Chen et al. Chemistry Central Journal (2015) 9:65 DOI 10.1186/s13065-015-0143-y

REVIEW Open Access Using C. elegans to discover therapeutic compounds for ageing‑associated neurodegenerative diseases Xi Chen1,2, Jeff W. Barclay1, Robert D. Burgoyne1 and Alan Morgan1*

Abstract Age-associated neurodegenerative disorders such as Alzheimer’s disease are a major public health challenge, due to the demographic increase in the proportion of older individuals in society. However, the relatively few currently approved for these conditions provide only symptomatic relief. A major goal of research is therefore to identify potential new therapeutic compounds that can slow or even reverse disease progression, either by impacting directly on the neurodegenerative process or by activating endogenous physiological neuroprotec- tive mechanisms that decline with ageing. This requires model systems that can recapitulate key features of human neurodegenerative diseases that are also amenable to compound screening approaches. Mammalian models are very powerful, but are prohibitively expensive for high-throughput screens. Given the highly conserved neurological pathways between mammals and invertebrates, Caenorhabditis elegans has emerged as a powerful tool for neuro- protective compound screening. Here we describe how C. elegans has been used to model various human ageing- associated neurodegenerative diseases and provide an extensive list of compounds that have therapeutic activity in these worm models and so may have translational potential. Keywords: Adult onset neuronal ceroid lipofuscinosis, Aging, Alzheimer’s disease, Amyotrophic lateral sclerosis, Caenorhabditis elegans, Compound screening, Frontotemporal dementia, Huntington’s disease, Neurodegeneration, Parkinson’s disease

Background of NDs in order to screen for potential new drugs. In this Despite decades of intense molecular research and the iden- review, we focus on compound screens performed in the tification of many specific causative mutations, debilitating nematode worm, Caenorhabditis elegans. We describe var- neurodegenerative diseases (NDs) including common dis- ious different NDs that have been modelled in worms and orders such as Alzheimer’s disease (AD) and Parkinson’s list the therapeutic compounds that have been identified disease (PD), afflict millions worldwide and remain a signif- for each. In some cases, these compounds have also been icant and unresolved financial and social burden. Indeed, as shown to be protective in mammalian ND models, suggest- ageing itself is by far the greatest risk factor for these dis- ing translational potential for human patients. We conclude eases, this burden is set to increase dramatically as a result that the combination of accurate genetic ND worm models of our increasingly ageing population. Given the urgent with high-throughput automated drug screening platforms need for therapies for these devastating and eventually fatal is a potentially very efficient strategy for early therapeutic disorders, many researchers have developed animal models drug discovery for NDs.

*Correspondence: [email protected] Review 1 Department of Cellular and Molecular Physiology, Institute An overview of human neurodegenerative diseases of Translational Medicine, University of Liverpool, Crown St, Liverpool L69 3BX, UK NDs are characterised by progressive neuropsychiat- Full list of author information is available at the end of the article ric dysfunction and the loss of structure and function of

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specific neuronal circuitry that in turn result in behav- and neuropathological defects, coupled with a surpris- ioural symptoms. NDs can occur on a completely heredi- ingly high degree of biochemical conservation compared tary basis (e.g. Huntington’s disease), or can be hereditary to humans. Remarkable similarities exist at the molecular and also appear sporadically in the majority of cases and cellular levels between nematode and vertebrate neu- (e.g. AD, PD). In spite of the diversity in the underlying rons. For example, ion channels, receptors, classic neu- genes involved, inheritance patterns, clinical manifes- rotransmitters [acetylcholine, glutamate, γ-aminobutyric tation and exact sites of neuropathology, the rare, early acid (GABA), serotonin, and dopamine (DA)], vesicular onset familial (also known as Mendelian) forms and the transporters and the neurotransmitter release machin- more prevalent late-onset sporadic forms of different ery are similar in both structure and function between NDs share some common genetic origins and patho- vertebrates and C. elegans [5, 6]. Importantly, the impact logical hallmarks, such as the progressive and chronic of different challenges such as genetic perturbations or nature of the disease, the extensive loss of specific neu- exposure to drugs on the survival and function of defined ronal subtypes, synaptic dysfunctions, the formation neuronal populations in the C. elegans nervous system and deposition of misfolded protein aggregates [1–3]. can be readily studied in vivo. Research and technological innovations over the past To date, various laboratories have developed and char- 10 years have made considerable progress in the elucida- acterised a diverse set of C. elegans models of various tion of mechanisms of ND initiation and progression that human NDs, including AD [7], PD [8] and polyglutamine lead to neurodegeneration. Emerging common themes in expansion diseases [9] (Table 1). These worm ND mod- the pathogenesis of neurodegeneration include: aberrant els have been developed by over-expressing human ND- phosphorylation, palmitoylation and acetylation of dis- associated genes (both wild type and mutant versions) ease-causing proteins, protein misfolding, deficient ubiq- and by mutating or altering the expression level of the uitin–proteasome system (UPS) or autophagic process to orthologous worm genes. Strong parallels were especially clear disease-causing proteins, altered RNA metabolism, observed in the genotype-to-phenotype correlations oxidative stress, mitochondrial dysfunction, excitotoxic- between the human NDs and the phenotypes of trans- ity, disrupted axonal transport, neuroinflammation and genic C. elegans ND models. This supports the validity of microglial activation [4]. Linkage analysis, high-through- the approach as expression of mutant human proteins in put sequencing and genome-wide association studies C. elegans can closely model a fundamental property of (GWAS) have also identified susceptibility genes in many these mutations in humans. NDs (Table 1) and promise to help unravel even more Nevertheless, there are also limitations to using C. ele- genes, novel loci and common genetic variants associated gans to model NDs that must be considered. Although with the diverse collection of human NDs. Thus devel- the worm offers huge potential for experimental manip- opments of therapeutic interventions that are applicable ulations, there are aspects of ND pathophysiology that across the broad spectrum of NDs and target the shared cannot easily be modelled in worms. For example, abun- pathogenic mechanisms may offer the best hope for a dant evidence supports an important role for brain future neuroprotective therapy. inflammation and microglial cell activation in several NDs, notably AD [10], but there is no microglial equiv- Caenorhabditis elegans as a model for human alent among the 56 glial cells of C. elegans. Clearly, the neurodegenerative disease very simplicity of the worm nervous system that makes it A major challenge to the identification of effective dis- so attractive for studying basic neurobiology is also a dis- ease-modifying therapies arises from an insufficient advantage in that the complexity of the mammalian brain knowledge about the contribution of multiple pathways cannot be adequately reflected, and so rodent models will to disease pathogenesis. Mammalian disease models continue to be required to validate any findings fromC. offer in vivo opportunities and extensive similarity to the elegans ND studies. There are also potential pitfalls of human brain, but testing the therapeutic value of small using C. elegans for drug screening, as many compounds molecules in mammalian model systems is extremely do not easily penetrate the worm’s protective cuticle [11] expensive and requires time-consuming experimen- and as biotransformation of compounds by the worms’ tal designs that can be prohibitive. Over the past dec- E. coli food source may give misleading pharmacological ades, C. elegans has increasingly been used as a model information [12]. Although these potential pitfalls can be system to study the underlying molecular mechanisms mitigated by combining predictive bioaccumulation algo- that give rise to neurodegeneration because of its well- rithms [11] with increased dose regimens, and by con- characterised and easily accessible nervous system, short firming drug effects using metabolically inactiveE. coli, generation time (≈3 days) and lifespan (≈3 weeks), trac- these issues need to be considered when performing drug tability to genetic manipulation, distinctive behavioural screens in worms. Chen et al. Chemistry Central Journal (2015) 9:65 Page 3 of 20 [ 31 ] [ 98 ] [ 7 , 28 89 – 91 ] [ 101 ] [ 96 ] References [ 15 , 96 97 ] [ 102 ] [ 28 , 92 – 95 ] [ 99 , 100 ] - 761 bacitracin; epigallocatechin gallate; gallate; bacitracin; epigallocatechin biloba extract Ginkgo ; glycitein; isoflavone EGb 761; soya rifampicin; aglycone oleuropein ferulic acid; T; curcumin; thioflavin JWB1-84-1 and JAY2-22- ; 33; NT219 related analogs; copper; related extract EGb 761 and Gink Dihuang golide A and J; Liuwei galanthamine; icariside II; (LWDH); (Baccharis cocoa peptide; Carqueja aglycone trimera) oleuropein PBT2 Clioquinol Ginkgo biloba extractCL2355: Ginkgo EGb CL2006: caffeine, tannic acid and CL2006: caffeine, CL2120: PBT2 Coffee extracts,tetracycline and Coffee Efficacious compounds identified/ Efficacious validated of aggregates under oxidative under oxidative of aggregates stress h after temperature shift within 48 h after temperature perinuclear deposits neurons in an age-related manner;neurons had 3 only 48 % of worms at day neurons, intactfive glutamatergic 7 only 25 % did and at day CL2355 is defective in chemotaxis CL2355 is defective associative benzaldehyde, toward and thrashing in liquid; learning, forms serotonin; to hypersensitive deposits; has partialamyloid steril - ity germline due to proliferation and embryonicdefects lethality reduced forward crawling, forward crawling, reduced resistance thrashings and strong H46R/H48Q-YFP aldicarb. to defect a movement produced than that seen in less prominent G85R-YFP forms amyloid deposits; increased deposits; increased amyloid forms stress oxidative deposits and of amyloid formation stress in oxidative no increase precedes amyloid deposition; amyloid precedes accumulation autophagosome and viability; chemotaxis severe and diminished touch defects habituation Paraquat hypersensitivity; Paraquat formation Severe and fully penetrant paralysis Severe Rapid of stable paralysis; formation Loss of GFP-marked glutamatergic glutamatergic of GFP-marked Loss CL2241 exhibit WT CL2241 exhibit movement. G85R and G85R-YFP: severely severely G85R and G85R-YFP: Age-dependent progressive paralysis; Age-dependentprogressive CL2109, CL3109 and CL3115: no Rapid paralysis; oxidative stress stress Rapid paralysis; oxidative Defects in brood size, movement, movement, size, Defects in brood Phenotypes muscles Heat shock inducible body wall Glutamatergic neurons Glutamatergic Inducible pan-neuronal Constitutive pan-neuronal Constitutive Constitutive muscles Constitutive Inducible body wall muscles Constitutive pan-neuronal Constitutive Expression in C. elegans Expression CL1119, CL1120, CL1121, CL2120; CL2109, CL3109; CL3115 UA166 CL2241, CL2355 iwIs8gf CL2005, CL2006, CL1019, CL1118, CL4176 LGIII, LGV, LGX LGV, LGIII, GMC101 CL2337 Strain/transgene name/(plasmid) Strain/transgene

