Tau phosphorylation at threonine-175 leads to fibril formation and enhanced cell death: implications for amyotrophic lateral sclerosis with cognitive impairment

Although amyotrophic lateral sclerosis (ALS) can be associated with cognitive impairment (ALSci) as a reflection of frontotemporal lobar degeneration, the basis of this process is unknown. The observation of neuronal and extraneuronal tau deposition in ALSci in addition to a unique tau phosphorylation at Thr175 has suggested that ALSci can be associated with alterations in tau metabolism. We have examined the association between phosphorylation at Thr175 and tau fibril formation. Both soluble and insoluble tau was purified from control, patients with Alzheimer's disease (AD), ALS without cognitive impairment, and ALSci and the tendency to fibril formation assayed ex vivo using the thioflavin S fluorescence assay. The extent of fibril formation was significantly greater in tau derived from ALSci, with ALS-derived tau being intermediate between control and AD-derived tau. Using both Neuro2A and human embryonic kidney (HEK293T) cells, we expressed full-length tau constructs harboring either a pseudophosphorylation at Thr175 (Thr175-Asp-tau), inhibition of Thr175 phosphorylation (Thr175-Ala-tau) or intact tau (wild-type tau). Both tau fibril formation and cell death were significantly enhanced in the presence of Thr175-Asp-tau, regardless of the tau isoform, suggesting that phosphorylation of Thr175 is associated with tau fibril formation in ALSci.     

Progranulin: normal function and role in neurodegeneration

Progranulin (PGRN) is a multifunctional protein that has attracted significant attention in the neuroscience community following the recent discovery of PGRN mutations in some cases of frontotemporal dementia. Most of the pathogenic mutations result in null alleles, and it is thought that frontotemporal dementia in these families results from PGRN haploinsufficiency. The neuropathology associated with PGRN mutations is characterized by the presence of tau-negative, ubiquitin-immunoreactive neuronal inclusions (frontotemporal lobar degeneration with ubiquitinated inclusions) that are also positive for the transactivation response DNA binding protein with M_r 43 kD. The clinical phenotype includes behavioral abnormalities, language disorders and parkinsonism but not motor neuron disease. There is significant clinical variation between families with different PGRN mutations and among members of individual families. The normal function of PGRN is complex, with the full-length form of the protein having trophic and anti-inflammatory activity, whereas proteolytic cleavage generates granulin peptides that promote inflammatory activity. In the periphery, PGRN functions in wound healing responses and modulates inflammatory events. In the CNS, PGRN is expressed by neurons and microglia; consequently, reduced levels of PGRN could affect both neuronal survival and CNS inflammatory processes. In this review, we discuss current knowledge of the molecular genetics, neuropathology, clinical phenotype and functional aspects of PGRN in the context of neurodegenerative disease.     

Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss

FUS is an RNA‐binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS‐containing aggregates are often associated with concomitant loss of nuclear FUS. Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell‐specific CRE‐mediated expression of wild‐type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.     

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.     

ALS‐associated fused in sarcoma (FUS) mutations disrupt Transportin‐mediated nuclear import

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C‐terminus of FUS show neuronal cytoplasmic FUS‐positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD‐FUS). We show that a non‐classical PY nuclear localization signal (NLS) in the C‐terminus of FUS is necessary for nuclear import. The majority of fALS‐associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease‐causing mutations within a PY‐NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co‐deposit with inclusions in fALS and FTLD‐FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS‐opathies.     

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation-associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full-length four-repeat human tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Results indicated that the mutants differ from each other and from wild-type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild-type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.     

Arginine methylation next to the PY‐NLS modulates Transportin binding and nuclear import of FUS

Fused in sarcoma (FUS) is a nuclear protein that carries a proline‐tyrosine nuclear localization signal (PY‐NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY‐NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN‐binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN‐mediated nuclear import of ALS‐associated FUS mutants. The unmethylated arginine–glycine–glycine domain preceding the PY‐NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS‐FUS patients contain methylated FUS, while inclusions in FTLD‐FUS patients are not methylated. Together with recent findings that FUS co‐aggregates with two related proteins of the FET family and TRN in FTLD‐FUS but not in ALS‐FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.     

The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP‐43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule‐associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over‐expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant‐negative Rab7‐interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.     

Structural brain imaging in frontotemporal dementia

Frontotemporal dementia (FTD) is the second commonest young-onset neurodegenerative dementia. The canonical clinical syndromes are a behavioural variant (bvFTD) and two language variants (progressive nonfluent aphasia, PNFA, and semantic dementia, SD) although there is overlap with motor neurone disease and the atypical parkinsonian disorders corticobasal syndrome (CBS) and progressive supranuclear palsy syndrome (PSPS). Characteristic patterns of atrophy or hypometabolism are described in each of the variants but in reality imaging studies are rather heterogeneous. This review attempts to address four key questions in the neuroimaging of FTD: 1) what are the early imaging features of the different FTD syndromes (and how do these change as the disease progresses); 2) what do studies of presymptomatic genetic cases of FTD tell us about the very early stages of the disease; 3) can neuroimaging help to differentiate the different FTD syndromes; and 4) can neuroimaging help to differentiate FTD from other neurodegenerative diseases? This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.