Fuyuki Kametani

Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

TAR DNA‐binding protein of 43kDa (TDP‐43) is deposited as cytoplasmic and intranuclear inclusions in brains of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD‐U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP‐43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP‐43.

Longer Forms of Amyloid β Protein: Implications for the Mechanism of Intramembrane Cleavage by γ-Secretase

γ-Cleavage of β-amyloid precursor protein (APP) in the middle of the cell membrane generates amyloid β protein (Aβ), and ϵ-cleavage, ∼10 residues downstream of the γ-cleavage site, releases the APP intracellular domain (AICD). A significant link between generation of Aβ and AICD and failure to detect AICD41-99 led us to hypothesize that ϵ-cleavage generates longer Aβs, which are then processed to Aβ40/42. Using newly developed gel systems and an N-end-specific monoclonal antibody, we have identified the longer Aβs (Aβ1-43, Aβ1-45, Aβ1-46, and Aβ1-48) within the cells and in brain tissues. The production of these longer Aβs as well as Aβ40/42 is presenilin dependent and is suppressed by {1S-benzyl-4R-[1S-carbamoyl-2-phenylethylcarbamoyl-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester, a transition state analog inhibitor for aspartyl protease. In contrast, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a potent dipeptide γ-secretase inhibitor, builds up Aβ1-43 and Aβ1-46 intracellularly, which was also confirmed by mass spectrometry. Notably, suppression of Aβ40 appeared to lead to an increase in Aβ43, which in turn brings an increase in Aβ46, in a dose-dependent manner. We therefore propose an α-helical model in which longer Aβ species generated by ϵ-cleavage is cleaved at every three residues in its carboxyl portion.

Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43

TAR DNA binding protein of 43 kDa (TDP-43) is a major component of the ubiquitin-positive inclusions found in the brain of patients with frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). Here, we report that expression of TDP-43 C-terminal fragments as green fluorescent protein (GFP) fusions in SH-SY5Y cells results in the formation of abnormally phosphorylated and ubiquitinated inclusions that are similar to those found in FTLD-U and ALS. Co-expression of DsRed-tagged full-length TDP-43 with GFP-tagged C-terminal fragments of TDP-43 causes formation of cytoplasmic inclusions positive for both GFP and DsRed. Cells with GFP and DsRed positive inclusions lack normal nuclear staining for endogenous TDP-43. These results suggest that GFP-tagged C-terminal fragments of TDP-43 are bound not only to transfected DsRed-full-length TDP-43 but also to endogenous TDP-43. Endogenous TDP-43 may be recruited to cytoplasmic aggregates of TDP-43 C-terminal fragments, which results in the failure of its nuclear localization and function. Interestingly, expression of GFP-tagged TDP-43 C-terminal fragments harboring pathogenic mutations that cause ALS significantly enhances the formation of inclusions. We also identified cleavage sites of TDP-43 C-terminal fragments deposited in the FTLD-U brains using mass spectrometric analyses. We propose that generation and aggregation of phosphorylated C-terminal fragments of TDP-43 play a primary role in the formation of inclusions and resultant loss of normal TDP-43 localization, leading to neuronal degeneration in TDP-43 proteinopathy.

The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease.

The actual presence of the predicted precursor of Alzheimers disease amyloid A4 protein, reported by Kang et al. (1987) in the Alzheimer brain, has yet to be verified. To identify the various regions of this precursor, antibodies were raised against three synthetic polypeptides, R35 (residues 274–286), R36 (residues 527–540), and R37 (residues 681–695), subsequences of the precursor protein; the specificity of these antibodies was ascertained by ELISA. Upon immunohistochemical examination, the antibody to R35 failed to react, but the antibody to R36 (the extracellular part) stained the amyloid of senile plaques and the staining pattern was identical to that of anti–A4 antibody. The antibody to R37 (the C–terminal intracellular part) stained what may be degenerating neurites in senile plaques whereas the amyloid remained unstained. An anti–neurofilament (NF) antibody reacted with some of the R37–positive grains, but R37–negative grains also were seen. Further, some R37–positive grains were not stained by the anti–NF antibody. The anti–GFAP antibody and the anti–macrophage antibody did not stain the R37–positive grains. These findings indicate that the amyloid protein in senile plaques actually contains a larger polypeptide than the A4 protein, and suggest that the intracellular C–terminal part of the precursor may exist in the degenerated neurites seen in senile plaques.

Reticulons RTN3 and RTN4‐B/C interact with BACE1 and inhibit its ability to produce amyloid β‐protein

β‐Secretase β‐site APP cleaving enzyme 1 (BACE1), is a membrane‐bound aspartyl protease necessary for the generation of amyloid β‐protein (Aβ), which accumulates in the brains of individuals with Alzheimers disease (AD). To gain insight into the mechanisms by which BACE1 activity is regulated, we used proteomic methods to search for BACE1‐interacting proteins in human neuroblastoma SH‐SY5Y cells, which overexpress BACE1. We identified reticulon 4‐B (RTN4‐B; Nogo‐B) as a BACE1‐associated membrane protein. Co‐immunoprecipitation experiments confirmed a physical association between BACE1 and RTN4‐B, RTN4‐C (the shortest isoform of RTN‐4), and their homologue reticulon 3 (RTN3), both in SH‐SY5Y cells and in transfected human embryonic kidney (HEK) 293 cells. Overexpression of these reticulons (RTNs) resulted in a 30–50% reduction in the secretion of both Aβ40 and Aβ42 from HEK293 cells expressing the AD‐associated Swedish mutant amyloid precursor protein (APP), but did not affect Aβ secretion from cells expressing the APP β‐C‐terminal fragment (β‐CTF), indicating that these RTNs can inhibit BACE1 activity. Furthermore, a BACE1 mutant lacking most of the N‐terminal ectodomain also interacted with these RTNs, suggesting that the transmembrane region of BACE1 is critical for the interaction. We also observed a similar interaction between these RTNs and the BACE1 homologue BACE2. Because RTN3 and RTN4‐B/C are substantially expressed in neural tissues, our findings suggest that they play important roles in the regulation of BACE1 function and Aβ production in the brain.

