Anterior horn cells with abnormal TDP-43 immunoreactivities show fragmentation of the Golgi apparatus in ALS

Recently, TAR DNA-binding protein of 43-kDa (TDP-43) was identified as a major component of ubiquitinated neuronal cytoplasmic inclusions observed in lower motor neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions. We herein investigated the relationship between TDP-43 immunoreactivities and fragmentation of the Golgi apparatus (GA). Each mirror section of spinal cord tissues in 10 ALS and 3 control cases were immunostained with polyclonal anti-TDP-43 and polyclonal anti-trans-Golgi-network (TGN)-46 antibodies. The neurons were divided into subtypes according to differences in TDP-43 immunoreactivities, and we examined the morphological changes of GA in each type. We divided the neurons into four subtypes according to the observed differences in TDP-43 immunoreactivities, Type A: neurons showing normal nuclear staining, Type B: neurons showing a loss of normal nuclear staining and a few granular cytoplasmic immunoreactivities, Type C: neurons showing a lot of granular immunoreactivities and no inclusions, Type D: neurons with inclusions. All of the neurons in Type A showed normal GA profiles, however, almost all of the neurons with abnormal TDP-43 immunoreactivities (Type B–D) showed GA fragmentation. These results suggest that neurons with abnormal TDP-43 immunoreactivities are associated with dysfunction of the secretory pathway in motor neurons.     

ALS and FTLD: two faces of TDP-43 proteinopathy

Major discoveries have been made in the recent past in the genetics, biochemistry and neuropathology of frontotemporal lobar degeneration (FTLD). TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, has been identified as the major pathological protein of FTLD with ubiquitin-immunoreactive (ub-ir) inclusions (FTLD-U) with or without amyotrophic lateral sclerosis (ALS) and sporadic ALS. Recently, mutations in the TARDBP gene in familial and sporadic ALS have been reported which demonstrate that abnormal TDP-43 alone is sufficient to cause neurodegeneration. Several familial cases of FTLD-U, however, are now known to have mutations in the progranulin ( GRN ) gene, but granulin is not a component of the TDP-43- and ub-ir inclusions. Further, TDP-43 is found to be a component of the inclusions of an increasing number of neurodegenerative diseases. Other FTLD-U entities with TDP-43 proteinopathy include: FTLD-U with valosin-containing protein ( VCP ) gene mutation and FTLD with ALS linked to chromosome 9p. In contrast, chromosome 3-linked dementia, FTLD-U with chromatin modifying protein 2B ( CHMP2B ) mutation, has ub-ir, TDP-43-negative inclusions. In summary, recent discoveries have generated new insights into the pathogenesis of a spectrum of disorders called TDP-43 proteinopathies including: FTLD-U, FTLD-U with ALS, ALS, and a broadening spectrum of other disorders. It is anticipated that these discoveries and a revised nosology of FTLD will contribute toward an accurate diagnosis, and facilitate the development of new diagnostic tests and therapeutics.     

肌萎缩侧索硬化症相关TDP-43降解机制的研究

目的探索肌萎缩侧索硬化症(ALS)相关TDP-43的降解机制。方法用瞬时转染的方法在运动神经元样细胞系NSC-34中过表达野生型(role of wild-type,WT)WT TDP-43,与家族型ALS相关的Q331K TDP-43、M337V TDP-43突变蛋白、TDP-43的两种C末端片段TDP-25和TDP-35,再给予自噬、蛋白酶体通路的特异性诱导剂和阻断剂,通过蛋白印迹方法检测5种TDP-43以及自噬标记物LC3-Ⅱ的表达水平。结果在自噬诱导剂作用下,各组LC3-Ⅱ的表达升高,同时两种突变TDP-43及其C末端片段的表达明显减少,在自噬通路和蛋白酶体阻断剂作用下突变TDP-43及其C末端片段表达水平明显增多,而WT TDP-43的蛋白表达水平仅在蛋白酶体阻断剂作用时增多。结论 WT TDP-43主要经由蛋白酶体途径降解,Q331K TDP-43、M337V TDP-43及其C末端片段经由蛋白酶体途径和自噬两种途径降解。     

Distinct TDP-43 pathology in ALS patients with ataxin 2 intermediate-length polyQ expansions

