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    Influence of Testosterone Deprivation and Replacement on Cognition and Oxidative Stress in Middle-Aged Male Rats
    (2017-03-14) Contreras, Jo; Metzger, Daniel; Oppong-Gyebi, Anthony; Kasanga, Ella; Vann, Philip; Sumien, Nathalie; Cunningham, Rebecca; Schreihofer, Derek; Smith, Charity B.S.
    Purpose: Data from aged men suggests a negative correlation between testosterone levels and cognitive function, including the development of mild cognitive impairment and Alzheimer’s disease. The purpose of this study was to 1) determine whether long-term testosterone deprivation (LTTD) impairs cognition and increases oxidative stress in the middle-aged male rat brain and 2) determine whether testosterone (T) replacement after LTTD can reverse these effects. Methods: Twelve-month old male Fischer 344 rats (13 per group) were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing T (short-term T deprivation; STTD). Additional groups were castrated for 10 weeks before being treated with T (long-term T deprivation; LTTD+T) or cholesterol (LTTD). Rats underwent cognitive testing with the Morris water maze (MWM). A 4-day acquisition phase was used for rats to learn the location of a hidden platform. A retention day was used to determine whether rats remembered the platform location after it was removed. A 2-day reversal trial in which the platform was moved to a new location was used to examine mental flexibility. These tests require both hippocampal and cortical areas of the brain. Following MWM rats were euthanized and brains were collected for immunoblotting for markers of cell death (Spectrin) and oxidative stress responses (NFkB, COX2, NOX2) in the hippocampus and cerebral cortex. Plasma advanced oxidative protein products (AOPP) were used as a peripheral marker of oxidative stress. Total testosterone was measured by ELISA. Results: Castration reduced total testosterone to 40% of intact levels whereas testosterone implants increased levels back to those of intact males. Overall, intact rats performed significantly worse on the MWM than STTD and LTTD with or without T replacement. We saw no significant changes in blood AOPP among treatment groups. Similarly, there were no significant differences in the expression of oxidative stress regulated genes or Spectrin cleavage in the hippocampus. Cortical measurements are on-going. Conclusions: These data suggest that castration with or without T replacement improves cognitive function in middle-aged rats, but does not significantly alter oxidative stress in the brain or periphery. These data support the safety profile of testosterone replacement to physiological levels and do not recapitulate correlative data observed in men.
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    Development Of Novel Muscle Relaxant Compounds
    (2017-03-14) Mishra, Nigam; Emmitte, Kyle; Dillon, Glenn; Kumar, Manish
    Purpose: Generation of a more potent muscle relaxant compound, with less abuse potential. Materials and Methods: Whole cell patch clamp electrophysiology technique, HEK-t stable cell line expressing H-α1β2γ2s, H-α2β2γ2s and R- α3β2γ2s, cell culture, gravity based rapid drug application system, drug synthesis via a matrix approach. Summary: The carbamate derivative carisoprodol (trade name Soma) is a widely prescribed skeletal muscle relaxant. Its recreational use is an increasing problem. Consequences of abuse include withdrawal symptoms, delusions, seizures and even death. Consequently, in 2012 carisoprodol was classified at the federal level as a schedule IV controlled substance. Its primary metabolite, meprobamate is also a controlled substance, and there remains a pressing need for efficacious muscle relaxants with reduced potential for abuse. Both carisoprodol and meprobamate act on GABAA receptors, the predominant inhibitory neurotransmitter receptor in the central nervous system, in a subunit-dependent manner. Work in recent years has shown that receptors expressing the α1 subunit are associated with anticonvulsive, sedative, and anxiolytic properties, whereas those expressing α2 and α3 subunits are associated with muscle relaxant properties. Here, using whole cell patch clamp electrophysiology, we are assessing the α subunit-related allosteric modulatory and direct gating effects of a series of compounds prepared via a matrix approach and surveying different alkyl substituents at the two positions of the carisoprodol molecule, with the goal of identification of a molecule likely to be efficacious for muscle relaxation, but with a reduced abuse potential profile. Conclusions: Studies to date indicate structural differences at the two positions of the carisoprodol molecule lead to differences in the allosteric modulatory and direct gating effects of the ligands on GABAA receptors. Subsequent testing in animal models will help to identify lead molecules for further development. As the GABAA receptor is a target for several therapeutic classes of drugs, other indications are also possible.
