Ubiquitin-positive protein inclusions are a hallmark of frontotemporal dementias and other neurodegenerative diseases. In some cases, the aggregating proteins are known, such as the tau protein in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer disease, or α-synuclein in Parkinson disease. But in many diseases, the makeup of the clumps of protein dotting the nucleus, cytoplasm or neurites is a mystery.

In a paper out today in Science, Virginia Lee and coworkers at University of Pennsylvania School of Medicine in Philadelphia report a positive ID of a new offender, responsible for inclusions in two different neurodegenerative diseases. The protein, TDP-43, is cleaved, hyperphosphorylated, and ubiquitinated in inclusion bodies in both frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). The results suggest that these two diseases may have a common pathological basis with TDP-43 at its root.

To identify their suspect, the first authors Manuela Neumann, Deepak Sampathu, and Linda Kwong made monoclonal antibodies to inclusion proteins. With the antibodies, they defined three subtypes of FTLD-U, each of which showed a disease-specific protein which reacted to a different monoclonal antibody after gel electrophoresis. By mass spectrometry analysis, they identified the protein recognized by two of the monoclonal antibodies as the TDP-43 protein. Subsequently, they showed that a polyclonal antibody raised against TDP-43 reacted with the inclusions in all three types of FTLD-U, and also inclusions in brain tissue from two different kindreds of FTDP-17U with progranulin mutations (see ARF related news story). In contrast, TDP-43-containing inclusions were not detected in either control or Alzheimer disease brains.

The TDP-43 protein in inclusions was both phosphorylated and ubiquitinated. In disease tissues, extracted protein appeared as fragments, with some full-length and high-molecular-weight aggregates as well. A product of the TARDP gene on human chromosome 1, TDP-43 is a highly conserved, ubiquitously expressed nuclear protein. It contains RNA-recognition motifs and may function in splicing. Pathological accumulation is accompanied by a movement of the protein into the cytoplasm, which may interfere with its nuclear functions.

Clinical and pathological evidence points to ALS being a different manifestation of the same neurodegenerative process as FTLD-U, and this idea was strengthened by the investigators’ finding that inclusions in ALS also contained TDP-43. All inclusions in motor neurons were stained with the TDP-43 antibody, as were formations in the hippocampus and cortex, from both sporadic and familial ALS cases.

“The identification of TDP-43 as the major component of UBIs [ubiquitin positive inclusions] specific to sporadic and familial FTLD-U as well as sporadic ALS resolves a long-standing enigma concerning the nature of the ubiquitinated disease proteins in these disorders. Thus, these diseases may represent a spectrum of disorders that share similar pathological mechanisms, culminating in the progressive degeneration of different selectively vulnerable neurons.” The identification of TDP-43 should lead to new insights into these diseases.—Pat McCaffrey

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  1. From a clinical perspective, the identification of TDP-43 protein represents a major breakthrough in our understanding of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The TDP-43 is the mystery protein that is associated with the ubiquitin-positive inclusions that are commonly found in many patients with FTLD and in most, if not all, patients with ALS.

    This finding is particularly important because several recent papers suggest that patients who have FTLD with ubiquitin inclusions at autopsy (FTLD-U) account for approximately 50 percent of all autopsy-confirmed FTLD cases (1-3). The remaining majority of FTLD cases are associated with the tau protein, but other neuropathological diagnoses exist. The finding that possibly one-half of all FTLD patients may have ubiquitin-positive neuropathology means that any breakthroughs in the biology of this protein could potentially translate into helping a large proportion of FTLD patients.

    In addition, the finding that the TDP-43 protein is also found in patients with ALS further supports the overlap between FTLD and ALS. Future research on the TDP-43 protein will likely also benefit ALS patients and help us understand how these two very different clinical phenotypes are related.

    References:

    . Frontotemporal lobar degeneration with motor neuron disease-type inclusions predominates in 76 cases of frontotemporal degeneration. Acta Neuropathol. 2004 Nov;108(5):379-85. PubMed.

