Jemeen Sreedharan

TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

Mutations in FUS, an RNA Processing Protein, Cause Familial Amyotrophic Lateral Sclerosis Type 6

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is familial in 10% of cases. We have identified a missense mutation in the gene encoding fused in sarcoma (FUS) in a British kindred, linked to ALS6. In a survey of 197 familial ALS index cases, we identified two further missense mutations in eight families. Postmortem analysis of three cases with FUS mutations showed FUS-immunoreactive cytoplasmic inclusions and predominantly lower motor neuron degeneration. Cellular expression studies revealed aberrant localization of mutant FUS protein. FUS is involved in the regulation of transcription and RNA splicing and transport, and it has functional homology to another ALS gene, TARDBP, which suggests that a common mechanism may underlie motor neuron degeneration.

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43–positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the ∼25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

Amyotrophic lateral sclerosis: Problems and prospects

Amyotrophic lateral sclerosis (ALS) is a lethal degenerative disorder of motoneurons, which may occur concurrently with frontotemporal dementia. Genetic analyses of the ∼10% of ALS cases that are dominantly inherited provide insight into ALS pathobiology. Two broad themes are evident. One, prompted by investigations of the SOD1 gene, is that conformational instability of proteins triggers downstream neurotoxic processes. The second, from studies of the TDP43, FUS, and C9orf72 genes, is that perturbations of RNA processing can be highly adverse in motoneurons. Several investigations support the concept that non‐neuronal cells (microglia, astroglia, oligodendroglia) participate in the degenerative process in ALS. Recent data also emphasize the importance of molecular events in the axon and distal motoneuron terminals. Only 1 compound, riluzole, is approved by the US Food and Drug Administration for ALS; several therapies are in clinical trials, including 2 mesenchymal stem cell trials. The challenges and unmet needs in ALS emphasize the importance of new research directions: high‐throughput sequencing of large DNA sets of familial and sporadic ALS, which will define scores of candidate ALS genes and pathways and facilitate studies of epistasis and epigenetics; infrastructures for candidate gene validation, including in vitro and in vivo modeling; valid biomarkers that elucidate causative molecular events and accelerate clinical trials; and in the long term, methods to identify environmental toxins. The unprecedented intensity of research in ALS and the advent of extraordinary technologies (rapid, inexpensive DNA sequencing; stem cell production from skin‐derived fibroblasts; silencing of miscreant mutant genes) bode well for discovery of innovative ALS therapies. Ann Neurol 2013;74:309–316

Age-dependent TDP-43-mediated Motor Neuron Degeneration Requires GSK3, Hat-trick, and Xmas-2

The RNA-processing protein TDP-43 is central to the pathogenesis of amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron (MN) disease. TDP-43 is conserved in Drosophila, where it has been the topic of considerable study, but how TDP-43 mutations lead to age-dependent neurodegeneration is unclear and most approaches have not directly examined changes in MN morphology with age. We used a mosaic approach to study age-dependent MN loss in the adult fly leg where it is possible to resolve single motor axons, NMJs and active zones, and perform rapid forward genetic screens. We show that expression of TDP-43(Q331K) caused dying-back of NMJs and axons, which could not be suppressed by mutations that block Wallerian degeneration. We report the identification of three genes that suppress TDP-43 toxicity, including shaggy/GSK3, a known modifier of neurodegeneration. The two additional novel suppressors, hat-trick and xmas-2, function in chromatin modeling and RNA export, two processes recently implicated in human ALS. Loss of shaggy/GSK3, hat-trick, or xmas-2 does not suppress Wallerian degeneration, arguing TDP-43(Q331K)-induced and Wallerian degeneration are genetically distinct processes. In addition to delineating genetic factors that modify TDP-43 toxicity, these results establish the Drosophila adult leg as a valuable new tool for the in vivo study of adult MN phenotypes.

Amyotrophic lateral sclerosis: recent genetic highlights.

PURPOSE OF REVIEW Amyotrophic lateral sclerosis (ALS), like other neurodegenerative diseases, remains incurable, but gene mutations linked to ALS are providing clues as to how to target therapies. It is important for researchers to keep abreast of the rapid influx of new data in ALS, and we aim to summarize the major genetic advances made in the field over the past 2 years. RECENT FINDINGS Significant variation in seven genes has recently been found in ALS: TBK1, CCNF, GLE1, MATR3, TUBA4A, CHCHD10 and NEK1. These have mostly been identified through large exome screening studies, though traditional linkage approaches and candidate gene screening remain important. We briefly update C9orf72 research, noting in particular the development of reagents to better understand the normal role of C9orf72 protein. SUMMARY Striking advances in our understanding of the genetic heterogeneity of ALS continue to be made, year on year. These implicate proteostasis, RNA export, nuclear transport, the cytoskeleton, mitochondrial function, the cell cycle and DNA repair. Functional studies to integrate these hits are needed. By building a web of knowledge with interlinked genes and mechanisms, it is hoped we can better understand ALS and work toward effective therapies.

