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Short Communications |
From the University of Southern California Neuromuscular Center, Department of Neurology, University of Southern California Keck School of Medicine, Good Samaritan Hospital, Los Angeles, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unknown in both IBM and AD are the factors leading to the abnormal accumulation of various proteins in the respective tissues, including the yet unidentified molecular pathways responsible for PHF formation. Recently it was demonstrated that AD neurofibrillary tangles (composed of PHFs) contain RNA13 and that RNA in vitro promotes assembly of tau into PHFs.14
We have now determined that clusters of IBM-PHFs contain RNA and a known RNA-binding protein, survival motor neuron (SMN).15 We previously demonstrated that in human muscle biopsies SMN is expressed at normal neuromuscular junctions postsynaptically16 in regenerating muscle fibers and in denervated, very atrophic, apoptotic-like fibers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies were performed on 10-µm transverse sections of fresh-frozen diagnostic muscle biopsies of 19 patients with these diagnoses: 11 s-IBM, 2 non-IBM vacuolar myopathies of unknown cause, 3 morphologically nonspecific myopathies, 1 polymyositis, 2 dermatomyositis, and 4 normal muscle biopsies. All IBM biopsies had muscle fibers containing tau-positive PHFs and congophilia.1
Acridine Orange (AO) Staining
This was performed as described by Miike et al.17 Sections were fixed in ether:95% ethanol (1:1) for 1 hour, rehydrated, rinsed, incubated in 1% acetic acid, rinsed, and stained for 3 minutes in 0.01% AO in 0.1 mol/L phosphate buffer, pH 6.0. The sections were then rinsed, incubated 1 minute in 0.1 mol/L calcium chloride, rinsed, and mounted in phosphate buffer. Fluorescence microscopy used a Zeiss Axiophot microscope with a BP 450–490 exciter filter, LP 520 barrier filter, and FT 510 chromatic beam-splitter.
Double Localization of AO and SMI-31 Immunoreactivity on the Same Section
Sections were incubated for 1 hour in SMI-31 mouse monoclonal antibody (Sternberger Monoclonals Inc., Baltimore, MD, diluted 1:1000), which recognizes phosphorylated tau in IBM PHFs,6,18 followed by incubation in a rabbit anti-mouse serum conjugated to 7-amino-4-methylcoumarin-3-acetic acid (AMCA) (DAKO, Carpinteria, CA). Subsequently the sections were rinsed, fixed in ether: 95% ethanol (1:1), and processed for AO staining as above. The blue color of AMCA was visualized with a G 365 exciter filter, LP 420 barrier filter and FT 395 chromatic beam-splitter, and the orange color of AO histofluorescence was visualized as above.
SMN Immunocytochemistry
For light microscopy, SMN was immunolocalized using two mouse monoclonal anti-SMN antibodies: 2B1, a gift from Dr. Gideon Dreyfuss, diluted 1:50, and an antibody directed against residues 14–174 of human SMN (Transduction Laboratories, Lexington, KY), diluted 1:20. We have previously described immunocytochemistry of SMN and its specificity in non-IBM human muscle biopsies.16 Electronmicroscopic immunocytochemistry of SMN was performed according to our previously established technique.4-6,18,19
Control Experiments
To evaluate whether orange AO histofluorescence was specific to RNA, before AO staining the sections were pretreated for 4 hours at 37°C with either 200 U/ml RNase-A solution (Ambion Inc., Austin, TX), containing 20 mmol/L of the protease-inhibitor phenylmethylsulfonyl fluoride (PMSF; Sigma, St. Louis, MO), or 10,000 U/ml RNase-free DNase solution (Roche Molecular Biochemicals, Indianapolis, IN). To determine whether SMN immunoreactivity reflects binding of the antigen to RNA, SMN immunohistochemistry was performed after the sections were pretreated with RNase solution as above. To block nonspecific binding of antibodies to Fc receptors, the sections were preincubated in normal goat serum diluted 1:10, as described.4-6,16,18,19 Omission of the primary antibody or replacement of the primary antibody with a nonrelevant serum controlled for specificity of the immunohistochemical reactions, as described.16,18,19
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AO positivity in this study refers only to the orange fluorescence
of RNA. In s-IBM, 70 to 80% of the vacuolated muscle fibers (VMFs),
and some of the non-VMFs, had AO positivity in the form of focal,
well-demarcated squiggly, rod-like, and dot-like cytoplasmic inclusions
(Figure 1, A
-D, F, H, J, and L).
Pretreatment with RNase-A, but not with DNase, completely abolished the
AO positivity of more than 95% of the inclusions (Figure 1, K
-M);
rare inclusions retained weakly positive AO staining.
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Double Localization of AO Positivity and SMI-31 Immunoreactivity
In all IBM muscle biopsies, AO positivity of the cytoplasmic
inclusions colocalized with SMI-31 immunoreactivity (Figure 1, D
-I).
Pretreatment with RNase-A did not influence the SMI-31
immunoreactivity.
