Objective TDP-43 is deposited as cytoplasmic and intranuclear inclusions in brains of subjects 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. Methods We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy and immunoblots. Additionally, we performed investigations aimed at identifying the responsible kinases and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization. Results We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially-available anti-TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15 nm diameter, and on immunoblots recognized hyperphosphorylated TDP-43 at 45 kDa, with additional 22–28 kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase 1 and 2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43. Interpretation These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders.

Synucleinopathies are human neurodegenerative diseases that include multiple system atrophy (MSA), Parkinson's disease, Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) (1). Existing treatments are at best symptomatic. These diseases are characterised by the presence in brain cells of filamentous inclusions of αsynuclein, the formation of which is believed to cause disease (2,3). However, the structures of α-synuclein filaments from human brain are not known. Here we show, using electron cryo-microscopy, that α-synuclein inclusions from MSA are made of two types of filaments, each of which consists of two different protofilaments. Non-proteinaceous molecules are present at the protofilament interfaces. By two-dimensional class averaging, we show that α-synuclein filaments from the brains of patients with MSA and DLB are different, suggesting that distinct conformers (or strains) characterise synucleinopathies. As was the case of tau assemblies (4-9), the structures of α-synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, with implications for understanding the mechanisms of aggregate propagation and neurodegeneration in human brain. These findings have diagnostic and potential therapeutic relevance, .

Ordered assembly of the tau protein into filaments characterizes multiple neurodegenerative diseases, which are called tauopathies. We previously reported that by electron cryo-microscopy (cryo-EM), tau filament structures from Alzheimer's disease (1,2), chronic traumatic encephalopathy (CTE) (3), Pick's disease (4) and corticobasal degeneration (CBD) (5) are distinct. Here we show that the structures of tau filaments from typical and atypical progressive supranuclear palsy (PSP), the most common tauopathy after Alzheimer's disease, define a previously unknown, three-layered fold. Moreover, the tau filament structures from globular glial tauopathy (GGT, Types I and II) are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs from the above and resembles the four-layered CBD fold. The majority of tau filaments from agingrelated tau astrogliopathy (ARTAG) also have the AGD fold. Surprisingly, tau protofilament structures from inherited cases with mutations +3/+16 in intron 10 of MAPT, the microtubule-associated protein tau gene, are identical to those from AGD, suggesting that a relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, tau filament structures from cases of familial British dementia (FBD) and familial Danish dementia (FDD) are the same as those from Alzheimer's disease and primary age-related tauopathy (PART). These structures provide the basis for a classification of tauopathies that also allows identification of new entities, as we show here for a case diagnosed as PSP, but with abundant spherical 4R tau inclusions in limbic and other brain areas. The structures of the tau fold of this new disease (Limbic-predominant Neuronal inclusion body 4R Tauopathy, LNT) were intermediate between those of GGT and PSP.

The so-called amyloid hypothesis, that the accumulation and deposition of oligomeric or fibrillar amyloid β (Aβ) peptide is the primary cause of Alzheimer's disease (AD), has been the mainstream concept underlying AD research for over 20 years. However, all attempts to develop Aβ-targeting drugs to treat AD have ended in failure. Here, we review recent findings indicating that the main factor underlying the development and progression of AD is tau, not Aβ, and we describe the deficiencies of the amyloid hypothesis that have supported the emergence of this idea.

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-phenylglycinet-butyl ester, a potent dipeptide ␥-secretase inhibitor, builds up A␤1-43 and A␤1-46 intracellularly, which was also confirmed by mass spectrometry. Notably, suppression of A␤40 appeared to lead to an increase in A␤43, which in turn brings an increase in A␤46, in a dose-dependent manner. We therefore propose an ␣-helical model in which longer A␤ species generated by ⑀-cleavage is cleaved at every three residues in its carboxyl portion.

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.

No abstract

Synucleinopathies are human neurodegenerative diseases that include multiple system atrophy (MSA), Parkinson’s disease, Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB) (1). Existing treatments are at best symptomatic. These diseases are characterised by the presence in brain cells of filamentous inclusions of α-synuclein, the formation of which is believed to cause disease (2, 3). However, the structures of α-synuclein filaments from human brain are not known. Here we show, using electron cryo-microscopy, that α-synuclein inclusions from MSA are made of two types of filaments, each of which consists of two different protofilaments. Non-proteinaceous molecules are present at the protofilament interfaces. By two-dimensional class averaging, we show that α-synuclein filaments from the brains of patients with MSA and DLB are different, suggesting that distinct conformers (or strains) characterise synucleinopathies. As was the case of tau assemblies (4–9), the structures of α-synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, with implications for understanding the mechanisms of aggregate propagation and neurodegeneration in human brain. These findings have diagnostic and potential therapeutic relevance, especially in view of the unmet clinical need to be able to image filamentous α-synuclein inclusions in human brain.