Insulin signalling promotes dendrite and synapse regeneration and restores circuit function after axonal injury

Abstract

Dendrite pathology and synapse disassembly are critical features of chronic neurodegenerative diseases. In spite of this, the capacity of injured neurons to regenerate dendrites has been largely ignored. Here, we show that, upon axonal injury, retinal ganglion cells undergo rapid dendritic retraction and massive synapse loss that preceded neuronal death. Human recombinant insulin, administered as eye drops or systemically after dendritic arbour shrinkage and prior to cell loss, promoted robust regeneration of dendrites and successful reconnection with presynaptic targets. Insulin-mediated regeneration of excitatory postsynaptic sites on retinal ganglion cell dendritic processes increased neuronal survival and rescued light-triggered retinal responses. Further, we show that axotomy-induced dendrite retraction triggered substantial loss of the mammalian target of rapamycin (mTOR) activity exclusively in retinal ganglion cells, and that insulin fully reversed this response. Targeted loss-of-function experiments revealed that insulin-dependent activation of mTOR complex 1 (mTORC1) is required for new dendritic branching to restore arbour complexity, while complex 2 (mTORC2) drives dendritic process extension thus re-establishing field area. Our findings demonstrate that neurons in the mammalian central nervous system have the intrinsic capacity to regenerate dendrites and synapses after injury, and provide a strong rationale for the use of insulin and/or its analogues as pro-regenerative therapeutics for intractable neurodegenerative diseases including glaucoma.

Figure 1

Insulin promotes dendritic regeneration in adult RGCs after axonal injury. (A–C) RGCs co-expressing YFP and SMI-32 (NF-H) with a clearly identifiable axon (arrowhead) were selected for dendritic arbour imaging and 3D reconstruction. (D and E) Three days after axotomy, RGCs had visibly smaller and simpler dendritic arbours relative to intact, non-injured neurons. (F) Human recombinant insulin or vehicle (PBS) were administered daily for four consecutive days by intraperitoneal injection (i.p.) or topically (eye drops) starting at 3 days post-axotomy, a time when there is already substantial dendrite retraction. RGC dendritic arbour analysis was carried out 7 days after injury. (G–J) Representative examples of dendritic arbours from axotomized retinas treated with vehicle or insulin, following the regimen described in (F), visualized at 7 days post-lesion (4 days of insulin treatment). (K–N) Quantitative analysis of dendritic parameters revealed that insulin-treated neurons had longer dendrites and markedly larger and more complex arbours than vehicle-treated controls (insulin i.p.: red, insulin eye drops: pink, PBS: dark grey). Values are expressed as the mean ± SEM. (ANOVA, ***P < 0.001, **P < 0.01, *P < 0.05, n = 4–6 mice/group, n = 28–46 RGCs/group, Table 1). The number of cells analysed in each group is indicated in Table 1. Scale bars: A–C = 25 µm, E–J = 50 µm.

Figure 2

mTORC1 activity is required for insulin-mediated dendritic branching. (A–D) Immunohistochemical analysis of retinal cross sections with an antibody against pS6^Ser240/244, a readout of mTORC1 function, and RBPMS, a selective marker of RGCs, revealed robust mTORC1 activity in these neurons. (E and F) Axonal injury induced a marked decrease of pS6^Ser240/244 labelling in RGCs, suggesting loss of mTORC1 activity, which was restored by insulin treatment. (G) Co-administration of insulin with siRNA against Raptor (siRaptor), an essential component of mTORC1 function, blocked the effect of insulin on RGC-specific pS6^Ser240/244 levels. (H) Quantification of the number of cells expressing both pS6^Ser240/244 and RBPMS relative to all RBPMS-positive cells confirmed that insulin fully restored mTORC1 activity in injured RGCs relative to vehicle- or siRaptor-treated retinas (ANOVA, ***P < 0.001, n = 5 mice/group). (I–K) Western blot and densitometry analysis confirmed that intravitreal delivery of siRaptor reduced retinal Raptor protein while a non-targeting control siRNA (siCtl) had no effect (Student’s t-test, **P < 0.05, n.s. = not significant, n = 6–7 mice/group). siRaptor did not alter the levels of Rictor, confirming the specificity of the siRNA. The lower panel is the same blot as in the upper panel but probed with an antibody that recognizes β-actin to ensure equal protein loading. (L–N) Co-administration of insulin and siRaptor resulted in a marked loss of dendritic branches and simpler arbours relative to insulin alone or insulin combined with siCtl at 7 days post-injury. (O–R) Quantitative analysis of dendritic parameters, including Sholl analysis (P), confirmed that selective mTORC1 knockdown blocked the ability of insulin to increase the number of regenerating branches, while no significant changes in process length or arbour area were observed (Q and R) (ANOVA, ***P < 0.001, **P < 0.01, *P < 0.05, n.s. = not significant; n = 4–5 mice/group, n = 32–43 RGCs/group, Table 1). Values are expressed as the mean ± SEM. Scale bars: A–C and L–N = 50 µm, D–G = 25 µm. GCL = ganglion cell layer; INL = inner nuclear layer; IPL = inner plexiform layer; OPL = outer plexiform layer.

