Gladstone scientists identify role of key protein in ALS and frontotemporal dementia
January 12, 2010 Scientists at the Gladstone Institute of Neurological Disease (GIND) have identified the reason a key protein plays a major role in two neurodegenerative diseases. In the current edition of the Journal of Neuroscience, researchers in the laboratory of GIND Associate Director Steven Finkbeiner, MD, PhD have found how the protein TDP-43 may cause the neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusion bodies (FTLDu). TDP-43, is the major component of protein aggregates in patients with these diseases. Mutations in the TDP-43 gene are also associated with familial forms of ALS and FTLDu.

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"TDP-43 is a very exciting protein. We found that its location in the cell is a good indicator of the damage it may cause," said Finkbeiner, senior investigator and senior author on the study. "Our findings and our experimental model will allow further studies of this protein and how it results in disease."

Under normal circumstances, TDP-43 is a common protein that stays mostly in the nucleus. It has several beneficial functions, including binding DNA and RNA, inhibiting retroviruses, and helping with RNA splicing and nuclear body formation. It also shuttles mRNA to the cytoplasm.

However, in patients with ALS and FTLDu, TDP-43 is redistributed from the nucleus to the cytoplasm and forms insoluble TDP-43 aggregates in the nucleus, cytoplasm, or neuronal processes.

The Finkbeiner team developed a model system to find out how TDP-43 might be involved in neurodegenerative diseases. They used genetic engineering to add a fluorescent tag to normal or wildtype and mutant TDP-43 in rat neurons. The tag allowed them to easily see the intracellular location of the protein.

To determine the effects of the mutant protein, the researchers used an automated microscope that can examine hundreds of thousands of neurons individually over several days. With this large amount of data, they could use sophisticated statistical analyses to follow the fate of each individual neuron and determine its risk of death at any given time.

Their experimental system used primary neurons. These neurons are taken directly from an animal to a culture dish and provide the best cells for experiments because they retain many of the features of cells in the intact brain. In fact, Dr. Finkbeiner's system showed many "normal" features of TDP-43 in neurons. For example, wildtype TDP-43 was found in the nucleus in healthy neurons. Mutant TDP-43 was also found in the nucleus, but there was more of the protein in the cytoplasm.

Several neurons developed aggregates of the protein called inclusion bodies, which are often found in diseased neurons. In addition, the system can be easily manipulated by the investigators, making it a valuable tool for dissecting the biological mechanisms underlying diseases associated with TDP-43 deposition.

"We expect this system to be very helpful to other investigators," explained Finkbeiner.

The researchers found that the mutant TDP-43 was toxic to neurons and that more of it was found in the cytoplasm. Although the mutant protein formed inclusion bodies, these did not affect the risk of cell death. However, the amount of cytoplasmic TDP-43 was a strong and independent predictor of neuronal death. Using genetic manipulations, they showed that targeting wild-type TDP-43 to the cytoplasm is sufficient to recreate the toxicity associated with mutant TDP43. On the other hand, the toxic effect of the mutant protein could be blunted by preventing its export from the nucleus. It seems as if the toxicity of the mutation depends on cytoplasmic mislocalization of TDP-43.

"Our results indicate that the mutant protein is mislocalized to the cytoplasm," Finkbeiner said. "Although we don't know the underlying mechanism, the protein seems to become toxic in the cytoplasm and then causes death of the neuron."

Hi ENV,

I'd asked in one topic whether Leptomycin B, an inhibitor of nuclear export may be beneficial.

It's of interest that lysyl-tRNA synthetase which interacts with mSOD1 at the cytoplasmic side of mitochondrial membranes is incorporated into HIV-1 and this study mentions that one of the functions of TDP-43 is to inhibit retroviruses. Perhaps that explains the association with ALS and high levels of reverse transcriptase. I'd like to see what's happening with those levels if ever they get around to trialling a TDP-43 therapy in patients.

There's more info here:

http://www.als.net/forum/Default.aspx?g=posts&t=47406

"Should SMN1, beta-catenin and COX-2 have accumulated in the cytoplasm in the SOD1 model due to increased cytoplasmic HuR, then leptomycin B, described here as an inhibitor of nucleocytoplasmic translocation of HuR may be beneficial."

Let me know if it's not what you're after?

