Chronic VEGFR-3 signaling preserves dendritic arborization and sensitization under stress

Abstract

Dendritic arborization is critical for the establishment and maintenance of precise neural circuits. Vascular endothelial growth factor D (VEGF-D), well-characterized as a "lymphangiogenic" growth factor, reportedly maintains dendritic arborization and synaptic strength in the hippocampus of adult mice through VEGF receptor (VEGFR-3) signaling. Here, we investigated the effect of chronic VEGFR-3-specific activation on adipose arbor morphometry using the Adipo-VD mouse, a model of inducible, adipose-specific VEGF-D overexpression. We examined whether adipose tissue innervation was preserved or functionally different in Adipo-VD mice during stress in vivo and if VEGFR-3 signaling afforded neuroprotection to challenged neurons in vitro. Chronic VEGFR-3 signaling in Adipo-VD subcutaneous adipose tissue resulted in a reduction in the dendrite length, dendritic terminal branches (filament length), and dendritic terminal branch volume (filament volume), but increased dendrite branching. We also identified reduced stimulus-evoked excitatory sympathetic nerve activity in Adipo-VD mice. Following 6-hydroxydopamine (6-OHDA) denervation, Adipo-VD dendritic arbors were preserved, including improved dendritic branch volume, length, and dendritic branches than in wildtype tissues. In vitro, we found that chronic elevation of VEGFR-3 signaling in developing mVC neurons changes the dendritic arbor complexity and improves stress-induced structure remodeling. Developing neurons are conferred neuroprotection against stress, potentially by upregulation of proteolytic conversion of pro-BDNF to mature BDNF. Mature neurons, however, display improved dendritic arbor complexity, and unaltered dendritic structural remodeling and improved resistance to stress with VEGFR-3 signaling. Overall, chronically increasing VEGFR-3 signaling in neurons has a synergistic impact on neurosensitization and neuroprotection during stress.

Keywords: Arborization; Dendrite; Neuroprotection; VEGF-C; VEGF-D.

Figure 1.. Chronically elevated VEGF-D levels impact subcutaneous adipose tissue innervation.

(A) Volume imaging of subcutaneous adipose neuronal structures in wild type: WT (−rtTA) and Adipo-VD (+rtTA) chow mice; (Map-2, set as grey scale for visual clarity). (B) Comparison of adipose dendrite length (measured from the cell body); (C) dendrite branching; (D) dendritic terminal filament length; and (E) dendritic terminal filament volume in adipose between WT and Adipo-VD mice. (F) RT-qPCR of neuronal phenotype genes in 1-month Adipo-VD mice normalized to ubiquitin and compared to WT tissue. (G) Norepinephrine within WT and Adipo-VD inguinal adipose per mg protein. (H) Serum norepinephrine levels in WT and Adipo-VD mice. (I) ATF3+ (red) positive nuclei (blue) in L3 dorsal root ganglia (DRG) of WT and Adipo-VD mice. (J) Comparison of the ratio of ATF3+ positive nuclei vs total DRG cell body nuclei of WT and Adipo-VD mice. (A) n=3,3; scale bar= 200 μm (B-E) n=3,3 (F) n=6,6 (G) n=4,5 (H) n=8,4 (I-J) n=4,4. *p<0.05 vs −rtTA (B-E) mean ± SEM (F, G, H, J) mean ± SD.

Figure 2.. Developing neurons differentiated in the presence of VEGFR-3 ligand results in minimal phenotypic change

(A) hiPSC-NSC control and mVC (VEGFR-3-specific mutant VEGF-C) neurons phenotypically characterized using PGP9.5 (green), MAP-2 (red), tyrosine-hydroxylase-TYR-H (green) and DAPI (blue). (B) RT-qPCR of neuronal phenotype genes and VEGFR-3 gene expression were compared between control and mVC neurons at day 31. (C) Sholl quantification of total dendritic intersections of hiPSC-NSC control and mVC neurons. (D) Area under the curve (AUC); (E) dendrite length; (F) dendrite intersection; and (G) critical radius of the dendrite processes calculated from Sholl intersections. (A) PGP 9.5 scale bar= 100 μm. MAP-2and TH scale bar= 20 μm n=2, (B) n=10 Bars represent mean ± SD (C-G) n=10,10 Statistically significant differences are indicated with asterisks, *p < 0.005. Bars represent mean ± SEM.

Figure 3.. Developing neurons differentiated in the presence of chronic VEGFR-3 ligand display improved dendritic arbor restructuring in-vitro

(A) hiPSC- Neural Stem Cell (NSC) control and mVC (VEGFR-3-specific mutant VEGF-C) neurons after 2 hrs. of 6-hydroxydopamine (6-OHDA represented as OHDA in figures) treatment PGP9.5 (green). (B) PI/trypan Blue exclusion assay based % viable cells calculated across various treatments. (C) RT-qPCR fold change of TYR expression between control, mVC, 6-OHDA and mVC-6-OHDA treated neurons. (D) RT-qPCR of VEGFR-3 gene expression compared between control, mVC, 6-OHDA and mVC-6-OHDA day 31 neurons. (E) RT-qPCR of BDNF gene expression. (F) BNDF immunoblot. (G) pro-BDNF represented as fold change and (H) mature-BDNF represented as raw densitometric arbitrary unit values across neurons with various treatments groups. (I) Quantification of total dendritic intersections through Shall analysis of hiPSC-NSC control, mVC, 6-OHDA and mVC-6-OHDA neurons. (J) Area under the curve (AUC); (K) dendrite length; (L) dendrite intersections; and (M) critical radius of the dendrite processes. (A) PGP 9.5 scale bar= 100 μm, n=2, (B-E) n=10,10 (F, G, H) n=3, (I, J, K, L, M) n=10. Statistically significant differences are indicated with asterisks, *p < 0.005, (B, C, D, E, G, H) Bars represent mean ± SD, (J, K, L, M) Bars represent mean ± SEM.