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    NADPH Oxidase (NOX1) Mediates Testosterone-Induced Neurodegeneration (2017)
    (2017-03-14) Cunningham, Rebecca; Tenkorang, Mavis
    Purpose: One of the primary characteristics of Parkinson’s disease (PD) is oxidative stress (OS). Men have a higher risk for PD than women. Testosterone, a primary male sex hormone has been implicated in PD, and is a known oxidative stressor. Previous studies in our lab have shown that testosterone exacerbates OS damage in dopaminergic neurons. However, the mechanism by which testosterone increases OS is unknown. We hypothesize that in dopaminergic cells, testosterone increases OS by activating NOX 1, a major OS generator in cells. Methods: To test our hypothesis, we used a dopaminergic cell line (N27 cells). For an oxidative stressor, we used tert-butyl-hydrogen peroxide (H2O2) to induce 20% cell loss prior to testosterone (100nm) administration. NOX1 inhibitors (Apocynin, Diphenyleneiodonium-DPI) were administered before H2O2 exposure. Cell viability was quantified using the MTT assay. Results: Testosterone is only damaging in the presence of OS. DPI, alone, was damaging to N27 cells, hence this was no longer used as a NOX1 inhbitor. Unlike DPI, Apocynin had no effect on cell viability. Further, Apocynin did not alter H2O2-induced cell loss, indicating that H2O2 increases OS via a non-NOX1 mechanism. However, Apocynin blocked testosterone’s damaging effects in an oxidative stress environment. Conclusions: Testosterone-induced cell loss is mediated by NOX1, indicating that NOX1 is involved in testosterone induced OS generation. By understanding testosterone’s mechanism of action, potential therapeutic targets for Parkinson’s disease can be explored.
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    The Influence of Estrogen on a Potential Memory Gene, RbAp48
    (2017-03-14) Rybalchenko, Nataliya; Singh, Meharvan; Toofan, Jessica
    Purpose: With aging, there is a tendency for humans to experience cognitive decline. Known variations in cognitive function with age provide an opportunity to investigate the reasons why some individuals age successfully while others do not. In some women, the postmenopausal period is associated with a decline in cognitive function. While hormone (replacement) therapy may have merit, its current use for treating cognitive dysfunction is controversial. At best, we recognize that there are responders and non-responders. Given that the histone binding protein, RbAp48, was recently implicated as a key determinant of cognitive dysfunction with age, we sought to determine the role of RbAp48 as a mediator of estrogen’s influence on cognitive function. As an initial investigation into the role of RbAp48 in mediating estrogen’s effect on cognitive function, we sought to determine if, in animal models of aging currently being used in our laboratory, RbAp48 declines with age, and if estrogen treatment influences RbAp48 expression. Methods: We evaluated the expression of RbAp48 in the hippocampus of female Sprague Dawley rats that were 4 months and 10 months of age, representing young adult and middle-aged rats. Within these two groups, we had two treatment groups: ovariectomized (OVX) and ovariectomized + estradiol treatment (OVX + E2). RbAp48 mRNA was assessed using semi-quantitative real-time PCR (rtPCR). GAPDH was used as a loading control, as it is stably expressed at high levels. Differences in expression of RbAp48 were based on the delta-delta CT methodology published by Livak and Schmittgen (2001). Statistical evaluation of differences between experimental groups was determined using a two-tailed t-test. Results: Our data revealed a statistically significant (n=5, p=0.0079) reduction in the levels of hippocampal RbAp48 mRNA in the 10 month mice, compared to the 4 month mice. Interestingly, E2 reduced RbAp48 in young OVX rats (n=5, p=0.0079), but had no effect on RbAp48 mRNA levels in middle-aged ovariectomized (n=5, p=0.1508). Conclusions: These studies confirm the reduction of RbAp48, a presumptive “memory gene”, with with age, but failed to implicate RbAp48 as a mediator of E2’s effects. Instead, we suggest that RbAp48 is permissive for E2’s effects. Ongoing studies will determine whether knockdown of RbAp48 expression abrogates estrogen’s positive effects on those measures relevant to cognitive function.
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    Androgen Receptors in the Middle Aged Male Rat Brain: Influence of Testosterone Deprivation on Expression
    (2017-03-14) Contreras, Jo; Cunningham, Rebecca; Fort*, Callie; Cuellar*, Elric; Lopez, Gladys; Metzger, Daniel; Oppong-Gyebi, Anthony; Schreihofer, Derek; Smith, Charity B.S.
