Wencheng Yang

Lack of TDP-43 abnormalities in mutant SOD1 transgenic mice shows disparity with ALS.

Mislocalization of the TAR-DNA binding protein (TDP-43) from the nucleus to the cytoplasm of diseased motor neurons and association with intraneuronal ubiquitinated inclusions has recently been reported in amyotrophic lateral sclerosis (ALS). Here, we have investigated TDP-43 immunoreactivity in three lines of mutant SOD1 transgenic mice, G93A, G37R and G85R and compared with labeling in one sporadic ALS case and two familial ALS cases carrying mutations in SOD1, A4T and I113T. Our findings show that there is no mislocalization of TDP-43 to the cytoplasm in motor neurons of mutant SOD1 transgenic mice, nor association of TDP-43 with ubiquitinated inclusions. In contrast, mislocalization of TDP-43 to the cytoplasm and association with ubiquitinated inclusions was found in the ALS cases, including those carrying mutations in SOD1. Interestingly, there was no association of TDP-43 with ubiquitinated hyaline conglomerate inclusions, pathology closely associated with ALS cases carrying mutations in SOD1. Our findings indicate that the process of motor neuron degeneration in mutant SOD1 transgenic mice is unlikely to involve the abnormalities of TDP-43 described in the human disease.

Cognitive impairment, frontotemporal dementia, and the motor neuron diseases

Traditionally, amyotrophic lateral sclerosis (ALS) has been considered to be a disorder in which the primary pathology resides in the degeneration of motor neurons, giving rise to progressive, diffuse muscle wasting, weakness, and spasticity. This view is rapidly being challenged to include a more widespread disease process in which motor system degeneration remains the core feature but not the sole system affected. Although its incidence remains to be determined, the evidence of cognitive impairment in ALS is overwhelming. However, it is important to recognize that there is likely to exist a continuum of cognitive dysfunction in ALS, ranging from mild cognitive impairment (ALSci) to a more fulminant progressive dementia of the frontotemporal type (FTD) with a small subset with an aphasic pattern. Most commonly, ALSci is reported as a subtle neurological presentation marked by deficits primarily in frontal and temporal functions. Although in the Japanese population, alterations in cognition have been reported to precede the motor system manifestations, typically the cognitive dysfunction follows the onset of neuromuscular deficits. Individuals with bulbar-onset disease appear to be at greater risk for the development of cognitive impairment, although these deficits are found to a lesser degree in limb-onset patients. Typical impairments include alterations in mental flexibility, verbal and nonverbal fluency, abstract reasoning, and memory, for both verbal and visual material. Less often, there is a rapidly progressive aphasic dementia. ALSci language characteristics include word-finding difficulty, lexical disorganization, and reliance on stereotypical sentences. Anterior-based language functions (eg, fluency, syntax, and grammar) are compromised. The relationship between FTD, FTD with ALS, and FTD with the neuropathological features of ALS in the absence of clinical findings remains to be clarified. There is likely to be significant overlap among these entities, as illustrated in a study of 36 FTD patients with no known diagnosis of ALS, 5 of whom met clinical and electrophysiological criteria for a definite diagnosis of ALS and an additional 13 who met criteria for possible ALS, of whom 1 subsequently developed ALS. The converse is also true. A study of word generation tests in 100 consecutive ALS patients identified a subset of 44 patients with impairments. Of these, diminished word generation was found in one third. Of this latter group, nearly all were shown to meet research criteria for FTD. In addition, one quarter of the patients with normal word generation who agreed to further evaluation met research criteria for FTD; these patients had new-onset personality changes. In addition to the precentral gyrus atrophy that is well recognized in ALS, neuroimaging in ALSci demonstrates a more widespread frontal and temporal cortical atrophy, with variable involvement of the precentral and postcentral gyrus, the anterior cingulate gyrus, corpus callosum, and brainstem tegmentum. Functional neuroimaging techniques, including single-positron emission tomography studies with either I-Nisopropyl-p-iodoamphetamine or Tc-d,l-hexamethylproplene–amine oxime (HM-PAO), and positron emission tomography, are sensitive to reduced frontal and temporal cortical blood flow in ALSci. Reductions in the N-acetylaspartase to creatine ratio of anterior cingulate gyrus neurons with the use of proton magnetic resonance spectroscopy has been shown to be a sensitive marker of ALSci. The typical neuropathological features of ALS include degeneration of the descending supraspinal motor pathways and their neurons of origin, and of brainstem and spinal motor neurons in addition to