Figure 4.. Effects of acute VEGFR-3 activation in hiPSC-Neural Stem Cell (NSC) derived neurons

(A) Representative raw and skeletonized Sholl intersection micrograph of hiPSC-NSC (control, VEGFR-3-specific mutant VEGF-C (mVC), 6-hydroxydopamine (6-OHDA represented as OHDA in figures) and mVC+6-OHDA) neurons. (B) Quantification of total dendritic length, intersections through Sholl analysis. (C, D) Immunoblot and quantified ratio of cleaved:pro caspase across the treatment conditions. (E) Area under the curve (AUC); (F) dendrite length; (G) dendritic intersections; (H) and critical radius of the dendrite processes calculated from Sholl intersections. (I) Immunoblot analysis by using phosphospecific antibodies of hiPSC-NSC neurons with or without mVC and 6-OHDA treatment. (J) Quantification of the immunoblot for VEGFR-3; (K) pERK/Total ERK; (L) pAKT/Total AKT; (M) GluN2B; and (N) BDNF (A) Original scale bar= 100 μm, images cropped with scales adjusted to 100 μm, n=10 (B,E,F,G,H) n=10 (C,D,I-N) n=3 Statistically significant differences are indicated with asterisks, *p < 0.005, (E-H) Bars represent mean ± SEM. (D,J-N) Bars represent mean ± SD.

Figure 5.. Chronically elevated VEGF-D levels protect inguinal adipose tissue innervation against injury.

(A) Volume imaging of subcutaneous adipose neuronal structures in wild type: WT (−rtTA) and Adipo-VD (+rtTA) chow mice after the injection of 6-hydroxydopamine (represented as OHDA in the figure) in the tissue; (Map-2, set as grey scale for visual clarity). (B) Comparison of adipose dendrite length (measured from the cell body); (C) dendrite branching; (D) dendritic terminal filament length; and (E) dendritic terminal filament volume in adipose between WT and Adipo-VD mice after OHDA. (F) Norepinephrine level within WT and Adipo-VD inguinal adipose per mg protein. (G) Serum Norepinephrine levels of WT and Adipo-VD mice. (H) ATF3+ (red) positive nuclei (blue) in L3 dorsal root ganglia (DRG) of WT and Adipo-VD mice. (I) Comparison of the ratio of ATF3+ positive nuclei vs total DRG cell body nuclei of WT and Adipo-VD mice. (J) Von-Frey pain threshold fold change comparison between WT and Adipo-VD quantified following 1 and 4-month chow diet feeding (+/− 6-OHDA) and all values normalized to WT reading at 1 month. (A) n=3,3 Grey Scale=MAP-2, scale bar= 200 μm (B-E) n=3,3 (F) n=4,4 (G) n=3,3 (H-I) n=4,4 (J) n=40. *p<0.05 vs WT (B-E) mean ± SEM (F, G, I, J) mean ± SD.

Figure 6.. Tissue innervation in Adipo-VD obese remains unchanged across normal and obese Adipo-VD mice.

(A) Volume imaging of subcutaneous adipose neuronal structures in wild type: WT (−rtTA) and Adipo-VD (+rtTA) high fed diet (HFD) mice; (Map-2, set as grey scale for visual clarity). (B) Comparison of adipose dendrite length (measured from the cell body); (C) dendrite branching; (D) dendritic terminal filament length; and (E) dendritic terminal filament volume in adipose between obese WT and Adipo-VD mice. (F) Norepinephrine concentration within obese WT and Adipo-VD inguinal adipose per mg protein. (G) Serum Norepinephrine levels in obese WT and Adipo-VD mice. (H) ATF3+ (red) positive nuclei (blue) in L3 dorsal root ganglia (DRG) of obese WT and Adipo-VD mice. (I) Comparison of the ratio of ATF3+ positive nuclei vs total DRG cell body nuclei of WT and Adipo-VD mice. (J) Von-Frey pain threshold fold change comparison between WT and Adipo-VD quantified following 1 and 4-month HFD diet feeding and all values normalized to WT reading at 1 month. (A) n=3,3 Grey Scale=MAP-2, scale bar= 200 μm (B-E) n=3,3 (F) n=4,4 (G) n=5,4 (H, I) n=4,4 (J) n=36. *p<0.05 vs WT (B-E) mean ± SEM (F, G, I, J) mean ± SD.

Figure 7.. Figure summarizing neuronal innervation changes in Adipo-VD mice.

Chronic over expression of the VEGFR-3 ligand VEGF-D in the tissue of Adipo-VD mice alters neuronal dendrite morphology, length, branching, dendritic terminal filament volume and dendritic terminal filament length. This dense arborization response is protective against 6-hydroxydopamine (6-OHDA) neurotoxic stress in vivo and also in cultured neurons. Created with Biorender.com.