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 relief1,2. 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 pathways2-6. The VTA consists of a major population of DA neurons which primarily innervates the NAc2,5. 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.


The structure and function of DAergic neurons in VTA and the DA receptor 1 (D1R)- and D2R-expressing NAc neurons are remodeled during CSDS2,8-16. 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 change17,18. 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 synapses8,10-12,19,20.

JulyIllustratonFourthDraft-100
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 function2,12,21,22. 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 spines24-28. In general, Rho inhibits, whereas Rac/Cdc42 promote, spine morphogenesis and development2,24,29,30. 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 methylation2,19. 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 cytoskeleton2,24,28-30. 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 remission1,2. 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.


References

1. Holtzheimer P.E. and Mayberg H.S. 2011. Stuck in a rut: rethinking depression and its treatment. Trends Neurosci34, 1-9.

2. Fox M.E. and Lobo M.K. 2019. The molecular and cellular mechanisms of depression: a focus on reward circuitry. Mol. Psychiatry. doi: 10.1038/s41380-019-0415-3.

3. Drevets W.C. 2001. Neuroimaging and neuropathological studies of depression: implications for the cognitive-emotional features of mood disorders. Curr. Opin. Neurobiol11, 240-249.

4. Liotti M. and Mayberg H.S. 2001. The role of functional neuroimaging in the neuropsychology of depression. J. Clin. Exp. Neuropsychol23, 121-136.

5. Nestler E.J. et al. 2002. Neurobiology of depression. Neuron34, 13-25.

6. Drysdale A.T. et al. 2017. Resting-state connectivity biomarkers define neurophysiological subtypes of depression. Nat. Med23, 28-38.

7. Czeh B. et al. 2016. Animal models of major depression and their clinical implications. Prog. Neuropsychopharmacol. Biol. Psychiatry64, 293-310.

8. Muhammad A. et al. 2012. Stress during development alters dendritic morphology in the nucleus accumbens and prefrontal cortex. Neuroscience216, 103-109.

9. Krishnan V. et al. 2007. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell131, 391-404.

10. Christoffel D.J. et al. 2011a. IkappaB kinase regulates social defeat stress-induced synaptic and behavioral plasticity. J. Neurosci31, 314-321.

11. Christoffel D.J. et al. 2011b. Structural and synaptic plasticity in stress-related disorders. Rev. Neurosci22, 535-549.

12. Christoffel D.J. et al. 2012. Effects of inhibitor of κB kinase activity in the nucleus accumbens on emotional behavior. Neuropsychopharmacology37, 2615-2623. 

13. Cao J.L. Et al. 2010. Mesolimbic dopamine neurons in the brain reward circuit mediate susceptibility to social defeat and antidepressant action. J. Neurosci30, 16453-16458.

14. Chaudhury D. et al. 2013. Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons. Nature493, 532-536.

15. Wook K.J. et al. 2016. Essential role of mesolimbic brain-derived neurotrophic factor in chronic social stress-induced depressive behaviors. Biol. Psychiatry80, 469-478.

16. Qu Y. et al. 2018. Regional differences in dendritic spine density confer resilience to chronic social defeat stress. Acta Neuropsychiatr. 30, 117-122.

17. Lim B.K. et al. 2012. Anhedonia requires MC4R-mediated synaptic adaptations in nucleus accumbens. Nature487, 183-189.

18. Francis T.C. et al. 2015. Nucleus accumbens medium spiny neuron subtypes mediate depression-related outcomes to social defeat stress. Biol. Psychiatry77, 212-222.

19. Golden S.A. et al. 2013. Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression. Nat. Med19, 337-344.

20. Francis T.C. et al. 2017. Molecular basis of dendritic atrophy and activity in stress susceptibility. Mol. Psychiatry22, 1512-1519.

21. Manji H.K. et al. 2003. Enhancing neuronal plasticity and cellular resilience to develop novel, improved therapeutics for difficult-to-treat depression. Biol. Psychiatry53, 707-742.

22. Vidal R. et al. 2011. New strategies in the development of antidepressants: towards the modulation of neuroplasticity pathways. Curr. Pharm. Des17, 521-533.

