Data from: Impact of genetic reduction of NMNAT2 on chemotherapy-induced losses in cell viability in vitro and peripheral neuropathy in vivo

Nicotinamide mononucleotide adenylyl transferases (NMNATs) are essential neuronal maintenance factors postulated to preserve neuronal function and protect against axonal degeneration in various neurodegenerative disease states. We used in vitro and in vivo approaches to assess the impact of NMNAT2 reduction on cellular and physiological functions induced by treatment with a vinca alkaloid (vincristine) and a taxane-based (paclitaxel) chemotherapeutic agent. NMNAT2 null (NMNAT2-/-) mutant mice die at birth and cannot be used to probe functions of NMNAT2 in adult animals. Nonetheless, primary cortical cultures derived from NMNAT2-/- embryos showed reduced cell viability in response to either vincristine or paclitaxel treatment whereas those derived from NMNAT2 heterozygous (NMNAT2+/-) mice were preferentially sensitive to vincristine-induced degeneration. Adult NMNAT2+/- mice, which survive to adulthood, exhibited a 50% reduction of NMNAT2 protein levels in dorsal root ganglia relative to wildtype (WT) mice with no change in levels of other NMNAT isoforms (NMNAT1 or NMNAT3), NMNAT enzyme activity (i.e. NAD/NADH levels) or microtubule associated protein-2 (MAP2) or neurofilament protein levels. We therefore compared the impact of NMNAT2 knockdown on the development and maintenance of chemotherapy-induced peripheral neuropathy induced by vincristine and paclitaxel treatment using NMNAT2+/- and WT mice. NMNAT2+/- did not differ from WT mice in either the development or maintenance of either mechanical or cold allodynia induced by either vincristine or paclitaxel treatment. Intradermal injection of capsaicin, the pungent ingredient in hot chili peppers, produced equivalent hypersensitivity in NMNAT2+/- and WT mice receiving vehicle in lieu of paclitaxel. Capsaicin-evoked hypersensitivity was enhanced by prior paclitaxel treatment but did not differ in either NMNAT2+/- or WT mice. Thus, capsaicin failed to unmask differences in nociceptive behaviors in either paclitaxel-treated or paclitaxel-untreated NMNAT2+/- and WT mice. Moreover, no differences in motor behavior were detected between genotypes in the rotarod test. Our studies do not preclude the possibility that complete knockout of NMNAT2 in a conditional knockout animal could unmask a role for NMNAT2 in protection against detrimental effects of chemotherapeutic treatment

The chemotherapeutic agent paclitaxel selectively impairs learning while sparing source memory and spatial memory

Chemotherapeutic agents are widely used to treat patients with systemic cancer. The efficacy of these therapies is undermined by their adverse side-effect profiles such as cognitive deficits that have a negative impact on the quality of life of cancer survivors. Cognitive side effects occur across a variety of domains, including memory, executive function, and processing speed. Such impairments are exacerbated under cognitive challenges and a subgroup of patients experience long-term impairments. Episodic memory in rats can be examined using a source memory task. In the current study, rats received paclitaxel, a taxane-derived chemotherapeutic agent, and learning and memory functioning was examined using the source memory task. Treatment with paclitaxel did not impair spatial and episodic memory, and paclitaxel treated rats were not more susceptible to cognitive challenges. Under conditions in which memory was not impaired, paclitaxel treatment impaired learning of new rules, documenting a decreased sensitivity to changes in experimental contingencies. These findings provide new information on the nature of cancer chemotherapy- induced cognitive impairments, particularly regarding the incongruent vulnerability of episodic memory and new learning following treatment with paclitaxel

NMNAT Vonfrey Acetone Vincristine 9 29 14

Contains raw data for the Vincristine graphs and experiments. Group A is the NMNAT2 +/- group and B is the wild-type group

Data from: Impact of genetic reduction of NMNAT2 on chemotherapy-induced losses in cell viability in vitro and peripheral neuropathy in vivo

