) and branch number (C, E) of newborn DGCs at 14 dpi. Each symbol represents data from a single DGC at 14 dpi. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post hoc test).</p

Knockdown of downstream mediators of semaphorin signaling, Cdk5 and FAK, induced defective dendritic morphologies of newborn DGCs in adult brain.

<p>(A) Representative images and tracings of dendrites of control, Cdk5 or FAK-shRNA expressing DGCs at 14 dpi (Scale bar, 20 µm). Graphs showing quantification of total dendritic length (B) and branch number (C) of newborn DGCs. Each symbol represents a single DGC at 14 dpi. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post-hoc test).</p

Expression of FAK phosphorylation-defective mutants in primary neurons impaired semaphorin-induced dendritic growth.

<p>(A) Representative images of primary hippocampal neurons transfected with vector, wild-type FAK (WT), Y397F FAK (Y397F), S732A FAK (S732A) and Y397F/S732A FAK (Y397F/S732A). Neurons were treated with AP control (AP), 3A-AP (3A) or 3F-AP (3F). Scale bar = 20 µm. (B) HEK293T cells transfected with the indicated constructs encoding FAK or its mutants validating the phosphorylation status of these constructs. (C, D and E) Graphs showing percentage dendritic growth of primary hippocampal neurons expressing FAK mutant constructs as indicated, treated with AP control (C), Sema3A (D) or 3F (E), with percentages normalized to Vector+AP. 30–100 neurons from each group were used for analysis. Values represent mean ± s.e.m. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post-hoc test).</p

NRP1 and NRP2 are expressed in adult neural progenitor cells and can be knocked down by NRP1 and NRP2 shRNA respectively.

<p>(A) Representative western blots showing that NRP1 and NRP2 are expressed in cortical and hippocampal primary neuronal culture and adult hippocampal neural progenitor cells (NPCs). HEK 293 cells were overexpressed with NRP1 (‘+’, upper panel) and NRP2 (‘+’, lower panel) and probed with anti-NRP1 and anti-NRP2 respectively. Untransfected HEK 293 cell lysate (‘−’, upper and lower panels). (B) A schematic diagram of retroviral constructs co-expressing GFP under EF1-α promoter and shRNA driven by the U6 promoter. (C) Retroviral constructs expressing different shRNAs were co-transfected with the indicated myc-tagged overexpression constructs into 293T cells. Western Blot analysis showed effective knockdown of wild-type NRP1 and NRP2 by shRNAs for NRP1 and NRP2. pcDNA3.1-nrp1-782 and pcDNA3.1-nrp1-2231 express NRP1 that harbor silent mutations rendering them are resistant to knockdown by pUEG-nrp1-782 and pUEG-nrp1-2231, respectively. pcDNA3.1-nrp2-238 and pcDNA3.1-nrp2-1076 are resistant to knockdown by pUEG-nrp2-238 and pUEG-nrp2-1076, respectively.</p

Roscovitine inhibition of Sema3A or 3F-induced dendritic growth was rescued by overexpression of wild-type FAK.

<p>(A) Representative images of primary hippocampal neurons transfected with vector or wild-type FAK (WT). Neurons were treated with AP control (AP), 3A-AP (3A), 3F-AP (3F), AP with roscovitine pre-treatment (AP+R), 3A-AP or 3F-AP with roscovitine pre-treatment (3A+R). Scale bar = 20 µm. (B) Representative blots showing phosphorylation of FAK in primary hippocampal neurons transfected with vector or WT-FAK treated with AP, Sema3A or Sema 3F together with vehicle (−R) or roscovitine (+R). (C, D and E) Graphs showing total dendritic length of primary hippocampal neurons expressing GFP (vector) or WT-FAK and treated with vehicle or roscovitine together with AP control (C), Sema3A (D) or 3F (E). 30–100 neurons from each group were used for analysis. Values represent mean ± s.e.m. (**P<0.01, 2-way ANOVA with Bonferroni’s post-hoc test).</p