Huntingtin Aggregates in the Olfactory Bulb in Huntington's Disease

Abstract

Olfactory deficits are an early and prevalent non-motor symptom of Huntington's disease (HD). In other neurodegenerative diseases where olfactory deficits occur, such as Alzheimer's disease and Parkinson's disease, pathological protein aggregates (tau, β-amyloid, α-synuclein) accumulate in the anterior olfactory nucleus (AON) of the olfactory bulb (OFB). Therefore, in this study we determined whether aggregates are also present in HD OFBs; 13 HD and five normal human OFBs were stained for mutant huntingtin (mHtt), tau, β-amyloid, TDP-43, and α-synuclein. Our results show that mHtt aggregates detected with 1F8 antibody are present within all HD OFBs, and mHtt aggregate load in the OFB does not correlate with Vonsattel grading scores. The majority of the aggregates were located in the AON and in similar abundance in each anatomical segment of the AON. No mHtt aggregates were found in controls; 31% of HD cases also contained tau neurofibrillary tangles within the AON. This work demonstrates HD pathology in the OFB and indicates that disease-specific protein aggregation in the AON is a common feature of neurodegenerative diseases that show olfactory deficits.

Keywords: Huntington’s disease; anterior olfactory nucleus; huntingtin aggregates; olfactory bulb; tau.

FIGURE 1

Fluorescent staining for Huntingtin (Htt) aggregates in human olfactory bulbs (OFBs). Confocal imaging of nuclear (yellow arrows) and cytoplasmic (magenta arrows) mHtt aggregates identified with 1F8 and 1C2 antibodies (A,B). No overlap is seen between 1F8 and TFIID, indicating 1F8 solely detects mHtt aggregates (C). Cells with 1F8⁺ and 1C2⁺ aggregates were only found in olfactory bubs from cases with Huntington’s Disease (HD). No 1F8⁺ aggregates were found in normals (D). 1C2 and 1F8 staining were done on sequential sections that detect similar amounts of cells with mHtt aggregates (yellow arrows). The inserts show representative images of NeuN⁺ cells with mHtt aggregates (E,F). Distribution of 1F8⁺ cells (green dots) throughout a sagittal olfactory bulb section. Majority of cells are found within the anterior olfactory nucleus (AON) (G). 1F8⁺ mHtt aggregates (yellow arrows) are found in PGP9.5⁺neurons within the AON (H). Double labelling of 1F8⁺ mHtt cells in the anterior olfactory nucleus with neuronal markers. Yellow arrows indicate that 1F8⁺ mHtt aggregates are found in AON neurons that stain for calbindin (I), somatostatin (J), tyrosine hydroxylase (K) and calretinin (L). Mitral cells (orange arrows) co-express PGP9.5 and Kif5a (M,N). 1F8⁺ mHtt aggregates were found in mitral cells (O). Scale bars represent 10 μm except for (D–G) 500 μm and M 20 μm.

FIGURE 2

Schematic overview of human olfactory bulb and 1F8⁺ mHtt cell density counts. Representative diagram of a sagittal human olfactory bulb sections indicating the location of the bulbar (AONb), interpeduncular (AONi), and retrobulbar AON (AONr) (A). 1F8⁺ mHtt cell density across Vonsattel grades for the AONb (B) and all AON regions combined (C). 1F8⁺ mHtt cell density across the AON segments for each HD case (D). Correlations between 1F8+ mHtt cell density for the total AON and age, CAG repeat length, and post-mortem delay (PMD) (E–G).

FIGURE 3

Fluorescent staining for tau and Huntingtin (Htt) aggregates in human olfactory bulbs (OFBs). Overview staining of tau aggregates in the olfactory bulb (A). Representative image of tau (red) and 1F8⁺ mHtt aggregates (green, yellow arrows) in AON neurons (B). Confocal Z-stack projection with orthongal projections of tau and 1F8⁺ mHtt aggregates occupying different subsections of the neurons (C). Scale bars represent 100 μm for (A) and 10 μm for (B,C).