Hippocampal subfield vulnerability to α-synuclein pathology precedes neurodegeneration and cognitive dysfunction

Abstract

Cognitive dysfunction is a salient feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The onset of dementia reflects the spread of Lewy pathology throughout forebrain structures. The mere presence of Lewy pathology, however, provides limited indication of cognitive status. Thus, it remains unclear whether Lewy pathology is the de facto substrate driving cognitive dysfunction in PD and DLB. Through application of α-synuclein fibrils in vivo, we sought to examine the influence of pathologic inclusions on cognition. Following stereotactic injection of α-synuclein fibrils within the mouse forebrain, we measured the burden of α-synuclein pathology at 1-, 3-, and 6-months post-injection within subregions of the hippocampus and cortex. Under this paradigm, the hippocampal CA2/3 subfield was especially susceptible to α-synuclein pathology. Strikingly, we observed a drastic reduction of pathology in the CA2/3 subfield across time-points, consistent with the consolidation of α-synuclein pathology into dense somatic inclusions followed by neurodegeneration. Silver-positive degenerating neurites were observed prior to neuronal loss, suggesting that this might be an early feature of fibril-induced neurotoxicity and a precursor to neurodegeneration. Critically, mice injected with α-synuclein fibrils developed progressive deficits in spatial learning and memory. These findings support that the formation of α-synuclein inclusions in the mouse forebrain precipitate neurodegenerative changes that recapitulate features of Lewy-related cognitive dysfunction.

Fig. 1. Timeline and experimental design.

Adult mice (C57BL/6J) were subjected to bilateral stereotactic injection into the forebrain with either α-synuclein fibrils or vehicle (PBS). Cohort were assessed at either 1 MPI, 3 MPI, or 6 MPI.

Fig. 2. Formation of α-synuclein pathology in the hippocampal subfields.

A Schematic representation of the mouse hippocampal subfields, including the dentate gyrus, CA1 and CA2/3 subfields. Digital pathology quantification of pS129-α-synuclein immunostaining in the (B) dentate gyrus, (C) CA1 subfield, and (D) CA2/3 subfield at 1, 3, and 6 MPI. Data are expressed as boxplots depicting the median, interquartile range, and individual data points of the % area occupied of pS129-α-synuclein immunostaining (n = 11–12 animals/group/time point). Dentate gyrus, CA2/3 subfield: *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test, as indicated. CA1 subfield: *p < 0.05 by Kruskal–Wallis test with Dunn’s multiple comparisons test, as indicated. E Representative images of pS129-α-synuclein immunostaining at 1, 3, and 6 MPI including the dorsal hippocampus and each subfield. Scale bar: 250 μm (hippocampus) or 100 μm (dentate gyrus, CA1 subfield, CA2/3 subfield).

Fig. 3. Divergent susceptibility of hippocampal projections to pathologic spread of α-synuclein pathology.

A Schematic representation of the septo-hippocampal and entorhinal-hippocampal pathways with neuronal populations labeled by a dot and a line with arrow projecting to the hippocampus. B Digital pathology quantification of pS129-α-synuclein immunostaining in the entorhinal cortex at 1, 3, and 6 MPI. Data are expressed as boxplots depicting the median, interquartile range, and individual data points of % area occupied of pS129-α-synuclein immunostaining (n = 11–12 animals/group/time point). ***p < 0.001 or ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test, as indicated. C Representative images of pS129-α-synuclein immunostaining at 1, 3, and 6 MPI in the medial septum/diagonal band (left column) and entorhinal cortex (right column). Scale bar: 250 μm.

Fig. 4. Evidence of axonal damage concurrent with the formation of α-synuclein pathology.

A Schematic representation of the “tri-synaptic circuit”, depicting the perforant path and Schaffer collaterals spanning the entorhinal cortex and hippocampus. B Schematic representation of the regions of interest depicted in histology images (C): the CA1 subfield and the entorhinal cortex. C Representative images of Gallyas Silver staining (gray/black fibers or puncta) at 1, 3, and 6 MPI in fibril-injected mice. Data are representative n = 4 animals/group/time point. Scale bar: 100 μm.

Fig. 5. Neuronal loss is observed in the CA2/3 hippocampal subfield.

Quantification of NeuN-positive immunostaining in the (A) dentate gyrus, (B) CA1 subfield, and (C) CA2/3 subfield. Data are expressed as boxplots depicting the median, interquartile range, and individual data points for NeuN-positive area normalized to control at 1, 3, and 6 MPI in the (A) dentate gyrus, (B) CA1 subfield, and (C) CA2/3 subfield (n = 6–12 animals/group/time point). ****p < 0.0001 by two-way ANOVA with Bonferroni’s multiple comparisons test, as indicated. D Representative images of NeuN immunostaining in the hippocampus and CA2/3 subfield in control (left columns) and fibril-injected mice (right columns) at 1, 3, and 6 MPI. Scale bar: 500 μm (hippocampus) or 100 μm (CA2/3 subfield).

Fig. 6. Mice injected with α-synuclein fibrils develop cognitive dysfunction.

A Schematic representation of the Barnes maze platform in which the target and reversal target are 180° apart. Quantification of the average latency across acquisition trials at (B) 1 MPI, (C) 3 MPI, and (D) 6 MPI. Data are expressed as boxplots depicting the median, interquartile range, and individual data points for the average latency of control and fibril-injected mice (n = 10–11 animals/group). **p < 0.01 or ***p < 0.001 by unpaired Student’s t test. E Spaghetti plot depicting the average latency of mice at 1, 3, and 6 MPI. Each line represents the performance trajectory of an individual mouse. Quantification of performance trajectory from (F) 1 MPI to 3 MPI and (G) 3 MPI to 6 MPI. Data are expressed as boxplots depicting the individual data points for the net change in average latency of each group from 1 MPI to 3 MPI and from 3 MPI to 6 MPI. **p < 0.01 by unpaired Student’s t test. H Boxplots depicting individual data points for the percentage of time in which mice occupied the target quadrant during the probe session. I Boxplots depicting the individual data points for the average speed during the probe session. J Boxplots depicting individual data points for the average latency of each group during the reversal segment of the Barnes maze. **p < 0.01 or ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test, as indicated. K Boxplots depicting individual data points for the percentage of time in which mice occupied the target quadrant during the probe session during the reversal segment of the Barnes maze. *p < 0.05 by unpaired Student’s t test.

Fig. 7. Cognitive performance is associated with neuronal loss in the CA2/3 subfield but not residual α-synuclein burden.

A, C, E Association between average latency in the Barnes maze at 6 MPI and (A, C, E) NeuN levels in the (A) dentate gyrus, (C) CA1 subfield, and (E) CA2/3 subfield in the 6 MPI cohort. **p < 0.01 by Spearman correlation, r values listed by region, as indicated. B, D, F Association between average latency at 6 MPI and (B, D, F) pS129-α-synuclein immunostaining in the (B) dentate gyrus, (D) CA1 subfield, and (F) CA2/3 subfield at 6 MPI. Not significant by Spearman correlation, r values listed by region, as indicated.