SMN Immunoreactivity
By light microscopy, 70 to 80% of the IBM-VMFs, and some of the
non-VMFs, had strongly SMN-immunoreactive squiggly, rod-like, and
dot-like inclusions (Figure 2, A
-C and
E). RNase pretreatment performed on serial sections either abolished or
prominently diminished SMN immunoreactivity (Figure 2, D and F)
.
Ultrastructural immunocytochemistry of the IBM-VMFs revealed that SMN
was immunolocalized only to the clusters of PHFs (Figure 3, A and B)
. PHFs appear as
tubulofilaments when horseradish peroxidase is used as the
immunohistochemical marker.5,6,18,19
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under ultraviolet excitation, AO that is intercalated into RNA gives a bright orange fluorescence, whereas AO staining of DNA gives a yellow fluorescence.20 Cytoplasm lacking abundant RNA and DNA gives a pale green background. Individual subtypes of accumulated RNA (ie, mRNA, rRNA, tRNA) cannot be distinguished by this method. The IBM muscle biopsies emitted orange AO fluorescence of squiggly, rod-like, and dot-like inclusions against a pale green background of nonstained cytoplasm. RNase, but not DNase, pretreatment abolished the AO labeling of more than 95% of the inclusions, indicating that their AO positivity was specific for RNA. (The RNase pretreatment did not totally abolish orange fluorescence of the occasional inclusions, perhaps because the RNase solution did not completely penetrate some tight clusters of PHFs, or because, in them, a fraction of RNA was more tightly complexed with PHF proteins.) The close colocalization of AO positivity with the immunoreactivity achieved with SMI-31 antibody recognizing IBM-PHFs suggests that the AO positivity reflects RNA on or within clusters of PHFs.
The mechanism of RNA accumulation within the clusters of IBM-PHFs is not known. In normal cells, some RNA associates with proteins of the cellular cytoskeleton.21 For example, the microtubule-associated protein MAP 1A is one component of the RNA-cytoskeleton complex.22 Normal tau is a microtubule-associated protein. When associated with tau, tubulin polymerizes into microtubules and by doing so prevents tau aggregation.14,23 Phosphorylated tau (P-tau) has a decreased ability to interact with tubulin, a phenomenon proposed to be important in causing P-tau aggregation into PHFs in AD brain.23 Free RNA can compete with tubulin for the cellular pool of tau, and in vitro RNA stimulates aggregation of tau into PHFs.14 In IBM muscle fibers, possibilities include: i) that phosphorylation of tau diminishes its interaction with tubulin and increases its association with RNA, and this process facilitates formation of PHFs; ii) that RNA has an abnormal propensity to associate with tau, resulting in the pathological phosphorylation of tau and aggregation of it into PHFs; or iii) that RNA is being sequestered in the PHFs due to the presence there of a non-tau RNA-binding protein, eg, SMN.
SMN is a recently discovered protein, encoded by a gene on chromosome 5q13, mutations of which are associated with, and putatively responsible for, more than 98% of the autosomal-recessive spinal muscular atrophy patients.24 Normal biological functions of SMN are largely unknown, but SMN is known to bind directly to RNA and to RNA-binding proteins.15,25 A role of SMN has also been proposed in RNA processing, in the biogenesis of small nuclear ribonucleic proteins, and in pre-mRNA splicing.25 In human muscle, SMN is developmentally regulated and, based on its immunolocalization, it probably plays a yet unknown role at normal nerve-muscle postsynaptic regions, in regenerating muscle fibers, and in post-denervation apoptosis-like processes of human muscle fibers.16 In IBM muscle the immunoreactivity of SMN was localized to PHFs, and it was abolished or greatly diminished after pretreatment with RNase. We therefore postulate that on PHFs the SMN is present, at least partially, through its association with RNA, and/or another protein already bound to PHF-linked RNA. For example, it would be interesting to determine whether small nuclear ribonucleoproteins (snRNPs) involved in spliceosome assembly are also accumulated within the clusters of PHFs, which might represent presumably inactive foci of these splicing elements.
PHFs in both AD brain and IBM muscle contain accumulation of several proteins, including markers of oxidative stress.12,26 Molecular mechanisms leading to accumulation of those proteins and their role in the pathogenic cascade of both IBM and AD remain unknown. Several mechanisms, including RNA binding and oxidative damage, have been proposed in relation to PHF formation in AD brain.14 RNA has been shown to be oxidized in AD brain.27 Whether RNA is oxidized in IBM muscle fibers is not yet known.
Our present study demonstrates two novel components of IBM PHFs, and it raises the possibility that RNA and possibly the RNA-binding protein SMN participate in PHF formation in IBM.
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Acknowledgements |
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Footnotes |
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Supported in part by grants from the National Institutes of Health (NS34103 and AG16768) and the Muscular Dystrophy Association (all to V. A.).
Accepted for publication December 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in inclusion-body myositis muscle and at normal human neuromuscular junctions. Neurosci Lett 1998, 254:77-80[Medline]
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