- Email: [email protected]
Intracellular trafficking of TDP-43 and its potential role in disease
Symposia S5-01: Other Dementias
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findings the term FTLD-U has become obsolete and a revised nomenclature on FTLD was recently recommended. FTLD with tau or TDP-43 pathology are renamed as FTLD-tau and FTLD-TDP, respectively, while FTLD cases with inclusions that can only be demonstrated by immunohistochemistry against proteins of the ubiquitin proteasome system (UPS) are designated as FTLD-UPS until the ubiquitinated protein(s) in these conditions will be identified. S5-01-02
INTRACELLULAR TRAFFICKING OF TDP-43 AND ITS POTENTIAL ROLE IN DISEASE
Emanuele Buratti, ICGEB, Trieste, Italy. Contact e-mail: buratti@icgeb. org Background: Nuclear protein TDP-43 is a multifunctional RNA binding protein that plays a role in several mRNA processes such as splicing, stability, and transport. Recently, it has been described to play an important role in neurodegenerative diseases that include FTLD-U, ALS, and Alzheimer disease. In many cases, TDP-43 is the principal protein component of the insoluble cytoplasmic inclusions found in affected neuronal tissues. In these inclusions, TDP-43 has been found to be aberrantly hyperphosphorylated, ubiquitinated, and degraded. Methods: TDP-43 function in the nucleus has been investigated using a wide range of molecular biology approaches that include: protein mutagenesis, microarray assays, immunohistochemistry. In parallel, we have also developed a Drosophila melanogaster strain deficient in TBPH, the homologue of the human TDP-43 gene. Results: Mutagenesis analysis has revealed that normal TDP-43 localization is the result of many factors embedded in the protein sequence (NLS and Cterminal) sequences and also correct maintenance of functional properties (ie. RNA binding). Using a coupled minigene/siRNA cellular system, we have finely mapped the interaction between TDP-43 and the hnRNP proteins to the region comprised between residues 321 and 366. This system has also been used to study the effects of several disease-associated mutations that fall within this region. Finally, with the aim of understanding the biological and pathological roles of TDP 43 we have constructed a Drosophila strain where the homologue of human TDP 43 gene (TBPH) has been deleted. Flies lacking TBPH appear externally normal but present deficient locomotor behaviors, reduced life span and anatomical defects at the neuromuscular junctions. Importantly, the phenotype can be rescued by expression of human TDP-43 in the motoneurons of TBPHdeficient Drosophila. Conclusions: The results obtained so far have highlighted the great complexity of TDP-43 regulation with regards to both its cellular localization and normal cellular functions. This is consistent with the preliminary data obtained from the Drosophila animal model that indicate the potential role of TDP-43 in neuronal development and thus suggest that disease pathogenesis is a consequence of lack of TDP-43 in the nucleus rather than direct toxic effect of the cytoplasmic aggregates. S5-01-03
MICRORNA DYSREGULATION IN FRONTOTEMPORAL LOBAR DEGENERATION
findings suggest that translational regulation by miRNAs may be an important mechanism underlying FTLD. Methods: In this study, we aimed to identify additional miRNAs that are dysregulated in FTLD using a miRNA array. As an initial miRNA profiling study, expression levels of >13,000 known and predicted miRNAs were measured in autopsy brain tissue of 25 FTLD patients, 5 control individuals and 5 Alzheimer’s disease patients selected from the extensive Mayo Clinic Jacksonville brain bank. The 25 FTLD patients comprise 10 FTLD-tau and 15 FTLD-U patients and were selected to present 5 patients with each of the major FTLD pathological subtypes: 5 patients with 3R tau pathology (Pick’s disease), 5 patients with 4R tau pathology (corticobasal degeneration) and 5 patients with each of the three FTLD-U pathological subtypes. Results: We are currently in the process of performing the miRNA profiling study. An overview of differentially expressed miRNAs in each of the pathological FTLD subtypes will be presented at the meeting. Conclusions: The identification of specific miRNAs implicated in FTLD pathogenesis is expected to broaden our biological understanding of FTLD and its underlying disease pathways. S5-01-04
