Dopamine neuron-specific LRRK2 G2019S effects on gene expression revealed by translatome profiling.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of late-onset Parkinson's disease. The pathogenic G2019S mutation enhances LRRK2 kinase activity and induces neurodegeneration in C. elegans, Drosophila and rodent models through unclear mechanisms. Gene expression profiling has the potential to provide detailed insight into the biological pathways modulated by LRRK2 kinase activity. Prior in vivo studies have surveyed the effects of LRRK2 G2019S on genome-wide mRNA expression in complex brain tissues with high cellular heterogeneity, limiting their power to detect more restricted gene expression changes occurring in a cell type-specific manner. Here, we used translating ribosome affinity purification (TRAP) coupled to RNA-seq to profile dopamine neuron-specific gene expression changes caused by LRRK2 G2019S in the Drosophila CNS. A number of genes were differentially expressed in the presence of mutant LRRK2 that represent a broad range of molecular functions including DNA repair (RfC3), mRNA metabolism and translation (Ddx1 and lin-28), calcium homeostasis (MCU), and other categories (Ugt37c1, disp, l(1)G0196, CG6602, CG1126 and CG11068). Further analysis on a subset of these genes revealed that LRRK2 G2019S did not alter their expression across the whole brain, consistent with dopamine neuron-specific effects uncovered by the TRAP approach that may yield insight into the neurodegenerative process. To our knowledge, this is the first study to profile the effects of LRRK2 G2019S specifically on DA neuron gene expression in vivo. Beyond providing a set of differentially expressed gene candidates relevant to LRRK2, we demonstrate the effective use of TRAP to perform high-resolution assessment of dopamine neuron gene expression for the study of PD.

Natural Variation in Age-Related Dopamine Neuron Degeneration is Glutathione-Dependent and Linked to Life Span.

Aging is the biggest risk factor for Parkinson's disease (PD), suggesting that age-related changes in the brain promote dopamine neuron vulnerability. It is unclear, however, whether aging alone is sufficient to cause significant dopamine neuron loss and if so, how this intersects with PD-related neurodegeneration. Here, through examining a large collection of naturally varying Drosophila strains, we find a strong relationship between life span and age-related dopamine neuron loss. Naturally short-lived strains exhibit a loss of dopamine neurons but not generalized neurodegeneration, while long-lived strains retain dopamine neurons across age. Metabolomic profiling reveals lower glutathione levels in short-lived strains which is associated with elevated levels of reactive oxygen species (ROS), sensitivity to oxidative stress and vulnerability to silencing the familial PD gene parkin . Strikingly, boosting neuronal glutathione levels via glutamate-cysteine ligase (GCL) overexpression is sufficient to normalize ROS levels, extend life span and block dopamine neurons loss in short-lived backgrounds, demonstrating that glutathione deficiencies are central to neurodegenerative phenotypes associated with short longevity. These findings may be relevant to human PD pathogenesis, where glutathione depletion is frequently reported in idiopathic PD patient brain. Building on this evidence, we detect reduced levels of GCL catalytic and modulatory subunits in brain from PD patients harboring the LRRK2 G2019S mutation, implicating possible glutathione deficits in familial LRRK2-linked PD. Our study across Drosophila and human PD systems suggests that glutathione plays an important role in the influence of aging on PD neurodegeneration.

SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis.

Huntington's disease (HD), an incurable neurodegenerative disorder, has a complex pathogenesis including protein aggregation and the dysregulation of neuronal transcription and metabolism. Here, we demonstrate that inhibition of sirtuin 2 (SIRT2) achieves neuroprotection in cellular and invertebrate models of HD. Genetic or pharmacologic inhibition of SIRT2 in a striatal neuron model of HD resulted in gene expression changes including significant down-regulation of RNAs responsible for sterol biosynthesis. Whereas mutant huntingtin fragments increased sterols in neuronal cells, SIRT2 inhibition reduced sterol levels via decreased nuclear trafficking of SREBP-2. Importantly, manipulation of sterol biosynthesis at the transcriptional level mimicked SIRT2 inhibition, demonstrating that the metabolic effects of SIRT2 inhibition are sufficient to diminish mutant huntingtin toxicity. These data identify SIRT2 inhibition as a promising avenue for HD therapy and elucidate a unique mechanism of SIRT2-inhibitor-mediated neuroprotection. Furthermore, the ascertainment of SIRT2's role in regulating cellular metabolism demonstrates a central function shared with other sirtuin proteins.

Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease.

We have previously demonstrated amelioration of Huntington's disease (HD)-related phenotypes in R6/2 transgenic mice in response to treatment with the novel histone deacetylase (HDAC) inhibitor 4b. Here we have measured the selectivity profiles of 4b and related compounds against class I and class II HDACs and have tested their ability to restore altered expression of genes related to HD pathology in mice and to rescue disease effects in cell culture and Drosophila models of HD. R6/2 transgenic and wild-type (wt) mice received daily injections of HDAC inhibitors for 3 days followed by real-time PCR analysis to detect expression differences for 13 HD-related genes. We find that HDACi 4b and 136, two compounds showing high potency for inhibiting HDAC3 were most effective in reversing the expression of genes relevant to HD, including Ppp1r1b, which encodes DARPP-32, a marker for medium spiny striatal neurons. In contrast, compounds targeting HDAC1 were less effective at correcting gene expression abnormalities in R6/2 transgenic mice, but did cause significant increases in the expression of selected genes. An additional panel of 4b-related compounds was tested in a Drosophila model of HD and in STHdhQ111 striatal cells to further distinguish HDAC selectivity. Significant improvement in huntingtin-elicited Drosophila eye neurodegeneration in the fly was observed in response to treatment with compounds targeting human HDAC1 and/or HDAC3. In STHdhQ111 striatal cells, the ability of HDAC inhibitors to improve huntingtin-elicited metabolic deficits correlated with the potency at inhibiting HDAC1 and HDAC3, although the IC50 values for HDAC1 inhibition were typically 10-fold higher than for inhibition of HDAC3. Assessment of HDAC protein localization in brain tissue by Western blot analysis revealed accumulation of HDAC1 and HDAC3 in the nucleus of HD transgenic mice compared to wt mice, with a concurrent decrease in cytoplasmic localization, suggesting that these HDACs contribute to a repressive chromatin environment in HD. No differences were detected in the localization of HDAC2, HDAC4 or HDAC7. These results suggest that inhibition of HDACs 1 and 3 can relieve HD-like phenotypes in model systems and that HDAC inhibitors targeting these isotypes might show therapeutic benefit in human HD.

Pcaf modulates polyglutamine pathology in a Drosophila model of Huntington's disease.

Huntingtin peptides with elongated polyglutamine domains, the root causes of Huntington's disease, hinder histone acetylation, which leads to transcriptional dysregulation. However, the range of acetyltransferases interacting with mutant Huntingtin has not been systematically evaluated. We used genetic interaction tests in Drosophila to determine whether specific acetyltransferases belonging to distinct protein families influence polyglutamine pathology. We found that flies expressing a mutant form of the Huntingtin protein (Httex1pQ93) exhibit reduced viability, which is further decreased by partial loss of Pcaf or nejire, while the tested MYST family acetyltransferases did not affect pathology. Reduced levels of Pcaf also led to the increased degeneration of photoreceptor neurons in the retina. Overexpression of Pcaf, however, was not sufficient to ameliorate these phenotypes, and the level of soluble Pcaf is unchanged in Httex1pQ93-expressing flies. Thus, our results indicate that while Pcaf has a significant impact on Huntington's disease pathology, therapeutic strategies aimed at elevating its levels are likely to be ineffective in ameliorating Huntington's disease pathology; however, strategies that aim to increase the specific activity of Pcaf remain to be tested.