23. Fox M.E. et al. 2018. Dendritic remodeling of D1 neurons by RhoA/Rho-kinase mediates depression-like behavior. Mol. Psychiatry. DOI: 10.1038/s41380-018-0211-5.

24. Nakayama A.Y. et al. 2000. Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons. J. Neurosci20, 5329-5338.

25. Luo L. 2002. Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu. Rev. Cell Dev. Biol18, 601-635.

26. Newey S.E. et al. 2005. Rho GTPases, dendritic structure, and mental retardation. J. Neurobiol64, 58-74.

27. Kiraly D.D. et al. 2010. Synaptic plasticity, a symphony in GEF. ACS Chem. Neurosci1, 348-365.

28. Tolias K.F. et al. 2011. Control of synapse development and plasticity by Rho GTPase regulatory proteins. Prog. Neurobiol94, 133-148.

29. Kuhn T.B. et al. 2000. Regulating actin dynamics in neuronal growth cones by ADF/cofilin and rho family GTPases. J. Neurobiol44, 126-144.

30. Tashiro A. and Yuste R. 2004. Regulation of dendritic spine motility and stability by Rac1 and Rho kinase: evidence for two forms of spine motility. Mol. Cell Neurosci26, 429-440.

31. Francis T.C. et al. 2019. The selective RhoA inhibitor rhosin promotes stress resiliency through enhancing D1-medium spiny neuron plasticity and reducing hyperexcitability. Biol. Psychiatry85, 1001-1010


Related Products


G-LISA Kits


RhoA G-LISA Activation Assay (Luminescence format) (Cat. # BK121)

RhoA G-LISA Activation Assay Kit (Colorimetric format) (Cat. # BK124)

RhoA G-LISA Activation Assay Kit (Colorimetric format) (Cat. # BK124-S)

Rac1,2,3 G-LISA Activation Assay (Colorimetric format) (Cat. # BK125)

Rac1 G-LISA Activation Assay (Luminescence format) (Cat. # BK126)

Cdc42 G-LISA Activation Assay (Colorimetric format) (Cat. # BK127)

Cdc42 G-LISA Activation Assay (Colorimetric format) (Cat. # BK127-S)

Rac1 G-LISA Activation Assay Kit (Colorimetric Based) (Cat. # BK128)

Rac1 G-LISA Activation Assay Kit (Colorimetric Based) (Cat. # BK128-S)

RalA G-LISA Activation Assay Kit (Colorimetric Based) (Cat. # BK129)

Ras G-LISA Activation Assay Kit (Colorimetric Based) (Cat . # BK131)

Arf1 G-LISA Activation Assay Kit (Colorimetric Based) (Cat. # BK132)

Arf6 G-LISA Activation Assay Kit (Colorimetric Based) (Cat. # BK133)

RhoA / Rac1 / Cdc42 G-LISA Activation Assay Bundle 3 Kits (Cat. # BK135)

Total RhoA ELISA (Cat. # BK150)


Pull-Down Activation


Ras Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK008)

Ras Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK008-S)

RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) (Cat. # BK030)

Arf1 Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK032-S)

Arf6 Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK033-S)

Cdc42 Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK034)

Cdc42 Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK034-S)

Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK035)

Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK035-S)

RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK036)

RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) (Cat. # BK036-S)

RalA Activation Assay Biochem Kit (bead pull-down format) (Cat . # BK040)

GGA3-PBD Beads (Binds active Arf1 and Arf6 proteins) (Cat. # GGA07)

PAK-PBD beads (binds active Rac/Cdc42 proteins) (Cat. # PAK02)

Rhotekin-RBD beads (binds active Rho proteins) (Cat. # RT02)


Acti-Stain Phalloidins

Acti-stain 488 Phalloidin (Cat. # PHDG1)

Acti-stain 555 Phalloidin (Cat. # PHDH1)

Acti-stain 670 Phalloidin (Cat. # PHDN1)

Rhodamine Phalloidin (Cat. # PHDR1)


Live Cell Imaging Products

Spirochrome SiR-Actin Kit (Cat. # CY-SC001)

Spirochrome Cytoskeleton Kit (Cat. # CY-SC006)

Spirochrome SiR700-Actin Kit (Cat. # CY-SC013)