    Purpose: 1) To determine whether long-term testosterone deprivation (LTTD) alters the levels and/or distribution of androgen receptors in the middle-aged male rat brain and 2) to determine whether testosterone replacement after LTTD influences androgen receptor levels. Methods: Twelve-month old male Fischer 344 rats were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing testosterone (STTD). Additional groups were castrated for 10 weeks before being treated with testosterone (LTTD+T) or cholesterol (LTTD). Four weeks later, rats were euthanized and brains were collected for immunoblotting and immunohistochemistry (IHC) for androgen receptors (AR) using antibodies targeting the N-terminus or C-terminus of the protein. The cerebral cortex, hippocampus, thalamus, hypothalamus were examined, and testes were used as positive control tissue. Results: Contrary to expectations, the full-length AR (116 kDa) was barely detectable in the hippocampus and cerebral cortex by immunoblotting with antibodies directed to either end of the AR. Rather, smaller fragments were readily detected. Examination of the size of these fragments (~30, 37, 50, and 80 kDa) and consultation of the literature for the human AR, suggested that they represented calpain-dependent cleavage fragments. A series of control experiments was performed in an attempt to extract the full-length AR using rat testes as a positive control tissue. Protease inhibitors, EDTA, and the AR agonist dihydrotestosterone failed to reduce the appearance of fragments. Interestingly, the pattern of fragments from the hippocampus (80 [greater than] 37 kDa) differed from that from the cortex (37 [greater than] 80 kDa) suggesting differential processing. IHC of coronal brain sections though the forebrain revealed nuclear AR staining consistent with full-length AR in regions of high expression, including the hypothalamus. In agreement with immunoblotting AR staining in the cortex and hippocampus appeared to be cytoplasmic, rather than nuclear. No significant differences were observed between treatment groups. Conclusions: These data suggest that AR protein in some areas of the middle-aged male rat brain is rapidly degraded into fragments with altered localization and potential for transcriptional activity and/or signaling functions. Although no differences in expression were apparent between treatment groups, the differential processing of AR in the rat brain is a novel finding warranting further investigation.
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    A Case Study of Deafness-Dystonia-Optic Neuropathy & Treatments
    (2017-03-14) Hamby, Tyler; Marks, Warren; Reed, Mary-Ann; Aalbers, Brian; Honeycutt, John; Durand, Christopher
    Purpose: DDON, also known as Mohr-Tranebjaerg syndrome is an inherited disorder of an Xp22 mutation of the mitochondrial transport protein TIMM8A. This mutation leads to progressive dystonia, otic neuropathy, and visual disturbances. Most patients will progress to dementia by early adulthood. Methods: We report on a teenage male with DDON and the treatments he received at Cook Children’s Medical Center (CCMC). Results: A 15-year-old patient with a prior diagnosis of DDON presented to CCMC for treatment of worsening dystonia. He communicated by gesture and sign language, and relied on a iPad. His increasing dystonia made him incapable of communicating effectively, particularly dystonia of the upper extremities. He was was previously treated with botulinum toxin chemodenervation, however, that treatment had lost efficacy as a relief of dystonia. He received Deep Brain Stimulation (DBS) of the globus pallidus internus (GPi). Since receiving DBS implants, several programming adjustments have been made and have led to a decrease in severity of symptoms. The patient was also started on a trial of baclofen to decrease muscle dystonia. Following a good response to trial, the patient was implanted with an intra-thecal baclofen pump (ITB) to decrease the dystonia of the trunk and lower extremities that had progressed. Over five years of treatment, scores on disability scales such as the Burke-Fahn-Marsden dystonia scale have slowly worsened as the disease has progressed. Concern was expressed about efficacy of the treatment, so a trial of observed time without DBS was performed. Marked worsening of disability was noted, showing that therapy has slowed the progression of this patient’s disease. Future care includes monitoring of DBS implants and transitioning care to another provider closer to the patient’s home. Conclusions: This is the fourth known case of DBS used in DDON treatment. This is also the first reported combination of ITB and DBS. Compared to other cases, this patient’s outcomes with DBS have not been as marked, due to some unique brain anatomy. ITB is a unique treatment for DDON and has shown some of the most efficacious results for this patient.
