Seeding and transgenic overexpression of alpha-synuclein triggers dendritic spine pathology in the neocortex

Abstract

Although misfolded and aggregated α-synuclein (α-syn) is recognized in the disease progression of synucleinopathies, its role in the impairment of cortical circuitries and synaptic plasticity remains incompletely understood. We investigated how α-synuclein accumulation affects synaptic plasticity in the mouse somatosensory cortex using two distinct approaches. Long-term in vivo imaging of apical dendrites was performed in mice overexpressing wild-type human α-synuclein. Additionally, intracranial injection of preformed α-synuclein fibrils was performed to induce cortical α-syn pathology. We find that α-synuclein overexpressing mice show decreased spine density and abnormalities in spine dynamics in an age-dependent manner. We also provide evidence for the detrimental effects of seeded α-synuclein aggregates on dendritic architecture. We observed spine loss as well as dystrophic deformation of dendritic shafts in layer V pyramidal neurons. Our results provide a link to the pathophysiology underlying dementia associated with synucleinopathies and may enable the evaluation of potential drug candidates on dendritic spine pathology in vivo.

Keywords: alpha‐synuclein; dendritic spines; in vivo imaging; seeding; synucleinopathies.

Figure 1. α‐Synuclein overexpression alters spine density and dynamics in vivo

1. Representative in vivo two‐photon recordings of eGFP‐labeled apical tuft dendrites in the somatosensory cortex in h‐α‐syn and control animals. Arrowheads mark representative spines that were stable (white, present > 7 days), newly formed (green), or lost (magenta). Gained spines that do not stabilize (yellow/green, present < 7 days) are defined as transient. Scale bar, 5 μm.

2. Spine density is reduced in both 6‐ (**P = 0.0012) and 12‐month‐old (***P = 0.0001) h‐α‐syn animals.

3. The fractions of both gained (P _6 months = 0.0527; P _12 months = 0.0678) and lost spines (*P _6 months = 0.0461; *P _12 months = 0.0206) are elevated in h‐α‐syn mice compared to controls; the fraction of transient spines is significantly higher (*P _6 months = 0.0315; **P _12 months = 0.0017).

4. Consequently, the daily turnover ratio (TOR) is significantly increased in both 6‐ and 12‐month‐old h‐α‐syn mice (*P _6 months = 0.0491; *P _12 months = 0.0338).

Data information: n = 5 (h‐α‐syn), n = 4 (control) animals, mean with s.e.m.; two‐way ANOVA genotype main factor, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. α‐Synuclein overexpression alters spine density, dynamics, and morphology differently depending on age

1. In young, 3‐month‐old h‐α‐syn mice, progressive decrease in spine density is observed in vivo.

2. The loss of spines in young h‐α‐syn animals is driven by a reduced fraction of newly gained spines, while the fraction of lost spines remains unchanged and the fraction of stable spines is increased (*P _{gained/stable} = 0.0256).

3. The daily turnover of spines shows no significant difference between groups (P = 0.4062).

4. Ex vivo confocal data in young mice confirm synapse loss in h‐α‐syn mice between 2 and 4.5 months of age (**P _{syn 2/4.5 months} = 0.0064; **P _{ctrl/syn 4.5 months} = 0.0012) and show a shift in spine morphology toward relatively more stubby (*p_stubby = 0.0418) and less thin spines (**P _{h‐α‐syn 2/4.5 months} = 0.0044; *P _{ctrl/syn 4.5 months} = 0.0155).

5. Ex vivo confocal data in aged h‐α‐syn mice show a decrease in total spine density (***P _7.5 months = 0.0005; **P _13.5 months = 0.0039) as well as in the fraction of mushroom spines (***P _7.5 months = 0.0004; *P _13.5 months = 0.0298), whereas the fraction of thin spines is increased (**P _7.5 months = 0.0097; *P _13.5 months = 0.0155).

Data information: (A–C) n = 4 animals per group; (D, E) n = 3–4 animals per group as illustrated; mean with s.e.m.; Bonferroni's post hoc test (A): ^# P < 0.05, ^## P < 0.01; two‐way ANOVA genotype main factor (B, C): *P < 0.05, **P < 0.01, ***P < 0.001; Student's t‐test (D, E): *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Injection of PFFs into the dorsal striatum triggers cortical α‐synuclein aggregation

1. A–C

   The quality of the injection material was verified using ThT fluorescence assay (A), sucrose‐gradient fractionation (B), and electron microscopy (C). Scale bar: 0.2 μm.

2. D

   Injection site, 0.2 mm anterior of the bregma (star) and imaging area (box) as depicted in (E).

3. E–I

   Representative images of the cortical layers I–VI in the somatosensory cortex of mice at different time points postinjection. Controls were injected with sterile PBS. Neurons in the layers IV and V of the somatosensory cortex contain aggregates of α‐synuclein phosphorylated at S129 (F), which are ubiquitin‐positive (G) as well as thioflavin S‐positive (H, I). Image stacks (E–I) are depicted as maximum intensity projections. Scale bars: 50 μm (E), 10 μm (F, G), 20 μm (H), 5 μm (I).

Figure 4. The presence of accumulated α‐synuclein induced by seeding 5 months prior to analysis causes spine loss and malformation in layer V apical dendrites

1. α‐Synuclein aggregates occur as intrasomal (arrowheads) and neuritic (arrows) accumulations and are present predominantly in upper layer V and layer IV of the cortex. In layer I, pS129‐positive structures are much less dense. Lines exemplarily mark dendrites used for analysis.

2. Spine analysis was performed on apical dendrites located in the cortical layers IV/V and I.

3. In layer I, spine density is reduced relative to PBS‐injected controls (**P = 0.0028), with the fraction of stubby spines being increased (*P = 0.0247) and the fraction of thin spines being decreased (*P = 0.0385). In layer IV/V, spine density is reduced as well (**P = 0.004), without a significant effect on spine morphology.

4. In apical tuft dendrites of PFF‐injected mice, dendrites display dystrophic swellings and parts of very small diameter; white lines: measurement positions.

5. Histograms of the dendritic shaft diameter.

6. Variation in the diameter of single dendrites in seeded mice compared to controls (*P = 0.011).

Data information: n = 6 (control), n = 7 (PFF) animals, mean with s.e.m.; Student's t‐test: *P < 0.05, **P < 0.01. Scale bars: 20 μm (A), 2 μm (B, D).

Figure 5. Cortical α‐synuclein accumulation and long‐term in vivo imaging of PDGF‐h‐α‐syn × GFP‐M mice

1. Immunostaining with 15G7 antibody shows cortical layer V overexpression of α‐synuclein (magenta) including accumulation in cell bodies (arrows).

2. Experimental timeline: 4 weeks after window implantation (white arrowhead), imaging was performed over 6 weeks, once every 7 days (gray arrowheads) and finally followed by perfusion and tissue fixation directly after the last imaging session (black arrowhead).

3. Overview and detailed projections of eGFP‐labeled layer V apical dendrites in the somatosensory cortex, imaged through a chronic cranial window.

Data information: Scale bars: 20 μm (A), 50 μm (C), 5 μm (C, inset).