1 – 42 - 1 – ::let myo

1 – 42 1 - 42 ::GFP] ::SOD - 1 (WT, :: Aβ - 3 :: Aβ mtl - 2 - 3 - :: gfp + P (N- myo - 6(su1006) ] myo myo snb - 1 1 – 42 ::apl - 1 ::GFP] 1 - 42 eat - 4 :APL-1] - 3 rab ::SOD - 1 (WT, A4 V, (wild type); Dimer Aβ snb - 1 :: ssAβ Cys Aβ Cys ::GFP] 1 – 42 35 eat - 4 ::GFP + rol hsp - 16.2 :: Aβ mtl - 2 ::SOD1(WT,G85R) - YFP ) + pCL26( mtl - 2 : GFP)]. + P ::GFP::degron :: mCherry ] terminus) + P 2 - 6(su1006) ] UTR) + (rol A4 V) or Met + P 42 3 G37R, G93A) + P unc - 54 snb - 1 [P smg - 1(cc546);Is P N2; ynIs13 [P N2; ynIs104 [P :DA- Aβ dvIs100 [pCL354( unc - 54 :DA- P 1(cc546); Is [P smg - 1(cc546); Model N2; Is [P 1(cc546); Is [P smg - 1(cc546); N2; Is [P models of human neurodegenerative diseases and drugs that were shown to confer neuroprotection to confer shown that were diseases and drugs modelsA list of published C. elegans of human neurodegenerative AD ALS NDs Transgenic overexpression of human neurodegeneration-associated protein/peptide of human neurodegeneration-associated overexpression Transgenic 1 Table Chen et al. Chemistry Central Journal (2015) 9:65 Page 4 of 20 [ 109 ] [ 103 ] [ 104 ] [ 110 ] [ 108 ] References [ 107 ] [ 34 , 37 38 ] [ 105 , 106 ] [ 62 ] - glumide, , , perphenazine, nefopam, glumide, ethosuximide ; , benz, and phenazine; resveratrol, benz, and phenazine; resveratrol, propyl trolox, reserpine, rolipram, and ethosuximide gallate, PHA767491; LDN-0130436 Azaperone, clofazimine, isoniazid, lor isoniazid, Azaperone, clofazimine, , salubrinal, guana - salubrinal, blue, Methylene Efficacious compounds identified/ Efficacious validated - SOD1(G85R) heterodimeric worms heterodimeric SOD1(G85R) loco impaired significantly have lifespan motion and reduced aggregates; R522G, P525L, FUS513 aggregates; shorterand FUS501: significantly P525L, FUS513 and lifespan. FUS501: partially or completely shrunken by severely paralysed, of age 8 days synapses SOD1 aggregates in neural cell SOD1 aggregates guidance bodies and causes axon defects locomotion defects and protein and protein locomotion defects aggregation locomotion; degeneration of neurons motor GABAergic progressive uncoordination and uncoordination progressive accumulation of insoluble tau; presynaptic neurodegeneration; transmission defect; cholinergic lifespan reduced % decrease in motil - causes 25–30 % decrease and ity 2 of adulthood, on day 10 % further approx. by decrease 6 of adulthood on day adult-onset, age-dependent loss paralysis, progressive of motility, degeneration, accumula - neuronal and TDP-43 tion of highly insoluble FUS proteins Increased aggregation formaton; formaton; Increased aggregation Formation of cytoplasmic FUS Formation Unc and abnormal motor neuron Unc and abnormal neuron motor Age-dependent paralysis; G93A Transgenic models developed robust robust models developed Transgenic Mutant TDP-43: significantly impaired impaired significantly TDP-43: Mutant Mutant tau: strong age-dependentMutant tau: strong Accumulation of mutant SOD1 Accumulation Have normal lifespan, but displayed but displayed normal lifespan, Have Phenotypes GABAergic motor neurons motor GABAergic Constitutive pan-neuronal Constitutive Constitutive pan-neuronal Constitutive Constitutive pan-neuronal Constitutive Constitutive muscles Constitutive GABAergic motor neurons motor GABAergic Expression in C. elegans Expression CK426 IW63, IW33, IW20, IW46, IW31, IW8 CK405, CK406, CK410; CK422; CK423; pJH897 CL2609, CL1681, CL1682 CK10, CK49, CK1301, CK1310 AM263; AM265 AM263; xqIs132, xqIs133, xqIs173, xqIs98 xqIs173, xqIs133, xqIs132, Strain/transgene name/(plasmid) Strain/transgene unc – snb - ::GFP ] - 2 - (WT,::FUS ::TDP - 43 (WT, myo ::FUS (WT,::FUS :GFP] unc - 47 :: mcherry; lin - 15( + ) ] :: DsRed2; P - 1 rgef unc - 47 snb - 1 pab - 1 - 1 regf V, G37R, ::SOD - 1 (WT, A4 V, 43 (WT, G290A, A315T, ::TDP - 43 (WT, G290A, A315T, :: 4R1 N human tau (WT, + P ::SNB-1::GFP] + Ex [P ], ::TDP - C25 YFP(iwIs22) Q331 K(iwEx20) ], ::TDP - 43 YFP M337 V(iwEx28) ], ::TDP - 43 YFP WT(iwIs27)::SOD1 - YFP ] and G85R(iwIs8) ] ::SOD1 - YFP sng - 1 snb - 1 aex - 3 :: SOD1(WT, G85R, G93A, ::G93A SOD1 - GFP YFP WT(iwIs26)::TDP - 43 YFP ], :RFP] snb - 1 snb - 1 snb - 1 snb - 1 snb - 1 unc - 25 :TDP-43; P :TDP-43; V337 M, P301L) + P [P [P [P [P [P 122 M337 V) G93C) –EGFP] R514G, R521G, R522G, R524S and P525L) + P ::YFP G127insTGGGstop) 1 S57Δ) + unc - 119( )] A315T) + unc - 119( )] snb - 1 unc - 54 unc - 25 Is [P lin - 15(n765ts); [P P N2; Is [P N2; Is [P 119(ed3); Is[P unc- 119(ed3); N2; Is [P Model P unc - 119(ed3);Is[P [P continued U U ALS/FTLD- FTDP-17 ALS/FTLD- NDs 1 Table Chen et al. Chemistry Central Journal (2015) 9:65 Page 5 of 20 [ 36 ] [ 114 , 115 ] [ 42 , 43 ] [ 40 ] [ 9 , 111 112 ] References [ 41 ] [ 33 ] [ 35 ] [ 113 ] cmp16 trichostatin; rotenone, oligomycin oligomycin trichostatin; rotenone, offici - and 2,4-dinitrophenol; D. narum extracts; salidroside Methylene blue, BSc3094, bb14 and blue, Methylene Lithium chloride, mithramycin, mithramycin, Lithium chloride, Resveratrol β-Lapachone Icariside II; NG-094; aspirin Efficacious compounds identified/ Efficacious validated - - - - showed showed 352 1 of adulthood, accelerated aggre accelerated 1 of adulthood, severe Tau, gation of insoluble of nervous defects developmental presynaptic impaired syetem, transmission and minimum perturbation of of the nervousthe development system neurodegeneration. Vast Vast majorityneurodegeneration. undergo (>90 %) of ASH neurons cell death in less than 3 days response insensitivity, significant anterior significant insensitivity, Mec phenotype; depos - significant abnormaliits and morphological - ties in PLM cell axons age-dependent unco progressive and neurodegeneration; ordination viabil - neuron no change in motor motor Mutant PHP tau: altered ity. Ala10 tau: development. neuron defects early onset of movement lifespan and reduced tion; overt dysfunction; neuronal polyQ length-dependent decrease pharyngealof thrashing, pumping cycleand erratic defecation impairment in touch response; impairment response; in touch tau strong neurodegeneration; accumulation in PLM neuron ment in touch response; neuro response; ment in touch WT4R:degeneration; tau little tau accumulation in PLM neuron aggregates; growth rates slowed slowed rates growth aggregates; motility reduced down; aggregates Strongly defective locomotion at day locomotion at day defective Strongly No obvious locomotion defects locomotion defects No obvious Accelerated polyQ mediated polyQ mediated Accelerated