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    AT1aR dependent GABAa inhibition in the MnPO
    (2017-03-14) Little, Joel; Bachelor, Martha; Cunningham, Tom; Farmer, George Jr.
    Background: The median preoptic nucleus (MnPO) receives input from other circumventricular organs (e.g. SFO and the OVLT) sensitive to circulating Angiotensin II (Ang II) and plasma Na+ concentrations suggesting an involvement in hydromineral balance and blood pressure regulation. Additionally, evidence suggest the SFO synthesizes Ang II and releases it on the MnPO as a neurotransmitter suggesting the role of the MnPO in hydromineral balance and blood pressure regulation is mediated in part by Ang II. The Ang II activation of AT1aR has also been shown to influence the function of GABAaRs though the mechanisms are still unclear. Here we investigate the role of Ang II signaling via the AT1aR in the MnPO and its influence on excitatory/inhibitory balance. Methods: Male Sprague-Dawley rats received infusions of an AAV construct containing GFP reporter and shRNA against AT1aR (shAT1a) or a shRNA scramble (shScr) targeted to the MnPO. Two weeks following AAV infusion, slices containing the MnPO were cut using standard in vitro slice procedures followed by loose patch recordings obtained from GFP labeled neurons. Spontaneous action potential firing was recorded in response to focal application of Ang II or muscimol. Additionally, activity of MnPO neurons in response to muscimol was observed in the presence of a PLC or PKC activator. The GABAa mediated effects in AT1a KD were compared to acute blockade of AT1aRs in rats that did not receive AAV infusions. Western blot and RT-qPCR analyses were used to investigate the effect of AT1a KD on GABAa and KCC2 protein and mRNA expression. Results: Brief focal application of Ang II produced a time dependent increase in spontaneous firing of MnPO neurons. The Ang II dependent enhancement of spontaneous activity was blocked by bath application of the AT1aR antagonist Losartan. Additionally, Ang II failed to alter firing rate of MnPO neurons in shAT1a KD rats. In control animals, the GABAa agonist muscimol decreased action potential activity. In rats that received microinjections of the shAT1a muscimol failed to decrease action potential activity. In AT1a KD rats, RT-qPCR analysis shows a reduction in AT1a and KCC2 mRNA but no reduction in GABAa Beta subunit mRNA. Conclusions: The current findings demonstrate Ang II dependent increases in the excitability of MnPO neurons are mediated by activation of AT1aRs. Moreover, AT1aRs activation also mediates the inhibitory effects of GABAaR activation. The current study suggests the reduction in GABAa dependent inhibition following AT1a KD is mediated by a down regulation of KCC2 and subsequent disruption of intracellular Cl- homeostasis. AT1aR function can modulate the balance of excitatory and inhibitory activity within the MnPO and efferent nuclei involved in the regulation of blood pressure and hydromineral balance. However, mechanisms underlying the dual excitatory/inhibitory functions of AT1aR activation remain unclear.
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    Artemisinin Protects Oxidative Stress-induced Neuronal Apoptosis Via Up-Regulation of Akt/Bcl-2 Signaling
    (2017-03-14) Liu, Ran; Xie, Luokun; Li, Wenjun; Winters, Ali; Chaudhari, Kiran; Prah, Jude; Yang, Shao-Hua; Lin, Shao-Peng
    Purpose: Artemisinin is a powerful anti-malarial drug that has been in use for decades. Recently, the novel biological effects of artemisinin on cancer, inflammation-related disorders, and cardiovascular disease were reported. The aim of this study was to explore the neuroprotective actions of artemisinin. Methods: The model of glutamate-induced oxidative injury in HT22 hippocampal cells was established to simulate cellular ischemic model. We investigated the effect of artemisinin on oxidative stress-induced cell apoptosis death and the activity of Akt/Bcl-2 pathway in HT22 cells. Results: Pretreatment with artemisinin attenuated reactive oxygen species (ROS) generations, preventing the decline of mitochondrial membrane potential and rescued the HT22 cells form glutamate-induced apoptosis death. The Akt/Bcl-2 pathway was activated by artemisinin in time dependent manner. Furthermore, the artemisinin inhibitor MK2206 blocked the neuroprotective effect of artemisinin. Conclusions: Artemisinin protects neuronal HT22 cell from glutamate-induced oxidative injury and apoptosis via Akt/Bcl-signaling, thereby might be applicated for clinical neurological therapy.
