A cytoskeletal-based approach to understanding antidepressant-like mechanisms and depression-related behaviors Pubblico
Shapiro, Lauren (Spring 2018)
Abstract
Adolescence is a dynamic period of neurodevelopment in which neurons in the prefrontal cortex (PFC) are remodeled. This structural reorganization is conserved across species and is critical for the maturation to adulthood. Yet evidence suggests that it may also contribute to the onset of neuropsychiatric disease. This dissertation considers the impact of cytoskeletal regulatory factors that maintain neuronal structure on neurodevelopmental disorders. I begin with a discussion of proteins associated with the b1-integrin — Arg kinase — Rho-kinase 2 (ROCK2) signaling cascade, a pathway critical for regulating neuronal structure, including during adolescence. I compare proteins levels and dendritic spine densities in two subregions of the PFC, the medial prefrontal cortex (mPFC) and orbital prefrontal cortex (OFC), to illustrate that these regions mature at different rates. I subsequently test the hypothesis that expedited neuronal remodeling during adolescence has therapeutic-like effects. I find that inhibition of ROCK2 has antidepressant-like effects in adolescent mice, but not adult mice, and enhances dendritic spine elimination in the mPFC. These findings identify ROCK2 as a novel therapeutic target for the treatment of adolescent-onset depression. Next, I test whether interfering with proteins involved in neuronal remodeling during adolescence contributes to depressive-like behaviors. I report that reducing b1-integrin levels in the mPFC during adolescence, but not adulthood, interferes with motivational processes, mimicking a hallmark symptom of depressive disorders (amotivation). Finally, I focus on the relationship between cytoskeletal regulatory proteins and the primary stress hormone, corticosterone (CORT), given that stress is a primary predictive factor in depression. I demonstrate that elevated CORT decreases levels of Arg kinase and simplifies dendrite structure in the hippocampus. Conversely, pharmacologically stimulating Arg kinase rescues CORT-induced structural and behavioral deficits. These findings indicate that cytoskeletal regulatory proteins may underlie both behaviors and structural impairments that result from elevated CORT levels. The experimental results reported in this dissertation ultimately reinforce the idea that the b1-integrin — Arg kinase — ROCK2 signaling pathway is essential for normative adolescent development and interrupting this signaling cascade may contribute to adolescent-onset disease.
Table of Contents
Table of Contents
Chapter 1: A framework and context for the dissertation
1.1 Context for the dissertation
1.2 Structural changes that occur during adolescence
1.3 Development of hormonal systems
1.3.1 Gonadal systems.
1.3.2 Hypothalamic-Pituitary-Adrenal axis.
1.4 Synaptic plasticity
1.4.1 Long-term potentiation (LTP).
1.4.2 Long-term depression (LTD).
1.5 Depression
1.5.1 Adolescent-onset depression: an overview.
1.5.2 The monoamine theory of depression.
1.5.3 The development of selective serotonin reuptake inhibitors.
1.5.4 The neurotrophic theory of depression and antidepressant efficacy.
1.5.5 Ketamine.
1.6 A framework for the dissertation
Chapter 2: Differential expression of cytoskeletal regulatory factors in the adolescent prefrontal cortex: Implications for cortical development
2.1 Context, Author’s Contribution, and Acknowledgement of Reproduction
2.2 Abstract
2.3 General Introduction
2.3.1 Connections of the PFC
2.3.2 Brain-derived neurotrophic factor (BDNF)-TrkB and β1-integrin-mediated signaling influence neuronal morphology
2.4 Methods and results associated with original research findings
2.4.1 Subjects.
2.4.2 Dendritic spine analysis.
2.4.3 Immunoblotting.
2.4.4 Statistical analyses.
2.5 Results
2.5.1 Dendritic spines in the mPFC and OFC are eliminated during adolescence.
2.5.2 Regional and age-dependent patterns in synaptic, neurotrophic, and cytoskeletal regulatory factors during adolescence.