Melanoma, HIV infection and rheumatoid arthritis have all been associated with ALS and increased HERV-K levels are reported in these diseases. I see that they mention that HERV-K encodes an enzyme - deoxyuridine triphosphate pyrophosphatase. Does activity of that enzyme appear to be increased in ALS, and if so, might an inhibitor of the same be worthwhile?

Mary

Exp Cell Res. 2009 Mar 10;315(5):849-62. Epub 2009 Jan 8.

The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation.
Serafino A, Balestrieri E, Pierimarchi P, Matteucci C, Moroni G, Oricchio E, Rasi G, Mastino A, Spadafora C, Garaci E, Vallebona PS.

Institute of Neurobiology and Molecular Medicine-ARTOV, CNR via Fosso del Cavaliere 100, 00133-Rome, Italy. annalucia.serafino@artov.inmm.cnr.it

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

PMID: 19167380 [PubMed - indexed for MEDLINE]

Neuroepidemiology. 2005;25(4):176-80. Epub 2005 Aug 15.

Amyotrophic lateral sclerosis mortality in 1.9 million US cancer survivors.
Freedman DM, Travis LB, Gridley G, Kuncl RW.

Division of Epidemiology and Genetics, National Cancer Institute, NIH/DHHS, Rm. 7036 Executive Plaza South, 6120 Executive Boulevard, Bethesda, MD 20892-7238, USA. mf101e@nih.gov

BACKGROUND: Large cancer registries offer the opportunity to explore and generate hypotheses about the pathogenesis of cancer and other diseases, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). METHODS: Using data from nine population-based cancer registries of the Surveillance, Epidemiology, and End Results (SEER) Program of the US National Cancer Institute (NCI) and death certificates, we followed 1.9 million cancer survivors who were diagnosed between 1973 and 2000 and who survived at least 1 year, through the year 2000. The outcome of interest was the standardized mortality ratio (SMR) of observed to expected ALS deaths among cancer survivors. To assess the validity of the study design, we also examined associations with Parkinson's disease mortality, which we expected to be inversely associated with smoking-related cancers. RESULTS: There was no significantly increased risk or deficit of ALS mortality for all cancer sites combined (SMR = 1.0). Parkinson's disease mortality was, as expected, significantly and inversely associated with smoking-related cancers. Both ALS and Parkinson's disease mortality were significantly elevated following melanoma (SMR = 1.6; 95% CI = 1.1-2.2; SMR = 1.5; 1.2-1.8, respectively). Contrary to previous hypotheses, ALS was unrelated to lymphomas or lymphoproliferative malignancies and was not associated with smoking-related cancers. CONCLUSIONS: In this exploratory study, we observed a modest, significant association between melanoma and both ALS and Parkinson's disease mortality. It would be useful to explore these findings in other large national databases that are able to link cancer and ALS and Parkinson's disease. Copyright (c) 2005 S. Karger AG, Basel.

PMID: 16103728 [PubMed - indexed for MEDLINE]

AIDS Res Hum Retroviruses. 2008 May;24(5):717-23.

Serological response to human endogenous retrovirus K in melanoma patients correlates with survival probability.
Hahn S, Ugurel S, Hanschmann KM, Strobel H, Tondera C, Schadendorf D, Löwer J, Löwer R.

Retroelement Section, Paul-Ehrlich-Institut, Langen, Germany.

A few years ago, reactivation of human endogenous retrovirus K (HERV-K) proviruses in melanoma was described. The expression of HERV-K proteins induces humoral immune responses. The aim of the present study was to elucidate the prognostic relevance of serological anti-HERV-K reactivity in melanoma patients. In a retrospective study, anti-HERV-K Gag and Env antibodies were detected in 51 of the 312 randomly selected and blinded sera from melanoma patients, but not in any of the 70 sera from healthy controls. Comparing serological HERV-K reactivity with established melanoma markers revealed a significant correlation (p = 0.018, Chi-square test) with the stage of disease classified according to the American Joint Committee on Cancer (AJCC). Anti-HERV-K reactivity was elevated in patients with acrolentiginous/mucosal/uveal melanoma (tumor subtypes developing at sun-protected sites) compared to patients with lentigo/nodular/superficial spreading melanoma (p = 0.011, Chi-square test). Patients with anti-HERV-K antibodies had a significantly decreased disease-specific overall survival (stage I-IV, p < 0.001; stage I-III, p = 0.005, log-rank test). Significantly, multivariate Cox regression analysis including prognostic markers in clinical use (e.g., AJCC stage, T-class, serum level of S100-beta) revealed serological HERV-K reactivity as an independent marker of reduced survival probability (p = 0.027) in melanoma patients with the early stages of the disease (AJCC I-III). This is the first report that the humoral anti-HERV-K immune response may provide additional prognostic information to that of established melanoma markers.