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    Graded Mild Head Injury as a Model for Sports Injury
    (2017-03-14) Metzger, Daniel; Oppong-Gyebi, Anthony; Vann, Philip; Sumien, Nathalie; Luedtke, Robert R.; Schreihofer, Derek; Sun, Fen
    Purpose: To develop a graded model of mild head injury that produces graded behavioral deficits in the mouse. This model will be used to test neuroprotective effects of novel compounds. This study was designed to determine the severity of injury required to cause different behavioral deficits in motor function and cognition. Methods: Young adult male C56/B6J mice were anesthetized daily with isoflurane (20 sec) and 15 sec later were subjected to a weight drop head injury using a tethered steel bar (43 grams) dropped through an acrylic tube from a height of 28 inches. Mice were placed prone on a scored aluminum foil stage 2 cm below the end of the tube. The blow was directed to a 5 mm midline area of the head rostral to the aural canals. The blow causes a break in the scored aluminum foil and allows the mouse to flip 180 degrees and land supine on a foam cushion. This model was chosen to model a hit to the head followed by rotational acceleration indicative of closed head injuries occurring in contact sports. Five groups of mice were randomized to receive 0, 5, 10, 15, 20, or 25 blows, 1 per day M-F. Five days after the final hit, mice then underwent cognitive and behavioral testing consisting of an accelerating Rotorod, Morris water maze, and active avoidance T-maze. Following testing brains will be examined for cell death and inflammation. Results: A total of 30 mice (5 per group) were used for this study. Body weight did not differ among the groups over the course of the study, however waking time after anesthesia was increased in all groups subjected to injury compared to mice anesthetized and not injured. Coordinated movement on an accelerating Rotorod revealed a linear trend for decreased performance with increasing number of head impacts suggesting that a graded approach is possible with this model. Time to fall was significantly shorter than controls at 15 and 25 hits. Water maze and T-maze tests are ongoing. Conclusions: These data suggest that a graded injury regimen can lead to graded behavioral responses in the young male mouse and will provide a useful model for testing the effectiveness of neuroprotective compounds that have the potential to be used as prophylactic agents for those involved in contact sports.
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    Sexual Dimorphism in Mouse Age-Related Motor Impairments
    (2017-03-14) Wong, Jessica; Vann, Phillip; Davis, Delaney; Forster, Michael; Sumien, Nathalie; Mock, J.
    Purpose: Clinical measures such as frailty, disability, and strength loss are correlated with decreased survival and are more prevalent in women, yet men have a higher mortality risk at all ages. This contradictory sexual dimorphism in mortality versus morbidity is not fully understood. Furthermore, many pre-clinical studies using rodents have combined sexes or tested only males which limits the possible inferences regarding sex-dependent changes in function across the lifespan as well as inferences relating to interventions. Therefore, the purpose of this study was to examine murine sexual dimorphism in age-related motor function decline. Our hypothesis was that motor impairments would increase with age, and that these deficiencies would be exacerbated in females. Methods: Male and female C57BL/6J mice were tested at 5, 10, and 20 months of age. Animals underwent a battery of behavioral tests measuring difference aspects of motor function, including tests measuring coordinated running and motor learning (rotorod), strength (wire suspension), and balance (bridge walking). Resulting dependent measures were analyzed using two-way analyses of variance with Age and Sex as between-groups factors and two-way repeated measures analyses of variance with Session as the within group factor. Results: Rotorod performance (learning and maximum) declined with age in both sexes, however declines were smaller for females at 10 and 20 months compared to the males. Latency to fall from the wire was significantly shorter in adult and old males compared to their young counterpart, while there was no significant differences in the females. Bridge walking performance declined in both males and females, but there was a larger decline in the males. Furthermore, age-related decline in balance was observed in the females only on the most difficult bridge, while age-related declines were apparent on all the bridges for the males. Conclusions: These data support that age-related decline leads to measurable changes in mouse motor function. However these deficits occur primarily in the males whereas females displayed fewer and smaller declines in motor function. This suggests that combining sexes or testing only a single sex could lead to limited results. Interestingly, women are typically more sensitive to age-related functional decline, while female mice in our study were less affected by age-related decline. In conclusion, this study highlights the importance of including both sexes in rodent pre-clinical research.
