Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disease. The causes of ALS are poorly understood, although the protein TDP-43 has been suggested to play a critical role in disease pathogenesis. Here we show that Ataxin-2, a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2 (SCA2), is a potent modifier of TDP-43 toxicity in animal and cellular models. The proteins associate in a complex that depends on RNA. Ataxin-2 is abnormally localized in spinal cord neurons of ALS patients. Likewise, TDP-43 shows mislocalization in SCA2. To assess a role in ALS, we analyzed the Ataxin-2 gene (ATXN2) in 915 ALS patients. We found intermediate-length polyQ expansions (27–33 Qs) in ATXN2 significantly associated with ALS. These data establish ATXN2 as a relatively common ALS disease susceptibility gene. Further, these findings indicate that the TDP-43/Ataxin-2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.

ALS is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most ALS patients. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of Dbr1, which encodes RNA lariat debranching enzyme. We show that in the absence of Dbr1 enzymatic activity intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43 away from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rodent neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43 cytotoxicity and suggest decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

The mechanisms that govern the formation of ␣-synuclein (␣-syn) aggregates are not well understood but are considered a central event in the pathogenesis of Parkinson's disease (PD). A critically important modulator of ␣-syn aggregation in vitro is dopamine and other catechols, which can prevent the formation of ␣-syn aggregates in cell-free and cellular model systems. Despite the profound importance of this interaction for the pathogenesis of PD, the processes by which catechols alter ␣-syn aggregation are unclear. Molecular and biochemical approaches were employed to evaluate the mechanism of catechol-␣-syn interactions and the effect on inclusion formation. The data show that the intracellular inhibition of ␣-syn aggregation requires the oxidation of catechols and the specific noncovalent interaction of the oxidized catechols with residues 125 YEMPS 129 in the C-terminal region of the protein. Cell-free studies using novel near infrared fluorescence methodology for the detection of covalent proteinortho-quinone adducts showed that although covalent modification of ␣-syn occurs, this does not affect ␣-syn fibril formation. In addition, oxidized catechols are unable to prevent both thermal and acid-induced protein aggregation as well as fibrils formed from a protein that lacks a YEMPS amino acid sequence, suggesting a specific effect for ␣-syn. These results suggest that inappropriate C-terminal cleavage of ␣-syn, which is known to occur in vivo in PD brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated ␣-syn inclusion formation and dopaminergic neurodegeneration. ␣-Synuclein (␣-syn)2 (NACP, synelfin), a small, neuron-specific protein, was first linked to PD by genetic analysis of families with autosomal dominant inheritance of the disease. Genetic analysis discovered a point mutation in the ␣-syn gene (SNCA), resulting in an amino acid conversion of Ala 53 to Thr (1). Subsequently, ␣-syn protein was detected in Lewy bodies within the dopaminergic neurons of the substantia nigra pars compacta (2), the intracellular proteinaceous inclusions characteristic of PD and related disorders. Since this discovery, the process of ␣-syn aggregation has been proposed to underlie dopaminergic degeneration that occurs in PD. Therefore, delineating the mechanisms of ␣-syn aggregation and its pathophysiological role in neurodegeneration has been the focus of many investigations.Although the in vivo factors that regulate ␣-syn aggregation are not well understood, mechanisms involving genetic and environmental factors have been proposed. Genetic analysis has uncovered two additional missense mutations in SNCA (A30P and E46K) (3, 4) as well as triplication of the SNCA genomic region (5). Mutations of ␣-syn or gene triplication may increase the rate of ␣-syn aggregation (6, 7), impair cellular degradation (8), or increase the amount of cytosolic ␣-syn beyond the critical concentration required to initiate polymerization (9). Most PD cases are sporadic and involve aggregation...

The budding yeast Saccharomyces cerevisiae is an emerging tool for investigating the molecular pathways that underpin several human neurodegenerative disorders associated with protein misfolding. Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disease primarily affecting motor neurons. The protein TDP-43 has recently been demonstrated to play an important role in the disease, however the mechanisms by which TDP-43 contributes to pathogenesis are unclear. To explore the mechanistic details that result in aberrant accumulation of TDP-43 and to discover potential strategies for therapeutic intervention, we employed a yeast TDP-43 proteinopathy model system. These studies allowed us to determine the regions of TDP-43 required for aggregation and toxicity and to define the effects of ALS-linked mutant forms of TDP-43. We have also been able to harness the power of yeast genetics to identify potent modifiers of TDP-43 toxicity using high-throughput yeast genetic screens. Here, we describe the methods and approaches that we have used in order to gain insight into TDP-43 biology and its role in disease. These approaches are readily adaptable to other neurodegenerative disease proteins.