2.5.3 Casting a spotlight on early and mid-adolescence.
2.6 General Review
2.6.1 PFC dendritic spine density and synaptic marker levels change during adolescence
2.6.2 BDNF-TrkB signaling regulates cell structure in the postnatal brain
2.6.3 Does TrkB.T1 impact neuron structure?
2.6.4 β1-integrin-mediated cell adhesion systems regulate postnatal neural development
2.6.5 Abl2/Arg kinase and cortactin determine cell structure
2.6.6 p190RhoGAP-p120RasGAP complex – Rho interactions
2.6.7 ROCK2 and LIMK2: Key cytoskeletal regulatory elements in the postnatal brain
2.6.8 Limitations of our current studies
2.7 Conclusions
Figure 2.1. Subregions of the rodent PFC and dendritic spine pruning during adolescence.
Figure 2.2. Levels of several synaptic and cytoskeletal regulatory factors change during adolescence.
Figure 2.3. Regional differences in proteins associated with β1-integrin signaling during early adolescence.
Figure 2.4. Regional differences in synaptic marker and neurotrophic factor levels at P42.
Figure 2.5. Regional differences in synaptic markers and neurotrophic factors in the hippocampus during adolescence.
Table 2.1. Antibodies used in this study
Chapter 3: Rho-kinase inhibition has antidepressant-like efficacy and expedites dendritic spine pruning in adolescent mice
3.1 Context, Author’s Contribution and Acknowledgement of Reproduction
3.2 Abstract
3.3 Introduction
3.4 Methods
3.4.1 Subjects.
3.4.2 Drugs and timing of injections.
3.4.3 Behavioral testing.
3.4.4 Immunoblotting.
3.4.5 Dendritic spine analyses.
3.4.6 ROCK2 shRNA and stereotaxic surgery.
3.4.7 Histology.
3.4.8 Electrophysiology.
3.4.9 Statistical analyses.
3.5 Results
3.5.1 ROCK inhibition has rapid antidepressant-like effects in adolescent mice.
3.5.2 ROCK inhibition alters signaling factors associated with antidepressant efficacy.
3.5.3 ROCK inhibition has differential effects in adolescent and adult mice.
3.5.4 Acute ROCK inhibition prunes vmPFC dendritic spines in adolescence.
3.5.5 Fasudil does not alter PFC- and hippocampal-dependent learning and memory.
3.5.6 vmPFC-selective ROCK2 silencing has antidepressant-like effects.
3.6 Discussion
3.6.1 ROCK2 inhibition has rapid antidepressant-like effects in adolescent mice.
3.6.2 Fasudil modulates antidepressant-related proteins.
3.6.3 ROCK inhibition expedites dendritic spine pruning in the vmPFC during adolescence.
3.6.4 Selective ROCK2 inhibition in the vmPFC has therapeutic-like effects.
Figure 3.1. ROCK inhibition has antidepressant-like efficacy in adolescent mice.
Figure 3.2. Acute fasudil increases Akt, an antidepressant-associated protein, in the vmPFC of adolescent mice.
Figure 3.3. Fasudil has differential effects on adult and adolescent mice, enriching TrkB and PSD-95 in adolescents.
Figure 3.4. Fasudil expedites dendritic spine pruning in the adolescent vmPFC.
Figure 3.5. Fasudil does not impact performance on a PFC-dependent reversal task. A
Figure 3.6. ROCK2 inhibition selectively in the vmPFC has antidepressant-like effects.
Table 3.1. Antibodies used in this study.
Table 3.2. Time spent immobile (raw values) for adult and adolescent mice in the forced swim test.
3.6 Supplementary documents
Supplementary Figure 3.1. Fasudil has dose-dependent antidepressant-like effects in adolescent male and female mice without inducing sedation at antidepressant-like dosing
Supplementary Figure 3.2. Ketamine has variable behavioral effects in adolescent mice.
Supplementary Figure 3.3. Effects of SLx-2219 on locomotor activity
Supplementary Figure 3.4. Representative western blot images illustrate effects of fasudil, fluoxetine and ketamine on signaling factors.
Supplementary Figure 3.5. Fasudil does not have antidepressant-like efficacy in adult mice.