Amyotrophic lateral sclerosis (ALS) is a progressive, adult-onset neurodegenerative disease characterized by degeneration of motor neurons, resulting in paralysis and death. A pathological hallmark of the degenerating motor neurons in most ALS patients is the presence of cytoplasmic inclusions containing the protein TDP-43. The morphology and type of TDP-43 pathological inclusions is variable and can range from large round Lewy body-like inclusions to filamentous skein-like inclusions. The clinical significance of this variable pathology is unclear. Intermediate-length polyglutamine (polyQ) expansions in ataxin 2 were recently identified as a genetic risk factor for ALS. Here we have analyzed TDP-43 pathology in a series of ALS cases with or without ataxin 2 intermediate-length polyQ expansions. The motor neurons of ALS cases harboring ataxin 2 polyQ expansions (n = 6) contained primarily skein-like or filamentous TDP-43 pathology and only rarely, if ever, contained large round inclusions, whereas the ALS cases without ataxin 2 polyQ expansions (n = 13) contained abundant large round and skein-like TDP-43 pathology. The paucity of large round TDP-43 inclusions in ALS cases with ataxin 2 polyQ expansions suggests a distinct pathological subtype of ALS and highlights the possibility for distinct pathogenic mechanisms.     

Optineurin inclusions occur in a minority of TDP-43 positive ALS and FTLD-TDP cases and are rarely observed in other neurodegenerative disorders

Optineurin (OPTN) is a multifunctional protein involved in vesicular trafficking, signal transduction and gene expression. OPTN mutations were described in eight Japanese patients with familial and sporadic amyotrophic lateral sclerosis (FALS, SALS). OPTN-positive inclusions co-localising with TDP-43 were described in SALS and in FALS with SOD-1 mutations, potentially linking two pathologically distinct pathways of motor neuron degeneration. We have explored the abundance of OPTN inclusions using a range of antibodies in postmortem tissues from 138 cases and controls including sporadic and familial ALS, frontotemporal lobar degeneration (FTLD) and a wide range of neurodegenerative proteinopathies. OPTN-positive inclusions were uncommon and detected in only 11/32 (34%) of TDP-43-positive SALS spinal cord and 5/15 (33%) of FTLD-TDP. Western blot of lysates from FTLD-TDP frontal cortex and TDP-43-positive SALS spinal cord revealed decreased levels of OPTN protein compared to controls (p < 0.05), however, this correlated with decreased neuronal numbers in the brain. Large OPTN inclusions were not detected in FALS with SOD-1 and FUS mutation, respectively, or in FTLD-FUS cases. OPTN-positive inclusions were identified in a few Alzheimer’s disease (AD) cases but did not co-localise with tau and TDP-43. Occasional striatal neurons contained granular cytoplasmic OPTN immunopositivity in Huntington’s disease (HD) but were absent in spinocerebellar ataxia type 3. No OPTN inclusions were detected in FTLD-tau and α-synucleinopathy. We conclude that OPTN inclusions are relatively rare and largely restricted to a minority of TDP-43 positive ALS and FTLD-TDP cases. Our results do not support the proposition that OPTN inclusions play a central role in the pathogenesis of ALS, FTLD or any other neurodegenerative disorder.     

反式激活应答DNA结合蛋白-43及其相关疾病的研究进展

反式激活应答DNA结合蛋白-43(TDP-43)是近年发现的一种病理沉积蛋白,常见于多种神经变性疾病,如肌萎缩侧索硬化、额颞叶变性、阿尔茨海默病等,这一类以病理性TDP-43沉积为主的神经变性疾病统称为TDP-43蛋白病。研究发现TDP-43在这些疾病中既有相同(均有TDP-43异常沉积),又有差异(不同的形态、分布而导致不同的症状),其作用机制至今仍有许多未明。这一异常蛋白的发现不仅为研究TDP-43蛋白病的发病机制及相关疾病间的联系提供了新途径,同时为探索新的诊断方法、治疗方案提供了方向。     

TDP-43 and amyloid precursor protein processing: implications for Alzheimer's disease

正In this perspective article, I will discuss our recent publication(Hicks et al., 2020), specifically its major findings and integration with the published literature.Alzheimer 's disease(AD) is a progressive neurodegenerative     