Severe defect in the nose touch in the nose touch defect Severe Highly penetrant posterior touch Both WTBoth and PHP tau PolyQ length-dependent- aggrega PolyQ Strong age-dependent Strong progressive Age-dependent progressive impair Age-dependent progressive Length-dependent formation of Length-dependent formation 40: formation of cytoplasmicQ > 40: formation Phenotypes PLML/R, PVM); weak in FLP, PVD, PVD, PLML/R, PVM); in FLP, weak BDU Chemosensory neurons Mechanosensory neurons Constitutive pan-neuronal Constitutive Constitutive pan-neuronal Constitutive Touch neurons (ALML/R, AVM, AVM, (ALML/R, neurons Touch Constitutive muscles Constitutive Expression in C. elegans Expression VH1014, VH1015; VH418, VH421 (Q67) AM308; (Q86) AM313. YFP AM313. (Q86) AM308; (Q67) (Q40) and AM80; AM78 lines: (Q35) and AM81 (Q67) and AM87; AM85 and AM324 AM322 (Q86) AM83; tmIs147, tmIs148, tmIs149 tmIs110, tmIs173 pCKX2001 HA659 BR5270, BR5485, BR5944, BR5706 BR5271, BR5486, BR6516, BR6427 ID24, ID1 HA759 VH255, VH1016, VH1018; VH254, CFP lines: (Q35) AM303; (Q40) AM305; AM305; (Q40) AM303; CFP lines: (Q35) tmIs81, tmIs178, tmIs179; tmIs146, tmIs82, tmIs83, tmIs84, tmIs85, tmIs171; pEGFP - N1 Q19, Q82 pCKX2004, pCKX2003, pCKX2002, Strain/transgene name/(plasmid) Strain/transgene mec - - - - (WT, - myo rab 352 rab :: HttQ150 :: Tau osm - 10 osm - ::mCherry] - 2 - 1 rgef :: HttQ150 + Dpy myo - 6(su1006) ] ::HttQ (0,19,35,40,67,86) - - 6(su1006) ] osm - 10 ::GFP + P :: tau WT(0N4R, :: tau (P301L, - 1 rgef + rol - 20 ( + )] mec - 7 mec - 7 osm - 10 ::polyQ -GFP/YFP/CFP ::DRPLAP - Q(32, 40, 56, 79) -GFP ::htt57Q(19, 88, 128) -GFP ::htt57Q(19, 88, 128) ::CFP + P ::GFP + P :: F3ΔK280 + P :: F3ΔK280(I277P)(I308P) + P ::mCherry] :YFP + Dpy 7 20 ( + )] 3 10 PHP, Ala10) + pha - 1( ) ] PHP, CFP/YFP] 0N3R) + rol R406 W) 2 3 unc - 54 unc - 54 mec - 3 mec - 3 Anti-aggregant lines: N2; Is [P Anti-aggregant N2; Is [ P N2; rmEx [P rtIs11 [P P P Pro-aggregant lines: N2; Is [P Pro-aggregant Model pqe - 1(rt13) III ; rtIs11 [P P ; Ex [P pha - 1(e2123ts) N2; Is [ P P continued HD NDs 1 Table Chen et al. Chemistry Central Journal (2015) 9:65 Page 6 of 20 [ 119 ] [ 126 ] [ 117 ] [ 106 ] References [ 120 ] [ 125 ] [ 53 ] [ 116 ] [ 120 – 124 ] [ 118 ] [ 127 ] geldanamycin (17-AAG), valproic valproic (17-AAG), geldanamycin acid and quinpirole; valproic acid; valproic and quinpirole; spermidine - p -Coumaroylcatalpol 10- O - trans 17-(allylamino)-17-demethoxy - Acetaminophen; bromocriptine bromocriptine Acetaminophen; AM140: ML346; celecoxib; NT219 ML346; celecoxib; AM140: salidroside AM141: Efficacious compounds identified/ Efficacious validated - in growth aggregation and toxicity are not are and toxicity aggregation by aging modulated significantly ergic neuron loss and dendritic neuron ergic breaks or A53T: failure in modulation of failure or A53T: locomotory to in response rate and markedly DA food reduced (~1 ng/g vs N2 ~5 ng/g). content in DA- impaired more A56P: dependent behaviour none-induced toxicity, exhibiting none-induced toxicity, survival68.4 % lower of after 4 days treatment 50 μM rotenone tion and motor dysfunctiontion and motor sion have similar aggregation similar aggregation sion have and have in their neurons profiles motility defects severe more response non-Tg, WT, and A53T α-synuclein- WT, non-Tg, strains; significant expressing loss and dendritic neuron DAergic breaks cells causes a significant motilitycells causes a significant defect throughout development and development throughout and retardation, growth aging, SNB-1::GFP synaptic abnormality. dimin - was broadly fluorescence ished in the nerve cord No motor deterioration or retardation deterioration or retardation No motor Formation of inclusions Formation PolyQ length-dependent toxicity;PolyQ Motility deficits, significant dopamin - Motility significant deficits, Increased neurodegeneration; A30P Increased neurodegeneration; A53T: greater vulnerability rote to greater A53T: PolyQ length-dependent- aggrega PolyQ - expres ATXN3 truncated with Worms Moderate impairments in touch Mean life span was similar among the Mean life α-Syn misfolding and accumulation α-Syn misfolding Q35 and Q37 aggregation in muscle Q35 and Q37 aggregation Strikingly defects motor severe Phenotypes Motor neurons Constitutive pan-neuronal Constitutive Constitutive muscles Constitutive Constitutive pan-neuronal Constitutive Dopaminergic neuron Dopaminergic Constitutive pan-neuronal Constitutive Constitutive pan-neuronal Constitutive Mechanosensory neurons Dopaminergic neuron Dopaminergic Constitutive muscles Constitutive Constitutive muscles Constitutive Constitutive pan-neuronal Constitutive Expression in C. elegans Expression AM519, AM520, AM666, AM685, AM685, AM666, AM520, AM519, AM599 AM428, AM419, AM420, AM684, AM684, AM420, AM419, AM428, AM702 AM683, AM141 AM491, AM513, AM509, AM494, AM494, AM509, AM513, AM491, AM396, AM416, AM422, AM391, AM391, AM422, AM416, AM396, BY273, UA18, UA31, UA44 UA31, BY273, UA18, NL5901 UA49 Q37) AM470; ( Q40) ; ( Q37) AM470; AM140 (Q35) Strain/transgene name/(plasmid) Strain/transgene : - 6 dat - unc - aex - unc - unc - 25 ::GFP/ ::GFP - aex - 3 dat - 1 ::EGFP] ::polyQ(0, 24, 35, 37, ::GFP syn (WT, A53T, ::α - syn (WT, A53T, unc - 51 ::α - syn:: GFP + rol ::α - syn (WT, A53T, ::α - syn (WT, A53T) + rol ::α - syn (WT, A53T) + P ::YFP] or P 257cAT3(Q45) dat - 1 unc - 54 ::α - syn:: YFP + unc 119( ) ] YFP AT3q130:: AT3q75, ::AT3q14, 257cAT3q75, ::257cAT3q14, unc - 54 unc - 119 unc - 51 mec - 7 ::GFP ::S129A or S129D α - syn + P ::S129A or S129D α - syn + P unc - 54 ::α - syn (WT, A53T) + P ::α - syn (WT, A53T) + P A56P) + P A53T, ::α - syn (A30P, ::YFP 257cAT3(Q63) :EGFP - 1 rgef - 1 rgef dat - 1 unc - 54 ::GFP/P :: mCherry β - syn) + pDPSU006 -GFP] YFP 257cAT3q128:: 257cAT3q80, P 54 (su1006) ] 3 51 1 6(su1006) ] A30P) + P 40)::YFP] P SNB-1::GFP P aex - 3 (acr - 2, unc 30) dat - 1 dat - 1 unc - 51 unc - 51 P N2; Is [P N2; Is [P P N2; Is [P Full-length ATXN-3 expressing lines: expressing ATXN-3 Full-length N2; rmIs[P Model P N2; Is [P P N2; [P C-terminal ATXN-3 expressing lines: expressing C-terminal ATXN-3 P P Is [P continued PD MJD NDs 1 Table Chen et al. Chemistry Central Journal (2015) 9:65 Page 7 of 20 [ 38 , 71 ] [ 128 ] [ 60 ] [ 61 , 129 ] [ 130 ] [ 99 ] [ 134 , 135 ] [ 133 ] [ 61 ] References [ 128 ] [ 131 ] [ 132 ] Resveratrol, rolipram; ethosuximide rolipram; Resveratrol, GW5074, indoline; sorafenib TTT-3002 and LRRK2-IN1 Quinacrine, resveratrol Quinacrine, Efficacious compounds identified/ Efficacious validated - - sensitive worms exhibiting L4 molting problems and morphogenesis impairment in locomotion, severe impairment in locomotion, severe chemosensory defects progressive neurodegeneration which precede of sensory- and signifi neurons cantly shorter lifespan defects and neurodegeneration and neurodegeneration defects R1441C- and G2019S- similar to LRRK2 models. DAergic neurons, behavioural behavioural neurons, DAergic dysfunctiondeficit, locomotor and depletion of dopamine(~72 % rapid loss). G2019S causes more deficits of behavioural progression than others ability of dopaminergic neurons neurons ability of dopaminergic Reduced mitochondrial stress. to in mutant LRRK2 (G2019S lifespan or R1441C) genic phenotypes and remarkable reductions in lifespan morphology, strikingmorphology, neuropatho touch at the tail caused by mutant at the tail caused by touch without expression PrP (PG13-PrP) causing cell death non-autonomous disruption of non-autonomous embryonic mitochondrial integrity, and larval developmental arrest, tissue defects, widespread delay, proteostasis and loss of organismal Mitochondrial stress, ER stress ER stress Mitochondrial stress, Exhibit thermotaxis defects Reduced body size, with some Reduced body size, Larval in molting defects lethality, Age-dependent progressive Age-dependent progressive Double mutants displayed DAergic DAergic Double mutants displayed Age-dependent degeneration of G2019S LRRK2 increased vulner G2019S LRRK2 increased High PrP levels cause abnormal levels High PrP Progressive loss of response to to loss of response Progressive Profound cell autonomous and cell cell autonomous Profound Phenotypes Dopaminergic neuron Dopaminergic Constitutive pan-neuronal Constitutive Constitutive pan-neuronal Constitutive Mechanosensory neurons Constitutive muscles Constitutive Expression in C. elegans Expression UA118 SGC722, SGC851, SGC856, SGC862 UA215, UA216 UA215, wlzIs1 - 7 cgIs51, cgIs52, cgIs53 GS1894 AM801, AM803, AM806 AM803, AM801, RM2754 TM3223 SG900, SGC910 Strain/transgene name/(plasmid) Strain/transgene dat - -1::GFP, -1::GFP, dat ::GFP + lin - ::LRRK2 mec - 7 ::LRRK2 (WT, ::LRRK2(G2019S/ ::PrP(WT, dat - 1 ::GFP] snb - 1 dat - 1 ::GFP ::LRRK2 mec - 7 : GFP; P : GFP; dat - 1 ::GFP; lin - 15 ( + ) ] ::GFP; ::sup35(rΔ2-5, nm, ric - 19 dat - 1 Str - 1 mec-4 unc - 54 -1:: LRRK2(R1441C/A2016T), + P ::PrP dat :GFP + lin - 15 ( ) ] (RNAi) + P (G2019S) D1994A)] (WT,R1441C, G2019S, K1347A) + P P G2019S, R1441C, KD, R1441C/ G2019S, R1441C, KD, KD ) + P 1 15 ( + ) ] PG13) + P r2e2 )::yfp], lin - 15( + ) ] ric - 19 lrk - 1(km17), (km41), (tm1898) and BY250 ; baEx129 [P lin - 15(n765ts) X; cwrEx900 [P lin - 15(n765ts) X; Is [P P sel - 12(ar131) and (ar171) apl - 1( RNAi) dnj - 14(ok237) dnj- 14(tm3223) apl - 1(yn10) lin - 15(n765ts); [P lin - 15(n765ts); Is [P Model N2; baIn20 [P rmIs319 [P continued PD ANCL AD Prion NDs Mutant/RNAi 1 Table Chen et al. Chemistry Central Journal (2015) 9:65 Page 8 of 20 [ 138 ] [ 77 ] [ 53 , 128 ] [ 142 ] [ 122 , 139 – 141 ] [ 54 ] [ 53 ] [ 137 ] References [ 53 , 136 ] [ 121 ] [ 137 ] - chloride, N-acetylneuraminicchloride, acid with tauroursodeoxycholic acid with tauroursodeoxycholic memantine; acetaminophen; Chondrus crispus extract P7C3, P7C3A20; polysaccharides speciosa; acetyl from - corynoline; n -butylidenephthalide 4-Aminopyridine, gaboxadol hydro gaboxadol 4-Aminopyridine, D-α-hydroxybutyrate in combination D-α-hydroxybutyrate Bromocriptine, quinpirole and quinpirole Bromocriptine, Lisuride, apomorphine Lisuride, and rottlerin; Riluzole Efficacious compounds identified/ Efficacious validated - cantly shorter than that of the WT;cantly shorter than that of the and egg-laying motility, defective hatching viability rounding with process loss and cell with process rounding appear in body loss reproducibly hours within a few this order and DA neurodegeneration and DA rotenone treatment than control than control treatment rotenone nematodes and almost completely ceased and almost completely Pharyngeal post-L1. after 5 days a rapid and showed pumping rates Mean lifespan decline. progressive N2 for is 6.0 vs 17.7 days sure to 150 mM paraquat to sure defects and lethalitydefects at early larval of ER stressors. stages in presence Majority at, or died or arrested the larvalprior 15.4 % to, L3 stage. shorter span than that of non- life strain Tg enhanced DA neurodegeneration, neurodegeneration, enhanced DA levels DA reduced - was signifi Body length and lifespan Mitochondrial stress, reduced reduced Mitochondrial stress, DA neurodegeneration DA Neuronal process blebbing, cell body blebbing, process Neuronal Reduced mobility, increased lethality increased Reduced mobility, Significantly more sensitive to sensitive more Significantly Thrashing rate progressively declined progressively rate Thrashing Increased sensitivity a 3-day to expo Display severe developmental developmental severe Display Oxidative stress Oxidative Oxidative stress, mitochondrial stress, mitochondrial stress, stress, Oxidative Phenotypes Expression in C. elegans Expression LL2073 BY250, BY200 BZ555 LM99 Strain/transgene name/(plasmid) Strain/transgene - ) 2 + ::GFP] ::GFP] sub dat - 1 dat - 1 ::α - syn WT, snb - 1 ::GFP] subjected to ::GFP] subjected to ::α-syn + P ::GFP], egIs1 [P ::α - syn A53T , N2, lrk 1(km17), - 2 ::LRRK2 (WT, R1441C, G2019S) dat - 1 cat dat - 1 Streptomyces Streptomyces ::GFP subjected to unc - 119 snb - 1 subjected to Rotenone subjected to secondaryvenezuelae metabolite 6-hydroxydopamine (6-OHDA) 6-hydroxydopamine P P jected to Manganesejected to (Mn subjected to 1-methyl-4-phenyl- subjected to (MPTP) 1,2,3,6-tetrahydropyridine ?(q782) qIs48 ] (I;III) ?(q782) (RNAi) dat - 1 vtIs7 [P smn - 1(cb131)I P N2; [P N2; [P 4(e937) let - [ bli - 4(e937) I/hT2 smn - 1(ok355) djr - 1.1 (RNAi) pdr - 1(lg103), (XY1046, KO3) and Parkin Model pink - 1(tm1779) subjected to Paraquat pink - 1(tm1779) subjected to pdr - 1(XY1046), P continued PD SMA Chemical treatment 1 Table NDs dementia Frontotemporal FTDP-17 disease, CJD Creutzfeldt-Jakob sclerosis, lateral ALS amyotrophic lipofuscinosis, ANCL adult-onset ceroid disease, neuronal diseases (NDs): AD Alzheimer’s Human neuorodegenerative SMA disease, type ataxia 3), PD Parkinson’s MJD Machado–Joseph disease (or spinocerebellar disease, HD Huntington’s inclusions, with ubiquitinated with parkinsonism-17, lobar degeneration FTLD-U frontotemporal spinal muscular atrophy Chen et al. Chemistry Central Journal (2015) 9:65 Page 9 of 20