Supplementary Figure 3.6. The antidepressant-like effects of fasudil in the forced swim test (FST) are TrkB-independent.
Supplementary Table 3.1. Electrophysiological properties of ROCK2+/- pyramidal neurons in the vmPFC.
Chapter 4: Early-life b1-integrin is necessary for reward-related motivation
4.1 Context, Author’s Contribution, and Acknowledgement of Reproduction
4.2 Abstract
4.3 Introduction
4.4 Methods
4.4.1 Subjects.
4.4.2 Stereotaxic surgery.
4.4.3 Immunoblotting.
4.4.4 Behavioral testing
4.4.5 Histology.
4.4.6 Statistical analysis.
4.5 Results
4.5.1 b1-integrin in the mPFC is involved in reward-related motivation in adolescent mice.
4.5.2 b1-integrin in the mPFC does not obviously affect other depression- and anxiety-related behavior.
4.6 Discussion
4.6.1 Reducing b1-integrin in the mPFC during adolescence blunts reward-related motivation.
4.6.2 Our model: mPFC-selective Itgb1 knockdown during adolescence may induce depressive-like behavior by interfering with neuronal maturation.
4.6.3 Adolescent-onset Itgb1 knockdown spares other depressive- and anxiety-related behaviors.
4.7 Conclusion
Figure 4.1. Itgb1 knockdown reduces b1-integrin protein levels, which correlate with phosphorylation of the Arg substrate p190RhoGAP.
Figure 4.2. mPFC-selective Itgb1 knockdown reduces progressive ratio responding in adolescent but not adult mice.
Figure 4.3. mPFC-selective Itgb1 knockdown increases post-reinforcement pausing in adolescent mice.
Figure 4.4. Adolescent-onset Itgb1 knockdown does affect other depression- and anxiety-related behaviors.
Chapter 5: Corticosteroid-induced dendrite loss and behavioral deficiencies can be blocked by activation of Abl2/Arg kinase
5.1 Context, Author’s Contribution and Acknowledgment of Reproduction
5.2 Abstract
5.3 Introduction
5.4 Materials and Methods
5.4.1 Subjects.
5.4.2 CORT exposure.
5.4.3 Gland harvesting.
5.4.4 Biocytin injection of hippocampal neurons.
5.4.5 Morphometric analysis of dendrites.
5.4.6 Immunoblotting.
5.4.7 Immunoprecipitation.
5.4.8 DPH treatment in vivo.
5.4.9 Behavioral testing.
5.4.10 Statistics.
5.5 Results
5.5.1 CORT simplifies hippocampal CA1 dendrites
5.5.2 An Arg kinase activator induces p190RhoGAP phosphorylation
5.5.3 DPH recovers dendrite arbor structure
5.5.4 DPH confers behavioral resilience to CORT
5.6 Discussion
5.6.1 Stress-related structural reorganization of hippocampal neurons
5.6.2 The Arg stimulator DPH yields behavioral resistance to CORT
Figure 5.1. Basal hippocampal CA1 dendrites progressively regress with repeated CORT exposure.
Figure 5.2. CORT exposure regulates cytoskeletal regulatory elements in the hippocampus.
Figure 5.3. Bi-directional regulation of hippocampal p190RhoGAP phosphorylation by CORT and the Arg activator DPH.
Figure 5.4. DPH corrects CORT-induced deficiencies in dendrite arborization.
5.7 Supplementary documents
Supplementary Figure 5.1. Exogenous CORT exposure decreases adrenal and thymus gland weights.
Supplementary Table 5.1. Antibodies used in this report.
Supplementary Table 5.2. CORT modifies the levels and activities of cytoskeletal regulatory factors in CA1-rich vs. CA3-rich tissue samples.
Chapter 6: Summary and future directions
6.1 Chapter 1
6.2 Chapter 2
6.3 Chapter 3
6.4 Chapter 4
6.5 Chapter 5
6.6 Conclusion
Figure 6.1. The b1-integrin — Arg kinase — ROCK signaling cascade is involved in antidepressant-like efficacy and expression of depression-related behaviors.
Appendix: Additional publications
Works Cited
About this Dissertation
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