Pathologic Thr175 tau phosphorylation in CTE and CTE with ALS.

OBJECTIVE: To investigate whether chronic traumatic encephalopathy (CTE) and CTE with amyotrophic lateral sclerosis (CTE-ALS) exhibit features previously observed in other tauopathies of pathologic phosphorylation of microtubule-associated protein tau at Thr175 (pThr175 tau) and Thr231 (pThr231 tau), and glycogen synthase kinase-3ß (GSK3ß) activation, and whether these pathologic features are a consequence of traumatic brain injury (TBI). METHODS: Tau isoform expression was assayed by western blot in 6 stage III CTE cases. We also used immunohistochemistry to analyze 5 cases each of CTE, CTE-ALS, and 5 controls for expression of activated GSK3ß, pThr175 tau, pThr231 tau, and oligomerized tau within spinal cord tissue and hippocampus. Using a rat model of moderate TBI, we assessed tau pathology and phospho-GSK3ß expression at 3 months postinjury. RESULTS: CTE and CTE-ALS are characterized by the presence of all 6 tau isoforms in both soluble and insoluble tau isolates. Activated GSK3ß, pThr175 tau, pThr231 tau, and oligomerized tau protein expression was observed in hippocampal neurons and spinal motor neurons. We observed tau neuronal pathology (fibrillar inclusions and axonal damage) and increased levels of pThr175 tau and activated GSK3ß in moderate TBI rats. CONCLUSIONS: Pathologic phosphorylation of tau at Thr175 and Thr231 and activation of GSK3ß are features of the tauopathy of CTE and CTE-ALS. These features can be replicated in an animal model of moderate TBI.

C9orf72 mutations do not influence the tau signature of amyotrophic lateral sclerosis with cognitive impairment (ALSci).

OBJECTIVE: C9orf72 mutations are associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and ALS-FTD. In addition to ALS-FTD, ALS patients may develop a spectrum of neuropsychological and neuropsychiatric deficits including ALS with cognitive impairment (ALSci). Here we examine the extent to which C9orf72 mutations are associated with ALSci and whether this alters the tau molecular signature. METHODS: We identified 16 ALSci cases within a post-mortem archive of 94 fully genotyped ALS cases, eight of which harboured a C9orf72 mutation, in addition to three cognitively-intact ALS cases with a C9orf72 mutation. Tau was fractionated into soluble and insoluble fractions, with or without dephosphorylation, and immunoblots for tau phospho-isoforms performed. RESULTS: Regardless of cognitive state or the presence of C9orf72 mutation, all ALS cases demonstrated six tau isoforms in both soluble and insoluble tau isolates. This pattern was unaffected by dephosphorylation. pThr175tau isoforms, a molecular signature of ALSci, were present regardless of C9orf72 genetic status. The pathognomic paired helical triplet in the insoluble tau fraction of Alzheimer's disease was not observed, regardless of cognitive or C9orf72 status. CONCLUSIONS: These findings suggest that the presence of a C9orf72 mutation does not influence the tau signature of ALS or ALSci.

Thr175-phosphorylated tau induces pathologic fibril formation via GSK3ß-mediated phosphorylation of Thr231 in vitro.

We have previously shown that amyotrophic lateral sclerosis with cognitive impairment can be characterized by pathologic inclusions of microtubule-associated protein tau (tau) phosphorylated at Thr(175) (pThr(175)) in association with GSK3ß activation. We have now examined whether pThr(175) induces GSK3ß activation and whether this leads to pathologic fibril formation through Thr(231) phosphorylation. Seventy-two hours after transfection of Neuro2A cells with pseudophosphorylated green fluorescent protein-tagged 2N4R tau (Thr(175)Asp), phosphorylated kinase glycogen synthase kinase 3 beta (active GSK3ß) levels were significantly increased as was pathologic fibril formation and cell death. Treatment with each of 4 GSK3ß inhibitors or small hairpin RNA knockdown of GSK3ß abolished fibril formation and prevented cell death. Inhibition of Thr(231) phosphorylation (Thr(231)Ala) prevented pathologic tau fibril formation, regardless of Thr(175) state, whereas Thr(231)Asp (pseudophosphorylated at Thr(231)) developed pathologic tau fibrils. Ser(235) mutations did not affect fibril formation, indicating an unprimed mechanism of Thr(231) phosphorylation. These findings suggest a mechanism of tau pathology by which pThr(175) induces GSK3ß phosphorylation of Thr(231) leading to fibril formation, indicating a potential therapeutic avenue for amyotrophic lateral sclerosis with cognitive impairment.

