IGF2 prevents dopaminergic neuronal loss and decreases intracellular alpha-synuclein accumulation in Parkinson's disease models

Abstract

Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.

Fig. 1. IGF2 expression prevents cytotoxicity effect of α-syn PFF in PD cellular models.

A, B SN4741 cells were treated with human recombinant IGF2 (rIGF2) 5 ng/ml or Leu27 5 ng/ml for 1 h. and exposed to human α-syn monomers (Mono) or α-syn PFF. PBS 1× was used as a control. After 24 h, cell death was measured by Sytogreen (A) and LDH assay (B). FC correspond to fold change. C Primary culture of cortical neurons was treated at 6 DIV with rmIGF2 100 ng/mL 1 h previously to incubation with mouse α-syn monomers and α-syn PFF. PBS 1× was used as a treatment control. After 72 h of incubation, cellular damage was evaluated by LDH released assay. D SHSY5Y cells were treated with human recombinant IGF2 (rIGF2) 5 ng/ml for 1 h and exposed to human α-syn monomers and α-syn PFF. After 24 h, cell death was measured by quantification of PI-positive cells. E, F SHSY5Y cells were transfected with IGF2-containing plasmid to overexpress IGF2 (IGF2 OE) or empty vector (EV) as control. After 24 h cells were incubated with human α-syn monomers or α-syn PFF and IGF2 receptor antibody (IGF2 OE + Ab IGF2R). Followed 24 h, cell viability was measured by cresyl violet staining (E) and by quantification of PI-positive cells (F). Scale bar 100 um. Data are presented as mean and SEM of at least three independent experiments. Statistically significant differences detected by two-tailed unpaired t test (**p < 0.01).

Fig. 2. IGF2 expression decrease α-syn aggregates in PD cellular models.

A SN4741 cells were treated with human recombinant IGF2 (rIGF2) 5 ng/ml or Leu27 5 ng/ml for 1 h and exposed to α-syn monomers (Mono) or α-syn PFF. PBS 1× was used as a control. After 24 h, α-syn levels were evaluated in whole cells extracts by western blot (left panel), using anti-α-syn antibody (upper panel). HSP90 levels were monitored as loading control (bottom panel). Bottom panel. α-syn levels were quantified and normalized to HSP90 levels. B SHSY5Y cells were transiently co-transfected with pAAV-α-syn-RFP expression vector and pAAV-IGF2-HA vector (IGF2 OE) or pAAV-empty vector (EV). 48 h later, α-syn levels were analyzed in whole cells extracts by western blot analysis using anti-α-syn antibody (upper panel). IGF2 levels were also determined (middle panel). Hsp90 levels were monitored as loading control (bottom panel). Bottom panel. α-syn levels were quantified and normalized to HSP90 levels. C Primary culture of cortical neurons 1 DIV were infected with AAV-IGF2 or AAV-control. 7 days later, neurons were treated with PFF or monomer (control) of α-syn. 7 days later, α-syn levels were evaluated in whole cells extracts by western blot (left panel) using anti-α-syn phosphorylated antibody (upper panel). IGF2 levels were determined (middle panel). b-actin levels were monitored as loading control (bottom panel). Bottom panel. α-syn levels were quantified and normalized to actin levels. Data are presented as mean and SEM of at least three independent experiments. Statistically significant differences detected by two-tailed unpaired t test (*p < 0.05, **p < 0.01).

Fig. 3. IGF2 expression increase α-syn secretion in PD cellular models.

A SN4741 cells were treated with human recombinant IGF2 (rIGF2) 5 ng/ml or Leu27 5 ng/ml for 1 h and exposed to α-syn monomers (Mono) or α-syn PFF. PBS 1× was used as a control. After 24 h, culture media was analyzed by dot blot using an anti-α-syn antibody (upper panel). α-syn levels were quantified and plotted in each condition (bottom panel). B SHSY5Y cells were transiently co-transfected with pAAV-α-syn-RFP expression vector and pAAV-IGF2-HA vector (IGF2 OE) or pAAV-empty vector (EV). After 48 h culture media was analyzed by dot blot using an anti-α-syn antibody (upper panel). α-syn levels were quantified and plotted in each condition (bottom panel). C Primary culture of cortical neurons (1 DIV) were infected with AAV-IGF2 or AAV-control. 7 days later, neurons were treated with PFF or monomer (Mono) of α-syn. 7 days later, α-syn levels was analyzed by dot blot using an anti-α-syn antibody (upper panel). α-syn levels were quantified and plotted in each condition (bottom panel). Data are presented as mean and SEM of at least three independent experiments. Statistically significant differences detected by two-tailed unpaired t test (*p < 0.05).