Cytoplasmic accumulation of TDP-43 in circulating lymphomonocytes of ALS patients with and without TARDBP mutations

TDP-43, encoded by TARDBP, is a ubiquitously expressed, primarily nuclear protein. In recent years, TDP-43 has been identified as the major pathological protein in ALS due to its mislocalisation in the cytoplasm of motor neurons of patients with and without TARDBP mutations and expression in forms that do not match its predicted molecular weight. In this study, the TDP-43 profile was investigated using western immunoblot analysis in whole lysates, nuclei and cytoplasm of circulating lymphomonocytes from 16 ALS patients, 4 with (ALS/TDP+) and 12 without (ALS/TDP-) TARDBP mutations in the protein C-terminal domain, and thirteen age-matched, healthy donors (controls). Three disease-unaffected first-degree relatives of an ALS/TDP+ patient were also included: one carried the parent mutation (Rel/TDP+) whereas the other two did not (Rel/TDP-). In all ALS patients, relatives and controls, TDP-43 retained the predicted molecular weight in whole cell lysates and nuclei, but in the cytoplasm its molecular weight was slightly smaller than expected. In quantitative terms, TDP-43 was expressed at approximately the same levels in whole cell lysates of ALS patients, relatives and controls. In contrast, TDP-43 accumulated in the cytoplasm with concomitant nuclear depletion in all ALS/TDP+ patients, in about 50% of ALS/TDP- patients and in the Rel/TDP+ subject compared to the controls. In the remaining ALS/TDP- patients and in the two Rel/TDP- subjects, TDP-43 matched the control levels in both subcellular compartments. Were these findings further confirmed, circulating lymphomonocytes could be informative of TDP-43 mislocalisation in nervous tissue and used as a biomarker for future disease risk.     

TDP-43-based animal models of neurodegeneration: new insights into ALS pathology and pathophysiology

The clinical and pathological overlap between amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) suggests these diseases share common underlying mechanisms, a suggestion underscored by the discovery that TDP-43 inclusions are a key pathologic feature in both ALS and FTLD. This finding, combined with the identification of TDP-43 mutations in ALS, directly implicates this DNA/RNA binding protein in disease pathogenesis in ALS and FTLD. However, many key questions remain, including what is the normal function of TDP-43, and whether disease-associated mutations produce toxicity in the nucleus, cytoplasm or both. Furthermore, although pathologic TDP-43 inclusions are clearly associated with many forms of neurodegeneration, whether TDP-43 aggregation is a key step in the pathogenesis in ALS, FTLD and other disorders remains to be proven. This review will compare the features of numerous recently developed animal models of TDP-43-related neurodegeneration, and discuss how they contribute to our understanding of the pathogenesis of human ALS and FTLD.     

Comparison of phosphorylated TDP-43-positive inclusions in oculomotor neurons in patients with non-ALS and ALS disorders

TDP-43 has been identified as a major component of the pathological inclusions in most forms of frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). In the present study, paraffin sections of the midbrain in 112 patients with various non-ALS disorders and 27 patients with sporadic ALS were immunostained with antibody against phosphorylated TDP-43 (pTDP-43). pTDP-43-positive inclusions in oculomotor neurons were detected in 18 of 112 patients with non-ALS disorders (16.1%). The appearance of the inclusions showed fine filamentous structures rather than the skein-like inclusions seen in the anterior horn cells of ALS spinal cords. The incidence was increased in the age range of 80-89 years old (10/37 cases; 27.0%), in which 6 of 10 cases demonstrated AD pathology in the temporal lobes. Twenty-seven ALS patients were examined and the findings were compared with those of non-ALS patients. There were 13 cases demonstrating pTDP-43-positive inclusions (48.1%) which showed stronger immunoreactivities in ALS cases. This is the first report demonstrating fine filamentous pTDP-43-positive inclusions in oculomotor neurons in non-ALS disorders. Although the mechanisms underlying pTDP-43 in oculomotor neurons are currently unknown, its detection is of interest, and the expression may occur not only in ALS but also during the aging process.     