AIDS Res Hum Retroviruses. 2007 Jan;23(1):116-22.

HIV-1 infection increases the expression of human endogenous retroviruses type K (HERV-K) in vitro.
Contreras-Galindo R, López P, Vélez R, Yamamura Y.

AIDS Research Program, Ponce School of Medicine, Ponce, Puerto Rico, 00716. rafaelc@med.umich.edu

Antibodies to HERV-K antigens have been linked to HIV-1 infection and expression of HERV-K proteins generates T-cell cytotoxic responses in many cancers. HERV-K RNA and protein abundance was measured in HIV-1-infected and control cells. In vitro exposure of HIV-1 laboratory-adapted and primary isolates on U87MG cells increased the expression of HERV-K RNA in a dose-dependent manner. HERV-K RNA and protein burdens were significantly increased in HIV-1-producing H9 cell lines compared to H9 cells. The expression of HERV-K was synergistically increased in HIV-1-infected PBMCs after stimulation with PMA/ionomycin. Furthermore, the expression of HERV-K in PBMCs, and particularly in CD4(+) T cells, was higher in HIV-1 patients compared to control subjects. The expression of HERV-K might be related to HIV-1 pathogenesis and AIDS-associated cancers.

PMID: 17263641 [PubMed - indexed for MEDLINE]

AIDS Res Hum Retroviruses. 2007 Sep;23(9):1083-6.

Comparative longitudinal studies of HERV-K and HIV-1 RNA titers in HIV-1-infected patients receiving successful versus unsuccessful highly active antiretroviral therapy.
Contreras-Galindo R, Almodóvar-Camacho S, González-Ramírez S, Lorenzo E, Yamamura Y.

AIDS Research Program, Ponce School of Medicine, Ponce, Puerto Rico 00716. rafaelc@med.umich.edu

The viral kinetics of HERV-K in HIV-1-infected patients receiving highly active antiretroviral therapy (HAART) is not unknown. HERV-K kinetic modeling may provide insight into factors altering the effectiveness of HAART in suppressing HIV-1 burden. We conducted a longitudinal study measuring the HERV-K RNA titers in four patients with successful HIV-1-suppressive HAART and in six patients undergoing HAART failure. HERV-K titers were usually undetectable in patients with successful HAART, and when detected, HERV-K titers remained below 5000 copies/ml. On the other hand, HERV-K RNA was consistently detected in patients who failed to respond to HAART before and after HIV-1 rebounds (p < 0.001). Elevated HERV-K RNA titers frequently preceded HIV-1 rebounds. These results suggest that HERV-K viral load may predict HIV-1 reactivation. HERV-K RNA testing might be clinically useful in predicting the onset of HIV-1 resistance due to suboptimal antiretroviral drug levels and/or poor adherence to treatment.

PMID: 17919102 [PubMed - indexed for MEDLINE]



Scand J Immunol. 2009 Sep;70(3):295-9.
Increase in human endogenous retrovirus HERV-K (HML-2) viral load in active rheumatoid arthritis.
Reynier F, Verjat T, Turrel F, Imbert PE, Marotte H, Mougin B, Miossec P.

Joint Unit Hospices Civils de Lyon - bioMérieux, and Department of Immunology and Rheumatology, Immunogenomics and inflammation research Unit EA 4130, University of Lyon, Lyon, France.

