Inducible dopaminergic glutathione depletion in an α-synuclein transgenic mouse model results in age-related olfactory dysfunction

Abstract

Parkinson's disease (PD) involves both motor and non-motor disturbances. Non-motor features include alterations in sensory olfactory function which may constitute a viable biomarker for the disorder. It is not clear what causes olfactory dysfunction but it appears to coincide with the development of synucleopathy within the olfactory bulb (OB). Elevation in alpha-synuclein (a-syn) is indeed a risk factor for development of the sporadic disorder. The multifactorial nature of the idiopathic disease combined with variability in its presentation suggests that it is likely to be influenced by several factors and that in vivo models that explore the synergistic effect of alpha-synuclein elevation with other potential contributing factors are likely to be of importance in understanding the disease etiology. Using a dual transgenic (DTg) mouse model of dopaminergic alpha-synuclein overexpression coupled with doxycycline (Dox)-inducible glutathione (GSH) depletion in these same cells, we demonstrate an age-related loss in behavioral olfactory function coupled with a significant neurodegeneration of glomerular dopaminergic neurons. This is accompanied by increase in alpha-synuclein levels in non-dopaminergic cells in the granule cell layer (GCL). In addition, isolated olfactory bulb synaptosomes from dual transgenic lines with Dox consistently showed a slight but significant reduction in maximum mitochondrial respiration compared to controls. These results suggest that in the presence of increased oxidative stress, increased alpha-synuclein expression within dopaminergic OB neurons results in neurodegeneration in the glomerular layer (GL) and increased alpha-synuclein levels in the granular cell layer which coincide with olfactory dysfunction.

Fig. 1

Buried pellet odor detection test at 8 months (A) and at 12 months of age (B) over a 6 day repeat trial period. The latency to find and eat a pellet was measured and reported as mean in seconds (s) ± SEM. Comparisons between DTg + Dox (black filled bar) and three control groups (DTg – Dox and a-syn +/- Dox) are reported (*: p<0.05, **: p<0.01, and #: p<0.001, Mann-Whitney U-test).

Fig 2

Nose poke odor detection/identification test at 8 months (A) and at 12 months of age (B) over a 4 day repeat trial period. The percentage of nose pokes in the target versus other holes was calculated and reported as mean ± SEM. 6.25% represents a random nose-poke (1/16 chance). DTg + Dox (black filled bar) as compared to DTg - Dox and a-syn + Dox. (*: p<0.05, **: p<0.01, and #: p<0.001, Mann-Whitney U test).

Fig. 3

Wooden block test to assess odor discrimination at 8 months (A) and at 12 months of age (B). Bars represent mean investigatory time (time spent sniffing block scented with a foreign animal's scent minus the time spent sniffing self-odored blocks) ± SEM. DTg + Dox (black filled bar) was compared and analyzed with DTg - Dox and a-syn + Dox (Wilcoxon signed rank test).

Fig. 4

Stereological analyses of TH⁺ or NeuN⁺ cells in the glomerular layer (GL) at 8 months (A-C) and at 12 months of age (D-F for TH and G-I for NeuN). Absolute number (A, D, and G), measured GL area (B, E, and H), and the density (C, F, and I) of TH⁺ or NeuN⁺ cells in the GL area were analyzed in dual Tg + Dox versus a-syn + Dox (n=7 each, Mean ± SEM, **: p< 0.01, *: p< 0.05, Paired t-test).

Fig. 5

Immunohistochemistry of alpha-synuclein (A, C, E, G, and I) and TH (B, D, F, H, and J) labeled OB cells. Boxes in A and B were shown enlarged in C and D, respectively. Top row and bottom row are neighboring sections at the same scale. Red arrows in C, E, G, and I represent alpha-synuclein labeled cells in the granule cell layer. Alpha-synuclein was detected in the GCL at 8 months (G and I) and 12 months (A, C, and E). Abb: GL: glomerular layer, EPL:external plexiform layer, GCL:granule cell layer, MCL: mitral cell layer, IPL:internal plexiform layer. Size bar: 50 μm.

Fig. 6

FCCP-stimulated maximal respiration (4 μM) in presynaptic nerve terminals (synaptosomes) isolated from the entire olfactory bulb. Rates were calculated relative to basal respiration level in medium containing glucose but not pyruvate (n=7 each, Mean ± SEM, *: p< 0.05, Paired t-test). Maximum respiration was presented as percent basal level. Three independently executed results were analyzed for statistical analysis (n=3, Paired t-test).