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. 2018 Aug 29;6(1):84.
doi: 10.1186/s40478-018-0586-1.

Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis

L McGurk  1 , J Mojsilovic-Petrovic  2   3 , V M Van Deerlin  4 , J Shorter  5 , R G Kalb  2   3 , V M Lee  4 , J Q Trojanowski  4 , E B Lee  4   6 , N M Bonini  7
Affiliations

Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis

L McGurk et al. Acta Neuropathol Commun. .

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2-5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis.

Keywords: ABT-888/Veliparib; Motor neuron disease, primary neuron; PAR; PARylation; Parp; Poly(ADP-ribose); TDP-43.

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Figures

Fig. 1
Fig. 1
Case demographics. a. Spinal cord tissue from 16 patients with no history of neurodegenerative disease was examined in this study; 7 were female and 9 were male. b. The spinal cord from 27 patients diagnosed with ALS were examined in this study; 11 were female and 16 were male. c. There was no statistical difference in the age of death between the normal and ALS patients. The graph represents the median with interquartile range. A Mann-Whitney test was used to test for significance. d. Compared to the no-mutation carriers, the presence of a mutation in C9orf72 or an intermediate polyQ expansion in ATXN2 did not cause a significant change in disease duration in these pre-selected cohorts. The graph represents the median with interquartile range. A Kruskal-Wallis test was used to test for significance
Fig. 2
Fig. 2
ALS motor neurons have elevated levels of nuclear PAR. a. Spinal cord sections from a neurologically normal case showing a motor neuron with no nuclear PAR immunoreactivity (arrow). An ALS-no mut case with three motor neurons with nuclear PAR (arrows). An ALS-ATXN2 case with two motor neurons presenting with nuclear PAR (arrows). An ALS-c9 case with one motor neuron with nuclear PAR (arrow). Sections were immunostained for PAR and counterstained with Hematoxylin. b. The presence of cytoplasmic PAR in the motor neurons of the spinal cord was quantified on a semi-quantitative scale (0 no detectable cytoplasmic PAR; + cytoplasmic PAR detected in 1 motor neuron; ++ cytoplasmic PAR detected in > 1 motor neuron), see also Tables 3 and 4. The data was charted as a percentage. c. The presence of nuclear PAR in the motor neurons of the spinal cord was quantified on a semi-quantitative scale (0 no detectable nuclear PAR; + nuclear PAR detected in 1 motor neuron; ++ nuclear PAR detected in > 1 motor neuron), see also Tables 3 and 4. The data were charted as a percentage. Slides were fully blinded and examined independently by two researchers, images for figures were captured with a 20X objective and an optivar magnification of 1.6
Fig. 3
Fig. 3
PAR does not form protein aggregates in the cytoplasm of motor neurons. a. Serial sections from ALS spinal cord tissue were stained for either PAR or phosphorylated TDP-43 (pS409/10). Motor neurons with phosphorylated TDP-43 aggregates did not also have cytoplasmic aggregates labelled with PAR. Arrowheads indicate the same neurons in each serial section. Scale bar: 50 μm. b. Serial sections of ALS spinal cord tissue were stained for either PAR or phosphorylated TDP-43 (pS409/10). The motor neurons shown with elevated nuclear PAR did not have cytoplasmic aggregates of phosphorylated TDP-43. Arrowheads indicate the same neuron in each section. Arrows indicate neuron with nuclear PAR. Scale bar: 50 μm
Fig. 4
Fig. 4
Small molecule inhibition of PARP-1/2 reduces the formation of stress-induced TDP-43 foci in mammalian cells. a Veliparib is a small molecule inhibitor of PARP-1/2 activity reported to inhibit the formation of G3BP1-labelled foci in the cytoplasm upon UV treatment [49]. b Exposure to arsenite leads to the formation of TIAR-labelled stress granules in the cytoplasm (arrows). Co-treatment with Veliparib inhibits the formation of TIAR-labelled stress granules. COS-7 cells transfected with TDP-43-YFP were immunostained for TIAR and counterstained with Hoescht. Cells were imaged for TIAR and Hoescht. c Cells were quantified for the presence of cytoplasmic TIAR-labelled stress granules. Mean (± SEM) is presented. One-way ANOVA followed by a Tukey’s test was used for significance. d Under normal conditions (ctrl), TDP-43-YFP diffusely localizes to the nucleus of COS-7 cells. Upon treatment with arsenite, TDP-43-YFP forms foci in the cytoplasm (arrows). The formation of cytoplasmic TDP-43-YFP foci is inhibited by treatment with Veliparib. Cells were counterstained with Hoescht. e Veliparib reduces the accumulation of TDP-43-YFP foci in the cytoplasm. Cells were quantified for the presence of cytoplasmic TDP-43-YFP foci. Mean (± SEM) is presented. One-way ANOVA (p = 0.0002) followed by a Tukey’s test was used for significance. f Hypothetical schematic showing that inhibition of PARP-1/2 activation by Veliparib inhibits the formation of stress-induced TDP-43-YFP foci
Fig. 5
Fig. 5
Veliparib inhibits TDP-43-associated neuronal loss in rat spinal cord cultures. a. The spinal cord was isolated from Sprague Dawley embryos (E16-E18), dissociated with protease and DNase, and seeded onto astrocyte coated 12-well plates. After 1 day in vitro (1 DIV) cell proliferation was stopped by the addition of 5 μM cytosine arabinoside (AraC). At 14 DIV cultures were infected with a LacZ control or TDP-43 attenuated herpes simplex virus alongside DMSO or Veliparib. At 19 DIV the neurons were fixed and immunostained for the neuronal marker neurofilament-H (NF-H) and counterstained with Hoescht. Five images (10X magnification) were captured from each condition and neuronal cell bodies were counted. Each condition was repeated three times from 3 independent cultures. b. Viral infection of TDP-43 leads to the loss of neuronal cell bodies in dose-dependent manner. Co-treatment with 1 μM or 5 μM Veliparib inhibits TDP-43-induced neuronal cell loss. Mean (± SEM) is presented, each data point represents three technical repeats from an independent culture. 1X represents a virus titer of 3-5 × 104 pfu/ml. Two-way ANOVA (p < 0.0001) and a Dunnett’s test for significance was performed. NS: not significant. c. Example images (magnification 10X), of rat spinal-cord cultures infected with 1X LacZ or 1X TDP-43 and incubated with DMSO or 5 μM Veliparib. Cultures were immunolabeled for Neuro filament-H (NF-H) and counterstained with Hoescht. d. Schematic showing that motor neuron loss induced by virally expressed TDP-43 in spinal cord cultures and that this loss is suppressed by the PARP-1/2 inhibitor Veliparib
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