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.

TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein.

The human TAR DNA-binding protein (TDP43) colocalizes with ubiquitinated inclusions in motor neurons in amyotrophic lateral sclerosis (ALS). TDP43 is both a DNA-binding protein with a nuclear export sequence that interacts with (TG)nTm elements in DNA and an RNA-binding protein that interacts with (UG)(6-12) motifs in single-stranded RNA. In control motor neurons, TDP43 was almost exclusively nuclear, whereas in ALS spinal motor neurons, TDP43 was predominantly localized to the cytosol and not the nucleus. TDP43 was observed as punctuate immunoreactivity and as dense skeins, with and without ubiquitinization. We observed that TDP43 stabilizes the human low molecular weight (hNFL) mRNA through a direct interaction with the 3'UTR. TDP43 is a unique hNFL mRNA-binding protein that is altered in its somatotopic localization in ALS spinal motor neurons and potentially contributes to the formation of NF aggregates in ALS through alterations in NF stoichiometry.

Neuronal tissue-specific ribonucleoprotein complex formation on SOD1 mRNA: alterations by ALS SOD1 mutations.

Amyotrophic lateral sclerosis (ALS) is a fatal disease of unknown etiology. Mutations in copper/zinc superoxide dismutase (SOD1) are the most commonly associated genetic abnormality. Given that SOD1 is ubiquitously expressed, the exclusive vulnerability of motor neurons is one of the most puzzling issues in ALS research. We here report that wild-type SOD1 mRNA forms ribonucleoprotein (RNP) complexes with protein homogenates of neuronal tissue but not with homogenates of non-neuronal tissues. 3' Untranslated region of SOD1 mRNA-dependent RNP complexes functioned to stabilize SOD1 mRNA. Moreover, SOD1 mRNAs harboring ALS-associated mutations, including silent mutations, were deficient in forming RNP complexes. In contrast, SOD1 mRNAs harboring artificial mutations, not known to be associated with ALS, demonstrated preserved RNP complex formation. This paper reports RNP complex formation on SOD1 mRNA as a neuronal tissue-specific and ALS-associated mutation sensitive feature.

Activated microglial supernatant induced motor neuron cytotoxicity is associated with upregulation of the TNFR1 receptor.

We have previously reported that supernatant derived from LPS-activated BV-2 cells, an immortalized microglial cell line, induces death of NSC-34 cells (a motor neuron hybridoma) through a TNFalpha and nitric oxide synthase (NOS) dependant mechanism. In this study, we have observed that LPS-activated BV-2 supernatant induces NSC-34 cell death in association with an upregulation of the TNF receptor 1 (TNFR1) expression on NSC-34 cells, both at the transcription level and at the cell surface protein level. The upregulation of TNFR1 receptor was independent of TNFalpha, and could be partly inhibited by the inhibition of iNOS activation in the BV-2 cells. The TNFR2 receptor was not involved. These observations have important implications in understanding the mechanism by which microglial activation contributes to the motor neuron degeneration.

Mutant copper-zinc superoxide dismutase binds to and destabilizes human low molecular weight neurofilament mRNA.

The mechanism by which mutated copper-zinc superoxide dismutase (SOD1) causes familial amyotrophic lateral sclerosis is believed to involve an adverse gain of function, independent of the physiological antioxidant enzymatic properties of SOD1. In this study, we have observed that mutant SOD1 (G41S, G85A, and G93A) but not the wild type significantly reduced the stability of the low molecular weight neurofilament mRNA in a dosage-dependent manner. We have also demonstrated that mutant SOD1 but not the wild type bound directly to the neurofilament mRNA 3'-untranslated region and that the binding was necessary to induce mRNA destabilization. These observations provide an explanation for a novel gain of function in which mutant SOD1 expression in motor neurons alters an intermediate filament protein expression.