Lauren Elman

Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS

The causes of amyotrophic lateral sclerosis (ALS), a devastating human neurodegenerative disease, are poorly understood, although the protein TDP-43 has been suggested to have a critical role in disease pathogenesis. Here we show that ataxin 2 (ATXN2), a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2, is a potent modifier of TDP-43 toxicity in animal and cellular models. ATXN2 and TDP-43 associate in a complex that depends on RNA. In spinal cord neurons of ALS patients, ATXN2 is abnormally localized; likewise, TDP-43 shows mislocalization in spinocerebellar ataxia type 2. To assess the involvement of ATXN2 in ALS, we analysed the length of the polyQ repeat in the ATXN2 gene in 915 ALS patients. We found that intermediate-length polyQ expansions (27–33 glutamines) in ATXN2 were significantly associated with ALS. These data establish ATXN2 as a relatively common ALS susceptibility gene. Furthermore, these findings indicate that the TDP-43–ATXN2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways

New players in Lou Gehrigs disease Amyotrophic lateral sclerosis (ALS), often referred to as “Lou Gehrigs disease,” is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Cirulli et al. sequenced the expressed genes of nearly 3000 ALS patients and compared them with those of more than 6000 controls (see the Perspective by Singleton and Traynor). They identified several proteins that were linked to disease in patients. One such protein, TBK1, is implicated in innate immunity and autophagy and may represent a therapeutic target. Science, this issue p. 1436; see also p. 1422 Analysis of the expressed genes of nearly 2900 patients with amyotrophic lateral sclerosis and about 6400 controls reveals a disease predisposition–associated gene. [Also see Perspective by Singleton and Traynor] Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

Stages of pTDP-43 pathology in amyotrophic lateral sclerosis

To see whether the distribution patterns of phosphorylated 43kDa TAR DNA‐binding protein (pTDP‐43) intraneuronal inclusions in amyotrophic lateral sclerosis (ALS) permit recognition of neuropathological stages.

A yeast functional screen predicts new candidate ALS disease genes

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.

Evidence of Multisystem Disorder in Whole-Brain Map of Pathological TDP-43 in Amyotrophic Lateral Sclerosis

BACKGROUND Pathological 43-kDa transactivating responsive sequence DNA-binding protein (TDP-43) has been identified recently as the major disease protein in amyotrophic lateral sclerosis (ALS), and in frontotemporal lobar degeneration with ubiquitinated inclusions, with or without motor neuron disease, but the distribution of TDP-43 pathology in ALS may be more widespread than previously described. OBJECTIVE To determine the extent of TDP-43 pathology in the central nervous systems of patients with clinically confirmed and autopsy confirmed diagnoses of ALS. DESIGN Performance of an immunohistochemical whole-central nervous system scan for evidence of pathological TDP-43 in ALS patients. SETTING An academic medical center. PARTICIPANTS We included 31 patients with clinically and pathologically confirmed ALS and 8 control participants. MAIN OUTCOME MEASURES Immunohistochemistry and double-labeling immunofluorescence to assess the frequency and severity of TDP-43 pathology. RESULTS In addition to the stereotypical involvement of upper and lower motor neurons, neuronal and glial TDP-43 pathology was present in multiple areas of the central nervous systems of ALS patients, including in the nigro-striatal system, the neocortical and allocortical areas, and the cerebellum, but not in those of the controls. CONCLUSIONS These findings suggest that ALS does not selectively affect only the pyramidal motor system, but rather is a multisystem neurodegenerative TDP-43 proteinopathy.

Enrichment of C-Terminal Fragments in TAR DNA-Binding Protein-43 Cytoplasmic Inclusions in Brain but not in Spinal Cord of Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis

TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimers disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies.