Despite the above caveats, C. elegans remains a widely Liuwei Dihuang [27] successfully protected the CL4176 used animal model to identify genes that modify neuro- inducible Aβ1–42 muscle-specific expression model by degeneration in vivo. Indeed, genetic screens performed inhibiting Aβ1–42 oligomerisation and reducing superox- on worm models have identified a wide variety of con- ide production. Oleuropein aglycone, the main polyphe- served genes that can suppress or increase disease pro- nol in extra virgin olive oil, was recently shown to protect gression and are thus potential therapeutic drug targets. against amyloid toxicity in both constitutive and induc- However, relatively few of these genetic modifiers are ible Aβ1–42 models [28]. In addition, two recent large, common to more than one disease model, despite the unbiased yeast-based screens of pharmacological modi- shared feature of protein misfolding/aggregation [13, fiers identified the 8-hydroxyquinoline chemical scaffold 14]. In addition to its utility for screening for genetic (8-OHQ), a class of clinically relevant bioactive contributors to NDs, C. elegans is a useful pharmaco- chelators as neuroprotective compounds that reduced logical model for testing potential neuroprotective com- proteotoxicity associated with the aggregation of several pounds. Numerous well-characterised ND models have ND-specific proteins including TDP-43, α-synuclein, been readily exploited for triaging compounds from large polyglutamine proteins, or Aβ1–42 [29, 30]. Notably, two libraries consisting of novel and pre-approved drugs, and closely related 8-OHQs–PBT2 and clioquinol, which for testing the effects of individual drugs, prior to valida- conferred neuroprotective benefits in mouse models of tion in vertebrate models. Potential therapeutics identi- AD, were further shown to rescue Aβ1–42 toxicity in C. fied via such compound screens using specific worm elegans body wall muscle cells [31] and glutamatergic ND models are shown in Figs. 1, 2, listed in Table 1 and neurons [30]. PBT2 was also effective in improving cog- described in detail below. nition and reducing Aβ in cerebrospinal fluid in a small Phase IIA trial in AD patients [31]. Alzheimer’s disease: amyloid‑β (Aβ) models β-Amyloid is the main component of the extracellu- Tauopathies lar plaques found in the brains of Alzheimer’s disease In addition to amyloid plaque deposition, Alzheimer’s patients. It is widely (though not universally) believed disease is associated with intraneuronal accumulation that aggregation of Aβ into oligomeric forms is the main of neurofibrillary tangles containing the microtubule- driver of neurodegeneration in Alzheimer’s disease. This associated protein Tau, which aggregates into insoluble has been modelled in nematodes by expressing human fibrillar deposits when it is hyperphosphorylated [32]. Aβ constructs in worm muscle cells [7]. The Aβ-induced Pathological Tau deposits are also observed in Pick’s dis- paralysis observed in the well-characterised muscle- ease, corticobasal degeneration, Down’s syndrome and specific strains has provided a valuable phenotype for specific types of frontotemporal dementia (FTD) such straightforward quantification of the effects of treatments as frontotemporal dementia with parkinsonism chro- on Aβ toxicity and validation of potential therapeutic mosome 17 type (FTDP-17) and frontotemporal lobar interventions for Alzheimer’s disease. The C. elegans dementia (FTLD). Various worm transgenic Tauopathy strain CL2006, which constitutively expresses human models expressing mutant human Tau constructs have Aβ1-42, has been elegantly used to demonstrate the neu- therefore been generated and yielded complementary roprotective effects of a diverse range of compounds findings in regards to the effects of neuronal Tau expres- (Table 1; Figs. 1, 2). These include natural products such sion [33–35]. Neurodegeneration in worms expressing as specific gingkolides [15], soya isoflavone glycitein [16], transgenic human mutant Tau can be assessed indirectly, the green tea component epigallocatechin gallate [17, using phenotypes such as impaired locomotion and 18] and coffee extract [19]; FDA-approved drugs such as reduced lifespan, but also directly by visualising loss of tannic acid, bacitracin, rifampicin [20], thioflavin T [21], neuronal cell bodies and neuronal processes in vivo. An reserpine [22] and the fluoxetine; and example of the latter is shown in Fig. 3, where a human polyphenolic compounds such as curcumin and ferulic Tau construct containing the FTDP-17-associated acid [23, 24]. These treatments conferred considerable V337 M mutation is expressed in all 302 worm neurons life-span extension and cellular stress tolerance [15, 16]. via a pan-neuronal C. elegans promoter. In addition, This was a consequence of most compounds attenuat- the 26 GABAergic neurons of the worm are specifi- ing the rate of toxic human Aβ1–42 mediated paralysis, cally labelled by driving green fluorescent protein (GFP) to suppress the Aβ1–42 induced increase in toxic reac- expression from GABA-specific C. elegans promoter. In tive oxygen species and hydrogen peroxide levels, and to control worms, a continuous, intact line of GFP fluores- inhibit Aβ1–42 oligomerisation and deposition [15, 25]. cence is seen running along both the ventral and dorsal Recent studies have also demonstrated how the antibiotic nerve cords on opposite sides of the animal. In contrast, tetracycline and its analogues [26], and ethanol extract of the mutant Tau transgenic strains exhibits large gaps in Chen et al. Chemistry Central Journal (2015) 9:65 Page 10 of 20