Nicotinamide mononucleotide adenylyl transferases (NMNATs) are essential neuronal maintenance factors postulated to preserve neuronal function and protect against axonal degeneration in various neurodegenerative disease states. We used in vitro and in vivo approaches to assess the impact of NMNAT2 reduction on cellular and physiological functions induced by treatment with a vinca alkaloid (vincristine) and a taxane-based (paclitaxel) chemotherapeutic agent. NMNAT2 null (NMNAT2-/-) mutant mice die at birth and cannot be used to probe functions of NMNAT2 in adult animals. Nonetheless, primary cortical cultures derived from NMNAT2-/- embryos showed reduced cell viability in response to either vincristine or paclitaxel treatment whereas those derived from NMNAT2 heterozygous (NMNAT2+/-) mice were preferentially sensitive to vincristine-induced degeneration. Adult NMNAT2+/- mice, which survive to adulthood, exhibited a 50% reduction of NMNAT2 protein levels in dorsal root ganglia relative to wildtype (WT) mice with no change in levels of other NMNAT isoforms (NMNAT1 or NMNAT3), NMNAT enzyme activity (i.e. NAD/NADH levels) or microtubule associated protein-2 (MAP2) or neurofilament protein levels. We therefore compared the impact of NMNAT2 knockdown on the development and maintenance of chemotherapy-induced peripheral neuropathy induced by vincristine and paclitaxel treatment using NMNAT2+/- and WT mice. NMNAT2+/- did not differ from WT mice in either the development or maintenance of either mechanical or cold allodynia induced by either vincristine or paclitaxel treatment. Intradermal injection of capsaicin, the pungent ingredient in hot chili peppers, produced equivalent hypersensitivity in NMNAT2+/- and WT mice receiving vehicle in lieu of paclitaxel. Capsaicin-evoked hypersensitivity was enhanced by prior paclitaxel treatment but did not differ in either NMNAT2+/- or WT mice. Thus, capsaicin failed to unmask differences in nociceptive behaviors in either paclitaxel-treated or paclitaxel-untreated NMNAT2+/- and WT mice. Moreover, no differences in motor behavior were detected between genotypes in the rotarod test. Our studies do not preclude the possibility that complete knockout of NMNAT2 in a conditional knockout animal could unmask a role for NMNAT2 in protection against detrimental effects of chemotherapeutic treatment

DRGEXP

DRG quantification data from western blots and NAD/NADH activity

NMNATGraphPad

The primary graph file used to generate figures 2-5. Contains averages of each subject throughout the necessary timecourse (via the spreadsheets)

Capsascin

The capsaicin spreadsheet contains raw data for each timepoint following capsaicin administration, as well as a tab for nocifensive behavior. The animals are the same as the paclitaxel spreadsheet

Vonfrey Acetone and RotaRod Data Paclitaxel

Contains raw data for the paclitaxel graphs and expiriments as well as rotarod data averages. Group A is the NMNAT2 +/- group and B is the wild-type group

MTT data

Contains average output for three experiments for WT vs HET vs KO as well as the graph template for Figure 1

Treatment of NMNAT2-/- cortical neurons with vincristine and paclitaxel renders them hypersensitive to neurotoxicity.

<p>A) DIV14 cortical neurons (i.e. 14 days in vitro) from wildtype (WT), NMNAT2+/- (HET) and NMNAT2-/- (KO) mice were treated with increasing concentrations of vincristine for 12 hours. Vincristine dose-dependently reduced the viability of both NMNAT2+/- and NMNAT2 -/- neurons. NMNAT2-/- neurons were especially hypersensitive to vincristine-mediated toxicity. B) Treatment of these neurons with increasing concentrations of paclitaxel significantly reduced cell viability in NMNAT2-/- at higher concentrations only. Panels C) and D) represent normalized absorbance (OD) in MTT-treated primary neurons that were used to calculate EC₅₀ values for chemotherapy-induced reductions in cell viability in WT, HET and KO conditions. (n = 3 in at least three independent experiments; *p<0.05, **p<0.001, ***p<0.0001 vs. each corresponding genotype; +p < 0.05 vs. WT).</p