Nitric oxide induces pathological synapse loss by a protein kinase G-, Rho kinase-dependent mechanism preceded by myosin light chain phosphorylation

Abstract

The molecular signaling that underpins synapse loss in neuropathological conditions remains unknown. Concomitant upregulation of the neuronal nitric oxide (NO) synthase (nNOS) in neurodegenerative processes places NO at the center of attention. We found that de novo nNOS expression was sufficient to induce synapse loss from motoneurons at adult and neonatal stages. In brainstem slices obtained from neonatal animals, this effect required prolonged activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and RhoA/Rho kinase (ROCK) signaling. Synapse elimination involved paracrine/retrograde action of NO. Furthermore, before bouton detachment, NO increased synapse myosin light chain phosphorylation (p-MLC), which is known to trigger actomyosin contraction and neurite retraction. NO-induced MLC phosphorylation was dependent on cGMP/PKG-ROCK signaling. In adulthood, motor nerve injury induced NO/cGMP-dependent synaptic stripping, strongly affecting ROCK-expressing synapses, and increased the percentage of p-MLC-expressing inputs before synapse destabilization. We propose that this molecular cascade could trigger synapse loss underlying early cognitive/motor deficits in several neuropathological states.

Figure 1.

nNOS expression in adult motoneurons is sufficient to induce synapse loss. A, Top, Av injection into the tip of the tongue retrogradely transfected HMNs. Bottom, An illustrative example of retrogradely cotransduced HMN. B, One week after injection of Av-eGFP/Av-mRFP, a high degree of cotransfection was observed. C–F, Syn- (C, D) or VGAT- (•) and VGLUT2-ir (*) (E, F) puncta around eGFP-expressing HMNs 7 d after injection of the indicated Avs. Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of syn/VGAT and VGLUT2, respectively. G, Average number of the indicated vesicular transporters-ir (VT-ir) puncta per 100 μm of eGFP-identified HMN perimeter at the indicated conditions (Av-eGFP, n = 38 HMNs from 4 rats; Av-eGFP/Av-nNOS, n = 44 HMNs from 5 rats). *p < 0.005, unpaired two-tailed Student's t test. H, A transfected HMN processed by immunogold against eGFP. This motoneuron was first selected by epifluorescence (inset). I, J, Synaptic boutons attached to the plasma membrane of a HMN identified by deposits of gold (arrows), containing F/P (I) or S (J) vesicles. K, Average bouton frequency, characterized by the type of vesicles, attached to transfected motoneurons (Av-eGFP/Av-mRFP, n = 4 HMNs from 4 rats; Av-eGFP/Av-nNOS, n = 3 HMNs from 3 rats). *p < 0.05, nonparametric Mann–Whitney U test. Error bars indicate SEM. Scale bars: A, H, inset, 50 μm; B, 100 μm; C, D, E, F, 10 μm; H, 5 μm; I, J, 0.5 μm.

Figure 2.

The NO/cGMP/PKG pathway reduces syn-ir puncta and the evoked EPSP in neonatal motoneurons in a RhoA/ROCK-dependent way. A–D, Syn-ir puncta around FG-identified HMNs obtained from slices incubated for 6 h as indicated. Scale bars, 10 μm. Secondary antibody was labeled with Cy5 for immunolabeling of syn. E, Average number of Syn-ir puncta per 100 μm of FG-identified HMN perimeter at the indicated conditions (aCSF, n = 99 HMNs from 7 pups; DETA/NO, n = 100 HMNs from 7 pups; DETA/NO+ODQ, n = 100 HMNs from 7 pups; DETA/NO+PKG-i, n = 50 HMNs from 7 pups; Y27632, n = 50 HMNs from 4 pups; DETA/NO+Y27632, n = 50 HMNs from 6 pups; H1152, n = 30 HMNs from 3 pups; DETA/NO+H1152, n = 30 HMNs from 3 pups). *p < 0.0001, one-way ANOVA test relative to the aCSF condition. F, Recordings (Rec) of EPSPs evoked in HMNs by electrical stimulation (St.) of the VLRF were performed by whole-cell patch clamp. G, Representative EPSPs (10 traces overlapped) recorded in HMNs after the indicated treatments. Membrane potential was held at −64 mV in all recorded motoneurons. The arrowheads point to stimulus artifact. H, Average EPSP amplitude recorded in HMNs under the indicated treatments (aCSF, n = 12 HMNs from 5 pups; DETA/NO, n = 19 HMNs from 4 pups; DETA/NO+ODQ, n = 15 HMNs from 4 pups; DETA/NO+PKG-i, n = 20 HMNs from 7 pups; DETA/NO+Exo C3, n = 14 HMNs from 5 pups; DETA/NO+Y27632, n = 20 HMNs from 6 pups; DETA/NO+H1152, n = 14 HMNs from 4 pups). *p < 0.0001, one-way ANOVA test relative to the aCSF condition. Error bars indicate SEM.