Alexander disease with hypothermia, microcoria, and psychiatric and endocrine disturbances

A recent article on Alexander disease (AxD)1 made note of the clinical and MRI phenotypic variation that is increasingly seen in this rare progressive leukodystrophy, particularly in late-onset and slowly progressive cases. AxD is caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP), resulting in Rosenthal fiber deposition in astrocytes. It is most commonly seen in infants with sporadic mutations, who present with macrocephaly, seizures, and developmental delay. The disease progresses rapidly to death within 10 years. Adult-onset cases are phenotypically different with more brainstem involvement and slower progression and more likely to demonstrate autosomal dominant inheritance.1-3 The following case highlights previously undescribed features of adult-onset AxD. A 38-year-old woman was referred with a 2-year history of progressive difficulty reading with oscillopsia, slurring dysarthria, choking, and stumbling. Her early motor and intellectual development were normal, and she achieved a university degree. At age 32 she developed amenorrhea lasting 9 months, having previously had normal periods, and was …

Neuronal death in amyotrophic lateral sclerosis (ALS): what can we learn from genetics?

Amyotrophic lateral sclerosis (ALS) is a difficult disease to study as it is mostly sporadic and rapidly progressive. Identification of genes causing familial ALS (FALS) has been instrumental in advancing our understanding of ALS pathogenesis, most notably with the use of mutant superoxide dismutase 1 (SOD1) models of disease. For 15 years SOD1 models have been the backbone of ALS research, but no effective reatments have been developed. However, recent advances have been made in the genetics of ALS with the identification of mutations in TAR DNA binding protein (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS), both of which have roles in RNA-processing and gene expression. Molecular links between ALS and frontotemporal dementia (FTD) are also suggested by linkage of ALS-FTD to chromosome 9. The study of the genetics of sporadic ALS (SALS) has been less fruitful, although this may change as we enter the era of resequencing. Further important clues as to the causes of ALS will come from the identification of other gene mutations that cause FALS, variants that increase susceptibility to SALS, and genetic factors that modify the ALS phenotype.

TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD

Amyotrophic lateral sclerosis–frontotemporal dementia (ALS-FTD) constitutes a devastating disease spectrum characterized by 43-kDa TAR DNA-binding protein (TDP-43) pathology. Understanding how TDP-43 contributes to neurodegeneration will help direct therapeutic efforts. Here we have created a TDP-43 knock-in mouse with a human-equivalent mutation in the endogenous mouse Tardbp gene. TDP-43Q331K mice demonstrate cognitive dysfunction and a paucity of parvalbumin interneurons. Critically, TDP-43 autoregulation is perturbed, leading to a gain of TDP-43 function and altered splicing of Mapt, another pivotal dementia-associated gene. Furthermore, a new approach to stratify transcriptomic data by phenotype in differentially affected mutant mice revealed 471 changes linked with improved behavior. These changes included downregulation of two known modifiers of neurodegeneration, Atxn2 and Arid4a, and upregulation of myelination and translation genes. With one base change in murine Tardbp, this study identifies TDP-43 misregulation as a pathogenic mechanism that may underpin ALS-FTD and exploits phenotypic heterogeneity to yield candidate suppressors of neurodegenerative disease.TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD, leading to aberrant Mapt splicing and a paucity of parvalbumin interneurons. Phenotypic heterogeneity is exploited to yield modifiers of disease.

Optimizing reproducibility of operant testing through reinforcer standardization: identification of key nutritional constituents determining reward strength in touchscreens

Reliable and reproducible assessment of animal learning and behavior is a central aim of basic and translational neuroscience research. Recent developments in automated operant chamber technology have led to the possibility of universal standard protocols, in addition to increased translational potential, reliability and accuracy. However, the impact of regional and national differences in the supplies of available reinforcers in this system on behavioural performance and inter-laboratory variability is an unknown and at present uncontrolled variable. Therefore, we aimed to identify which constituent(s) of the reward determines reinforcer strength to enable improved standardization of this parameter across laboratories. Male C57BL/6 mice were examined in the touchscreen-based fixed ratio (FR) and progressive ratio (PR) schedules, reinforced with different kinds of milk-based reinforcers to directly compare the incentive values of plain milk (PM, high-calorie: high-fat/low-sugar), strawberry-flavored milk (SM, high-calorie: low-fat/high-sugar), and semi-skimmed low-fat milk (LM, low-calorie: low-fat/low-sugar) on the basis of differences in caloric content, sugar/fat content, and flavor. Use of a higher caloric content reward was effective in increasing operant training acquisition rate. Total trial number completed in FR and breakpoint in PR were higher using the two isocaloric milk products (PM and SM) than the lower caloric LM, with comparable outcomes between PM and SM conditions, suggesting that total caloric content determines reward strength. Analysis of within-session changes in response rate revealed that overall outputs in FR and PR primarily depend on the response rate at the initial phase of a session, which itself was dependent on reinforcer caloric content. Interestingly, the rate of satiation, indicated by decay in response rate within a FR session, was highest when reinforced with SM, suggesting a rapid satiating effect of sugar. The key contribution of reward caloric content to operant performance was confirmed in a multi-laboratory study using the touchscreen 5-choice serial reaction time task (5-CSRTT) reinforced by two isocaloric milk-based liquid rewards with different countries of origin, which yielded consistent performance parameters across sites. Our results indicate that milk-based liquid reinforcer standardization can be facilitated by matching caloric content across laboratories despite regional or national differences in other non-caloric aspects of the reinforcers.