Molecular link between inefficient GluA2 RNA editing and TDP-43 pathology in ALS motor neurons

The motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients exhibit several molecular abnormalities, including 2 that are specific to ALS motor neurons: (1) pathological changes related to the mislocalization of the TAR DNA-binding protein (TDP-43), including both the appearance of phosphorylated TDP-43-containing inclusions in the cytoplasm and the loss of TDP-43 from the nucleus; and (2) inefficient RNA editing at the Q/R site of GluA2, a subunit of the AMPA receptor. TDP-43-related pathological features are closely associated with ALS in most ALS patients and with significant behavioral and pathological changes in genetically engineered mice; therefore, abnormal TDP-43 processing is believed to play a role in the pathogenesis of ALS. The extent of GluA2 RNA editing decreases in the motor neurons of sporadic ALS patients in a disease-specific and motor neuron-selective manner. Importantly, this molecular abnormality is a direct cause of death of motor neurons in conditional knockout mice for adenosine deaminase acting on RNA 2 (ADAR2), the enzyme that specifically catalyzes RNA editing at the GluA2 Q/R site. Notably, these molecular abnormalities, i.e., TDP-43-related pathological features and inefficient GluA2 RNA editing, are found in approximately half of the motor neurons in sporadic ALS patients and both of them always occur in the same motor neurons. Because TDP-43-related pathological features and inefficient GluA2 RNA editing are highly disease specific in ALS motor neurons, investigation into the molecular link between these abnormalities is likely to provide new insights into ALS pathogenesis.     

Conjoint pathologic cascades mediated by ALS/FTLD-U linked RNA-binding proteins TDP-43 and FUS

The RNA-binding proteins TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) play central roles in neurodegeneration associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Normally localized in the nucleus, in sites affected by ALS and FTLD-U they are mislocalized to the cytoplasm and form cytoplasmic inclusions. TDP-43 and FUS are transported to the nucleus in a Ran-GTPase-dependent manner via nuclear import receptors, but they also contribute to the formation of stress granules (SGs), which are intracytoplasmic structures incorporating RNA. C-terminal truncations of TDP-43 eliminate the nuclear transport signal and cause mislocalization of the protein to the cytoplasm, where it accumulates and forms SGs. ALS-associated FUS mutations impair nuclear transport and cause mislocalization of FUS to the cytoplasm, where it also contributes to assembly of SGs. Furthermore, the ALS susceptibility factor ataxin-2, recently identified as a potent modifier of TDP-43 toxicity, is also a predicted cytoplasmic RNA-binding protein and a constituent protein of SGs, suggesting that it is a part of the common pathologic cascade formed by TDP-43 and FUS. Thus, we propose that excessive mislocalization of the RNA-binding proteins TDP-43, FUS, and ataxin-2 into the cytoplasm leads to impairment of the RNA quality control system, forming the core of the ALS/FTLD-U degenerative cascade. In this review, we discuss the molecular basis of the novel disease spectrum of ALS/FTLD-U, including the neurodegenerative mechanism of the cytoplasmic RNA-binding proteins TDP-43 and FUS and the possibility of a novel therapeutic strategy.     

TDP-43 pathology in sporadic ALS occurs in motor neurons lacking the RNA editing enzyme ADAR2

Both the appearance of cytoplasmic inclusions containing phosphorylated TAR DNA-binding protein (TDP-43) and inefficient RNA editing at the GluR2 Q/R site are molecular abnormalities observed specifically in motor neurons of patients with sporadic amyotrophic lateral sclerosis (ALS). The purpose of this study is to determine whether a link exists between these two specific molecular changes in ALS spinal motor neurons. We immunohistochemically examined the expression of adenosine deaminase acting on RNA 2 (ADAR2), the enzyme that specifically catalyzes GluR2 Q/R site-editing, and the expression of phosphorylated and non-phosphorylated TDP-43 in the spinal motor neurons of patients with sporadic ALS. We found that all motor neurons were ADAR2-positive in the control cases, whereas more than half of them were ADAR2-negative in the ALS cases. All ADAR2-negative neurons had cytoplasmic inclusions that were immunoreactive to phosphorylated TDP-43, but lacked non-phosphorylated TDP-43 in the nucleus. Our results suggest a molecular link between reduced ADAR2 activity and TDP-43 pathology.