Figure 3.

NO/cGMP/PKG induces p-MLC in synaptic puncta in a ROCK-dependent way before synapse loss. A, Immunoblots for p-MLC, MLC, and α-tub performed from microdissected HN after incubation of slices for the indicated times with aCSF or DETA/NO-supplemented aCSF. B, Time course of the p-MLC/MLC ratio in HNs incubated with DETA/NO versus aCSF (aCSF and DETA/NO, n = 3 Western blots from 6 animals per time point and condition). aCSF values was taken as 1. *p < 0.05, nonparametric Mann–Whitney U test. ^#p < 0.0001, unpaired two-tailed Student's t test. C, Average of the indicated ratios after DETA/NO treatment for 4 h compared with the control condition taken as 1. *p < 0.05, nonparametric Mann–Whitney U test. D–F, Syn- and p-MLC-ir puncta around FG-identified HMNs from slices incubated for 3 h under the indicated conditions. Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of syn and p-MLC, respectively. G, Average number of syn-ir puncta colocalizing with p-MLC-ir puncta (left y-axis) and syn-ir puncta per 100 μm of HMN perimeter (right y-axis) after receiving the indicated treatments (aCSF, n = 20 HMNs; DETA/NO, n = 25 HMNs; 8-Br-cGMP, n = 42 HMNs; H1152, n = 24 HMNs; DETA/NO+PKG-i, n = 23 HMNs; DETA/NO+H1152, n = 20 HMNs; 8-Br-cGMP+H1152, n = 21 HMNs). Experiments were performed in four pups per condition. H–J, ROCK- and p-MLC-ir puncta around FG-identified HMNs from slices incubated for 3 h under the indicated conditions. Details of the z-projection of a ROCK-ir puncta colocalizing with p-MLC (J). Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of ROCK and p-MLC, respectively. *p < 0.05, one-way ANOVA test relative to the aCSF condition. Error bars indicate SEM. Scale bars: D–J, 10 μm; J, high-magnification photomicrographs, 1 μm.

Figure 4.

NO induced synapse detachment in neonatal motoneurons. A, B, VGAT-ir (•) and VGLUT2-ir (*) puncta around FG-identified HMNs from slices incubated for 6 h under the indicated conditions. Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of VGAT and VGLUT2, respectively. C, Average number of VT-ir puncta per 100 μm of HMN perimeter after receiving the indicated treatments (aCSF, n = 50 HMNs from 5 pups; DETA/NO, n = 50 HMNs from 5 pups). *p < 0.0001, unpaired two-tailed Student's t test. D, E, Illustrative examples of synaptic boutons attached to the plasma membrane of a HMN with F/P (D) or S (E) vesicles. F, G, Segments of plasma membranes of HMNs obtained from slices receiving the indicated treatments for 6 h. The filled arrows point to synaptic boutons attached to the plasma membrane. In DETA/NO-treated HMNs, large segments of plasma membrane frequently were covered by glial-like processes (connected arrows). m, Mitochondrion. H, Average linear density of boutons, characterized by the type of vesicles, attached to motoneurons in slices receiving the indicated treatments (aCSF, n = 15 HMNs from 3 pups; DETA/NO, n = 15 HMNs from 3 pups). *p < 0.001, **p < 0.0001, unpaired two-tailed Student's t test. Error bars indicate SEM. Scale bars: A, B, 10 μm; D, E, 0.5 μm; F, G, 1 μm.

Figure 5.