    . Frontotemporal lobar degeneration: demographic characteristics of 353 patients. Arch Neurol. 2005 Jun;62(6):925-30. PubMed.

    . Frontotemporal dementia: clinicopathological correlations. Ann Neurol. 2006 Jun;59(6):952-62. PubMed.

  2. In this paper, Drs. Lee and Trojanowski and colleagues have at long last identified the mystery protein hiding within the ubiquitinated inclusions that characterize certain histological forms of frontotemporal lobar degeneration (FTLD), termed FTLD-U. This task has challenged neuroscientists for well over a decade, with all prior attempts at identification using immunohistochemical or biochemical methods proving fruitless. The culprit protein is a TAR DNA-binding protein, known as TDP-43. This protein is present within all the ubiquitinated structures in FTLD-U, viz., the neuronal cytoplasmic inclusions, the neuronal intranuclear inclusions, and the neuritic changes, though whether this is the sole component of these structures (other than ubiquitin) remains uncertain. Some previous studies reported the presence of p62 protein within neuronal cytoplasmic inclusions, but such findings have been inconsistent. Moreover, Lee and Trojanowski have shown that the ubiquitinated neuronal cytoplasmic inclusions seen within spinal and cranial nerve nuclear motor neurons in motor neuron disease (amyotrophic lateral sclerosis) also contain TDP-43.

    This is an immensely important study with huge implications for neurobiology.

    Firstly, it pinpoints a key biochemical constituent in the pathogenesis of FTLD-U and motor neuron disease (MND), and one which previous work would never have regarded as a likely candidate protein.
    Secondly, although an association between FTLD and MND had long been known on account of some cases showing defined clinical features of both disorders, sharing pathological features of both disorders, and families being known where some members had FTLD, others MND, and others the combined disorder, it was never clear whether this association was coincidental or causal. Now we can see causality, and the implication that FTLD and MND are part and parcel of the same disease spectrum will have major ramifications for understanding pathogenesis, and eventual treatment.
    Thirdly, the finding of TDP-43 pathological changes in FTLD patients with mutations in the newly identified progranulin (PGRN) gene, who typically show FTLD-U pathological changes, firmly brings together a causal relationship in these two fundamental proteins in driving the pathogenesis of the disorder, and opens up untapped vistas of neurobiological research.

    Therefore, in rapid time, two major (protein) pieces in the jigsaw puzzle of FTLD have been identified. The challenge now will be to fit the pieces around these and eventually identify the linking processes that bring these together into the fuller picture. Nonetheless, it is clear that even within FTLD-U there are different histological and clinical phenotypes, and it will be necessary to dissect out biochemical or other factors that might determine where the TDP-43 pathological changes take place in the brain to produce the clinical phenotype. That is, why is it that in some patients the most common clinical manifestation of FTLD-U, frontotemporal dementia, is present in association with bilateral involvement of the frontal and temporal lobes, yet in others only the temporal lobes are affected—producing semantic dementia—and in others the left hemisphere is preferentially affected to give progressive non-fluent aphasia. Also, what determines whether TDP-43 changes will be in the brainstem and spinal cord to give MND, or in the cerebral cortex to give FTLD? Lastly, in all this flurry of excitement, it should not be forgotten that tauopathy is still a major cause of FTLD, and it is not immediately apparent how pathological changes in the expression or function of tau might link in with progranulin and TDP-43. Clearly, changes in all three molecules can produce the same disorder of FTLD either separately or collectively: it is not possible to unequivocally discriminate FTD patients with MAPT mutations from those with PGRN mutations, or others without mutations in either. Interrelationships within this Bermuda triangle of tau, progranulin, and TDP-43 will need to be addressed.

