Michael P. Hart

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.

Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.

A combination of yeast genetics and protein biochemistry define how the fused in sarcoma (FUS) protein might contribute to Lou Gehrigs disease.

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.

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.

Inhibition of Monocarboxylate Transporter 2 in the Retrotrapezoid Nucleus in Rats: A Test of the Astrocyte–Neuron Lactate-Shuttle Hypothesis

The astrocyte-neuronal lactate-shuttle hypothesis posits that lactate released from astrocytes into the extracellular space is metabolized by neurons. The lactate released should alter extracellular pH (pHe), and changes in pH in central chemosensory regions of the brainstem stimulate ventilation. Therefore, we assessed the impact of disrupting the lactate shuttle by administering 100 μm α-cyano-4-hydroxy-cinnamate (4-CIN), a dose that blocks the neuronal monocarboxylate transporter (MCT) 2 but not the astrocytic MCTs (MCT1 and MCT4). Administration of 4-CIN focally in the retrotrapezoid nucleus (RTN), a medullary central chemosensory nucleus, increased ventilation and decreased pHe in intact animals. In medullary brain slices, 4-CIN reduced astrocytic intracellular pH (pHi) slightly but alkalinized neuronal pHi. Nonetheless, pHi fell significantly in both cell types when they were treated with exogenous lactate, although 100 μm 4-CIN significantly reduced the magnitude of the acidosis in neurons but not astrocytes. Finally, 4-CIN treatment increased the uptake of a fluorescent 2-deoxy-d-glucose analog in neurons but did not alter the uptake rate of this 2-deoxy-d-glucose analog in astrocytes. These data confirm the existence of an astrocyte to neuron lactate shuttle in intact animals in the RTN, and lactate derived from astrocytes forms part of the central chemosensory stimulus for ventilation in this nucleus. When the lactate shuttle was disrupted by treatment with 4-CIN, neurons increased the uptake of glucose. Therefore, neurons seem to metabolize a combination of glucose and lactate (and other substances such as pyruvate) depending, in part, on the availability of each of these particular substrates.

Evaluating the prevalence of polyglutamine repeat expansions in amyotrophic lateral sclerosis

Objective: Given the recent finding of an association between intermediate-length polyglutamine (polyQ) expansions in ataxin 2 and amyotrophic lateral sclerosis (ALS), we sought to determine whether expansions in other polyQ disease genes were associated with ALS. Methods: We assessed the polyQ lengths of ataxin 1, ataxin 3, ataxin 6, ataxin 7, TBP, atrophin 1, and huntingtin in several hundred patients with sporadic ALS and healthy controls. Results: Other than ataxin 2, we did not identify a significant association with the other polyQ genes and ALS. Conclusions: These data indicate that the effects of ataxin 2 polyQ expansions on ALS risk are likely to be rooted in the biology of ataxin 2 or ataxin 2-specific interactions, rather than the presence of an expanded polyQ repeat per se. These findings have important consequences for understanding the role of ataxin 2 in ALS pathogenesis and provide a framework for future mechanistic studies.

ALS-Associated Ataxin 2 PolyQ Expansions Enhance Stress-Induced Caspase 3 Activation and Increase TDP-43 Pathological Modifications

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by the loss of motor neurons. The degenerating motor neurons of ALS patients are characterized by the accumulation of cytoplasmic inclusions containing phosphorylated and truncated forms of the RNA-binding protein TDP-43. Ataxin 2 intermediate-length polyglutamine (polyQ) expansions were recently identified as a risk factor for ALS; however, the mechanism by which they contribute to disease is unknown. Here, we show that intermediate-length ataxin 2 polyQ expansions enhance stress-induced TDP-43 C-terminal cleavage and phosphorylation in human cells. We also connect intermediate-length ataxin 2 polyQ expansions to the stress-dependent activation of multiple caspases, including caspase 3. Caspase activation is upstream of TDP-43 cleavage and phosphorylation since caspase inhibitors block these pathological modifications. Analysis of the accumulation of activated caspase 3 in motor neurons revealed a striking association with ALS cases harboring ataxin 2 polyQ expansions. These findings indicate that activated caspase 3 defines a new pathological feature of ALS with intermediate-length ataxin 2 polyQ expansions. These results provide mechanistic insight into how ataxin 2 intermediate-length polyQ expansions could contribute to ALS—by enhancing stress-induced TDP-43 pathological modifications via caspase activation. Because longer ataxin 2 polyQ expansions are associated with a different disease, spinocerebellar ataxia 2, these findings help explain how different polyQ expansions in the same protein can have distinct cellular consequences, ultimately resulting in different clinical features. Finally, since caspase inhibitors are effective at reducing TDP-43 pathological modifications, this pathway could be pursued as a therapeutic target in ALS.

Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.

TDP-43 toxicity in yeast

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.

Neurexin controls plasticity of a mature, sexually dimorphic neuron

During development and adulthood, brain plasticity is evident at several levels, from synaptic structure and function to the outgrowth of dendrites and axons. Whether and how sex impinges on neuronal plasticity is poorly understood. Here we show that the sex-shared GABA (γ-aminobutyric acid)-releasing DVB neuron in Caenorhabditis elegans displays experience-dependent and sexually dimorphic morphological plasticity, characterized by the stochastic and dynamic addition of multiple neurites in adult males. These added neurites enable synaptic rewiring of the DVB neuron and instruct a functional switch of the neuron that directly modifies a step of male mating behaviour. Both DVB neuron function and male mating behaviour can be altered by experience and by manipulation of postsynaptic activity. The outgrowth of DVB neurites is promoted by presynaptic neurexin and antagonized by postsynaptic neuroligin, revealing a non-conventional activity and mode of interaction of these conserved, human-disease-relevant factors.