Transgenic mouse models of Huntington’s disease

JC5 Research Articl2: Draft

Marzia Bamiani

Career Choice

Undecided

Group Theme

Huntington’s Disease

Combined treatment with the mood stabilizers lithium and valproate produces multiple beneficial effects in transgenic mouse models of Huntington’s disease.

Brief Summary In Your Own Words: Goal, Experimental Design, Results, Conclusion

This research analyzes impacts of mixed-drug therapy on BDNF (Brain Derived Neurotrophic Factor) protein levels among transgenic and wild mice indicated under Huntington’s Disease (HD’s) N171-82Q mouse mutation. The mice’s diet was chow, lithium and chow, valproate and chow, or a valproate-lithium mixture and chow. These test mice were surrendered following 14, 28, and 56 days of treatment in order to match BDNF protein levels in the cortex between different treatment time intervals. Brain cortex samples were acquired for assessment through the Western Blotting method of analysis. Investigation-specified mutual treatment using valproate and lithium proved most effective in cumulative BDNF protein stages during every treatment period. (1)

INTRODUCTION and BACKGROUND:

What Is Being Studied? Topic, Problem, Previous Studies, Gap in Knowledge

Why Are These Experiments Important? What is the scope of problem? How will these studies help?

What Must We Know To Understand the Experiments? Anatomy/Physiology, Subjects, Treatments, Outcomes

Huntington’s Disease (HD) is a hereditary, dominantly autosomal, usually fatal neurodegenerative affliction, which usually presents later in life (ranging from mid-30s to mid-40s). The ailment is triggered by a Chromosome 4 gene malfunctioning; this gene is important as it generates the Huntington protein. Individuals afflicted with HD (because of the defective protein), exhibit a broad variety of symptoms, which may include impaired movement, psychiatric disorders, functional ability loss, and impairment of cognitive functioning. The symptoms surface due to degeneration of neuronal cell linked to the defective Huntington protein. The function of BDNF proteins is maintaining neuronal cell growth, survival and increase. In people suffering from HD, the neuronal cells gradually reduce HD symptoms and signs. Individuals with Huntington’s disease depict lower BDNF protein levels. A combined treatment strategy using valproate and lithium has demonstrated a growth in HD patients’ BDNF proteins, and it is thought to diminish the implication caused by it and inhibit the progress of HD and its symptoms. The monovalent cation — Lithium — has been typically used as a pharmacological agent for treating bipolar disorder for over half a period. It is the first treatment course alongside acute mania, whilst prophylactically being employed for persistent depressive and manic periods. Valproate, which functions as anticonvulsant, also works well in bipolar disorder treatment. The specific way these medications work is yet to be properly understood. Still, considerable attention has been given to these mood stabilizers’ power in protecting against different abuses. As a validation, VPA and lithium may be used for co-treatment to generate largely reliable behavioral advantages in both of the HD models as well as, notably, prolonged N171-82Q mice survival time. Additionally, HDAC and GSK-3b hyperactivity was suppressed constantly using this combined treatment program, also linked to up-regulation of a couple of key neuronal protection and growth proteins – heat shock protein and the BDNF, or neurotrophic factor derived from the brain. Valproate and lithium have already been approved by the Food and Drug Administration and can be used by doctors for the treatment of this disease, even though, this is not a curable disease; the doctors frequently use these drugs to stall the progress of this fatal disease. The Gap in knowledge is that the cause of this disease is unknown and the cure is not yet discovered. Hence, scientists and health professionals are working and investigating by the use of animal models to find out the best treatment and absolute cure for Huntington’s Disease. (1) (2)

EXPERIMENTAL DESIGN and DATA ANALYSIS:

What Was Done? Procedures, Treatments, Sample Collection and Method. Include Relevant Times.

How Was Data Analyzed? Statistical Tests, Descriptive Statistics, N, Significance Level

In this experiment, Transgenic mice model was used to express HD N171-82Q mouse model mutation for analyzing the impacts of mood-stabilizing medications valproate and lithium upon BDNF protein levels within brain cortex. The test mice’s diet was one of the following combinations: lithium/lithium-carbonate and control chow, sodium valproate and chow, or a mixture of chow, sodium valproate and lithium carbonate. These test mice were surrendered following 14, 28, and 56 days of treatment, and this was followed by dissection of their cerebral cortex for examination (western blot analysis). Homogenization of cortex samples was conducted in T-PER protein (tissue protein) obtained from solution. A ten-minute centrifugation process was performed, following which the supernatants’ western blotting commenced.

Loading control used was beta-actin, and protein level detection was performed by employing LI-COR (Odyssey infrared imaging system). Outcomes were standardized to the beta-actin. Cortex was examined for ascertaining if mice treatment using valproate, lithium, or both in combination, increased BDNF protein levels in the animals’ brains, as well as for quantifying the drug treatment that effected greatest BNDF protein increase.