To study the viral loads of human endogenous retrovirus HERV-K (HML-2) type 1 and type 2 in rheumatoid arthritis (RA), we measured the viral loads of HERV-K (HML-2) type 1 and type 2 using nucleic acid sequence-based amplification (NASBA) technology. We analyzed plasma samples from RA patients (n = 79) and healthy volunteers (HV, n = 46) and synovial fluid samples from RA (n = 10) and osteoarthritis (OA, n = 10) patients. HERV-K type 1 and type 2 viruses were detected and quantified for the majority of plasma and synovial fluid samples from RA patients. HERV-K type 1 and type 2 viral loads were significantly elevated in RA patients compared with HV in plasma (P < 0.0001) and from RA patients compared with OA patients in synovial fluid (type 1: P = 0.0007; type 2: P = 0.023). Moreover, an association was observed between the HERV-K type 1 viral load in plasma and the disease activity in RA patients (RA patients with low activity versus high activity P = 0.0129; RA patients with intermediate activity versus high activity P = 0.037). Our findings showed that HERV-K (HML-2) viral load can be detected in plasma samples from RA patients, with higher levels observed for those with active disease. There was an association of HERV-K type 1 levels with the disease activity.

PMID: 19703019 [PubMed - indexed for MEDLINE]

Expression and cytoplasmic localisation of deoxyuridine triphosphate pyrophosphatase encoded by a human endogenous retrovirus

J. M. Harris1, E. M. McIntosh2 and G. E. Muscat1

(1)  Centre for Molecular and Cellular Biology, Departments of Microbiology and Biochemistry, University of Queensland, St Lucia, Queensland, Australia, AU
(2)  Department of Biology, York University, Toronto, Canada, CA


Summary.  Many lentiviruses encode a dUTPase which may protect against toxic misincorporation of dUTP into cDNA during reverse transcription. However, the primate lentiviruses HIV and SIV do not express a dUTPase. Significantly, the host genomes of these lentiviruses contain a multicopy endogenous retrovirus which is absent in non-primate genomes. In humans, this endogenous retrovirus is known as HERV-K and encodes a potential dUTPase sequence. Previously, we have suggested that HIV infection is complemented by a cytosolic dUTPase derived from the dUTPase gene encoded by HERV-K. This study demonstrates expression of HERV-K dUTPase transcripts and protein in human cell lines using RT-PCR and western blot analysis. Immunocytochemistry showed that HERV-K dUTPase was predominantly located in cell cytoplasm when transiently expressed in COS-1 cells. These data provide substantiation and support for the hypothesis above and is the first documentation of expression of an enzyme of nucleotide metabolism expressed by an endogenous retrovirus.
Accepted July 21, 1999/Received January 13, 1999

As complement C4A contains HERV-K, is it possible that the increased HERV-K reported in mTDP-43 cells is actually C4A?

This from the second study:"We have observed elevated levels of C4a in patients with Lyme-associated motor neuron disease that resembles amyotrophic lateral sclerosis (data not shown)."

Mary



Anal Biochem. 2006 Oct 1;357(1):122-7. Epub 2006 Aug 1.

Identification of the size and antigenic determinants of the human C4 gene by a polymerase chain-reaction-based amplification method.
Lee HH, Chang SF, Tseng YT, Lee YJ.

Department of Medical Research, Mackay Memorial Hospital, No. 45 Min-Sheng Road, Tanshui, Taipei Co. 251, Taiwan. hhlee@ms1.mmh.org.tw

The human C4 complement components of the C4 locus are encoded by two genes, C4A and C4B, located on chromosome 6p21.3 of the major histocompatibility complex of the human leukocyte antigen class III region. The size difference between the two genes is due to the presence of HERV-K (C4), an endogenous retroviral sequence (6.7 kb long), in intron 9 of the long C4 gene. Whether the C4 is the long (L) or short (S) gene was determined by the Southern blot method, and the antigenic determinants in residues 1,054-1,106 of Rodgers and Chido were generally identified by immunoblot analysis. Herein, we explore a polymerase chain reaction (PCR) amplification method for directly determining the size of C4 loci adjacent to the respective RP1 and RP2 genes and antigenic determinants by DNA sequencing. From the results of this study, we concluded that all of the C4 genes adjacent to the RP1 gene presented the long gene. In addition, 47% of the C4 genes adjacent to the RP2 gene were the short gene and 53% were the long gene. This result was consistent with that of the Southern blot analysis. The PCR method is practical for identifying the C4 genotype and can be used to detect other polymorphisms among variants of C4 genes.

ARTICLE LINKS:
Fulltext | PDF (152 K) | Request Permission
Complement Split Products C3a and C4a in Chronic Lyme Disease.