17-AAG 4-Aminopyridine Acetaminophen Acetylcorynoline Apomorphine Aspirin (MJD) (SMA) (PD) (PD) (PD) (HD)

Azaperone Bacitracin Bromocriptine BSc3094 Caffeine Celecoxib (FTDP) (AD) (PD) (FTDP) (AD) (HD)

Clioquinol Clofiazimine Cmp16 Curcumin Dinitrophenol Epigallocatechin (AD) (FTDP) (FTDP) (AD, PD) (HD) (AD)

Ethosuximide Ferulic acid Fluoxetine Gaboxadol Galanthamine Ginkgolide (ALS,ANCL,FTDP) (AD) (AD) (SMA) (AD) (AD)

Glycitein Guanabenz GW5074 Icariside Indoline Isoniazid (AD) (ALS) (PD) (AD,HD) (PD) (FTDP)

JAY2-22-33 JWB1-84-1 Lisuride Lithium Lorglumide LRRK2-IN1 (AD) (AD) (PD) (HD) (FTDP) (PD) Chen et al. Chemistry Central Journal (2015) 9:65 Page 11 of 20

(See figure on previous page.) Fig. 1 Structures of compounds with therapeutic effects in C. elegans models of human neurodegenerative diseases. Chemical structures were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov) or MolBase (http://www.molbase.com). AD Alzheimer’s disease, ALS amyotrophic lateral sclerosis, ANCL adult-onset neuronal ceroid lipofuscinosis, FTDP frontotemporal dementia with parkinsonism-17, HD Huntington’s disease, MJD Machado–Joseph disease (spinocerebellar ataxia type 3), PD Parkinson’s disease, Prion prion disease, SMA spinal muscular atrophy these nerve cords where neuronal processes are missing, the impact of aggregation prone proteins on cellular thus directly demonstrating severe neurodegeneration in function and to identify novel disease modifiers [39– the living animal. 41]. The progressive nature of polyQ-mediated toxicity, Using such Tauopathy models, compounds with known protein aggregation and general severity of phenotype anti-aggregation activity like methylene blue, were shown demonstrated in these models is age- and polyQ-tract- to effectively ameliorate the worms’ motility and neu- length-dependent, recapitulating critical aspects of poly- ronal defects [36]. In addition, a novel compound belong- glutamine expansion diseases in patients. ing to the aminothienopyridazine class, cmp16, was also Voisine et al. [42] screened candidate pharmacologi- shown to rescue these phenotypes and to suppress Tau cal compounds utilising a HD model in which the pqe- aggregation in worms [36]. Importantly, aminothieno- 1 genetic mutant background greatly enhanced toxicity pyridazines are known to suppress Tau aggregation in induced by a human Huntingtin construct containing a mammalian cells and so the improved blood–brain bar- 150-residue glutamine tract (Htt-Q150). Both lithium rier permeability of cmp16 suggests that this compound chloride and mithramycin alleviated neuronal cell death, may have significant translational potential. In a recent while trichostatin A (a class I and class II HDAC inhibi- screen of a library of FDA-approved compounds, dopa- tor) provided significant neuroprotection. Using the mine D2 receptor antagonism was identified as a prom- same HD model, Varma et al. [43] discovered that small ising strategy for targeting tau-induced neurotoxicity, molecular inhibitors of metabolism (mitochondrial and as such as azaperone, perphenazine, and glycolytic function) such as rotenone, oligomycin and zotepine improved the phenotypic features of Tauopathy 4-dinitrophenol rescued neuronal loss and degenera- in worms (Table 1; Figs. 1, 2). Azaperone, in particular, tion by activating caspase inhibition and ERK and AKT effectively ameliorated mutant Tau-induced functional prosurvival signalling and their efficacy was further defects and reduced the level of insoluble Tau aggre- validated in cell culture and Drosophila HD models gation [37]. Finally, a recent study reported that the (Table 1; Figs. 1, 2). Resveratrol, a demonstrated acti- anti-epileptic drug, ethosuximide, could ameliorate the vator of sirtuin deacetylases, also effectively alleviated impaired motility and reduced lifespan phenotypes of Htt-Q128 toxicity in both worm and neuronal culture the Tau V337 M worm FTDP-17 model [38]. Interest- models [44]. Recently, treatment of a C. elegans model ingly, ethosuximide’s action in this worm Tau model was of SCA3 (spinocerebellar ataxia type 3; also known as independent of its main proposed target in epilepsy, the Machado-Joseph disease) with 17-(allylamino)-17-dem- T-type calcium channel. ethoxygeldanamycin (17-AAG), an HSP90 inhibitor, suc- cessfully decreased the mutant ATXN3 aggregation and Polyglutamine (polyQ) disorders improved locomotor activity [39]. Treatment of the same Expansion of trinucleotide CAG repeats in a variety of model with valproic acid (VA), another HDAC inhibi- different genes leads to neurodegenerative diseases such tor and a well-known anti-epileptic drug, also led to as Huntington’s disease and spinocerebellar ataxias due improved locomotor activity accompanied by a decrease to the expression of a polyglutamine tract within the in mutant ATXN3 aggregation. Therefore, HDAC inhibi- encoded protein. Diverse worm transgenic models where tors which promote histone acetylation over deacetyla- varying lengths of polyQ tracts are expressed in specific tion and which were also known to provide protection sets of neurons, muscle cells and even intestine cells have against polyQ mediated toxicity in vertebrate and Dros- been widely used to model several aspects of polyQ neu- ophila neurons may hold promise as a preventive ther- rotoxicity, notably to address the mechanisms underlying apy in polyQ diseases.

(See figure on next page.) Fig. 2 Structures of compounds with therapeutic effects in C. elegans models of human neurodegenerative diseases. Chemical structures were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov) or MolBase (http://www.molbase.com). AD Alzheimer’s disease, ALS amyotrophic lateral sclerosis, ANCL adult-onset neuronal ceroid lipofuscinosis, FTDP frontotemporal dementia with parkinsonism-17, HD Huntington’s disease, MJD Machado–Joseph disease (spinocerebellar ataxia type 3), PD Parkinson’s disease, Prion prion disease, SMA spinal muscular atrophy Chen et al. Chemistry Central Journal (2015) 9:65 Page 12 of 20

Memantine Methylene blue Mithramycin ML346 N-acetylneuraminic Nefopam (PD) (ALS,FTDP) (HD) (HD) acid (SMA) (FTDP)

Oleuropein Oligomycin P7C3 PBT2 Perphenazine PHA767491 aglycone (AD) (HD) (PD) (AD) (FTDP) (AD)

Phenazine Propylgallate Quinacrine Reserpine Resveratrol Rifampicin (ALS) (ALS) (Prion) (AD,ALS) (ALS,ANCL,HD,Prion) (AD)

Riluzole Rolipram Rotenone Rottlerin Salidroside Salubrinal (SMA) (ALS,ANCL) (HD) (PD) (HD) (ALS)

Sorafenib Spermidine Tannic acid Tauroursodeoxy- Tetracycline Thioflavin T (PD) (PD) (AD) cholic acid (PD) (AD) (AD)

Trazodone Trichostatin A Trolox TTT-3002 Valproic acid Zotepine (FTDP) (HD) (ALS) (PD) (MJD,PD) (FTDP) Chen et al. Chemistry Central Journal (2015) 9:65 Page 13 of 20

was also shown to significantly improve polyQ-medi- Control Tau V337M ated animal paralysis, reducing the number of Q35-YFP aggregates and delaying polyQ-dependent acceleration of aging [51].

