Expression of huntingtin-associated protein 1 in adult mouse dorsal root ganglia and its neurochemical characterization in reference to sensory neuron subpopulations

Abstract

Huntingtin-associated protein 1 (HAP1) is a polyglutamine (polyQ) length-dependent interactor with causal agents in several neurodegenerative diseases and has been regarded as a protective factor against neurodegeneration. In normal rodent brain and spinal cord, HAP1 is abundantly expressed in the areas that are spared from neurodegeneration while those areas with little HAP1 are frequent targets of neurodegeneration. We have recently showed that HAP1 is highly expressed in the spinal dorsal horn and may participate in modification/protection of certain sensory functions. Neurons in the dorsal root ganglia (DRG) transmits sensory stimuli from periphery to spinal cord/brain stem. Nevertheless, to date HAP1 expression in DRG remains unreported. In this study, the expression of HAP1 in cervical, thoracic, lumbar and sacral DRG in adult male mice and its relationships with different chemical markers for sensory neurons were examined using Western blot and immunohistochemistry. HAP1-immunoreactivity was detected in the cytoplasm of DRG neurons, and the percentage of HAP1-immunoreactive (ir) DRG neurons was ranged between 28-31 %. HAP1-immunoreactivity was comparatively more in the small cells (47-58 %) and medium cells (40-44 %) than that in the large cells (9-11 %). Double-immunostaining for HAP1 and markers for nociceptive or mechanoreceptive neurons showed that about 70-80 % of CGRP-, SP-, CB-, NOS-, TRPV1-, CR- and PV-ir neurons expressed HAP1. In contrast, HAP1 was completely lacking in TH-ir neurons. Our current study is the first to clarify that HAP1 is highly expressed in nociceptive/proprioceptive neurons but absent in light-touch-sensitive TH neurons, suggesting the potential importance of HAP1 in pain transduction and proprioception.

Keywords: CB, calbindin; CGRP, calcitonin gene-related peptide; CR, calretinin; DAB, diaminobenzidine; DRG, dorsal root ganglia; HAP1, Huntingtin-associated protein 1; Huntingtin-associated protein 1; Iba1, ionized calcium-binding adapter molecule 1; Immunohistochemistry; LTMRs, low-threshold mechanoreceptors; MRGPR, Mas-related G-protein-coupled receptor; NDS, normal donkey serum; NOS, nitric oxide synthetase; NeuN, neuronal nuclei; Neurodegeneration; Neuroprotection; PB, phosphate buffer; PV, parvalbumin; Peripheral nervous system; SBMA, spinal and bulbar muscular atrophy; SP, substance P; STB, stigmoid body; Sensory neurons; TBST, Tris-buffered saline with 0.1 % Tween; TH, tyrosine hydroxylase; TRPV1, transient receptor potential vanilloid 1; VGLUT, vesicular glutamate transporter; htt, huntingtin; polyQ, polyglutamine.

Fig. 1

Western blotting and immunohistochemistry for huntingtin-associated protein 1 (HAP1). (A) Western blot analysis using lysate from the dorsal root ganglion (DRG), spinal cord and whole brain showing bands of approximately 85 KDa for HAP1B and 75 KDa for HAP1A. (B) Pre-adsorption of the anti-HAP1 antibody with a blocking peptide against HAP1 antibody resulted in disappearance of the HAP1-positive bands. α tubulin was used for loading control. (C–F) Immunohistochemistry showing the presence of HAP1-immunoreactive (ir) cells in the DRG of different spinal level. (G–J) Preincubation with a blocking peptide against the anti-HAP1 antibody eliminated the HAP1-immunoreactivity. Arrowheads indicate the HAP1-ir stigmoid body in the cytoplasm. Scale bar = 100 μm in C–J and 10 μm in insets of. C–F.

Fig. 2

Double-label immunofluorescence immunohistochemistry for HAP1 with NeuN or Iba1. Photomicrograph showing double-label immunofluorescence staining of HAP1 and NeuN (A-C) or Iba1 (D-F) in the thoracic DRG. Arrows indicate cells for single-positive for HAP1. Arrowheads indicate cells single-positive for NeuN or Iba1. Double arrowheads indicate the cells positive for both HAP1 and NeuN. Scale bar = 50 μm in A-F.

Fig. 3

Analysis of cell profile and size distribution of HAP1-ir neurons in the DRG. Bar graphs showing (A) percentage of HAP1-ir neuron profile and (B-D) size distribution of HAP1-ir neurons in the DRG of different spinal level. Values represent the mean ± SEM (n = 6).

Fig. 4

Double-label immunofluorescence immunohistochemistry for HAP1 with SP or CGRP. Photomicrograph showing double-label immunofluorescence staining of HAP1 and SP (A-C) or CGRP (D-F) in the thoracic DRG. Arrows indicate cells for single-positive for HAP1. Arrowheads indicate cells single-positive for SP or CGRP. Double arrowheads indicate the cells positive for both HAP1 and SP or CGRP. Scale bar = 50 μm in A-F.

Fig. 5

Double-label immunofluorescence immunohistochemistry for HAP1 with CB, NOS, or TRPV1. Photomicrograph showing double-label immunofluorescence staining of HAP1 and CB (A-C), NOS (D-F) or TRPV1 (G-I) in the thoracic DRG. Arrows indicate cells for single-positive for HAP1. Arrowheads indicate cells single-positive for CB, NOS, or TRPV1. Double arrowheads indicate the cells positive for both HAP1 and CB, NOS or TRPV1. Scale bar = 50 μm in A-I.

Fig. 6

Double-label immunofluorescence immunohistochemistry for HAP1 with CR, PV or TH. Photomicrograph showing double-label immunofluorescence staining of HAP1 and CR (A-C), PV (D-F) or TH (G-I) in the thoracic DRG. Arrows indicate cells single-positive for HAP1. Arrowheads indicate cells single-positive for CR, PV, or TH. Double arrowheads indicate the cells positive for both HAP1 and CR or PV. Scale bar = 50 μm in A-L.

Fig. 7

Pseudo Venn-diagram indicating the relative extent to which nociceptive or mechanoreceptive markers express HAP1 in DRG. The extent to which nociceptive or mechanoreceptive markers themselves are co-expressed in DRG is not indicated. The sizes of the circles depicting the nociceptive or mechanoreceptive markers is not representative of the relative number of the neurons that express those markers.