Rho-kinase inhibition has antidepressant-like efficacy and expedites dendritic spine pruning in adolescent mice
Introduction
Depression is a major public health concern costing the United States $99 billion in medical expenses annually (Greenberg et al., 2015). According to the 2015 National Survey on Drug Use and Health, 12.5% of youth ages 12–17 had a major depressive episode within the past year, and only 45% of these individuals received treatment (Substance Abuse and Mental Health Services Administration, 2017). In 2004, the FDA issued a black box warning regarding the use of antidepressants for individuals under the age of 25 due to a heightened risk of suicidal ideation. Although some more recent studies have scrutinized this policy, physicians remain reticent to prescribe antidepressants to adolescents (Isacsson and Rich, 2014). As a result, there is a dire need for novel antidepressants suitable for adolescents and young adults.
During adolescence, the prefrontal cortex (PFC), a brain region that provides “top-down” control over emotion and behavior, undergoes dramatic structural reorganization and synaptic remodeling. Some dendritic spines and synapses are refined, whereas others are pruned (Bourgeois et al., 1994; Huttenlocher and Dabholkar, 1997; Rakic et al., 1994; Shapiro et al., 2017). Although the molecular mechanisms mediating these events are not fully understood, some research suggests that structural instability may contribute to vulnerability to neuropsychiatric diseases (Keshavan et al., 2014). Thus, expediting or otherwise enhancing the structural remodeling that occurs during adolescence may be therapeutic in the treatment of adolescent-onset depression.
An important factor in regulating dendritic spine structure, including during adolescence, is the RhoA GTPase substrate Rho-kinase (ROCK) (Koleske, 2013; Maekawa et al., 1999; Murakoshi et al., 2011; Schubert et al., 2006). There are two isoforms of ROCK: ROCK1 and ROCK2, the latter more highly expressed in brain tissue (Duffy et al., 2009; Julian and Olson, 2014; Nakagawa et al., 1996). ROCK2 inhibits cofilin-mediated actin cycling, the process by which the equilibrium between monomeric, globular-actin (G-actin) and polymeric, filamentous-actin (F-actin) alters the size and shape of dendritic spines (dos Remedios et al., 2003; Pontrello and Ethell, 2009). ROCK2 inhibits cofilin and can prevent changes in the morphology of the dendritic spine (Maekawa et al., 1999; Zhou et al., 2009). Pharmacological inhibition of ROCK2 can thus promote activity-dependent dendritic spine pruning or spine head enlargement (stabilization), depending on the extracellular milieu (Murakoshi et al., 2011; Schubert et al., 2006) and in other contexts, can enhance neurite outgrowth (discussed in (Harbom et al., 2018)). The ROCK inhibitor, fasudil, has a favorable pharmacological profile in humans: it is in clinical use, has oral bioavailability and minimal side effects, and readily crosses the blood brain barrier (Chen et al., 2013).
Here we report that the antidepressant-like potential of fasudil in adolescent mice is comparable to that of the commonly prescribed antidepressant fluoxetine (Prozac) and the novel antidepressant, subanaesthetic ketamine. We find that fasudil enhances the ratio of active/inactive TrkB and elevates Akt, signaling factors associated with antidepressant action. Fasudil also expedites dendritic spine pruning on excitatory neurons in the ventromedial prefrontal cortex (vmPFC). Further, shRNA-mediated silencing of the neuronal ROCK2 isoform selectively within the vmPFC also has antidepressant-like effects. Together, these findings suggest that inhibiting ROCK in the vmPFC enriches adolescent-typical dendritic spine pruning and has antidepressant-like potential. Importantly, systemic fasudil treatment was neither sedative, nor did it alter PFC-dependent learning and memory in adolescents, and it had virtually no antidepressant-like effects in adults. These findings identify ROCK inhibitors, including fasudil, as potential antidepressant agents well-suited to adolescent populations.
Section snippets
Results
The development of novel antidepressant agents suitable for adolescents is an urgent medical need. Here we characterize the effects of the ROCK inhibitor, fasudil, in tests of antidepressant-like efficacy designed for use in healthy rodents (as opposed to models of depression), and on PFC neurobiology in adolescent and adult mice.
Discussion
Adolescent-onset depression is an urgent medical concern due to increased resistance to typical antidepressants and a high risk of recurrence across the lifespan (DeFilippis and Wagner, 2014). Further, few viable treatment options exist. Based on evidence that mammalian PFC neurons undergo considerable structural maturation during adolescence, we investigated the antidepressant-like effects of pharmacological compounds that act on cytoskeletal regulatory, rather than classical neurotransmitter,
Methods
Subjects. Subjects were C57BL/6 mice (Jackson Labs). Mice used for dendritic spine analyses expressed thy1-derived YFP (H-line from: (Feng et al., 2000)) and were fully back-crossed onto a C57BL/6 background. Due to heightened risk of depression in women (Kuehner, 2017), female mice were used unless otherwise noted.
Mice referred to as “adolescent” were P41–42 (Spear, 2000), and adult mice were ~10–13 weeks old. Mice were maintained on a 12-h light cycle (0700 on) and provided food and water ad
Role of authors
Authors had access to the data in the study and take responsibility for the integrity of the data and accuracy of the data analyses. Study concept and design: LS and SG. Acquisition of data: LS, HK and JG. Analysis and interpretation of data: LS, HK, JG and SG. Manuscript composition: LS. Critical revision of the manuscript for important intellectual content: LS and SG. Statistical analyses: LS and SG. Obtained funding: LS, DR and SG. Administrative, technical, and material support: DR and SG.
Conflict of interest
None of the authors has any conflicts to disclose.
Acknowledgements
We thank Dr. Alonzo Whyte and Weibo Fu for their assistance in imaging and reconstructing dendritic spines. We thank A. Allen for assistance with the experiments and illustrations in Fig. 4, Elizabeth Hinton for her assistance with forced swim testing and Apoorva Gangavelli for her assistance with operant conditioning. The work in the SLG lab was supported by NIH (National Institutes of Health) 5T32GM008602-17, F31MH109208, R01MH101477 and R01MH117103. The work in the DGR lab was supported by
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