Fig. 4. IGF2 treatment improved motor performance in an idiopathic PD mice model.

A Experimental strategy to determine the effect of IGF2 gene therapy in idiopathic PD mice model. B Beam test was performed to evaluate spontaneous motor changes associated with dopamine depletion in the striatum of α-syn PFF injected mice. Number of errors for steps was measure and plotted for each condition at basal condition (0 weeks), 6, or 12 weeks. C Dopaminergic innervation was detected using anti-TH antibody (green), in striatum tissue. Total cells were visualized using DAPI staining (blue). FC correspond to fold change. Scale bar = 20 μm. D Medium spine neuron present in the striatum brain region was visualized using anti-DARPP32 antibody (capyso). Phospho -α-syn (p-α-syn) accumulation were visualized in striatum tissue sections by anti-p-α-syn immunostaining (red). Total cells were visualized using DAPI staining (blue). Scale bar = 20 μm. For all experiment mean and SEM were represented for seven independent experiments. Statistically significant differences were detected by two-tailed unpaired t test (*p < 0.05).

Fig. 5. Local delivery of IGF2 into the SNpc prevent dopaminergic neuronal loss and decrease the p-α-syn accumulation in idiopathic PD mice model.

A At 3 months-old WT animals were injected with α-syn PFF into the right striatum by brain stereotaxis. At 6 weeks after α-syn PFF delivery, the mice were injected with AAV carrying either IGF2 (AAV-IGF2) or empty (AAV-control) into the right SNpc by brain stereotaxis. After 6 weeks, dopaminergic neurons were visualized in midbrain tissue sections by anti-tyrosine hydroxylase (TH) immunostaining (red). Representative images of TH staining in midbrain tissue sections. Scale bar: 50 μm. B TH-positive neurons were quantified as the percentage of neurons in the injected side relative to the non-injected (control) in the Substancia Nigra (SN) (left panel). C At 3 months-old WT animals were injected with α-syn PFF into the right striatum by brain stereotaxis. At 6 weeks after PFF delivery, the mice were injected with AAV carrying either IGF2 (AAV-IGF2) or empty (AAV-control) into the right SNpc by brain stereotaxis. After 6 weeks, phosphor -α-syn (p-α-syn) accumulation were visualized in midbrain tissue sections by anti-p-α-syn immunostaining (brown). Hematoxylin-Eosin staining (blue/violet) was used to visualize the total cells. Representative images of p-α-syn staining in midbrain tissue sections. Scale bar: 20 μm. D p-α-syn positive neurons were quantified using integrated density per area and the percentage of p-α-syn staining on the injected side relative to the non-injected was determine in each condition. FC correspond to fold change. For all experiment mean and SEM were represented for 7 independent experiments. Statistically significant differences were detected by two-tailed unpaired t test (***p < 0.001; *p < 0.05).

Fig. 6. IGF2 treatment rescues dendritic spines loss in PD mice model.

A Immunofluorescence for dopaminergic neurons were detected using anti-TH antibody, anti-synaptophysin was used as a presynaptic marker. Image represents the selection of analyzed filaments (gray dendrite) and spots (red spots). Scale bar = 10 μm. B High magnification to show filaments and spots analyzed. Comparison between AAV-control and AAV-IGF2 conditions. Scale bar = 2 μm. C Quantification of number spots present in filaments of 10 μm. Spots present at 2.5 μm from the center of the filament were analyzed. Data represents ~2800 filaments analyzed for AAV-control condition and ~9400 filaments analyzed for AAV-IGF2. Images were analyzed using Imaris software. For all experiment mean and SEM were represented for three independent experiments. Statistically significant differences were detected by two-tailed unpaired t test (*p < 0.05).