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Recent discoveries have led researchers in a new direction in ALS and IBM research.
Background: DNA sequences contain the raw genetic information needed by the cell in order to carry out its functions and maintain proper protein levels. This information has to be extracted from the DNA inside the nucleus of the cell and moved into the cytoplasm where proteins are produced. These processes involve a large number of RNAs and RNA binding proteins, collectively known as ribonucleoprotein complexes (RNPs).
Processing of DNA occurs in two steps, transcription that occurs inside the nucleus of the cell and translation which occurs within the cytoplasm in the body of the cell. The information in DNA is contained in small bits called exons that will become part of the final genetic message, as well as introns which are pieces of sequence that are cut out of the final message. In step one, transcription, many RNAs assist in the process of splicing the DNA apart, separating out exon sequences and producing a strand of messenger RNA that will ultimately be transported out of the nucleus of the cell. When the messenger RNA arrived in the cytoplasm, the second step of DNA expression takes place -- translation. In translation, the code of the messenger RNA is used as a message template to manufacture different kinds of protein, again with the assistance of many different RNAs, including ribosomal RNA and transfer RNAs.
Ribonucleoprotein complexes consist of many different types of RNA including, mRNAs, noncoding RNAs, and proteins that either directly or indirectly associate with these RNAs. TAR DNA binding protein (TDP-43) is one such protein. These proteins are very widely distributed throughout the cells of the body.
Normally, proteins interact with each other and form larger complexes by joining together, processes that are usually well controlled. Normally, proteins that are damaged or somehow misfolded are digested by a cellular garbage disposal unit called the proteasome that breaks the proteins apart and recycles their parts. Problems can develop under the influence of environmental, genetic or epigenetic factors leading to the abnormal aggregation of protein -- lumps of protein that the proteasomes cannot digest. These abnormal accumulations of protein then continue to build in size, eventually disrupting the operation of the cell. A number of neurodegenerative and neuromuscular diseases are characterized by the accumulation of abnormal protein aggregations. Traditionally, beta amyloid, tau and alpha synuclein proteins have been associated with these defective accumulations of protein. Recently, TDP-43 aggregation has been associated with a number of neurodegenerative disorders, including; Alzheimer, ALS, and inclusion body myositis. Normally, TDP-43 is found in the nucleus of the cell but in these disorders TDP-43 is found in the cytoplasm implying a pathology related to the function of this protein.
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TDP-43, a DNA-binding protein, is usually found in the cell's nucleus and is presumed to have a role in managing the translation of DNA into RNA and thence into proteins.
Mutations in TDP-43, a DNA/RNA-binding protein, cause an inherited form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS).
Understanding of ALS pathogenesis began with the landmark discovery of dominant causative mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1) in ~20% of familial ALS cases and ~1% of sporadic cases. Most efforts to understand ALS pathogenesis over the past 15 years have focused on mutations in SOD1.
A shift in our understanding of ALS pathogenesis began with the identification of the TAR DNA-binding protein (TDP-43) as a major component of ubiquitinated protein aggregates found in many patients with sporadic ALS or the most common form of frontotemporal dementia called FTLD-U (frontotemporal lobar degeneration with ubiquitinated inclusions). In ALS and FTLD-U patients, TDP-43 immunoreactive inclusions are observed in the cytoplasm and nucleus of both neurons and glial cells.
TDP-43 inclusions are now recognized as a common characteristic of most ALS patients. It is unclear whether aggregation of TDP-43 is a primary event in ALS pathogenesis or whether it is a byproduct of the disease process.
Accumulation of intracellular or extracellular misfolded or misprocessed proteins in the central nervous system is a feature of many neurodegenerative conditions.
The gene encoding TDP-43 on chromosome 1 (chromosome 1p36), TARDBP, constituted an excellent candidate for direct sequencing in search of disease-causing mutations and starting in early 2008, dominant mutations in the TARDBP gene were reported as a primary cause of ALS. These studies collectively provided persuasive evidence that the aberrant form of TDP-43 can directly trigger neurodegeneration. The identification of TDP-43 in ALS fueled another discovery of additional ALS mutations in a gene encoding another DNA/RNA-binding protein called FUS (fused in sarcoma) or TLS (translocation in liposarcoma) and the identification of a dominant missense mutation (R521C) in the FUS/TLS gene. Like TDP-43, FUS/TLS is almost ubiquitously expressed. It is mainly localized in the nucleus, but cytoplasmic accumulation has been detected in most cell types.