Virally directed nNOS expression in neonatal HMNs reduces excitatory and inhibitory puncta by a paracrine/retrograde action of NO. A, B, VGAT- and VGLUT2-ir puncta around eGFP-transfected HMNs from animals at P6–P9 after injection of the indicated Avs into the tongue at P3. Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of VGAT and VGLUT2, respectively. C, Average number of VT-ir puncta per 100 μm of HMN perimeter in eGFP-identified motoneurons from animals receiving the indicated mixture of Avs (Av-eGFP, n = 28 HMNs from 3 pups; Av-eGFP/Av-nNOS, n = 33 HMNs from 3 pups). D–F, Syn-ir puncta around eGFP-transduced HMNs at P6–P9 in sections obtained from slices incubated for 6 h with aCSF (D), l-NAME (E), or C-PTIO (F) from animals receiving the injection of Av-eGFP/Av-nNOS at P3 in the tip of the tongue. All animals received daily a subcutaneous injection of l-NAME beginning on the day of Av administration. Secondary antibody was labeled with Cy5 for immunolabeling of syn. G, Average change in linear density of syn-ir puncta on eGFP-identified HMNs under the indicated treatments (aCSF, n = 14 HMNs from 3 pups; l-NAME, n = 11 HMNs from 5 pups; C-PTIO, n = 12 HMNs from 5 pups). In these experiments, the control condition was considered the same as in Figure 2E. H, Whole-cell patch-clamp recordings were performed from eGFP-identified HMNs. Inset, DIC-infrared photomicrograph of the same transfected HMN immediately before gigaseal formation with a glass pipette. I, Representative traces (10 overlapped) of evoked EPSPs recorded in HMNs under the indicated conditions. The arrowheads point to the stimulus artifact. J, Average mean amplitude of the EPSP recorded under the indicated treatments (aCSF, n = 8 HMNs from 3 pups; l-NAME, n = 10 HMNs from 4 pups; C-PTIO, n = 9 HMNs from 4 pups). In these experiments the control condition was considered the same as in Figure 2H. *p < 0.0001, one-way ANOVA relative to the FG-identified HMNs incubated in aCSF. Error bars indicate SEM. Scale bar, 10 μm.

Figure 6.

Axonal injury induces a NO/cGMP-dependent reduction of VGLUT2/ROCK-ir puncta apposed to adult motoneurons preceded by an increase in p-MLC. A–C, VGAT-ir (•) and VGLUT2-ir (*) puncta around FG-backlabeled HMNs obtained from intact animals (A) and 7 d after XIIth nerve crushing treated daily with the indicated drugs. Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of VGAT and VGLUT2, respectively. D, Average number of VT-ir puncta per 100 μm of HMN perimeter in FG-identified motoneurons 7 d after XIIth nerve crushing from animals receiving the indicated treatments (control/intact, n = 34 HMNs; d-NAME, n = 45 HMNs; l-NAME, n = 33 HMNs; 7-NI, n = 38 HMNs; ODQ, n = 30 HMNs; from 3 animals per experimental condition). *p < 0.0001, one-way ANOVA test relative to the control/intact group. E, Syn-ir and ROCKα- or ROCKβ-ir puncta adjacent to SMI32-immunolabeled HMNs in the intact and crushed sides 7 d after XIIth nerve crushing. Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of ROCK and VGLUT2, respectively. F, Average percentage of VGLUT2-ir puncta colocalizing with the indicated ROCK isoforms in the intact and crushed sides apposed to SMI32-identified HMNs 7 d after nerve injury (n = 30 HMNs per condition and ROCK isoform). *p < 0.01, **p < 0.001, unpaired two-tailed Student's t test. Error bars indicate SEM. G, H, Syn-ir colocalizing with p-MLC puncta apposed to SMI32-immunolabeled HMNs in intact and injured sides from animals 4 d after unilateral XIIth nerve crushing. Secondary antibodies were labeled with Cy5 and Cy3 for immunolabeling of syn and p-MLC, respectively. Scale bars, 10 μm.

Figure 7.

Proposed mechanism involved in pathological NO-induced synapse elimination. The emergence of new sources for the synthesis of pathological amounts of NO under neurodegenerative conditions is caused by upregulation of nNOS in sick neurons. Through a paracrine/retrograde signaling pathway, NO activates sGC in neighboring target presynaptic boutons. The sustained rise of cGMP within presynaptic terminals subsequently stimulates PKG, which either directly and/or indirectly through other mediators activates RhoA/ROCK signaling. Finally, ROCK can directly and/or indirectly phosphorylate MLC, thereby inducing actomyosin contraction underlying synaptic bouton retraction.