Methylene blue and dimebon inhibit aggregation of TDP-43 in cellular models

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD‐U) are major neurodegenerative diseases with TDP‐43 pathology. Here we investigated the effects of methylene blue (MB) and dimebon, two compounds that have been reported to be beneficial in phase II clinical trials of Alzheimers disease (AD), on the formation of TDP‐43 aggregates in SH‐SY5Y cells. Following treatment with 0.05 μM MB or 5 μM dimebon, the number of TDP‐43 aggregates was reduced by 50% and 45%, respectively. The combined use of MB and dimebon resulted in a 80% reduction in the number. These findings were confirmed by immunoblot analysis. The results indicate that MB and dimebon may be useful for the treatment of ALS, FTLD‐U and other TDP‐43 proteinopathies.

Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease

AA amyloidosis is one of the principal causes of morbidity and mortality in captive cheetahs (Acinonyx jubatus), which are in danger of extinction, but little is known about the underlying mechanisms. Given the transmissible characteristics of AA amyloidosis, transmission between captive cheetahs may be a possible mechanism involved in the high incidence of AA amyloidosis. In this study of animals with AA amyloidosis, we found that cheetah feces contained AA amyloid fibrils that were different from those of the liver with regard to molecular weight and shape and had greater transmissibility. The infectious activity of fecal AA amyloid fibrils was reduced or abolished by the protein denaturants 6 M guanidine·HCl and formic acid or by AA immunodepletion. Thus, we propose that feces are a vehicle of transmission that may accelerate AA amyloidosis in captive cheetah populations. These results provide a pathogenesis for AA amyloidosis and suggest possible measures for rescuing cheetahs from extinction.

Progressive Wild‐Type Transthyretin Deposition after Liver Transplantation Preferentially Occurs onto Myocardium in FAP Patients

To elucidate whether progressive wild‐type transthyretin (TTR) deposition can actually occur after liver transplantation (LT), amyloid fibrils were investigated in two familial amyloid polyneuropathy patients with TTR Val30Leu variant, who died 1 year after LT. Amyloid fibrils were extracted from cardiac muscles, sciatic nerves and kidney, which were investigated by the immunoprecipitation‐mass spectrometry method and liquid chromatography‐ion trap mass spectrometry analysis. The ratio of wild‐type to variant TTR in cardiac muscle was approximately 5:5 before LT, but greatly increased to about 9:1 after transplantation. The ratios in sciatic nerves and kidney obtained at autopsy were approximately 5:5. Wild‐type TTR was undetectable in kidney amyloid obtained before LT. Our results indicate that paradoxical wild‐type TTR deposition after LT can preferentially occur in myocardium, leading to fatal cardiac dysfunction, but it is quite likely that this phenomenon can also occur in other visceral organs.

Phosphorylated and cleaved TDP-43 in ALS, FTLD and other neurodegenerative disorders and in cellular models of TDP-43 proteinopathy.

Transactivation response (TAR) DNA‐binding protein of Mr 43 kDa (TDP‐43) is a major component of the tau‐negative and ubiquitin‐positive inclusions that characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration which is now referred to as FTLD‐TDP. Concurrent TDP‐43 pathology has been reported in a variety of other neurodegenerative disorders such as Alzheimers disease, forming a group of TDP‐43 proteinopathy. Accumulated TDP‐43 is characterized by phosphorylation and fragmentation. There is a close relationship between the pathological subtypes of FTLD‐TDP and the immunoblot pattern of the C‐terminal fragments of phosphorylated TDP‐43. These results suggest that proteolytic processing of accumulated TDP‐43 may play an important role for the pathological process. In cultured cells, transfected C‐terminal fragments of TDP‐43 are more prone to form aggregates than full‐length TDP‐43. Transfecting the C‐terminal fragment of TDP‐43 harboring pathogenic mutations of TDP‐43 gene identified in familial and sporadic ALS cases into cells enhanced the aggregate formation. Furthermore, we found that methylene blue and dimebon inhibit aggregation of TDP‐43 in these cellular models. Understanding the mechanism of phosphorylation and truncation of TDP‐43 and aggregate formation may be crucial for clarifying the pathogenesis of TDP‐43 proteinopathy and for developing useful therapeutics.

Granules in glial cells of patients with Alzheimer's disease are immunopositive for C-terminal sequences of β-amyloid protein

Granular structures that are recognized by antibodies specific for the C-terminal but not the N-terminal sequences of the beta-amyloid protein (A beta) fragments are present in a subset of microglia and astrocytes in Alzheimer brain tissue. The immunohistochemical profile indicates that the A beta in these granules is truncated between the residues 17 and 31 and terminates at the residue 42 or 43. Such granule-containing glia occur only in brain areas with the heavy A beta deposits. Whether the intraglial A beta fragments accumulate as a result of phagocytosis of extracellular A beta or are formed intracellularly by glial cells from amyloid precursor protein (APP) remains unknown.