J. Alex Parker

Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons.

We report that Sir2 activation through increased sir-2.1 dosage or treatment with the sirtuin activator resveratrol specifically rescued early neuronal dysfunction phenotypes induced by mutant polyglutamines in transgenic Caenorhabditis elegans. These effects are dependent on daf-16 (Forkhead). Additionally, resveratrol rescued mutant polyglutamine–specific cell death in neuronal cells derived from HdhQ111 knock-in mice. We conclude that Sir2 activation may protect against mutant polyglutamines.

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin-induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

Deletion of C9ORF72 Results in Motor Neuron Degeneration and Stress Sensitivity in C. elegans

An expansion of the hexanucleotide GGGGCC repeat in the first intron of C9ORF72 gene was recently linked to amyotrophic lateral sclerosis. It is not known if the mutation results in a gain of function, a loss of function or if, perhaps both mechanisms are linked to pathogenesis. We generated a genetic model of ALS to explore the biological consequences of a null mutation of the Caenorhabditis elegans C9ORF72 orthologue, F18A1.6, also called alfa-1. alfa-1 mutants displayed age-dependent motility defects leading to paralysis and the specific degeneration of GABAergic motor neurons. alfa-1 mutants showed differential susceptibility to environmental stress where osmotic stress provoked neurodegeneration. Finally, we observed that the motor defects caused by loss of alfa-1 were additive with the toxicity caused by mutant TDP-43 proteins, but not by the mutant FUS proteins. These data suggest that a loss of alfa-1/C9ORF72 expression may contribute to motor neuron degeneration in a pathway associated with other known ALS genes.

Pharmacological reduction of ER stress protects against TDP-43 neuronal toxicity in vivo.

C. elegans and D. rerio expressing mutant TAR DNA Binding Protein 43 (TDP-43) are powerful in vivo animal models for the genetics and pharmacology of amyotrophic lateral sclerosis (ALS). Using these small-animal models of ALS, we previously identified methylene blue (MB) as a potent suppressor of TDP-43 toxicity. Consequently here we investigated how MB might exert its neuroprotective properties and found that it acts through reduction of the endoplasmic reticulum (ER) stress response. We tested other compounds known to be active in the ER unfolded protein response in worms and zebrafish expressing mutant human TDP-43 (mTDP-43). We identified three compounds: salubrinal, guanabenz and a new structurally related compound phenazine, which also reduced paralysis, neurodegeneration and oxidative stress in our mTDP-43 models. Using C. elegans genetics, we showed that all four compounds act as potent suppressors of mTDP-43 toxicity through reduction of the ER stress response. Interestingly, these compounds operate through different branches of the ER unfolded protein pathway to achieve a common neuroprotective action. Our results indicate that protein-folding homeostasis in the ER is an important target for therapeutic development in ALS and other TDP-43-related neurodegenerative diseases.

Methylene blue protects against TDP-43 and FUS neuronal toxicity in C. elegans and D. rerio.

The DNA/RNA-binding proteins TDP-43 and FUS are found in protein aggregates in a growing number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and related dementia, but little is known about the neurotoxic mechanisms. We have generated Caenorhabditis elegans and zebrafish animal models expressing mutant human TDP-43 (A315T or G348C) or FUS (S57Δ or R521H) that reflect certain aspects of ALS including motor neuron degeneration, axonal deficits, and progressive paralysis. To explore the potential of our humanized transgenic C. elegans and zebrafish in identifying chemical suppressors of mutant TDP-43 and FUS neuronal toxicity, we tested three compounds with potential neuroprotective properties: lithium chloride, methylene blue and riluzole. We identified methylene blue as a potent suppressor of TDP-43 and FUS toxicity in both our models. Our results indicate that methylene blue can rescue toxic phenotypes associated with mutant TDP-43 and FUS including neuronal dysfunction and oxidative stress.

Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans

Mutations in the DNA/RNA binding proteins TDP-43 and FUS are associated with Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. Intracellular accumulations of wild type TDP-43 and FUS are observed in a growing number of late-onset diseases suggesting that TDP-43 and FUS proteinopathies may contribute to multiple neurodegenerative diseases. To better understand the mechanisms of TDP-43 and FUS toxicity we have created transgenic Caenorhabditis elegans strains that express full-length, untagged human TDP-43 and FUS in the worms GABAergic motor neurons. Transgenic worms expressing mutant TDP-43 and FUS display adult-onset, age-dependent loss of motility, progressive paralysis and neuronal degeneration that is distinct from wild type alleles. Additionally, mutant TDP-43 and FUS proteins are highly insoluble while wild type proteins remain soluble suggesting that protein misfolding may contribute to toxicity. Populations of mutant TDP-43 and FUS transgenics grown on solid media become paralyzed over 7 to 12 days. We have developed a liquid culture assay where the paralysis phenotype evolves over several hours. We introduce C. elegans transgenics for mutant TDP-43 and FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.