Importantly, TDP-43-positive inclusions are absent in ALS patients with FUS/TLS mutations, implying that neurodegenerative processes driven by FUS/TLS mutations are independent of TDP-43 aggregation. It will now be essential to assess FUS/TLS accumulation and localization in ALS patients with TDP-43 mutations, as well as in patients with other neurodegenerative diseases, especially those with mislocalized TDP-43.
The identification of causative TDP-43 and FUS/TLS mutations, along with TDP-43 pathology in most cases of ALS, represents a dramatic shift in our understanding of this disease.
The lessons to be learned for TDP-43 and FUS/TLS in triggering neurodegenerative disease will not be unique to ALS. Abnormal TDP-43 inclusions have been reported for several other neurodegenerative conditions, including in ~30% of Alzheimer's disease patients.
Discovery of the involvement of TDP-43 and FUS/TLS in ALS and other neurodegenerative diseases reinforces the role of altered RNA processing in neurodegeneration. Earlier well established examples of altered RNA processing in neurodegeneration include errors in RNA metabolism due to loss of the SMN (survival of motor neuron) protein in spinal muscular atrophy and of FMRP in fragile-X mental retardation.
In addition, an RNA gain-of-function mechanism has been implicated in a set of diseases including the myotonic dystrophies, where a transcript with an abnormal repeat expansion alters the function and localization of alternative splicing regulators.
Reference for above: Rethinking ALS: The FUS about TDP-43. Cell, Volume 136, Issue 6, 20 March 2009, Pages 1001-1004 Clotilde Lagier-Tourenne, Don W. Cleveland.
Using an in vitro yeast model, researchers now report that TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS and FTLD-U patients. The C-terminal domain of TDP-43 is critical for spontaneous aggregation. TDP-43 is intrinsically aggregation-prone and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations. This supports elevating TDP-43 to the ranks of disease-linked aggregators such as amyloid-beta and tau in Alzheimer disease and alpha-synuclein in Parkinson's. Although there is a correlation between TDP-43 mutations, aggregation, and disease, the experiments do not conclusively show that aggregation directly leads to disease.
Reference for above: TDP-43 is intrinsically aggregation-prone and ALS-linked mutations accelerate aggregation and increase toxicity. J Biol Chem. 2009 May 22. Johnson BS, Snead D, Lee JJ, McCaffery JM, Shorter J, Gitler AD.
TDP-43 multisystem proteinopathy Immunohistochemical scans of 64 patients strongly suggest that amyotrophic lateral sclerosis, frontotemporal lobar degeneration with amyotrophic lateral sclerosis or motor neuron disease, and frontotemporal lobar degeneration with ubiquitinated inclusions are different manifestations of a multiple-system TDP-43 proteinopathy linked to similar mechanisms of neurodegeneration.
Reference for above: Clinical and Pathological Continuum of Multisystem TDP-43 Proteinopathies. Arch Neurol. 2009;66(2):180-189. Felix Geser, MD, PhD; Maria Martinez-Lage, MD; John Robinson, BS; Kunihiro Uryu, PhD; Manuela Neumann, MD; Nicholas J. Brandmeir, MS; Sharon X. Xie, PhD; Linda K. Kwong, PhD; Lauren Elman, MD; Leo McCluskey, MD; Chris M. Clark, MD; Joe Malunda; Bruce L. Miller, MD; Earl A. Zimmerman, MD; Jiang Qian, MD; Vivianna Van Deerlin, MD, PhD; Murray Grossman, MD, EdD; Virginia M.-Y. Lee, PhD, MBA; John Q. Trojanowski, MD, PhD.
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TAR DNA binding protein-43 (TDP-43) is found in ubiquitinated inclusions (UBIs) in some frontotemporal dementias (FTD-U). One form of FTD-U, due to mutations in the valosin containing protein (VCP) gene, occurs with an inclusion body myopathy (IBMPFD). Since IBMPFD brain has TDP-43 in UBIs, we looked for TDP-43 inclusions in IBMPFD muscle. In normal muscle, TDP-43 is present in nuclei. In IBMPFD muscle, TDP-43 is additionally present as large inclusions within UBIs in muscle cytoplasm. TDP-43 inclusions were also found in 78% of sporadic inclusion body myositis (sIBM) muscles. In IBMPFD and sIBM muscle, TDP-43 migrated with an additional band on immunoblot similar to that reported in FTD-U brains. This study adds sIBM and hereditary inclusion body myopathies to the growing list of TDP-43 positive inclusion diseases.