Inhibition of specific HDACs and sirtuins suppresses pathogenesis in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is associated with transcriptional dysregulation, and multiple studies with histone deacetylase (HDAC) inhibitors suggest that global approaches for restoring transcriptional balance and appropriate protein acetylation are therapeutically promising. To determine whether more targeted approaches might be effective, we have tested the impact of all the HDACs in Drosophila on Huntingtin (Htt)-induced pathology. Among the zinc-dependent or 'classic' HDACs, we find that neurodegeneration is most sensitive to levels of Rpd3. We also find that among the NAD(+)-dependent class III deacetylases, genetic or pharmacological reduction of either Sir2 or Sirt2 provides neuroprotection to Htt-challenged animals and that even greater neuroprotection is achieved when Rpd3 and Sir2 are simultaneously reduced. Our experiments suggest that longevity promoting strategies may be distinct from those that protect against neurodegeneration in Drosophila challenged with mutant human Htt. These results highlight a novel therapeutic approach for HD in the form of Sir2 inhibition and possible combinatorial inhibition of Sir2 and Rpd3.

CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.

A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor.

Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.

SIRT2- and NRF2-Targeting Thiazole-Containing Compound with Therapeutic Activity in Huntington's Disease Models.

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in ex vivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactive oxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders.

SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation.

A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease.

Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

Biologically active molecules that reduce polyglutamine aggregation and toxicity.

Polyglutamine expansion in certain proteins causes neurodegeneration in inherited disorders such as Huntington disease and X-linked spinobulbar muscular atrophy. Polyglutamine tracts promote protein aggregation in vitro and in vivo with a strict length-dependence that strongly implicates alternative protein folding and/or aggregation as a proximal cause of cellular toxicity and neurodegeneration. We used an intracellular polyglutamine protein aggregation assay based on fluorescence resonance energy transfer (FRET) to identify inhibitors of androgen receptor (AR) aggregation in three libraries of biologically active small molecules: the Annotated Compound Library, the NINDS Custom Collection and a kinase inhibitor collection. In the primary screen 10 compounds reduced AR aggregation. While 10/10 also reduced huntingtin (Htt) exon 1 aggregation, only 2/10 reduced aggregation of pure polyglutamine peptides. In a PC-12 model 9/10 compounds reduced aggregation. Five out of nine compounds tested in an Htt exon 1 assay of neurodegeneration in Drosophila partially rescued the phenotype. Three of the five compounds effective in flies are FDA-approved drugs. These compounds provide new leads for therapeutic development for the polyglutamine diseases based on their efficacy in mammalian cells and a Drosophila model. The high predictive value of the primary screen suggests that the FRET-based screening assay may be useful for further primary and secondary screens for genes or small molecules that inhibit polyglutamine protein aggregation.

Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract within the huntingtin protein (Htt). Identifying the pathways that are altered in response to the mutant protein is crucial for understanding the cellular processes impacted by the disease as well as for the rational development of effective pharmacological interventions. Here, expression profiling of a cellular HD model identifies genes that implicate altered mitogen-activated protein kinase (MAPK) signaling. Targeted biochemical studies and pharmacological modulation of these MAPK pathways suggest that mutant Htt affects signaling at upstream points such that both ERK and JNK are activated. Modulation of the ERK pathway suggests that this pathway is associated with cell survival, whereas inhibition of JNK was found to effectively suppress pathogenesis. These studies suggest that pharmacological intervention in MAPK pathways, particularly at the level of ERK activation, may be an appropriate approach to HD therapy.

Identification of combinatorial drug regimens for treatment of Huntington's disease using Drosophila.

We explore the hypothesis that pathology of Huntington's disease involves multiple cellular mechanisms whose contributions to disease are incrementally additive or synergistic. We provide evidence that the photoreceptor neuron degeneration seen in flies expressing mutant human huntingtin correlates with widespread degenerative events in the Drosophila CNS. We use a Drosophila Huntington's disease model to establish dose regimens and protocols to assess the effectiveness of drug combinations used at low threshold concentrations. These proof of principle studies identify at least two potential combinatorial treatment options and illustrate a rapid and cost-effective paradigm for testing and optimizing combinatorial drug therapies while reducing side effects for patients with neurodegenerative disease. The potential for using prescreening in Drosophila to inform combinatorial therapies that are most likely to be effective for testing in mammals is discussed.

Fly models of Huntington's disease.

Can Drosophila models be engineered that accurately reflect Huntington's disease (HD) and other neurological diseases and can they contribute to the search for treatments and cures? A number of publications seem to provide a resounding yes to that question. Here we seek to review some of the salient features of these models.

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

SUMO modification of Huntingtin and Huntington's disease pathology.

Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that a pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation.