TAR DNA-binding Protein 43 Positive Inclusions Research Articles

An Elderly Woman With Difficulty Reading and Abnormal Eye Movements

An Elderly Woman With Difficulty Reading and Abnormal Eye Movements

Investigation of C9orf72 in 4 Neurodegenerative Disorders

OBJECTIVE To estimate the allele frequency of C9orf72 (G4C2) repeats in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Alzheimer disease (AD), and Parkinson disease (PD). DESIGN The number of repeats was estimated by a 2-step genotyping strategy. For expansion carriers, we sequenced the repeat flanking regions and obtained APOE genotypes and MAPT H1/H2 haplotypes. SETTING Hospitals specializing in neurodegenerative disorders. SUBJECTS We analyzed 520 patients with FTLD, 389 patients with ALS, 424 patients with AD, 289 patients with PD, 602 controls, 18 families, and 29 patients with PD with the LRRK2 G2019S mutation. MAIN OUTCOME MEASURE The expansion frequency. RESULTS Based on a prior cutoff (>30 repeats), the expansion was detected in 9.3% of patients with ALS, 5.2% of patients with FTLD, and 0.7% of patients with PD but not in controls or patients with AD. It was significantly associated with family history of ALS or FTLD and age at onset of FTLD. Phenotype variation (ALS vs FTLD) was not associated with MAPT, APOE, or variability in the repeat flanking regions. Two patients with PD were carriers of 39 and 32 repeats with questionable pathological significance, since the 39-repeat allele does not segregate with PD. No expansion or intermediate alleles (20-29 repeats) were found among the G2019S carriers and AD cases with TAR DNA-binding protein 43-positive inclusions. Surprisingly, the frequency of the 10-repeat allele was marginally increased in all 4 neurodegenerative diseases compared with controls, indicating the presence of an unknown risk variation in the C9orf72 locus. CONCLUSIONS The C9orf72 expansion is a common cause of ALS and FTLD, but not of AD or PD. Our study raises concern about a reliable cutoff for the pathological repeat number, which is important in the utility of genetic screening.

Activation of Transforming Growth Factor-beta/Smad Signaling Reduces Aggregate Formation of Mislocalized TAR DNA Binding Protein-43 (P03.182)

Objective: To investigate the possible pathophysiological linkage between TAR DNA binding protein-43 (TDP-43) positive inclusions and transforming growth factor (TGF)-beta/Smad signaling system. Background We have previously reported that phosphorylated Smad2/3 (pSmad2/3) proteins, the major intracellular mediators of TGF-beta signaling, were abnormally accumulated in TDP-43 positive inclusions of anterior horn cells (AHCs) in ALS patients. Moreover, the nuclear pSmad2/3 immunosignal was decreased in inclusion-bearing AHCs in comparison with those without inclusions. These findings suggested that TGF-beta/Smad signaling system and its neuroprotective effect might be disturbed in ALS. Design/Methods: We constructed a plasmid of the nuclear localization signal deletion mutant of TDP-43, and transfected it into HEK293T cells. TDP-43 positive inclusions were formd in cytoplasm of the HEK293T cell under proteasome inhibition. Plasmids of Smad2 protein and constitutive active or dominant negative forms of TGF type1 receptor were also transfected into the HEK293T cells. The direct effects of TGF-beta/Smad signaling on the aggregation formation were analyzed by immunocytochemistry and western blotting. Results: The aggregates formed in cytoplasm of the HEK293T cells contained ubiquitinated, phosphorylated, and fragmented TDP-43, consistent with the essential features of the human pathology. Moreover, the aggregates were colocalized with phosphorylated Smad2 under continuous TGF-beta stimulation. Overexpression of Smad2 reduced the amount of cytoplasmic aggregates in HEK293T cells, and TGF-beta stimulation augmented this reduction effect in a dose-dependent manner. Conclusions: Our data indicate that activation of TGF-beta/Smad signaling system is protective against aggregate formation of cytoplasmically mislocalized TDP-43 and may be a potential therapeutic approach to delay the progression of ALS. Disclosure: Dr. Nakamura has nothing to disclose. Dr. Kaneko has nothing to disclose. Dr. Ito has nothing to disclose. Dr. Fujisawa has nothing to disclose. Dr. Kusaka has nothing to disclose.

Rescue of Progranulin Deficiency Associated with Frontotemporal Lobar Degeneration by Alkalizing Reagents and Inhibition of Vacuolar ATPase

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue

Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein - RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway.

Proteolytic processing of TAR DNA binding protein‐43 by caspases produces C‐terminal fragments with disease defining properties independent of progranulin

Neuronal and glial deposition of misfolded, proteolytically processed, polyubiquitinated and abnormally phosphorylated C-terminal fragments (CTFs) of the TAR DNA binding protein-43 (TDP-43) is a pathological hallmark of frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) and certain cases of amyotrophic lateral sclerosis. We demonstrate that TDP-43 can be proteolytically processed by caspases upon induction of apoptosis to a major 35 kDa and a minor 25 kDa CTF. These fragments are initially soluble, but over time they accumulate as insoluble and pathologically phosphorylated derivatives. However, proteolytic processing appears not to be absolutely required for the deposition of insoluble TDP-43 species, since a caspase resistant mutant of TDP-43 is also converted into insoluble species. Phosphorylation at S409/410 apparently occurs late during the conversion of soluble to insoluble TDP-43, suggesting that phosphorylation is not a prerequisite for aggregation. Loss of function of the progranulin (PGRN) gene causes FTLD-U with TDP-43 positive inclusions and has been suggested to lead to caspase activation and subsequent TDP-43 processing. However, siRNA-mediated knockdown of PGRN in cell culture as well as a PGRN gene knockout in mice failed to cause the formation of the disease characterizing CTFs of TDP-43. Our findings therefore suggest that caspase-mediated processing generates CTFs of similar biochemical properties as those occurring in nuclear and cytoplasmic deposits of FTLD-U patients independent of PGRN levels.

Dual pathologies: Utility of TAR DNA‐binding Protein 43 (TDP‐43) Staining in Patients with Frontal and Temporal Lobe Abnormalities and Alzheimer disease

Prominent behavioral changes and language dysfunction are features of frontotemporal lobar degeneration (FTLD) but can also occur in AD. Recently, application of TAR DNA-binding protein 43 (TDP-43) antibody has been shown to be useful for demonstrating the pathologic lesions of FTLD-U and FTLD-MND. Since these cases might represent combined disorders (FTD and AD), we studied the utility of TDP-43 staining to demonstrate inclusion bodies in brains of patients with clinical features of frontal or temporal lobe dysfunction but with autopsy features of AD. METHODS: Eight patients from 1998 to 2005 were identified, including 1 with combined pathologic features of FTLD-U (previously identified with anti-ubiquitin staining) and mild AD (Braak stage IV). Sections of hippocampus and frontal and temporal lobes were stained with anti-TDP-43 antibody (Proteintech Group). RESULTS: Of the 8 patients, 1 had advanced AD (Braak stage VI) and scattered TDP-43 positive cytoplasmic inclusions in hippocampal dentate granular cells and frontal and temporal neocortical neurons. The previously identified patient with combined FTLD-U and mild AD also had TDP-43 positive cytoplasmic and nuclear inclusions. CONCLUSION: TDP-43 aids in the identification of case with dual pathologies, particularly in advanced AD where inclusion may be masked by overwhelming AD pathology.