Clinical and Pathological Continuum of Multisystem TDP-43 Proteinopathies

OBJECTIVE To determine the extent of transactivation response DNA-binding protein with a molecular weight of 43 kDa (TDP-43) pathology in the central nervous system of patients with clinically and autopsy-confirmed diagnoses of frontotemporal lobar degeneration with and without motor neuron disease and amyotrophic lateral sclerosis with and without cognitive impairment. DESIGN Performance of immunohistochemical whole-central nervous system scans for evidence of pathological TDP-43 and retrospective clinical medical record review. SETTING An academic medical center. PARTICIPANTS We included 64 patients with clinically and pathologically confirmed frontotemporal lobar degeneration with ubiquitinated inclusions with or without motor neuron disease and amyotrophic lateral sclerosis with or without cognitive impairment. MAIN OUTCOME MEASURE Neuronal and glial TDP-43 pathology. RESULTS We found evidence of neuronal and glial TDP-43 pathology in all disease groups throughout the neuraxis, albeit with variations in the frequency, morphology, and distribution of TDP-43 lesions. Moreover, the major clinical manifestations (eg, cognitive impairments, motor neuron signs, extrapyramidal symptoms, neuropsychiatric features) were reflected by the predominant distribution and burden of TDP-43 pathology. CONCLUSION These findings strongly suggest that amyotrophic lateral sclerosis, frontotemporal lobar degeneration with amyotrophic lateral sclerosis or motor neuron disease, and frontotemporal lobar degeneration with ubiquitinated inclusions are different manifestations of a multiple-system TDP-43 proteinopathy linked to similar mechanisms of neurodegeneration.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

Plasma multianalyte profiling in mild cognitive impairment and Alzheimer disease

Objectives: While plasma biomarkers have been proposed to aid in the clinical diagnosis of Alzheimer disease (AD), few biomarkers have been validated in independent patient cohorts. Here we aim to determine plasma biomarkers associated with AD in 2 independent cohorts and validate the findings in the multicenter Alzheimers Disease Neuroimaging Initiative (ADNI). Methods: Using a targeted proteomic approach, we measured levels of 190 plasma proteins and peptides in 600 participants from 2 independent centers (University of Pennsylvania, Philadelphia; Washington University, St. Louis, MO), and identified 17 analytes associated with the diagnosis of very mild dementia/mild cognitive impairment (MCI) or AD. Four analytes (apoE, B-type natriuretic peptide, C-reactive protein, pancreatic polypeptide) were also found to be altered in clinical MCI/AD in the ADNI cohort (n = 566). Regression analysis showed CSF Aβ42 levels and t-tau/Aβ42 ratios to correlate with the number of APOE4 alleles and plasma levels of B-type natriuretic peptide and pancreatic polypeptide. Conclusion: Four plasma analytes were consistently associated with the diagnosis of very mild dementia/MCI/AD in 3 independent clinical cohorts. These plasma biomarkers may predict underlying AD through their association with CSF AD biomarkers, and the association between plasma and CSF amyloid biomarkers needs to be confirmed in a prospective study.

High-Dimensional Spatial Normalization of Diffusion Tensor Images Improves the Detection of White Matter Differences: An Example Study Using Amyotrophic Lateral Sclerosis

Spatial normalization of diffusion tensor images plays a key role in voxel-based analysis of white matter (WM) group differences. Currently, it has been achieved using low-dimensional registration methods in the large majority of clinical studies. This paper aims to motivate the use of high-dimensional normalization approaches by generating evidence of their impact on the findings of such studies. Using an ongoing amyotrophic lateral sclerosis (ALS) study, we evaluated three normalization methods representing the current range of available approaches: low-dimensional normalization using the fractional anisotropy (FA), high-dimensional normalization using the FA, and high-dimensional normalization using full tensor information. Each method was assessed in terms of its ability to detect significant differences between ALS patients and controls. Our findings suggest that inadequate normalization with low-dimensional approaches can result in insufficient removal of shape differences which in turn can confound FA differences in a complex manner, and that utilizing high-dimensional normalization can both significantly minimize the confounding effect of shape differences to FA differences and provide a more complete description of WM differences in terms of both size and tissue architecture differences. We also found that high-dimensional approaches, by leveraging full tensor features instead of tensor-derived indices, can further improve the alignment of WM tracts.