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Postural And Motor Dysfunctions In A Mouse Model Of Manganism

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Title:
Postural And Motor Dysfunctions In A Mouse Model Of Manganism
Series Title:
19th Annual Undergraduate Research Symposium
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Giraldo, Genesys
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English
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Undetermined

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Center for Undergraduate Research
Center for Undergraduate Research
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Conference papers and proceedings
Poster

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Abstract:
Manganese (Mn) is an essential trace mineral that plays a role in growth, bone formation, and other physiological processes. In humans, exposure to excess manganese can result in manganese accumulation in the brain as well as in neurological and motor disturbances resembling a parkinsonian-like disorder known as manganism. SLC39A14 is the metal transporter responsible for Mn homeostasis, and mutations in the Slc39a14 gene have been associated with elevated Mn concentrations in the blood and brain. Brain manganese accumulation in Slc39a14-/- mice was associated with motor deficits in a variety of motor tasks. Additionally, most Slc39a14-/- mice also displayed postural abnormalities, like torticollis. Consequently, we investigated whether this postural defect could compromise the performance of mice in the administered locomotor tests. In this study we compared the performance of Slc39a14-/- mice that showed torticollis with the performance of the Slc39a14-/- mice without signs of torticollis and with the performance of the control, wild type mice. The results of our comparative analyses across a variety of locomotor tests demonstrate the unequivocal locomotor impairment of Slc39a14-/- mice, regardless of the presence of torticollis, but do not provide any compelling evidence that torticollis significantly confounded the locomotor performance of Slc39a14-/- mice in the tests. ( en )
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Research authors: Genesys Giraldo, Mitchell Knutson, Christopher Janus - University of Florida
General Note:
Faculty Mentor: Manganese (Mn) is an essential trace mineral that plays a role in growth, bone formation, and other physiological processes. In humans, exposure to excess manganese can result in manganese accumulation in the brain as well as in neurological and motor disturbances resembling a parkinsonian-like disorder known as manganism. SLC39A14 is the metal transporter responsible for Mn homeostasis, and mutations in the Slc39a14 gene have been associated with elevated Mn concentrations in the blood and brain. Brain manganese accumulation in Slc39a14-/- mice was associated with motor deficits in a variety of motor tasks. Additionally, most Slc39a14-/- mice also displayed postural abnormalities, like torticollis. Consequently, we investigated whether this postural defect could compromise the performance of mice in the administered locomotor tests. In this study we compared the performance of Slc39a14-/- mice that showed torticollis with the performance of the Slc39a14-/- mice without signs of torticollis and with the performance of the control, wild type mice. The results of our comparative analyses across a variety of locomotor tests demonstrate the unequivocal locomotor impairment of Slc39a14-/- mice, regardless of the presence of torticollis, but do not provide any compelling evidence that torticollis significantly confounded the locomotor performance of Slc39a14-/- mice in the tests. - Center for Undergraduate Research,