    The identification of TDP-43 as a (major/sole) component of the ubiquitinated protein of FTLD and MND, in conjunction with the identification of mutations in PGRN, have opened up huge new fields within the neurobiology of neurodegenerative disease with tentacles that may stretch far wider than these two disorders themselves. Whether there is a role for either or both of these proteins in other disorders like Alzheimer disease and Parkinson disease remains to be seen. The gauntlet has been cast down—it is up to the neuroscience community to pick this up and address these issues. What is certain is that there will be a major change in the focus of neurobiological research as groups worldwide seek to investigate the implications of changes in proteins such as progranulin and TDP-43 in terms of health and disease. We can look forward within the near future to major advances in our understanding of how the brain works in respect of these molecules and why neurodegenerative disease occurs when they fail to function properly. Maybe even a treatment for neurodegenerative disease may come a little closer.

  3. Neumann, Sampathu, Kwong, and colleagues have resolved a long-standing issue in the research field of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). These authors have identified TDP-43 as a major component of ubiquitin-positive inclusions that characterize these disorders. They first extracted a fraction from the patients' brains using monoclonal antibodies and then analyzed it by mass spectrometry. Their findings have greatly facilitated the understanding of the molecular pathogenesis of FTLD and ALS.

    Independently, we have also found TDP-43 as a component of the inclusions in FTLD [1]. Following electrophoresis of the sarkosyl-insoluble brain extracts from FTLD, Alzheimer disease (AD) and dementia with Lewy bodies (DLB), we have done exhaustive analyses by mass spectrometry. Following identification of each molecule that is more abundant in FTLD than AD/DLB, we have studied FTLD brain samples immunochemically and immunohistochemically. The antibodies to TDP-43 have immuno-stained neuronal inclusions and dystrophic neurites in the hippocampus and the temporal cortex in FTLD, and skein-like inclusions in the spinal cord in FTLD and ALS. Immunoblotting of the sarkosyl-insoluble fraction has shown abnormal changes in TDP-43 including hyperphosphorylation, fragment formation, and smear-like staining, all of which are similar to abnormal tau in AD and suggest a central role for the formation of abnormal aggregates. These findings are comparable with those by Lee's group. This is not surprising, since both groups have employed principally the same polyclonal antibody which is the only commercially available rabbit polyclonal.

    In addition, however, we have found TDP-43-positive glial inclusions in the spinal cord in FTLD and ALS. These inclusions were also positive for tau. The distribution of glial inclusions was consistent with the degenerating areas, suggesting that glial abnormalities are involved in the pathological processes of ALS and FTLD. A difference between our results and theirs is the TDP-43-positive staining of some, but not all, tau-positive structures including Pick bodies and neurofibrillary tangles. The significance of these findings remains to be established, since immunoblot analysis did not show any abnormality in TDP-43 in Pick disease and Alzheimer disease. Our paper will appear shortly in Biochem Biophys Res Commun [1].

    In the case of tau and α-synuclein, detection of abnormally modified molecules has revealed far more extensive pathology than that seen by ubiquitin immunohistochemistry. While lesions immunohistochemically labeled for TDP-43 are a little more numerous than those labeled for ubiquitin, the difference is far less than that we have experienced for tau and α-synuclein immunohistochemistry. This may be a point that remains to be cleared up. Another issue that is open for further investigations is to prove, by protein chemistry, the ubiquitination of TDP-43.

    In any event, it has to be emphasized that two different approaches have come to the same conclusion, establishing with certainty that TDP-43 is the major component of the inclusions in FTLD and ALS. This further strengthens the hypothesis that these disorders are part of a clinicopathological spectrum that shares similar pathogenesis, and suggests the possibility that TDP-43 may be a common therapeutic target for these disorders. It is now necessary to investigate the relationship of TDP-43 to other molecules that have been reported to be associated with familial FTD, FTD with motor neuron disease, or ALS. Such molecules include progranulin, charged multivesicular body protein 2B (CHMP2B), valosin-containing protein, dynactin, and an unidentified protein in familial disease linked to chromosome 9.

    References:

    . TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun. 2006 Dec 22;351(3):602-11. PubMed.

References

News Citations

  1. Birds of a Feather…Mutations in Tau Gene Neighbor Progranulin Cause FTD

Further Reading

Papers

  1. . Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies. Am J Pathol. 2006 Oct;169(4):1343-52. PubMed.

Primary Papers

  1. . Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. PubMed.