Day 1 Parkinson’s disease (PD) Pathologically, PD is characterised by degeneration of dopaminergic neurons in the substantia nigra and accumulation of Lewy bodies containing aggregated α-synuclein protein. Although most cases are idiopathic, Day 5 PD can be caused by both environmental (e.g. pesti- cide exposure) and genetic (e.g. α-synuclein and LRRK2 mutation) effects. Multiple worm PD models, notably the toxin-induced models, have aided in the discovery and validation of potential pharmacological interventions for PD. An example of how dopaminergic neurodegeneration Day 10 can be directly assessed in vivo in C. elegans is shown in Fig. 4. Here, the eight dopaminergic neurons of the worm are specifically labelled by GFP expression from the pro- moter of the C. elegans . In control worms, fluorescent neuronal cell bodies extending long processes are clearly visible in the head (6 neurons) and Insets tail (2 neurons) of the animal. However, treatment with the PD-inducing toxin, 6-hydroxydopamine (6-OHDA), causes the loss of GFP-labelled dopaminergic neuronal Fig. 3 A C. elegans genetic model of the Tauopathy, FTDP-17. Triple cell bodies and/or processes, thus enabling direct visuali- transgenic worms expressing human V337M mutant Tau protein sation of neurodegeneration. (Paex-3::V337M Tau), a pharyngeal GFP marker (Pmyo-2::GFP) and Chemical screens have suggested that compounds a GFP reporter transgene marking the cell bodies and processes which protect mitochondria or increase autophagy pro- of all C. elegans GABAergic neurons (Punc-25::GFP) were compared with control single Punc-25::GFP transgenic worms. All panels are tect against α-synuclein toxicity [52, 53]. Braungart et al. micrographs of representative whole worms. Control (left panels) and [54] performed a focused compound screen using the Tau V337M expressing worms (right panel) were examined after 1, 5 C. elegans MPTP model of PD and found that lisuride and 10 days of age. In control worms, intact ventral and dorsal cords and apomorphine (dopamine receptor agonists), as well were observed at all ages. In contrast, the mutant Tau transgenic as rottlerin (protein kinase C inhibitor) ameliorated the GABAergic reporter strain exhibited severe degeneration of neuronal processes. Ventral and dorsal cord gaps (arrows) are disruptions in the MPTP-induced behavioural defects when present at a continuity of the ventral and dorsal nerve cords, respectively. Scale low concentration. In addition, (dopamine bar represents 200 μm for all panels except for the bottom two panels, transporter inhibitor), nicotine (acetylcholine recep- which are high magnifications of the boxed areas of day-10 worms tor agonist), selegiline (monoamine oxidase inhibitor), shown above MPEP (mGluR-5 inhibitor), , α-lipoic acid (antioxidant) and ascorbic acid (antioxidant) were effec- tive at higher concentrations [53]. In another screen, two mammalian dopamine D2 receptor agonists, bromocrip- Other pan-neuronal or neuron specific HD models tine and quinpirole, were identified to confer significant facilitated the identification of other potential thera- neuroprotection independent of dopamine receptors in peutic interventions, including the anti-cancer agent a 6-OHDA-induced dopaminergic neurodegeneration β-lapachone [45], D. officinarum root extracts [46] and a model of PD [55]. Similarly, a low concentration of aceta- phenol glycoside salidroside [47], which conferred pro- minophen (analgesic and antipyretic) was reported by tection against polyQ neuronal toxicity. Treating C. ele- Locke et al. [56] to protect significantly against 6-OHDA gans muscle polyQ models with hydroxylamine, icariin toxicity-induced dopaminergic neurodegeneration in and celecoxib derivatives (NG-094, icariside II and OSU- Pdat-1::GFP expressing worms. However, the protection 03012, respectively) ameliorated polyQ-mediated protein appears to be selective as acetaminophen was not neu- aggregation and protected against polyQ proteotoxicity roprotective against α-synuclein-induced neurodegen- [48–50] (Table 1; Figs. 1, 2). Aspirin, an analgesic agent, eration at any concentration tested. The anti-epileptic Chen et al. Chemistry Central Journal (2015) 9:65 Page 14 of 20

◂ Fig. 4 A C. elegans model of toxin-induced Parkinson’s disease. a a PDE Dopaminergic (DA) neuronal cell bodies and neurites in BY250 worms were visualised using an integrated Pdat-1::GFP dopamine transporter marker. C. elegans has eight DA neurons: six are located in the anterior CEP ADE region, which can be subclassified in pairs as two anterior deirid neu- rons (ADE), two dorsal cephalic neurons (CEP) which are postsynaptic to the ADE neurons and two ventral CEPs that are not postsynaptic WT Blebbing Rounding Cell loss to the ADEs; two posterior deirid neurons (PDE) located posteriorly b are also shown. Arrows depict the four CEP neuron processes and indicate the ADE and PDE cell bodies in a young worm. Anterior is to the left. b Representative examples of worms scored which display the three characteristic stages of DA neurodegeneration in response to 6-OHDA. Magnification of anterior region of C. elegans shows only the anterior-most DA neurons. WT: in this example, all six anterior DA neurons of this worm appear robust and the dendrites are intact and * fully extended. Neuronal process blebbing; cell body rounding: this * worm exhibited prominent cell body rounding (asterisk) and dendrite blebbing (arrows); cell body loss: this worm exhibited a complete loss of GFP in most DA neurons as CEP and ADE neurons have all degener- ated and are no longer visible in any focal plane, only retention of GFP expression in the remnants of neuron cell bodies and broken neurites. All scale bars represent 20 μm. c Representative images of worms c 24 h post-6-OHDA-exposure are presented. BY250 worms treated with ascorbic acid (AA) alone expressed intact and strong GFP in all six DA neurons and dendrites in the heads. However, the majority of BY250 worms incubated with 50 mM 6-OHDA showed a marked GFP expression reduction in the dendrites of ADEs and CEPs, many of the cell somas became round (asterisk) and blebs appeared along the dendrites of CEPs (arrows)

n-butylidenephthalide, curcumin, N-acetylcysteine and vitamin E on 6-OHDA-induced degeneration of dopaminergic neurons and their ability to attenuate BY250 + AA α-synuclein accumulation. n-butylidenephthalide, in particular, had the greatest neuroprotective capacity * and was shown to also restore food-sensing behaviour and dopamine levels in both pharmacological and trans- genic C. elegans PD models as well as enhancing the life * span of 6-OHDA-treated animals [58]. Acetylcorynoline, the major alkaloid component derived from Corydalis bungeana, a traditional Chinese medical herb demon- strated the same neuroprotective effects when applied to the same pharmacological and transgenic C. elegans PD * models [59]. Kinase-targeted inhibition of LRRK2 protein activ- BY250 + 6-OHDA + AA ity was recently established as an effective treatment for PD as LRRK2 kinase inhibitors consistently mitigated pathogenesis caused by different LRRK2 mutations. Liu et al. [60] showed that though GW5074, an indo- drug, valproic acid provided significant dopaminergic line compound, and sorafenib, a Raf kinase inhibitor, neuroprotection in a C. elegans PD model associated did not have protective effects against α-synuclein- and with human α-synuclein overproduction, which was 6-OHDA-induced toxicity, they increased survival and further shown to be mediated through ERK-MAPK sig- reduced dopaminergic neurodegeneration in G2019S- nalling [57]. A more recent study has also demonstrated LRRK2 transgenic C. elegans and Drosophila. Yao et al. the neuroprotective effects of the naturally occurring [61] further demonstrated the potency of kinase inhibi- polyamine spermidine and phytocompounds such as tors as they were able to pharmacologically rescue both Chen et al. Chemistry Central Journal (2015) 9:65 Page 15 of 20