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Results Results cont. 1 3 Assessment of Physical Conditions (SHIRPA) Figure 1. The body weight of Slc39a14 -/mice (n = 33) and wildtype (WT) mice (n = 30). Data represents mean s.e.m. Vertical Pole descending test F 15.0 17.0 19.0 21.0 23.0 25.0 27.0 29.0 10 11 12 13 14 Body Weight (g) Age (weeks) WT Slc39a14-/0 5 10 15 20 25 30 35 WT (n=30) Slc39a14-/(n=33) Number of Animals Genotype Righting Reflex No Righting Reflex Slc39a14 -/w/o torticollis Figure 2. Righting reflex response of WT and Slc39a14 -/mice (Picture 7) Dashed lines were attributed to Slc39a14-/mice without torticollis to distinguish them from Slc39a14-/mice with torticollis. Data represents mean s.e.m Figure 3. Time to turn head down in the pole descending test (Pictures 1 and 2) There was no significant difference in the time it took to turn the 50 cm pole between genotypes. Only Slc39a14-/mice that exhibit both torticollis (Tor) and Tail away activity (Ta) differ significantly in their turning times from WT mice. Data represents the individual time to turn the pole for every mouse. (No Tor/Ta = no torticollis or tail away activity exhibited, Ta = tail away activity exhibited but no Torticollis was shown, Tor = torticollis exhibited but no tail away activity shown, Tor/Ta = both Torticollis and tail away activity exhibited.) Figure 4. Time to descend the pole descending test (Pictures 1 and 2) Other than the outlier in the WT data (n = 30), Slc39a14-/mice (n = 33) took longer to descend the 50 cm pole than did WT mice. There was an outlier in the data for Slc39a14-/mice (not shown) who took 61.67s to descend the pole. Data represents the individual time to descend the pole for every mouse. Introduction Postural and Motor Dysfunctions in a Mouse Model of Manganism Conclusion Center for Translational Research in Neurodegenerative Diseases & Department of Neuroscience, University of Florida, Gainesville References Methods Mice Sixty-three Slc39a14 -/mice and control wild type (WT) littermates were used in the study; 30 WT (15 M and 15 F), and 33 Slc39a14 -/(16 M and 17 F). The mice were bred in house (Dr. Knutson Lab, UF Animal Facility), and kept in same sex groups of 2 5, under standard laboratory conditions (12-hour LD cycle (Lights ON: 6AM)), a room temperature of 22 1¡C, and water and food provided ad libitum. All tests were performed during the light phase between 09:00 and 14:00 hours, in accordance with AAALAS and institutional guidelines. IACUC UF approved the husbandry conditions and all experimental protocols used in the study. Experimental Design The mice were handled frequently before the first behavioral test. The body weight of the mice was recorded before and after the battery of tests. The battery of tests was administered between the 10 th and 14 th week of age. The tests were administered in the following sequence: SHIRPA screen, vertical pole descending test, beam traversing test, and Rotarod test. Primary behavioral observation screen (SHIRPA) The primary evaluation screening of the mice was accomplished using SHIRPA, ( S mithKline B eecham, H arwell, I mperial College, R oyal London Hospital, P henotype A ssessment) 8 a set of short evaluations used to characterize the mice. The assessment of each mouse included its posture, transfer arousal and basic reflexes, including righting reflex ( Pictures 7-10 ). Vertical Pole descending test The pole test evaluates spontaneous motor behavior of mice descending a vertical pole ( Pictures 1 and 2 ). Mice were placed head up near the top of the vertical wooden pole. The pole was 50 cm long, 1 cm in diameter, and wrapped by firmly glued wire mesh to facilitate grip and prevent sliding. The times to turn head down and descend the pole were recorded. The cut-off time of the test was 120 seconds. Elevated Beam traversing test The beam-traversing test evaluates locomotor behavior of a mouse while crossing an elevated narrow beam. Two beams; a wide beam (2.5 cm), and a narrow (1.7 cm), 110 cm long were used ( Pictures 3 and 4 ). Beams were fitted with evenly spaced obstacles (6x6mm) placed at every 10 cm. After habituation to the test, the mice were evaluated in two consecutive days (two trials per day) on a wide and narrow beams respectively. The time to reach the goal cage, the number of slips, falls, and pauses, were recorded. Rotarod test The test evaluates deficits in motor coordination and balance in rodents. The mice were placed on a horizontal rod that rotated along its long axis (Rotamex-5 apparatus; Columbus Inst. OH), forcing animals to walk forward in order to remain upright on a beam and not fall ( Picture 12 ). The mice were tested in three consecutive days, with two, 5-min trials. The speed of the rod accelerated gradually from 4 to 40 rpm within the 5-min trail. The time to fall in each trial was recorded. 