Figure 3

mTORC2 regulates process extension and length in regenerating dendritic arbours. (A and B) Western blot analysis of retinal homogenates demonstrated a substantial reduction of pAkt^Ser473, a readout of mTORC2 activity, in axotomized retinas treated with vehicle, while insulin administration rescued pAkt^Ser473 levels (ANOVA, **P < 0.01, *P < 0.05, n = 5 mice/group). The lower panel is the same blot probed with an antibody against total Akt for normalization. (C–F) Western blot and densitometry analyses demonstrated that intravitreal injection of siRNA against Rictor (siRictor) led to reduction of retinal Rictor protein while a non-targeting control siRNA (siCtl) had no effect. Rictor knockdown also resulted in pAkt^Ser473 downregulation, indicative of mTORC2 function loss, but did not alter the levels of Raptor, thus validating its specificity (Student’s t-test, **P < 0.01, *P < 0.05, n.s. = not significant, n = 6–7 mice/group). The lowest panel represents the same blot probed with an antibody against β-actin to ensure equal protein loading. (G and H) Co-administration of insulin and siRictor resulted in a dramatic reduction in dendritic length and arbour area at 7 days post-injury relative to control retinas. (I) Administration of insulin and KU0063794 (KU), dual mTORC1 and mTORC2 inhibitor, resulted in overt dendrite degeneration characterized by a dramatic loss of branches and much shorter processes. Scale bars = 50 µm. (J–M) Quantitative analysis of dendritic parameters, including Sholl analysis (M), confirmed that siRictor-mediated mTORC2 inhibition resulted in a substantial loss of regenerating branches, while no significant changes in process length or arbour area were observed (ANOVA, ***P < 0.001, **P < 0.01, *P < 0.05, n.s. = not significant, n = 4–5 mice/group, n = 38–43 RGCs/group, Table 1). Values are expressed as the mean ± SEM.

Figure 4

Insulin restores glutamatergic postsynaptic sites in injured neurons. (A) Glutamatergic synapses visualized in the inner plexiform layer (IPL) on retinal cross-sections using immunolabelling against VGLUT1 (green) and PSD95 (red), a pre- and a postsynaptic marker, respectively. (B and C) Axonal injury induced a pronounced loss of both VGLUT1 and PSD95 expression in the inner plexiform layer, which was completely restored by insulin treatment (analysis at 7 days post-axotomy). (D and E) High magnification of VGUT1 and PSD95 puncta in the inner plexiform layer at the level of the OFF sublamina in retinas treated with insulin or vehicle. (F) Quantitative analysis of pre- and postsynaptic co-localized voxels, which measured the 3D volume occupied by both VGLUT1 and PSD95 in the inner plexiform layer, confirmed that insulin promoted synaptic marker regeneration (insulin: red bar, vehicle: grey bar, ANOVA, ***P < 0.001, n = 4–6 mice/group). (G) Representative examples of biolistically labelled RGCs with plasmids encoding tdTomato (red, RGC dendrites) and PSD95-YFP (cyan, PSD95 puncta) at 7 days after axotomy. In the absence of treatment, many retraction bulbs and varicosities (yellow arrowheads) as well as abnormally large PSD95 puncta (white arrowheads) were observed. (H and I) Higher magnification of dendritic branch segments show abnormally large PSD95 clusters in vehicle-treated retinas relative to intact RGCs. (J–U) Analysis of 3D-reconstructed PSD95 puncta density along RGC dendrites demonstrated striking insulin-mediated regeneration of excitatory postsynaptic sites in ON-sustained, OFF-sustained, and OFF-transient alpha RGCs relative to vehicle-treated controls. (J’–T’) Higher magnification images of individual segments are provided to show PSD95-YFP puncta (blue) along dendrites (red) for each condition. Values are expressed as the mean ± SEM. (ANOVA, ***P < 0.001, **P < 0.01, n = 5–6 mice/group, n = 3–6 RGCs/group). Scale bars: A–C and G = 10 μm, D and E = 5 μm, H and I = 1 µm, J–S = 30 μm, J’–T’ = 2.5 μm. GCL = ganglion cell layer; INL = inner nuclear layer.

Figure 5

Insulin rescues retinal function and increases neuronal survival. (A–F) Representative examples of ERG recordings elicited by dim scotopic (A, C and E) or photopic (B, D and F) light stimulation prior to axotomy (pre-injury, black trace), or after axotomy and treatment with PBS (blue trace) or insulin (red trace). Pre- and post-axotomy recordings were normalized relative to the contralateral, non-injured eye (grey traces). (G and H) Quantitative analysis of the pSTR or PhNR amplitudes demonstrated restoration of RGC function in insulin-treated eyes relative to controls that received vehicle at 7 days post-axotomy (ANOVA, **P < 0.01, *P < 0.05, n = 4–6 mice/group). (I–K) Flat-mounted retinas from eyes treated with insulin displayed higher densities of RBPMS-positive RGCs compared to control retinas treated with vehicle at 1 week after optic nerve axotomy. (L) Co-administration of insulin with KU0063794 completely abrogated the pro-survival effect of insulin, strongly suggesting a role for both mTORC1 and mTORC2 in insulin-mediated RGC survival. Scale bars = 50 µm. (M) Quantitative analysis of RBPMS-positive cells confirmed that insulin (red) promoted significant RGC soma survival at 1, 2, 4, and 6 weeks after optic nerve lesion compared to control eyes treated with vehicle (grey), or a combination of insulin and KU0063794 (green). The densities of RGC soma in intact, non-injured animals is shown as reference (white, 100% survival). Systemic administration of KU0063794 alone did not induce RGC death in intact retinas ruling out any toxic effect of this compound. Values are expressed as the mean ± SEM. (ANOVA, ***P < 0.001, **P < 0.01, *P < 0.05, n = 5–6 mice/group).