CLINICAL IMMUNOLOGY

Scandinavian Journal of Immunology. 69(1):64-69, January 2009.
Stricker, R. B. *,+; Savely, V. R. *,+; Motanya, N. C. +; Giclas, P. C. ++
Abstract:
Complement split products C3a and C4a are reportedly elevated in patients with acute Lyme disease. We have now examined these immunologic markers in patients with chronic Lyme disease compared to appropriate disease controls. The study population consisted of 29 healthy controls, 445 patients with chronic Lyme disease, 11 patients with systemic lupus erythematosus (SLE) and six patients with AIDS. The Lyme disease patients were divided according to predominant musculoskeletal symptoms (324 patients) or predominant neurologic symptoms (121 patients). C3a and C4a levels were measured by radioimmunoassay. All patients with chronic Lyme disease and AIDS had normal C3a levels compared to controls, whereas patients with SLE had significantly increased levels of this marker. Patients with predominant musculoskeletal symptoms of Lyme disease and AIDS patients had significantly increased levels of C4a compared to either controls, patients with predominant neurologic symptoms of Lyme disease or SLE patients. Response to antibiotic therapy in chronic Lyme disease was associated with a significant decrease in the C4a level, whereas lack of response was associated with a significant increase in this marker. In contrast, AIDS patients had persistently increased C4a levels despite antiretroviral therapy. Lyme patients with positive single-photon emission computed tomographic (SPECT) scans had significantly lower C4a levels compared to Lyme patients with normal SPECT scan results. Patients with predominant musculoskeletal symptoms of Lyme disease have normal C3a and increased C4a levels. This pattern differs from the increase in both markers seen in acute Lyme disease, and C4a changes correlate with the response to therapy in chronic Lyme disease. C4a appears to be a valuable immunologic marker in patients with persistent symptoms of Lyme disease.

Copyright (C) 2009 Blackwell Publishing Ltd.

I thought it was especially interesting that they were able to take "normal" (wild-type) TDP-43 and alter its gene so it had more nuclear-export hooks on it, which caused more of it to be exported into the cytoplasm, and then that caused the same sort of toxicity that the mutant TDP-43 from the ALS family did.

Now, most PALS do not have TDP-43 mutations. But they do have inclusions (clumps) of TDP-43 in their cytoplasm. This article argued that it is not the clumps that are important, it is the raw level of TDP-43 in the cytoplasm. It wasn't clear if most PALS have high levels of TDP-43 in addition to the clumps, but let's suppose it is true.

So there are two directions to research. One, how do high levels of cytoplasmic TDP-43 lead to toxicity? Two, what causes the TDP-43 to be exported from the nucleus into the cytoplasm in sporadic ALS?

And then, could there be a drug that would destroy or block TDP-43 in the cytoplasm, without affecting what it does in the nucleus? That would sound like a promising therapy.

A study above mentions that C4A is increased in Lyme disease mimicing ALS. Does the RCCX module affect location of TDP-43?


What's happening with the RCCX module in ALS? It's interesting that they report that C4A and C4B are deleted or duplicated together with the adjacent genes including RP (serine-threonine kinase), CYP21 (steroid 21-hydroxylase) and TNX (tenascin-X).

http://www.biomedcentral.com/1471-2156/7/1
Background
The complement system is a major constituent of innate immunity. Complement C4 plays an essential role in the activation cascades of the classical complement pathway as a subunit of the C3 and C5 convertases. C4 genes, located on the short arm of chromosome 6, are present either in a long (21 kilobasepair, kb) or in a short (14.6 kb) form, the long variant contains a 6.36 kb endogenous retrovirus HERV-K in its intron 9 [1,2]. These genes are deleted or duplicated together with the adjacent genes including RP (serine-threonine kinase), CYP21 (steroid 21-hydroxylase) and TNX (tenascin-X). The set of the four genes (RP, C4A or C4B, CYP21 and TNX) is referred to as the RCCX module [3]. The variation of the number of RCCX modules and sizes of the C4 genes leads to different RCCX length forms (Fig (1)): besides the monomodular L (long) and S (short), the bimodular (LL, LS, SS) and trimodular (LLL, LSS, LLS, LSL) types, the quadrimodular version (LLLL) was also described with a very low frequency. These length variants create more than 20 different haplotype combinations