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    Sex Dependent Alteration in Psychomotor and Cognitive Functions After Chronic Metformin Treatment
    (2017-03-14) Winters, Ali; Shetty, Ritu; Li, Wenjun; Xie, Luokun; Prah, Jude; Liu, Ran; Sumien, Nathalie; Yang, Shaohua; Chaudhari, Kiran
    Purpose: Metformin, the most commonly used anti-hyperglycemic medication has been proposed to have delayed aging and longevity benefits. Without due consideration to gender/sex influence, metformin administration is being tested for non-diabetic benefits. Amid mixed reports on cognition, the purpose of the current study was to identify the influence of sex variation in the psychomotor and cognitive outcomes after long term metformin treatment. Materials and Methods: Young normo-glycemic male and female C57BL/6J mice (aged 4 mo, n=10 each; total n=40 mice) were randomly assigned to either a control group or metformin group (administered 2 mg/ml in drinking water). After 1 month of treatment, a battery of behavioral tests was initiated to assess the psychomotor and cognitive functions. Metformin treatment was continued during behavior assessment. Results: Overall female mice weighed lesser than male mice. Over the experiment time span, metformin neither altered the body weight nor decreased the blood glucose level significantly. There was no variation in muscle strength or reflexes between male and female mice on either treatment. Male mice were more anxious than female mice and metformin treatment decreased anxiety in male mice only. Female mice had better motor learning and maximum coordinated running performance than male mice. Metformin treatment improved motor learning only in male mice. Metformin treatment improved balance function irrespective of sex. Overall male mice had better retention of long term memory which was deteriorated after metformin treatment. Further, metformin impaired the short term memory and cognitive flexibility only in male mice. Conclusions: This study demonstrated that metformin affects psychomotor or cognitive function differently influenced by sex. Our results suggested that chronic metformin was beneficial for psychomotor function and detrimental for short term and long-term memory in male sex. While, in female sex, metformin had beneficial or no effects on brain functions.
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    The Effect of Music on Gait Pattern for Patients with Parkinson's Disease
    (2017-03-14) McEwen, Madeline; Cooper, Ralph; Liu, Howe; Guzman, Jamie
    Introduction: Reports have shown that music therapy can help patients with Parkinson’s Disease who often demonstrate difficulty ambulating. However, it is unclear how music therapy can work for this patient population. The purpose of this literature review is to identify specific music therapy parameters used such as genre of music, frequency, and length of treatment sessions and analyze how each were implemented to improve ambulatory ability in patients with Parkinson’s Disease. Methods: Research was conducted through PubMed resulting in nine applicable articles in the last ten years which included two systematic reviews, six randomized control trials, and one cohort study. Inclusion criteria are subjects with Parkinson’s Disease, ambulatory without physical assistance, and living in community. Exclusion criteria are visual or auditory deficits. Results: Music genre used in these studies were Renaissance (four studies), Classical (one study), German folk (three studies), jazz (one study) and music with an underlying metronome beat (three studies). The most frequently used type of music was instrumental with a definitive beat (six studies) that was familiar to the subject (three studies), resulting in significant improvements in gait stride, cadence, and step length (nine studies). Subjects listened to music via headphones or speaker (five headphones, one speaker, others not specified). Compared to their gait speed baseline (GSB), subjects performed best when the music frequency was increased by ten percent (five studies). One of the most important factors of music therapy is the use of familiar music, which results in minimal cognitive demand to synchronize gait (four studies). In terms of therapy parameters, the length of each intervention session lasted thirty to sixty minutes with thirty minutes being the most often used (seven studies); the frequency was one to three times per week with three times per week as the most selected (five studies); and the entire duration of the therapy ranged from one to thirteen weeks with three weeks as the most common. Conclusions: The quality of music that will promote gait initiation and improve gait parameters in patients with Parkinson’s Disease include the following: familiarity, instrumental music, definitive beat, headphone delivery, and GSB increased by ten percent. The most common selected intervention parameters are thirty minute sessions, three sessions per week, for a duration of three weeks.