Statistical Analysis: All of the statistical analyses for this research were carried out by employing Graph Pad Prism (Graph Pad, San Diego, CA). The data of female and male mice was combined, as gender did not prove to have any significant impact on their behavioral tests. This data is expressed in the mean ± SEM (standard error of mean). Data analysis is carried out through one-way ANOVA (analysis of variance) for one outcome. Where needed, assessment of treatment or genotype comparisons at distinct time points was done using post-hoc Student t-test. When assessment of transgenic HD and wildtype mice phenotypes was done across ages, the research used two-way ANOVA (genotype age), after which data was treated using Bonferroni correction. Kaplan — Meier analysis was utilized for assessing drug treatment’s significant impacts on survival. (1)

METHOD:

What Must We Know To Understand ONE Method: How does it work? Required Equipment, Reagents, Description.

Western Blot analysis is an evaluation technique employed for identifying and quantifying protein presence in any particular sample for the specific experiment. In this experiment, Western Blot has been utilized for quantifying BDNF protein in target mice’s brain cortex samples. Firstly, all proteins are detached via lysing from cortex tissues. They are subsequently separated through electrophoresis, a process by which proteins get organized on the basis of size. The first step in this process protein treatment with detergent for initiating denaturation, their unfolding, and creation of electrical charge. In the succeeding step, the proteins will be processed with electric current and injected into the gel “Nupage Bis-Tris.” This enables proteins movement through the Electrophoreses gel. Proteins that are smaller in size move more rapidly through this gel, and hence, sample proteins effectively separate depending on size. Results emerge in ladder formation, and are subsequently transported to nitrocellulose membrane, which is then subject to secondary anti-rabbit or goat anti-mouse antibodies containing fluorescent molecules, whose function is BDNF protein binding. Lastly, researchers subject this nitrocellulose membrane to a photographic film, and at this stage, light is emitted by the antibody. The light takes the form of a dark area of rectangular shape on the film. Western blot interpretation is done on the basis of visible bands (lighter-darker) produced. Darker areas imply higher protein concentration. Western Blotting is one of the best methods that researchers and scientist mostly use, because it gives an accurate result. Each of the bands shows a very clear and dark enough shade under it to figure out the main result, They give the best result while doing DNA or any other kinds of experiment in the Laboratory. (3) (4)

RESULTS:

How Can You Best Sum Up the Results? Write a Short, New Title

What Was Done? Purpose of Experiment, Subjects, N, Outcome with Units

What Does Data Analysis Show? Significant Difference(s) and p-value(s), Relevant Comparisons that are the Same.

What Do Results Mean? How does outcome relate to the overall goal of the study?

After two weeks, though level of BDNF proteins in the brain cortex was found to increase above control levels following monotherapy treatment using valproate and lithium, the increase wasn’t significant. Combined valproate and lithium treatment brought about a more significant rise in level of cortex BDNF proteins compared to treatment using only valproate or lithium. After four weeks, though level of BDNF proteins in the brain cortex was found to increase above control levels following monotherapy treatment using lithium, the change wasn’t significant. Individual valproate treatment and combined treatment using valproate and lithium brought about a significant rise in levels of BDNF proteins compared to control.

After eight weeks, though level of BDNF proteins in the brain cortex was found to increase above control levels following monotherapy treatment using lithium, the change wasn’t significant. Individual valproate treatment and combined treatment using valproate and lithium brought about a significant rise in levels of BDNF proteins compared to control. (1)

Valproate-Lithium Co-Treatment is successful in alleviating natural locomotor disorientation of HD mice. HD motor symptoms include weakened coordination of both involuntary and voluntary movements. For identifying drug treatment impacts on muscle movement and behavioral changes, YAC128 and N171-82Q mice were tested longitudinally in their motor tests, beginning from the age of 6-22 weeks and three to twelve months, respectively.

Mice’s spontaneous locomotor action was assessed using horizontal distance they travel in the course of an open-field test of 30 minutes’ duration. In case of N171-82Q mice, significant genotype effects were revelaed on their locomotor activity, through two-way ANOVA (genotype age). N171-82Q mice exhibited no significant difference compared to wild-type ones at six weeks, however, they displayed significant decrease in locomotor activity fairly early (at ten weeks) compared to age-matched mice of the wildtype group (Figure 2a; Bonferroni posttest). There was no decrease observed in overall distance traveled at the age of fourteen weeks, among N171-82Q mice. Likewise, locomotor activity among YAC128 mice didn’t significantly differ from wildtype mice at the age of three months. But, two-way ANOVA (genotype age) dispalyed significant age (F (3, 98) 5.64, p=0.0013) and genotype (F (1, 98) 24.40, P=0.0001) impacts on locomotor action. YAC128 mice depicted significantly locomotor activity impairment between 6 and 12 months (Figure 2d; Bonferroni posttest).