1. Jenkitkasemwong S., Akinyode A., Paulus, E., Weiskirchen R., Hojyo S., Giraldo, G., Knutson, M. D. (2018). SLC39A14 deficiency alters manganese homeostasis and excretion resulting in brain manganese accumulation and motor deficits in mice Proceedings of the National Academy of Siences of the United States of America I (1). 2. Tuschl K., Meyer, E., Valdivia, L. E., Zhao, N., Dadswell C., AbdulSada A., Wilson, S. W. (2016). Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia. Nature Communications 7 (May). https:// doi.org /10.1038/ ncomms11601 3. Chen, P., Chakraborty S., Peres, T. V., Bowman, A. B., & Aschner M. (2015). Manganese-induced neurotoxicity: from C. elegans to humans. Toxicol Res. 4 (2), 191202. https:// doi.org /10.1039/C4TX00127C 4. Hojyo S, Fukada T, Shimoda S, Ohashi W, Bin B-H, et al. (2011) The Zinc Transporter SLC39A14/ZIP14 Controls G-Protein Coupled Receptor-Mediated Signaling Required for Systemic Growth. PLoS ONE 6(3): e18059. doi:10.1371/journal.pone.0018059 5. Rogers, D. C., Fisher, E. M. C., Brown, S. D. M., Peters, J., Hunter, A. J., & Martin, J. E. (1997). Commentary Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment. Mamm Genome 8 (10), 711713. https:// doi.org /10.1007/s003359900551 Acknowledgements We thank Denisse Cancel, Shaina Wallach, Jeffry Berrier and Kristi Douglas (UF ACS) for excellent husbandry care of the mice, as well as Jessica Schrier and Armin Garcia for help the help provided during testing. Manganese ( Mn ) is an essential trace mineral that plays a role in growth, bone formation, immune response, and other physiological processes 2 Ingested Mn has a rapid turnover rate, and Mn levels are maintained through proper biliary excretion. However, since Mn is required for cellular activities, Mn levels are under strict homeostatic regulation and can result in toxicity at excessive levels 3 Specifically, elevated Mn concentrations are neurotoxic and lead to manganism a parkinsonian -like disorder caused by accumulation of Mn in the basal ganglia 2 Patients with manganism present symptoms of bradykinesia and rigidity, reduced response speed, irritability, and mood changes 3 In our study we investigated the effect of deregulation of Mn homeostasis in a mouse model. We focused on SLC39A14 protein, which is a metal transporter that mediates transport of zinc (Zn), iron (Fe), cadmium (Cd), and Mn in the body. Mutations in the Slc39a14 gene have been associated with elevated Mn concentrations in the blood and brain 1 Interestingly, the loss of function in Slc39a14 gene did not affect Zn, Fe, and Cd blood levels 2 In this study, we use knockout Slc39a14 -/mice, which manifest the loss of function in the Slc39a14 gene. This mouse model exhibits hepatic Mn deficiency and Mn accumulation in other tissues such as the bone, heart, kidney, blood, and brain. Slc39a14 -/mice also demonstrate motor deficits in a variety of motor tasks 1 We also observed that some Slc39a14 -/mice exhibited torticollis (known as wry neck), a condition in which the muscles in the neck contract, causing the head to twist slightly to one side. Torticollis has been previously described in Slc39A14 -/mice, indicating that the Slc39a14 gene may play a critical role in skeletal formation 4 Given our recently reported group findings that Slc39A14 -/mice present motor deficits 1 we investigated whether this postural defect could be primarily cause behind the compromised performance of mice in locomotor tests In this project we re-analyzed our data 1 focusing on individual performance of Slc39A14 -/mice with and without identifiable torticollis and compared them to the performance of the control, wild type mice across a variety of locomotor tests. The results of our analyses showed that torticollis did not significantly confounded the motor impairment of Slc39A14 -/mice in the tests. The inspection of the individual data for Slc39A14 -/mice with torticollis and Slc39A14 -/mice without torticollis revealed stochastic distribution of scores by mice with and without torticollis. Table 1. The Prevalence of Torticollis and Straub Tail Genotype Torticollis Straub Tail Tail Wrapped Total WT 0 1 29 30 Slc39a14-/27 26 7 33 Table 1. Frequency of straub tail (Picture 2) as compared to torticollis (Pictures 9-11). Tail