Sirtuin inhibition protects from the polyalanine muscular dystrophy protein PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is caused by polyalanine expansion in nuclear protein PABPN1 [poly(A) binding protein nuclear 1] and characterized by muscle degeneration. Druggable modifiers of proteotoxicity in degenerative diseases, notably the longevity modulators sirtuins, may constitute useful therapeutic targets. However, the modifiers of mutant PABPN1 are unknown. Here, we report that longevity and cell metabolism modifiers modulate mutant PABPN1 toxicity in the muscle cell. Using PABPN1 nematodes that show muscle cell degeneration and abnormal motility, we found that increased dosage of the sirtuin and deacetylase sir-2.1/SIRT1 exacerbated muscle pathology, an effect dependent on the transcription factor daf-16/FoxO and fuel sensor aak-2/AMPK (AMP-activated protein kinase), while null mutants of sir-2.1, daf-16 and aak-2 were protective. Consistently, the Sir2 inhibitor sirtinol was protective, whereas the Sir2 and AMPK activator resveratrol was detrimental. Furthermore, rescue by sirtinol was dependent on daf-16 and not aak-2, whereas aggravation by resveratrol was dependent on aak-2 and not daf-16. Finally, the survival of mammalian cells expressing mutant PABPN1 was promoted by sirtinol and decreased by resveratrol. Altogether, our data identify Sir2 and AMPK inhibition as therapeutic strategies for muscle protection in OPMD, extending the value of druggable proteins in cell maintenance networks to polyalanine diseases.

C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress

The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 μm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neurons that did not produce exophers. The extruded exopher transits through surrounding tissue in which some contents appear degraded, but some non-degradable materials can subsequently be found in more remote cells, suggesting secondary release. Our observations suggest that exopher-genesis is a potential response to rid cells of neurotoxic components when proteostasis and organelle function are challenged. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.

Restless legs syndrome‐associated MEIS1 risk variant influences iron homeostasis

Restless legs syndrome (RLS) is a frequent sleep disorder that is linked to disturbed iron homeostasis. Genetic studies identified MEIS1 as an RLS‐predisposing gene, where the RLS risk haplotype is associated with decreased MEIS1 mRNA and protein expression. We show here that RNA interference treatment of the MEIS1 worm orthologue increases ferritin expression in Caenorhabditis elegans and that the RLS‐associated haplotype leads to increased expression of ferritin and DMT1 in RLS brain tissues. Additionally, human cells cultured under iron‐deficient conditions show reduced MEIS1 expression. Our data establish a link between the RLS MEIS1 gene and iron metabolism. Ann Neurol 2011;

Huntingtin-Interacting Protein 1 Influences Worm and Mouse Presynaptic Function and Protects Caenorhabditis elegans Neurons against Mutant Polyglutamine Toxicity

Huntingtin-interacting protein 1 (HIP1) was identified through its interaction with htt (huntingtin), the Huntingtons disease (HD) protein. HIP1 is an endocytic protein that influences transport and function of AMPA and NMDA receptors in the brain. However, little is known about its contribution to neuronal dysfunction in HD. We report that the Caenorhabditis elegans HIP1 homolog hipr-1 modulates presynaptic activity and the abundance of synaptobrevin, a protein involved in synaptic vesicle fusion. Presynaptic function was also altered in hippocampal brain slices of HIP1−/− mice demonstrating delayed recovery from synaptic depression and a reduction in paired-pulse facilitation, a form of presynaptic plasticity. Interestingly, neuronal dysfunction in transgenic nematodes expressing mutant N-terminal huntingtin was specifically enhanced by hipr-1 loss of function. A similar effect was observed with several other mutant proteins that are expressed at the synapse and involved in endocytosis, such as unc-11/AP180, unc-26/synaptojanin, and unc-57/endophilin. Thus, HIP1 is involved in presynaptic nerve terminal activity and modulation of mutant polyglutamine-induced neuronal dysfunction. Moreover, synaptic proteins involved in endocytosis may protect neurons against amino acid homopolymer expansion.