One objective in this study was to construct a neuroanatomic and morphologic profile for polyglutamine antibody 1C2 immunoreactivity in the HD CNS. 1C2 immunoreactivity was present in diverse areas of the HD CNS, and a variety of cytoplasmic and nuclear staining patterns was identified in neurons, neuropil, and glia. All of the HD cases showed immunoreactivity in the neocortex, striatum, hippocampal pyramidal neurons/subiculum/entorhinal cortex, lateral geniculate body, substantia nigra, basal pontine nuclei, medullary reticular formation, and cerebellar dentate nucleus (). Some cases showed 1C2 immunoreactivity in the dentate gyrus, thalamus, locus coeruleus, and spinal cord. None of the HD cases demonstrated immunoreactivity in the cerebellar cortex or pituitary gland.
1C2 immunoreactivity was identified in normal and diseased control cases, a finding not reported to date in the HD immunohistochemistry literature. Neurons and occasional adjacent macrophages in the substantia nigra and locus coeruleus of these controls demonstrated aggregated cytoplasmic immunoreactivity. This immunoreactivity was not present when staining with an antibody to desmin was performed under similar conditions.
What is the epitope of 1C2 in these locations? Is it truly the long glutamine stretch? In addition to polyglutamine stretches, 1C2 has been shown to bind polyleucine- and polyalanine-containing proteins (22
). We cannot exclude the possibility that the 1C2 immunoreactivity in nigral neurons may represent detection of leucine or alanine residues present in neuromelanin. Neuromelanin is a pigmented material that accumulates in neuronal cytoplasm over time and is believed to arise from the spontaneous autoxidation of dopamine in the substantia nigra and of norepinephrine in the locus coeruleus (24
). Neuromelanin extracted from the nigras of 18 normal brains by Double et al (24
) contained a variety of amino acids, including leucine and alanine, but not glutamine.
Another possibility is that substantia nigra 1C2 immunoreactivity represents an accumulation of normal TBP. The detection of TBP by 1C2 was proposed as a possible explanation by Rudnicki et al (19
) for neuronal 1C2 immunoreactivity in cases of Huntington disease-like 2 (HDL2). HDL2 is an autosomal dominant disease with clinical features and neuropathology similar to that of HD but is not associated with a CAG expansion in the IT15 gene at 4p16.3. Rather, it is caused by a CAG/CTG repeat expansion in a variably spliced exon of the junctophilin-3 gene at 16p24.3. Intraneuronal protein aggregates immunoreactive for 1C2 have been identified in all reported cases to date, but it is not clear whether LGS are present within the aggregates. Besides TBP, polyalanine and polyleucine stretches were also proposed as possible antigens for 1C2 in these HDL2 cases. Fortunately, a difference in distribution of 1C2 immunoreactivity is one feature that distinguishes HD from HDL2. In the 5 cases of HDL2 studied, all lacked 1C2-immunoreactive intranuclear aggregates in the pons and medulla, 2 locations in which we and others have demonstrated reproducible 1C2 immunoreactivity in HD (18
Despite this 1C2 immunoreactivity in 3 limited areas of control cases, we feel that its consistent, reproducible staining in numerous CNS locations in HD cases provides a supplemental diagnostic tool in the postmortem diagnosis of HD. Our experience with 2B4, on the other hand, leads us to believe that it would be difficult to use in a diagnostic setting. Although our survey was small, the morphologic patterns of 2B4 staining were less reproducible in distinguishing between HD and controls.
Immunohistochemistry has been integral to the advancement of knowledge about HD with the work of Lunkes et al (13
) and Schilling et al (17
) as prime examples. They narrowed the mHtt cleavage site to an area between amino acids 81 and 90 and 115 and 129 using multiple anti-Htt antibodies. While the identity of the enzyme(s) performing this cleavage in the neuron and the exact location of the cleavage remain unknown, this information puts the results of the many immunohistochemical studies listed in into a clearer molecular framework. These anti-Htt antibodies (by no means an exhaustive list), segregate roughly into 2 groups: those that recognize the N-terminal fragment of mHtt and those that recognize the portion of mHtt on the carboxy end of the putative cleavage site. With cleavage between amino acids approximately 90 and 115 in HD, the latter antibodies will not label the aggregates that are characteristic of HD and thus will not differentiate between HD and non-HD CNS tissue. With rare exceptions, this holds true in the studies listed in and . The 1C2 antibody is unique in that its epitope should be present within an HD neuron regardless of where mHtt is cleaved and which fragments of the protein aggregate. But clearly, LGS and therefore 1C2 binding are not specific for HD; 8 other CAG repeat diseases (spinal bulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and spinocerebellar atrophy 1, 2, 3, 7, 8, and 17) are currently known to contain expanded CAG repeats that are translated in LGS (25
Immunohistochemical technique is also a worthwhile consideration when working with HD antibodies. Technical methods, especially for epitope retrieval, have repeatedly been shown to play an important role in the interpretability and reproducibility of immunohistochemical staining. Recent work by BrainNet Europe centers demonstrates that variability in alpha-synuclein staining between laboratories can significantly influence interrater reliability (26
). 1C2-immunoreactive intranuclear inclusions were a prominent finding in many of the CNS locations that we studied, but they have not been a consistent finding in other studies. Yamada et al (18
) and Rudnicki et al (19
), but not Gourfinkel-An et al (9
) or Sieradzan et al (12
), identified neuronal intranuclear inclusions in their studies. Technical differences in epitope retrieval and staining methods may explain these dissimilar results although Yamada et al did not describe their methods. We and others have found that formic acid pretreatment of CNS tissue improves antigenicity for 1C2 (19
, 29). Our use of chemical and heat pretreatment may have unmasked epitopes within the neuronal nucleus that had been undetectable in other studies.
Our finding of 1C2 immunoreactivity in neuromelanin-containing neurons and adjacent macrophages in normal and diseased controls raises interesting and important questions about the nature of these neurons and the epitope recognized by 1C2. Aside from these areas, we have identified widespread, reproducible 1C2 immunoreactivity in postmortem CNS tissue of individuals with HD that is absent in controls. Compared to staining with the 2B4 anti-huntingtin antibody, 1C2 may therefore be of diagnostic utility in postmortem CNS evaluation for HD when other CAG repeat diseases are not under consideration and when the substantia nigra, locus coeruleus, and pituitary gland are avoided.