Wrapped (Picture 1) is interpreted as the normal way in which mice descend the pole, with their tails wrapped around the pole. 2 SHIRPA and Vertical Pole descending test 4 Elevated Beam traversing test 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.75 1.25 1.75 Time to goal (s) Wide Narrow WT Slc39a14-/w/tor Slc39a14-/w/o tor Figure 5. Latency to reach the goal in beam traversing test (Pictures 3 and 4). Data represents mean s.e.m -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 0 20 40 60 80 100 120 140 160 Number of Slips WT Slc39a14-/w/ tor Slc39a14-/w/o tor Wide Narrow Figure 6. Number of slips in beam traversing test. WT mice (n = 30) exhibited the least number of slips. In the wide beam, Slc39a14-/mice without torticollis (n = 6) exhibited less slips than did Slc39a14-/mice with torticollis. By contrast, in the narrow beam, Slc39a14-/mice with torticollis (n = 27) exhibited less slips than did Slc39a14-/mice without torticollis. Data represents average number of slips for each individual mouse. -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 0 50 100 150 Number of Pauses WT Slc39a14-/w/tor Slc39a14-/w/o tor Wide Narrow Figure 7. Number of pauses in beam traversing test. On average, Slc39a14-/mice with torticollis (n = 27) exhibited the least amount of pauses on both the wide and narrow beams. In the wide beam, Slc39a14-/mice without torticollis (n = 6) exhibited more pauses than WT mice. However, in the narrow beam, WT mice (n = 30) exhibited more pauses than Slc39a14-/mice without torticollis. Data represents average number of pauses for each individual mouse. 5 Rotarod test 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 DAY 1 DAY 2 DAY 3 Latency to Fall (s) WT Slc39a14-/w/tor Slc39a14-/w/out tor Figure 8. Latency to fall off the rotarod in three consecutive days. Slc39a14-/mice without torticollis (n = 6) appear to have made a significant improvement on the third day of testing, but the data is affected by an outlier. Data represents mean s.e.m. 0.0 50.0 100.0 150.0 200.0 250.0 0 20 40 60 80 100 Latency to Fall (s) Day 3 WT Slc39a14-/w/tor Slc39a14-/w/o tor Figure 9. On day 3 of testing, individual scores for the latency to fall off the rotarod. Circled is the individual mouse with an unusually long latency to fall off the rod for Slc39a14-/mice without torticollis. Data represents the latency to fall of each individual mouse that was tested. Our results demonstrate significant differences between WT mice and Slc39a14 -/mice in motor behavior and physical condition 1 Most of the Slc39a14 -/mice developed torticollis (28 out of the 33 mice). Although the Slc39a14 -/mice with torticollis did not differ from their control WT littermates in respiration and tremor levels, transfer arousal, palpebral closure, piloerection, pinna reflex, limb grasping, wire maneuver, or negative geotaxis as per the SHIRPA screening, the mice did demonstrate severe difficulties in rearing and most of them did not exhibit the righting reflex. Overall, the Slc39a14 -/mice showed impaired performance in most locomotor tests, as compared to the control, wild type mice. However, based on the number of mice tested in this study, we did not discovered compelling evidence differentiating the performance of Slc39a14 -/mice with torticollis and those without. Our recently published study revealed that Mn accumulates most extensively in the bone and in the brain of Slc39a14 -/mice 1 suggesting that the skeleton is the main extrahepatic reservoir for excess Mn. Future studies should focus on the interaction between the central effect of Mn accumulation in the brain and peripheral, skeletal deformations. 6 Examples of Analyzed Behaviors 7. Absence of Righting Reflex 1. Tail Wrapped 2. Straub Tail 4. Mouse entering goal cage 3. Elevated beam traversing test 5. WT mouse traversing beam 6. Slc39a14 -/mouse traversing beam 9. Torticollis 10. Torticollis 11. Torticollis 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0 10 20 30 40 50 60 70 Time (s) WT Slc39a14-/! Ta Tor/Ta + Tor No Tor/No Ta 671M979 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0 10 20 30 40 50 60 70 Time (s) TA Ta Tor WT Slc39a14 -/ 671M979 Ta Tor/Ta + Tor No Tor/No Ta 8. Mouse showing normal behavior in a tube 12. RR test Genesys Giraldo Mitchell Knutson, Christopher Janus