Wendy Strong

Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis

The intraneuronal aggregation of phosphorylated high‐molecular‐weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper‐phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X‐100 insoluble protein fractions, with respect to migration patterns on 2D‐iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain‐mediated proteolysis. The rate of calpain‐mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.

Tau protein hyperphosphorylation in sporadic ALS with cognitive impairment

The authors have characterized frontal cortical tau protein in cognitively intact (4) and cognitively impaired (ALSci, 4) ALS patients and compared it with control (2) or Alzheimer disease (AD, 1)- derived tau. The authors observed expression of both 3R and 4R tau isoforms; increased insoluble tau protein; phosphatase resistance; and hyperphosphorylation at T175, S208, and S210. Soluble tau from both AD and ALSci was also phosphorylated at S237. Tau hyperphosphorylation is associated with ALS.

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.

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.

Reduced lipolysis of large apo E-poor very-low-density lipoprotein subfractions from type IV hypertriglyceridemic subjects in vitro and in vivo

Heparin-Sepharose chromatography was used to separate Sf 60-400 very-low-density lipoproteins (VLDL) from type IV hypertriglyceridemic subjects into apolipoprotein (apo) E-poor and apo E-rich subfractions. Since we have previously demonstrated that the apo E-poor fraction accumulates in plasma of type IV subjects, the aim of the present studies was to determine whether it was resistant to lipolysis in comparison to the apo E-rich fraction. The apo E-rich fraction was found to be 30% more effective than the apo E-poor fraction at competing with a glycerol tri[1-14C]oleate emulsion for in vitro lipolysis by normolipidemic human post-heparin plasma (P < .01), when assayed under conditions in which both lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were active. Similar results were obtained when bovine milk LPL was used as the source of lipolytic activity (P < .025 for apo E-rich relative to apo E-poor VLDL), while neither fraction competed effectively with the synthetic substrate for lipolysis by HTGL only. When equal amounts of triglyceride from VLDL subfractions were incubated with bovine milk LPL, 25% more free fatty acid was released from the apo E-rich fraction than from the apo E-poor fraction (P < .025). The effects of heparin-induced lipolysis in vivo in type IV subjects on the relative amounts and composition of these VLDL subfractions were also assessed. Heparin infusion was associated with a 50% reduction in plasma Sf 60-400 VLDL triglyceride concentration. In addition, heparin-induced lipolysis resulted in a marked decrease in the relative amount of apo E-rich VLDL, while the relative amount of apo E-poor VLDL was increased. These results demonstrate that the apo E-poor VLDL subfraction is resistant to lipolysis by LPL relative to its apo E-rich counterpart, suggesting that reduced lipolytic efficiency may contribute to its observed accumulation in plasma of type IV subjects.

Metabolism of apolipoproteins C-II, C-III, and B in hypertriglyceridemic men. Changes after heparin-induced lipolysis.

The C apolipoproteins are normally transferred to high density llpoprotelns (HDL) after lipolysis of very low density llpoproteln (VLDL) trlglycerlde. In previous studies, a loss of plasma C apolipoproteins was documented after heparin-induced lipolysis In hypertriglyceridemic subjects. The present studies were designed to determine If this decline In plasma C apolipoproteins was due to their clearance with VLDL remnants. Five Type IV hypertriglyceridemic and two normal subjects were Injected with125I-VLDL and1s1Mow density llpoprotelns (LDL) to document klnetically an excess of VLDL apollpoproteln (apo) B flux relative to LDL apo B flux In the Type IV subjects. A mean of 46% VLDL apo B was cleared from the circulation, without conversion to Intermediate density llpoprotein (IDL) or LDL. Heparln was then Infused (9000IU over 4 hours) to generate an excess of VLDL remnants that were not converted to IDL or LDL. VLDL trlglyceride, apo B, and apo C concentrations fell at a similar rate. VLDL apo B declined by 42% (p<0.01). However, no Increases were observed In IDL or LDL apo B In the Type IV subjects. This resulted in a 14% (p<0.01) decline In plasma apo B concentrations, Indicating a clearance of VLDL remnants. VLDL apo C-ll and C-lll concentrations fell by 42% (p<0.025) and 52% (p<0.01), respectively. During the first 2.5 hours of Infusion, they were almost quantitatively recovered In HDL Thereafter, the C apolipoproteins declined In HDL during which time VLDL apo C concentrations continued to decline. This resulted In a delayed fall (C-ll, 21%, p<0.025; C-lll, 26%, p<0.01) In plasma apo C concentrations. Thus, during enhanced lipolysis In Type IV subjects, C apolipoproteins were not cleared with VLDL remnants, but were initially transferred to HDL It Is possible that C apolipoproteins, along with VLDL surface llplds, form an HDL-like particle that Is destined for clearance rather than for recycling to newly formed VLDL.

Thr175-phosphorylated tau induces pathologic fibril formation via GSK3β-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 GSK3β activation. We have now examined whether pThr(175) induces GSK3β 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 GSK3β) levels were significantly increased as was pathologic fibril formation and cell death. Treatment with each of 4 GSK3β inhibitors or small hairpin RNA knockdown of GSK3β 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 GSK3β phosphorylation of Thr(231) leading to fibril formation, indicating a potential therapeutic avenue for amyotrophic lateral sclerosis with cognitive impairment.

Pathologic Thr175tau 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–3β (GSK3β) 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 GSK3β, 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-GSK3β 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 GSK3β, 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 GSK3β in moderate TBI rats. Conclusions Pathologic phosphorylation of tau at Thr175 and Thr231 and activation of GSK3β 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)

Abstract 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.