Microtubule-associated tau protein positive neuronal and glial inclusions in ALS

Background: The authors compared tau protein deposition in the frontal cortex of patients with cognitive impairment of amyotrophic lateral sclerosis (ALSci) (n = 6), cognitively intact patients with ALS (n = 6), and age-matched controls (n = 6) in order to determine the pathologic substrate of ALSci. Methods: Archival paraffin-embedded tissue was examined using Gallyas staining and immunostaining for tau-1 (phosphorylation-dependent tau epitope), tau-2 (phosphorylation independent), Alzheimer-specific tau phosphoepitopes (AT 8; ser396 phosphorylation), β-amyloid, glial fibrillary acid protein, SMI 31 (recognizing phosphorylated NFH), α-synuclein, or ubiquitin. Results: Tau immunoreactive astrocytic and dense neuronal inclusions were found in both ALS and ALSci, although to a greater extent in ALSci. Superficial linear spongiosis and Gallyas-positive intraneuronal aggregates, immunoreactive with tau-1 and AT 8 but rarely to ser396 tau, were unique to ALSci. Dense extracellular aggregates were observed by both Gallyas staining and tau-1 immunostaining. Tufted degenerating astrocytes containing tau-1 and AT 8 immunoreactive aggregates and, rarely, dense Gallyas positive neuritic plaques immunoreactive with tau-1 and AT 8, but not with ser396 tau or β-amyloid, were observed in ALSci. Tau positive glial coiled bodies were observed in the deep cortical layers and adjacent subcortical white matter in ALSci. Although 3R and 4R tau mRNA isoforms were expressed to similar levels in the frontal cortex of all cases, the total amount of tau mRNA was increased in both ALS and ALSci. Both gray and white matter soluble tau protein expression was similar among control, ALS, and ALSci cases. Conclusions: Cognitive dysfunction in ALS may reflect abnormal tau protein metabolism.

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.

Upregulation of GSK3β expression in frontal and temporal cortex in ALS with cognitive impairment (ALSci)

The deposition of highly phosphorylated microtubule-associated tau protein has been observed in ALS with cognitive impairment (ALSci). In these studies, we have examined whether the expression of two candidate protein kinases for mediating tau hyperphosphorylation (GSK3beta or CDK5) are also altered. The expression of GSK, CDK and p25/p35 was assayed in human frontal, hippocampal, cerebellar, cervical (dorsal and ventral) and lumbar (dorsal and ventral) tissue from neurologically intact control (5), ALS (5) or ALSci (5) patients using RT-PCR, Western blot or immunohistochemistry. To assess GSK-3beta activity, we examined GSK3beta, phospho-GSK3beta and phospho-beta-catenin expression. Expression levels relative to that of beta-actin were compared by ANOVA. The expression of GSK, GSK3beta and phospho-GSK3beta was increased in both ALS and ALSci compared to that of the control. This was accompanied by an increased expression of phospho-beta-catenin. No significant difference between control, ALS or ALSci was observed with respect to the expression of CDK5 or p25/p35. Both GSK3beta and phospho-GSK3beta immunoreactive neurons were mainly located in layer II and layer III in the frontal cortex and in layer II in the hippocampus. This was consistent with the previously described distribution of hyperphosphorylated tau bearing neurons in ALS and ALSci. These data suggest that GSK3beta expression is upregulated in ALS and ALSci and that GSK3beta activation is associated with the intraneuronal deposition of hyperphosphorylated tau protein. This supports the potential role for GSK3beta as a therapeutic target in ALS.

Widespread neuronal and glial hyperphosphorylated tau deposition in ALS with cognitive impairment.

