July 2019 Newsletter: Rho-family GTPases, Neuronal Plasticity, and Depression

Depression constitutes a spectra of symptoms that adversely affect an individual’s cognitive, emotional, motivational, and physiological well-being; this collection of heterogenous symptoms and pathologies is termed major depressive disorder (MDD), and for as many as 40% of individuals suffering from MDD, medications are not able to provide sufficient and/or long-lasting therapeutic relief^1,2. Functional imaging studies and neuropathological studies with post-mortem human brains implicate dysfunction in the nucleus accumbens (NAc) and ventral tegmental area (VTA), critical nuclei in the brain’s dopaminergic (DAergic) reward pathways^2-6. The VTA consists of a major population of DA neurons which primarily innervates the NAc^2,5. Dysfunctional neurophysiology and plasticity in these nuclei likely contribute to some of the symptoms of MDD, specifically loss of pleasure and motivation (anhedonia)^2-6. Anhedonia is studied using chronic social defeat stress (CSDS) which elicits depression-associated behaviors (e.g., reduced social interactions, increased anhedonia, negative body weight changes) in 70% of mice (i.e., termed stress-susceptible) while the remaining 30% do not undergo these adverse behavioral changes (i.e., termed stress-resilient)^2,7.

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The structure and function of DAergic neurons in VTA and the DA receptor 1 (D1R)- and D2R-expressing NAc neurons are remodeled during CSDS^2,8-16. Stress-induced anhedonia to model depression-like behavior results in reduced spontaneous excitatory inputs to D1R-expressing MSNs, whereas D2R-expressing MSNs undergo the opposite change^17,18. D1R-expressing (but not D2R) accumbal neurons also display reduced dendritic arborization (morphological plasticity), an important parameter in excitatory neurotransmission for the MSNs as dendritic spines are the primary site of excitatory synapses^{8,10-12,19,20}.

Figure 1. Dysfunctional plasticity in dopamine 1 receptor (D1R)-expressing medium spiny neurons (MSNs) in the nucleus accumbens (NAc) in response to chronic social defeat stress (CSDS). Following CSDS exposure, D1R-expressing MSNs in NAc display elevated levels of active RhoA, decreased excitatory inputs, hyperexcitability, dendritic atrophy (loss of complexity and mature spines, sometimes decreased density), increased social avoidance in animals containing these neurons, and decreased levels of active Rac1. The negative neurophysiological, behavioral, and structural spine changes are blocked or reversed by Rho and ROCK inhibitors (e.g., C3 transferase, rhosin, Y27632).

Stress-induced models of MDD involve changes (i.e., plasticity) in dendritic spine morphology, density, and/or synaptic function^2,12,21,22. Indeed, CSDS reduces the dendritic complexity and total dendritic length of D1R-expressing, but not D2R-expressing, MSNs in the NAc²³ (Fig. 1). Such changes in dendrite and spine morphologies require dynamic remodeling of the actin cytoskeleton. Rho-family GTPases (e.g., Rho, Rac, and Cdc42) regulate the morphogenesis and remodeling of actin-based neuronal structures such as spines^24-28. In general, Rho inhibits, whereas Rac/Cdc42 promote, spine morphogenesis and development^2,24,29,30. The above observations prompted the natural question: what role do Rho-family GTPases have in CSDS-induced neuronal plasticity in NAc neurons? In D1R-expressing MSNs, gene expression of RhoA, its primary downstream effector, Rho-kinase (ROCK), and the Rho GEF Arhgef1 significantly increased²³. Notably, no other GTPase’s gene profile changes. Intra-accumbal inhibition of RhoA with the C3 transferase enzyme prevents stress-induced social avoidance while intra-accumbal activation of RhoA enables a subthreshold stress treatment to produce social avoidance²³. Similarly, the small-molecule, selective RhoA inhibitor rhosin reverses chronic stress-induced increases in RhoA activity and hyperexcitability and reduces spontaneous excitatory inputs to D1R-expressing MSNs in NAc³¹ (Fig. 1). In addition, rhosin enhanced the density of dendritic spines (sites of excitatory neurotransmission)³¹. Prevention of RhoA over-activation confers resistance to stress-induced deficiencies in neuronal function and dendritic structure³¹ (Fig. 1). Concomitant with stress-induced social avoidance and altered electrophysiological activity in D1R-expressing MSNs is the loss of dendritic arborization, total dendritic length, and reduced number of branch points along the dendrites. Over-activation of RhoA following CSDS is also responsible for these deleterious changes in overall dendrite morphology²³. Furthermore, in CSDS-treated mice, similar restoration of social behavior and dendritic complexity to that of control mice is observed following systemic injection of the ROCK inhibitor, Y27632, for 7 days²³ (Fig. 1).

Contrary to a CSDS-mediated increase in RhoA expression and activity in the NAc, Rac1, another Rho-family GTPase, is down-regulated at the transcriptional and translational level specifically in the NAc after CSDS¹⁹. Of the Rho-family GTPases examined, only Rac1 displays stress-induced transcriptional regulation in CSDS-susceptible (but not resilient) mice that correlates with increased social avoidance behavior¹⁹ (Fig. 1). Rac1 transcript levels are also reduced in the NAc of post-mortem brains of non-medicated MDD patients¹⁹. The CSDS-mediated reduction in Rac1 transcript and protein levels is via epigenetic regulation with a decrease in permissive acetylation of the Rac1 promoter in susceptible, but not resilient, mice. Furthermore, susceptible mice also have enhanced methylation directly upstream of the promoter and resilient mice have decreased methylation^2,19. Intra-accumbal inhibition of class 1 HDACs reverses the Rac1 down-regulation and the associated social avoidance behavior. Similarly, in NAc from post-mortem brains of depressed human subjects, Rac1 gene expression is reduced¹⁹. Rac1 is responsible for the formation and maturation of spines (i.e., morphogenesis) on the MSNs of NAc via its regulation of cofilin-mediated remodeling of the actin cytoskeleton^2,24,28-30. Concomitant with down-regulation of Rac1 mRNA and protein, social avoidance, and anhedonia following CSDS is formation of immature stubby dendritic spines on accumbal MSNs that co-localize with cofilin¹⁹. Rescue of Rac1 protein levels is correlated with reversing social avoidance behavior and the development of such spines¹⁹.

Summary

Successful treatment of MDD is hampered by several factors, including treatment resistance, multi-symptoms and neurobiological systems involved, and a high rate of remission^1,2. It is unlikely that a one-size-fits-all-therapeutic-approach will yield success, thus necessitating the need to explore signaling cascades associated with all of the neurobiological systems involved in depression. To this end, Cytoskeleton offers purified cytoskeletal proteins, functional assays, signal transduction reagents, GTPase activation assays, antibodies, live cell imaging probes, and kits to quantify endogenous levels of post-translational modifications in cells and tissues.

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