HEREDITARY DIFFUSE LEUKOENCEPHALOPATHY WITH AXONAL SPHEROIDS
Zbigniew K. Wszolek, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is an autosomal dominant disorder characterized by progressive cognitive, behavioral and personality changes, and motor deficits. The original kindred was reported in Sweden (Axelsson R, et al. Acta Psychiatr Scand Suppl 1984;314:1-65). The sporadic cases have also been recognized. The objective of this lecture is to summarize the current knowledge about HDLS. Methods: Review of published HDLS families and sporadic cases, clinical features, and available radiological and pathological material. Comparison of clinical and pathological features with pigmentary orthochromatic leukodystrophy (POLD). Results: HDLS and POLD are most likely the same conditions. They both are underreported and frequently misdiagnosed as Alzheimer disease, frontotemporal dementia, atypical parkinsonism, multiple sclerosis, and others. They both occur throughout the world. There is no curative treatment available. Genetic defect is unknown. Conclusions: At the present time accurate diagnosis of both HDLS and POLD depends on a histological evaluation. Diagnostic brain biopsies were performed in some instances but most cases were diagnosed on autopsies.
S5-01-05
QUANTIFICATION OF TOTAL a-SYNUCLEIN IN CEREBROSPINAL FLUID BY ELISA: CROSSSECTIONAL INVESTIGATION OF SUBJECTS WITH DEMENTIA AND PARKINSONISM
Rosa Rademakers, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected]
Michael G. Schlossmacher, University of Ottawa, Ottawa, ON, Canada. Contact e-mail: [email protected]
Background: The identification of mutations in the microtubule associate protein tau (MAPT) and progranulin (GRN) as the two major genes involved in the genetic etiology of familial FTLD has led to important new insights into the molecular understanding of FTLD. In addition, recognition of two distinct pathologies underlying these genetic subtypes, tau depositions in MAPT mutation carriers (FTLD-tau) and TAR DNA-binding protein (TDP43)-positive inclusions in GRN mutation carriers (FTLD-U) provided support for the existence of multiple disease pathways leading to FTLD. Despite these recent advances, little is currently known about the molecular mechanisms leading to the disease in sporadic FTLD-tau and FTLD-U. We recently demonstrated that a common genetic variant (rs5848), located in the 3’ untranslated region of GRN in a binding-site for microRNA (miRNA)-659, is a major susceptibility factor for FTLD-U. Individuals carrying two copies of the rs5848 T-allele showed a 3.2-fold increased risk to develop FTLD-U compared to non T-allele carriers (95% CI: 1.50-6.73), most likely through suppressed translation of GRN by miRNA-659. These
Background: Alpha-Synuclein (aSyn) is a neuronal and hematological protein linked to heritable Parkinson disease (PD). The accumulation of aSyn aggregates is a histopathological feature of several incurable brain diseases that include familial and sporadic PD, dementia with Lewy bodies (DLB), heritable Alzheimer disease (AD) and the Lewy body variant of AD. These incurable disorders, which are collectively called synucleinopathies, are often difficult to diagnose early in their course. Methods: To explore the potential of quantifying aSyn in living subjects for diagnostic purposes, we built, validated and optimized a sandwich-type ELISA to measure total aSyn levels in unconcentrated CSF. We then carried out a cross-sectional study in 80 Caucasians who had previously undergone lumbar puncture. Results: Using a 384-well plate ELISA format, we monitored CSF aSyn concentrations (pg / ul) in subjects with advanced PD (mean þ SD, 3.0 þ/ 1.0), in cases of probable and definite DLB (3.8 þ/ 3.3); in sporadic AD patients (5.2 þ/ 4.2), and in a group of other neurological control persons (NCO, 6.0 þ/ 4.7). In