Long-term HD mice treatment using monotherapy (either valproate or lithium) displayed a trend of correcting reduced locomotor activity; however, effects proved to be inconsistent at different measurement points, and were statistically insignificant (Figures 2c and f). By contrast, drug treatment effects on mice’s locomotor activity were considerably enhanced when valproate and lithium were administered in combination.

Compared to transgenic mice that do not receive treatment (open bars; Fig. 2c and f), impacts of co-treatment using valproate and lithium were significant at age ten weeks among N171-82Q mice (Fig. 2c) and six months among YAC128 mice (Fig. 2f). Locomotor activity of wildtype mice remained unaffected when treated with a combination of valproate and lithium (closed symbol; Fig. 2a vs. 2b; 2d vs. 2e). Also, there were no significant differences observed with regard to locomotor activity, between transgenic HD and wild-type mice treated with a combination of valproate and lithium (Fig. 2b and e). (1)

DISCUSSION:

Are Results Believable? Were Results Conclusive? Do They Make Sense?

Is Problem Solved? Relate Results to Larger Problem. Suggest Future Studies

The aim in this research was to investigate the acknowledgment of the likely underlying co-treatment mechanisms (using valproate and lithium) in YAC128 as well as N171-82Q HD mice models. These two strains of mice display behavioral and neurological abnormalities akin to those displayed among humans with HD; however, they are characterized by different pathological progressions and genetic backgrounds. Long-term valproate-lithium co-treatment of HD mice may increase. Improvement in N171-82Q mice’s rotarod performance did not prove to be any secondary impact of weight loss, since the variables weren’t correlated. Further, it was unlikely that improvement in rotarod performance linked to co-treatment occurred because of drug induced enhancements in their locomotor activity in general, since N171-82Q mice’s locomotor impairment was clearly seen even at the age of ten weeks, while rotarod performance deficits were insignificant until age 18 weeks. A significant role is played by the striatum in motor functioning, and rotarod task; it influences complex cognitive actions such as administrative functions and memory in HD. Mutant Huntington leads to considerable degeneration of mid-sized spiny.

To conclude, current results revealed that valproate-lithium co-treatment improves motor deficits and psychiatric disturbances in both HD mouse models more consistently and robustly. These behavioral advantages most likely emerge due to potentiated HDACs and GSK-3b inhibition, for inducing critical molecules like HSP70 and BDNF. This explains more about the effects of valproate as well as lithium, as they are associated with an extensive history of being safe for human use. Further, HD’s devastating symptoms increase in intensity without remission till the patient succumbs, so this information provides strong reasoning for combined valproate and lithium usage for treating HD patients as early as possible, to stop the degree of progress and create a delay on it. (5)(6)

BIBLIOGRAPHY: At Least 7 Sources, Numbered. Articles in Harvard Format. Others in APA Format.

1. Chiu, C, Liu, G, Leeds, P, & Chuang, D 2011, ‘Combined treatment with the mood stabilizers lithium and valproate produces multiple beneficial effects in transgenic mouse models of Huntington’s disease’, Neuropsychopharmacology: Official Publication Of The American College Of Neuropsychopharmacology, 36, 12, pp. 2406-2421, MEDLINE with Full Text, EBSCOhost, viewed 10 October 2015.

1. Huntington’s disease. (2015). Retrieved 2015, from http://www.mayoclinic.org/diseases-conditions/huntingtons-disease/basics/definition/con-20030685

1. Cloe, A., (2015). How a Western Blot Test Works. Retrieved 2015, from http://www.livestrong.com/article/83858-western-blot-test-works/

1. Kramer, D., (2013, December 6). Western blotting (immunoblot): Gel electrophoresis for proteins. Retrieved October 28, 2015, from Kramer, D. (2013, December 6). Western blotting (immunoblot): Gel electrophoresis for proteins. Retrieved October 28, 2015, from http://www.antibodies-online.com/resources/17/1224/Western blotting immunoblot Gel electrophoresis for proteins/

1. Buitrago MM. Schulz JB. Dichgans J.Luft AR (2004). Short- and long-term motor skill learning in an accelerated rotarod training paradigm. Neurobiol Learn Mem 81, 211-216.

1. Feng HL, Leng Y, Ma CH, Zhang J, Ren M, Chuang DM (2008). Combined lithium and valproate treatment delays disease onset, reduces neurological deficits and prolongs survival in an amyotrophic lateral sclerosis mouse model. Neuroscience 155: 567-572.

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