Reference for above: TDP-43 accumulation in inclusion body myopathy muscle suggests a common pathogenic mechanism with frontotemporal dementia. J Neurol Neurosurg Psychiatry. 2008;79:1186-1189.Weihl CC, Temiz P, Miller SE, Watts G, Smith C, Forman M, et al.
Overexpression of TDP-43 has been reported in several conditions with cellular overexpression, including sporadic and hereditary IBM. FUS/TLS protein has not yet been studied in IBM. Nevertheless, alteration in RNA processing and protein aggregates appears to be the key event in these motor neuron and muscle diseases.
Over the past year, TDP-43 immunoreactive UBIs have been reported in sIBM 7, 15 [see references below] and in familial myopathies with or without vacuolar change.
In this [July 2009] issue of Muscle and Nerve, Salajegheh and colleagues [see reference below] confirm and extend the recent observations 7, 12, 15 of excessive TDP-43 in IBM muscle fibers.
First, the authors found an average of nearly 1 in 4 IBM muscle fibers were immunoreactive to sarcoplasmic TDP-43. This makes the TDP-43 marker positivity a far more frequent finding than the presence of rimmed vacuoles, fluorescent Congo red material or other previously described markers (mostly in less than 1% of fibers). Second, TDP-43 inclusions were also demonstrated in muscle fibers of hIBM and myofibrillar myopathies. Coupled with similar findings in other recent reports,7,12,15 this suggests a common element in the pathogenesis of these myopathies. TDP-43 is so far the most sensitive and specific diagnostic immunohistochemistry marker for IBM. Third, no TDP-43 gene mutation was discovered in this report. [However, no TDP-43 gene mutation was initially discovered in a small cohort of sALS cases. However, it is now well known that TDP-43 mutations have been identified in fALS and sALS cases.]
It is unclear whether the TDP-43 aggregates in IBM are just trashed proteins, or whether they are pathogenic through either gain of one or more toxic properties or loss of normal function from its sequestration in the cytoplasm and corresponding disruption of its interaction with nuclear protein partners and RNA targets.
The striking specificity and high degree of sensitivity, in concert with other recent reports, could make TDP-43 a standard diagnostic marker for IBM.
A number of questions remain unanswered, however. It is unknown if protein aggregates are a cause or consequence of the disease process. It will be interesting to see if FUS/TLC aggregates also occur in IBM muscle fibers, and if mutations in any of the DNA/RNA binding proteins (TDP-43, FUS/TLC, and others) are discovered in familial or sporadic IBM.
Finally, a better understanding of the role of TDP-43 in IBM may be crucial to the development of targeted therapeutic strategies in IBM.
Reference for above: TDP-43: A reliable immunohistochemistry marker for inclusion body myositis? Muscle Nerve 40: 8-9, 2009 Ashok Verma, and Rup Tandan
The nucleic acid binding protein TDP-43 was recently identified in normal muscle nuclei and also in nonnuclear sarcoplasm and around some rimmed vacuoles in IBM and inclusion body myopathy with Paget's disease and frontotemporal dementia (IBMPFD).
In this study we found that extranuclear sarcoplasmic immunoreactivity of the normally nuclear protein TDP-43 is a prominent and highly sensitive and specific feature of IBM among the inflammatory myopathies.
In IBM we found that sarcoplasmic accumulation of TDP-43 was accompanied by its nuclear depletion (present in 12% of myonuclei of such fibers compared to 99% of myonuclei in fibers lacking sarcoplasmic accumulation), suggesting redistribution of this molecule from the myonucleus to the sarcoplasm.
Reference for above: Sarcoplasmic redistribution of nuclear TDP-43 in inclusion body myositis. Muscle Nerve. 2009; 40(1):19 -31. Salajegheh M, Pinkus JL, Taylor JP, Amato AA, Nazareno R, Baloh RH, Greenberg SA.
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7. TDP-43 accumulation is common in myopathies with rimmed vacuoles. Acta Neuropathol. 2009;117:209-211. Kusters B, van Hoeve BJ, Schelhaas HJ, Ter Laak H, van Engelen BG, Lammens M.
12. TAR DNA-Binding protein 43 accumulation in protein aggregate myopathies. J Neuropathol Exp Neurol. 2009; 68:262-273. Olive M, Janue A, Moreno D, Gamez J, Torrejon-Escribano B, Ferrer I.
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