the behavioural deficit and neurodegeneration mani- neuroprotective synaptic chaperone cysteine string pro- fested by the expression of mutant LRRK2 G2019S and tein (CSP) [67–70]. Our lab has recently developed a R1441C in vivo using two LRRK2 inhibitors, TTT-3002 C. elegans model of ANCL by using null mutants of the and LRRK2-IN1, which also potently inhibited in vitro worm DNAJC5 orthologue, dnj-14 [71]. These worms kinase activities of LRRK2 wild-type, R1441C and have similar phenotypes to ANCL patients and also to G2019S at nanomolar to low micromolar concentrations CSP mutants in mice, in terms of reduced lifespan, pro- when administered either pre-symptomatically or post- gressive neuronal dysfunction and neurodegeneration symptomatically. Compounds that have been shown to [72]. This evolutionary conservation of CSP’s neuropro- be protective in the various worm PD models are listed tective function suggests that the worm dnj-14 model in Table 1 and their chemical structures shown in Figs. 1 could have potential for identifying generic neuropro- and 2. tective interventions rather than disease specific drug targets. Indeed, a focused screen of pharmacological Amyotrophic lateral sclerosis (ALS) compounds that ameliorated the dnj-14 lifespan and neu- A number of transgenic lines expressing mutant forms ronal defects identified the polyphenolic molecule res- of human SOD1 found in familial ALS patients under a veratrol, which has been shown to be neuroprotective in range of promoters have been generated and recapitu- a range of animal neurodegeneration models [71]. In con- lated the motor neuron degeneration and paralysis char- trast to other worm neurodegeneration models [44, 63, acteristic of ALS patients [102, 103, 105, 108]. Genes 73, 74], however, resveratrol acted in a sir-2.1-independ- recently shown to be mutated in ALS include the DNA/ ent manner, as sir-2.1; dnj-14 double mutants showed full RNA binding proteins TDP-43 and FUS, and C9ORF72, lifespan rescue by resveratrol. Instead, the mechanism of a novel familial and sporadic ALS causative gene. Treat- resveratrol action appeared to be via inhibition of cAMP ment with methylene blue, an aggregation inhibitor of phosphodiesterase, as the phosphodiesterase inhibi- the class, not only rescued toxic pheno- tor, rolipram was shown to mimic the effect of resvera- types (including neuronal dysfunction and oxidative trol in rescuing dnj-14 phenotypes [71]. More recently, stress) associated with mutant TDP-43 and FUS in C. ele- the anti-epileptic drug ethosuximide has been shown gans and zebrafish ALS models [62], but also ameliorated to be protective in the dnj-14 model, acting through a Tau mediated toxicity in a newly established C. elegans DAF-16/FOXO-dependent mechanism that is distinct model [36]. Using transgenic TDP-43 models, Tauffen- from its proposed in epilepsy [38]. berger et al. evaluated 11 compounds previously reported Ethosuximide also ameliorates the phenotypes of worm to enhance longevity in C. elegans and resveratrol (poly- models of FTDP-17 [38] and ALS [63] and reduces pro- phenol), rolipram (phosphodiesterase 4 inhibitor), reser- tein aggregation in a mouse neuronal cell culture model pine (antihypertensive), ethosuximide (anti-epileptic), of Huntington’s disease [38], suggesting that it may have trolox and propyl gallate (antioxidants) were revealed general and evolutionarily conserved neuroprotective as effective candidates that protected against mutant properties. Indeed, it has recently been shown that etho- TDP-43 toxicity in motor neurons [63] (Table 1; Figs. 1, suximide reverses cognitive decline in a rat model of 2). Recent genetic experiments by Kraemer’s group sug- Alzheimer’s disease [75]. Finally, a recent genome-wide gested that inhibiting cell division cycle kinase 7 (CDC7) transcriptional profiling study of dnj-14 mutants revealed kinase activity reduces phosphorylation of TDP-43 and a striking reduction in expression of ubiquitin protea- the consequent neurodegeneration. Small molecule inhi- some system (UPS)-related genes in comparison to wild bition of CDC-7 by PHA767491 was further shown to type control strains [76]. Genes encoding components of robustly reduce TDP-43 phosphorylation and prevent multimeric E3 ubiquitin ligases were especially over-rep- TDP-43 dependent neurodegeneration both in vitro and resented, suggesting that these may represent potential in vivo [64]. novel drug targets for treatment of ANCL and perhaps other neurodegenerative diseases. Autosomal dominant adult‑onset neuronal ceroid lipofuscinosis (ANCL) Translational implications of C. elegans chemical ANCL, also known as autosomal dominant Kufs’ dis- screens ease and Parry disease, is a rare hereditary disease The different screening strategies that have been applied characterised by intra-neuronal inclusions of auto- to C. elegans ND models have provided distinct insights fluorescent lipofuscin-like material and neurodegen- into potential therapeutic approaches in patients. These eration [65, 66]. Recently, four independent research strategies range from robotic automated imaging-based groups have reported that ANCL is caused by muta- approaches designed for high throughput compound tions in the DNAJC5 gene that encodes the endogenous library screening [77] to highly focused screens of a Chen et al. Chemistry Central Journal (2015) 9:65 Page 16 of 20

selected small group of compounds that target a com- and suitability for high-throughput compound screening mon pathological process such as protein aggrega- mean that both target-driven and phenotypic screening tion [21]. Large scale screens offer greater coverage of approaches can easily be performed (and potentially com- chemical space and so have potential to identify unify- bined). Although phenotypic screening became unfash- ing pharmacological themes amongst multiple hits from ionable as a drug discovery paradigm in the post-genomic compound libraries. For example, several different dopa- era, Swinney and Anthony have clearly shown that most mine D2 receptor antagonists were recovered as hits in new medicines still continue to be discovered via pheno- an unbiased library screen using a Tauopathy model, typic screening [85]. This influential work has forced a re- with genetic techniques then being used to confirm that evaluation in the pharma industry and a consequent shift reduced D2 receptor function is indeed neuroprotec- towards phenotypic screening that incorporates avail- tive [37]. Whilst this suggests that several currently pre- able knowledge of targets/mechanisms [86], for which C. scribed atypical anti-psychotic drugs could potentially be elegans is ideally suited. Furthermore, there is increasing re-purposed for treatment of human tauopathies, dosing evidence that using compound combinations designed to regimens would need to be carefully considered given act on multiple molecular targets can be an effective ther- reports that the relatively high doses of these medica- apeutic strategy—as exemplified by the spectacular suc- tions used to treat aggression and agitation in dementia cess of combination therapy for HIV [87]. Testing of many patients may increase the risk of death [78]. such drug combinations can be performed rapidly and One observation that emerges from our analysis of the cheaply using worm models, in contrast to rodent models. large number of studies to date is that very few compounds In addition, technical developments such as CRISPR [88] are therapeutic in multiple C. elegans ND models. Indeed, now offer the potential to rapidly create new and more out of the 72 compounds shown in Figs. 1 and 2, only etho- accurate C. elegans models of human neurodegenerative suximide and resveratrol are effective in more than two ND diseases, by precisely delivering single-copies of mutant models and therefore appear to have general neuroprotec- genes identified from patients to appropriate desired tive activity. This may be due in part to the fact that most locations in the worm genome. Although C. elegans has published studies have focused on relatively small sets of already facilitated the identification of potential novel compounds and so activity across multiple ND models therapeutics, the future combination of more accurate remains to be tested. Nevertheless, it seems certain that this genetic models with high-throughput automated drug also reflects disease-specific pharmacological actions—for screening platforms is a potentially very efficient strategy example, Raf kinase inhibition is therapeutic in LRRK2- for therapeutic drug discovery for NDs. based PD models, but ineffective in α-synuclein- and Authors’ contributions 6-OHDA-based PD models [60]. Clearly, effective clinical XC, JWB, RDB and AM conceived and designed the study. XC and AM wrote treatments with such highly disease-specific drugs requires the manuscript, with input from JWB and RDB. All authors read and approved the final manuscript. knowledge of the underlying pathophysiological mecha- nism, which is not always diagnosable in NDs. Drugs such Author details as ethosuximide and resveratrol are therefore potentially 1 Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown St, Liverpool L69 3BX, UK. 2 Present very useful, as they may provide general neuroprotective Address: Centre for Neurodegenerative Science, Van Andel Research Institute, activity regardless of uncertainties regarding molecular 333 Bostwick Avenue NE, Grand Rapids, Michigan, MI 49503, USA. pathology. The mechanism of action of ethosuximide and Acknowledgements resveratrol remains unclear and controversial [79–81], but We are grateful to the BBSRC and AgeUK for funding our research into worm both have been linked to increased longevity and healthspan models of neurodegeneration. We thank Dr. Brian Kraemer (University of in model organisms [82, 83]. Given that dietary restriction, Washington, USA) for providing the CK49 and CZ1200 strains shown in Fig. 3; and the Caenorhabditis Genetics Center for providing the BY250 strain created the best established intervention known to increase longev- by Dr. Randy Blakely (Vanderbilt University, USA) shown in Fig. 4. ity and healthspan, is therapeutic in multiple ND models from invertebrates to mice [84], it is clear that slowing the Competing interests The authors declare that they have no competing interests. ageing process can confer general neuroprotection. It may be that ethosuximide and resveratrol modulate some of the Received: 27 August 2015 Accepted: 15 November 2015 same conserved neuroprotective mechanisms that decline with age, thus potentially explaining their therapeutic effects in radically different ND models.

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