P166
findings the term FTLD-U has become obsolete and a revised nomenclature on FTLD was recently recommended. FTLD with tau or TDP-43 pathology are renamed as FTLD-tau and FTLD-TDP, respectively, while FTLD cases with inclusions that can only be demonstrated by immunohistochemistry against proteins of the ubiquitin proteasome system (UPS) are designated as FTLD-UPS until the ubiquitinated protein(s) in these conditions will be identified. S5-01-02
INTRACELLULAR TRAFFICKING OF TDP-43 AND ITS POTENTIAL ROLE IN DISEASE
Emanuele Buratti, ICGEB, Trieste, Italy. Contact e-mail: buratti@icgeb. org Background: Nuclear protein TDP-43 is a multifunctional RNA binding protein that plays a role in several mRNA processes such as splicing, stability, and transport. Recently, it has been described to play an important role in neurodegenerative diseases that include FTLD-U, ALS, and Alzheimer disease. In many cases, TDP-43 is the principal protein component of the insoluble cytoplasmic inclusions found in affected neuronal tissues. In these inclusions, TDP-43 has been found to be aberrantly hyperphosphorylated, ubiquitinated, and degraded. Methods: TDP-43 function in the nucleus has been investigated using a wide range of molecular biology approaches that include: protein mutagenesis, microarray assays, immunohistochemistry. In parallel, we have also developed a Drosophila melanogaster strain deficient in TBPH, the homologue of the human TDP-43 gene. Results: Mutagenesis analysis has revealed that normal TDP-43 localization is the result of many factors embedded in the protein sequence (NLS and Cterminal) sequences and also correct maintenance of functional properties (ie. RNA binding). Using a coupled minigene/siRNA cellular system, we have finely mapped the interaction between TDP-43 and the hnRNP proteins to the region comprised between residues 321 and 366. This system has also been used to study the effects of several disease-associated mutations that fall within this region. Finally, with the aim of understanding the biological and pathological roles of TDP 43 we have constructed a Drosophila strain where the homologue of human TDP 43 gene (TBPH) has been deleted. Flies lacking TBPH appear externally normal but present deficient locomotor behaviors, reduced life span and anatomical defects at the neuromuscular junctions. Importantly, the phenotype can be rescued by expression of human TDP-43 in the motoneurons of TBPHdeficient Drosophila. Conclusions: The results obtained so far have highlighted the great complexity of TDP-43 regulation with regards to both its cellular localization and normal cellular functions. This is consistent with the preliminary data obtained from the Drosophila animal model that indicate the potential role of TDP-43 in neuronal development and thus suggest that disease pathogenesis is a consequence of lack of TDP-43 in the nucleus rather than direct toxic effect of the cytoplasmic aggregates. S5-01-03
MICRORNA DYSREGULATION IN FRONTOTEMPORAL LOBAR DEGENERATION
findings suggest that translational regulation by miRNAs may be an important mechanism underlying FTLD. Methods: In this study, we aimed to identify additional miRNAs that are dysregulated in FTLD using a miRNA array. As an initial miRNA profiling study, expression levels of >13,000 known and predicted miRNAs were measured in autopsy brain tissue of 25 FTLD patients, 5 control individuals and 5 Alzheimer’s disease patients selected from the extensive Mayo Clinic Jacksonville brain bank. The 25 FTLD patients comprise 10 FTLD-tau and 15 FTLD-U patients and were selected to present 5 patients with each of the major FTLD pathological subtypes: 5 patients with 3R tau pathology (Pick’s disease), 5 patients with 4R tau pathology (corticobasal degeneration) and 5 patients with each of the three FTLD-U pathological subtypes. Results: We are currently in the process of performing the miRNA profiling study. An overview of differentially expressed miRNAs in each of the pathological FTLD subtypes will be presented at the meeting. Conclusions: The identification of specific miRNAs implicated in FTLD pathogenesis is expected to broaden our biological understanding of FTLD and its underlying disease pathways. S5-01-04
HEREDITARY DIFFUSE LEUKOENCEPHALOPATHY WITH AXONAL SPHEROIDS
Zbigniew K. Wszolek, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is an autosomal dominant disorder characterized by progressive cognitive, behavioral and personality changes, and motor deficits. The original kindred was reported in Sweden (Axelsson R, et al. Acta Psychiatr Scand Suppl 1984;314:1-65). The sporadic cases have also been recognized. The objective of this lecture is to summarize the current knowledge about HDLS. Methods: Review of published HDLS families and sporadic cases, clinical features, and available radiological and pathological material. Comparison of clinical and pathological features with pigmentary orthochromatic leukodystrophy (POLD). Results: HDLS and POLD are most likely the same conditions. They both are underreported and frequently misdiagnosed as Alzheimer disease, frontotemporal dementia, atypical parkinsonism, multiple sclerosis, and others. They both occur throughout the world. There is no curative treatment available. Genetic defect is unknown. Conclusions: At the present time accurate diagnosis of both HDLS and POLD depends on a histological evaluation. Diagnostic brain biopsies were performed in some instances but most cases were diagnosed on autopsies.
S5-01-05
QUANTIFICATION OF TOTAL a-SYNUCLEIN IN CEREBROSPINAL FLUID BY ELISA: CROSSSECTIONAL INVESTIGATION OF SUBJECTS WITH DEMENTIA AND PARKINSONISM
Rosa Rademakers, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected]
Michael G. Schlossmacher, University of Ottawa, Ottawa, ON, Canada. Contact e-mail: [email protected]
Background: The identification of mutations in the microtubule associate protein tau (MAPT) and progranulin (GRN) as the two major genes involved in the genetic etiology of familial FTLD has led to important new insights into the molecular understanding of FTLD. In addition, recognition of two distinct pathologies underlying these genetic subtypes, tau depositions in MAPT mutation carriers (FTLD-tau) and TAR DNA-binding protein (TDP43)-positive inclusions in GRN mutation carriers (FTLD-U) provided support for the existence of multiple disease pathways leading to FTLD. Despite these recent advances, little is currently known about the molecular mechanisms leading to the disease in sporadic FTLD-tau and FTLD-U. We recently demonstrated that a common genetic variant (rs5848), located in the 3’ untranslated region of GRN in a binding-site for microRNA (miRNA)-659, is a major susceptibility factor for FTLD-U. Individuals carrying two copies of the rs5848 T-allele showed a 3.2-fold increased risk to develop FTLD-U compared to non T-allele carriers (95% CI: 1.50-6.73), most likely through suppressed translation of GRN by miRNA-659. These
Background: Alpha-Synuclein (aSyn) is a neuronal and hematological protein linked to heritable Parkinson disease (PD). The accumulation of aSyn aggregates is a histopathological feature of several incurable brain diseases that include familial and sporadic PD, dementia with Lewy bodies (DLB), heritable Alzheimer disease (AD) and the Lewy body variant of AD. These incurable disorders, which are collectively called synucleinopathies, are often difficult to diagnose early in their course. Methods: To explore the potential of quantifying aSyn in living subjects for diagnostic purposes, we built, validated and optimized a sandwich-type ELISA to measure total aSyn levels in unconcentrated CSF. We then carried out a cross-sectional study in 80 Caucasians who had previously undergone lumbar puncture. Results: Using a 384-well plate ELISA format, we monitored CSF aSyn concentrations (pg / ul) in subjects with advanced PD (mean þ SD, 3.0 þ/ 1.0), in cases of probable and definite DLB (3.8 þ/ 3.3); in sporadic AD patients (5.2 þ/ 4.2), and in a group of other neurological control persons (NCO, 6.0 þ/ 4.7). In