Abstract Although the biological basis of frontotemporal syndromes associated with amyotrophic lateral sclerosis (ALS) is considered to be altered metabolism of TDP-43, in ALS with cognitive impairment (ALSci) the metabolism of tau protein is also altered. This includes neuronal hyperphosphorylation (pThr175). Using novel polyclonal phospho-tau antibodies (pSer208, 210, pThr217 and pThr175) and antibodies directed against PHF tau (pSer202), TDP-43 or ubiquitin, we characterized tau deposition in ALS and ALSci. In ALS, we observed pThr175 tau immunoreactive intraneuronal and neuritic aggregates throughout the amygdala and entorhinal cortex. In ALSci, this extended to the anterior cingulate gyrus, superior frontal cortex and substantia nigra. The pThr217 antibody detected widespread astrocytic tau deposition, including punctuate or fibrillary aggregates, or intensely immunoreactive tufted astrocytes in the superior frontal cortex, anterior cingulate gyrus, entorhinal cortex, amygdala and basal ganglia of ALS. In ALSci, a similar but more widely distributed pThr217 pathology was observed. There was no correlation between the extent of pathological tau deposition and TDP-43 pathology, although nuclear TDP-43 immunoreactivity was absent in neurons with tau pathology. In conclusion, ALSci is unique in possessing both tau and TDP-43 pathology. The presence of widespread astrocytic tau pathology suggests that ALSci may initially be characterized by astrocytic pathology.

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.

Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1.

Mutations in the RNA-binding protein FUS/TLS (FUS) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Although predominantly nuclear, this heterogenous nuclear ribonuclear protein (hnRNP) has multiple functions in RNA processing including intracellular trafficking. In ALS, mutant or wild-type (WT) FUS can form neuronal cytoplasmic inclusions. Asymmetric arginine methylation of FUS by the class 1 arginine methyltransferase, protein arginine methyltransferase 1 (PRMT1), regulates nucleocytoplasmic shuttling of FUS. In motor neurons of primary spinal cord cultures, redistribution of endogenous mouse and that of ectopically expressed WT or mutant human FUS to the cytoplasm led to nuclear depletion of PRMT1, abrogating methylation of its nuclear substrates. Specifically, hypomethylation of arginine 3 of histone 4 resulted in decreased acetylation of lysine 9/14 of histone 3 and transcriptional repression. Distribution of neuronal PRMT1 coincident with FUS also was detected in vivo in the spinal cord of FUS(R495X) transgenic mice. However, nuclear PRMT1 was not stable postmortem obviating meaningful evaluation of ALS autopsy cases. This study provides evidence for loss of PRMT1 function as a consequence of cytoplasmic accumulation of FUS in the pathogenesis of ALS, including changes in the histone code regulating gene transcription.

Threonine175, a novel pathological phosphorylation site on tau protein linked to multiple tauopathies

Microtubule associated protein tau (tau) deposition is associated with a spectrum of neurodegenerative diseases collectively termed tauopathies. We have previously shown that amyotrophic lateral sclerosis (ALS) with cognitive impairment (ALSci) is associated with tau phosphorylation at Thr175 and that this leads to activation of GSK3β which then induces phosphorylation at tau Thr231. This latter step leads to dissociation of tau from microtubules and pathological tau fibril formation. To determine the extent to which this pathway is unique to ALS, we have investigated the expression of pThr175 tau and pThr231 tau across a range of frontotemporal degenerations. Representative sections from the superior frontal cortex, anterior cingulate cortex (ACC), amygdala, hippocampal formation, basal ganglia, and substantia nigra were selected from neuropathologically confirmed cases of Alzheimer’s disease (AD; n = 3), vascular dementia (n = 2), frontotemporal lobar degeneration (FTLD; n = 4), ALS (n = 5), ALSci (n = 6), Parkinson’s disease (PD; n = 5), corticobasal degeneration (CBD; n = 2), diffuse Lewy body dementia (DLBD; n = 2), mixed DLBD (n = 3), multisystem atrophy (MSA; n = 6) and Pick’s disease (n = 1) and three neuropathologically-normal control groups aged 50–60 (n = 6), 60–70 (n = 6) and 70–80 (n = 8). Sections were examined using a panel of phospho-tau antibodies (pSer208,210, pThr217, pThr175, pThr231, pSer202 and T22 (oligomeric tau)). Across diseases, phospho-tau load was most prominent in layers II/III of the entorhinal cortex, amygdala and hippocampus. This is in contrast to the preferential deposition of phospho-tau in the ACC and frontal cortex in ALSci. Controls showed pThr175 tau expression only in the 7th decade of life and only in the presence of tau pathology and tau oligomers. With the exception of DLBD, we observed pThr175 co-localizing with pThr231 in the same cell populations as T22 positivity. This suggests that this pathway may be a common mechanism of toxicity across the tauopathies.

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.