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    Connexin 43 as a Mediator of Estrogen-induced Protection against Oxidative Stress
    (2017-03-14) Rybalchenko, Nataliya; Singh, Meharvan PhD; Kubelka, Nicholas
    Hypothesis: 17-β estradiol (E2) and the estrogenic metabolite of dihydrotestosterone (DHT), 5-α-androstane-3,17-β-diol (3βdiol), protect against oxidative stress by increasing the expression and function of Connexin 43 (Cx43) – containing gap junctions in cortical astrocytes. Methods: In order to assess the expression of Cx43 and cell viability, real time RTPCR and the MTT assay was used, respectively. Cerebral cortical astrocytes derived from postnatal day 2 female C57/Bl6 mice were treated with physiologically relevant concentrations of E2, DHT, 3βdiol, or the vehicle control, DMSO, and evaluated for Cx43 mRNA expression. For the cell viability assays, astrocytes were pre-treated with either E2, DHT or DMSO vehicle control and then exposed to iodoacetic acid (IAA) oxidative insult. To further determine the role of Cx43 gap junctions, either Gap19 (inhibitor of the Cx43 hemichannel) or Gap26 (inhibitor of the dimeric Cx43 containing gap junction) were co-applied with the insult. Results: E2 treatment (3 hr) significantly increased Cx43 mRNA expression relative to DMSO control, while both DHT and 3βdiol (also applied for 3 hr) did not. Longer treatment with E2 (18 hr) yielded a non-significant trend to increase Cx43 mRNA expression. In the viability assays, neither E2 or DHT alone (18 hours pre-treatment) nor the Cx43 hemichannel selective inhibitor peptide (Gap19) alone protected against IAA toxicity. However, Gap19 did increase the protective efficacy of not only E2, but DHT as well. Interestingly, inhibition of both Cx43 hemichannels and the dimeric Cx43-containing gap junctions using Gap26, in and of itself, conferred protection against IAA toxicity. Like Gap19, Gap26 also significantly enhanced the protective efficacy of E2. Conclusions: Both E2 and DHT showed some regulatory interaction with astrocyte Cx43 that impacted protection against oxidative stress. This is the first evidence that E2 or DHT regulate Cx43 in the brain and the mechanisms underlying these interactions remain to be further characterized.
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    Ceftriaxone, a Beta-Lactam Antibiotic, Reduces the Severity of L-DOPA-Induced Dyskinesia in a Rat Model of Parkinson’s Disease
    (2017-03-14) McInnis, Tamara; Chotibut, Tanya; Meadows, Samantha; Bishop, Christopher; Salvatore, Michael; Kasanga, Ella
    Purpose: Levodopa (L-DOPA) therapy remains the most pharmacologically used agent for the management of Parkinson’s disease (PD). However, chronic treatment with L-DOPA leads to debilitating dyskinesias in 50% of Parkinson’s disease patients after 5 years and ~90% after 10 years. Delineating the mechanisms of L-DOPA-induced dyskinesia (LID) is therefore a major priority for alleviating this debilitating side effect of L-DOPA. There is evidence for increased glutamate signaling in LID and in PD. However, glutamate receptor antagonists in the PD patient have achieved mixed clinical outcomes with untoward side effects. Therefore, an alternate intervention targeting the elevated glutamatergic signaling could prove useful. The beta-lactam antibiotic, ceftriaxone, increases the expression of glutamate transporter 1 (GLT-1), a transporter that plays a major role in glutamate clearance in the central nervous system. We have recently shown that ceftriaxone when given at the time of 6-hydroxydopamine (6-OHDA) injection resulted in an attenuation of tyrosine hydroxylase (TH) loss, an increase in GLT-1 expression and reduced serine-19 TH phosphorylation, a calcium-dependent target specific for nigrostriatal neurons. In this study, we determined if ceftriaxone therapy initiated 7 days after 6-OHDA, but prior to L-DOPA, could reduce L-DOPA-induced abnormal involuntary movements (AIMS) in an established L-DOPA-induced dyskinesia model. Methods: Ceftriaxone (200 mg/kg, i.p., once daily for 7 consecutive days) was initiated 7 days post-6-OHDA lesion (days 7-13) and then continued every other week (days 21-27, 35-38) until the end of the study (day 38 post-lesion, 20 consecutive days of L-DOPA). Results: Preliminary results show reduced AIMs at the time points 1, 4 and 7 days after the initiation of L-DOPA treatment upon the administration of ceftriaxone with a significant reduction (p Conclusions: Intermittent delivery of a ceftriaxone regimen prior to and after L-DOPA may reduce LID severity, possibly in conjunction with a reduction in nigrostriatal lesion severity during the time course of ceftriaxone administration.
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    Alcohol Influences HAND Via Astrocyte-TLR4 and cPLA2 Signaling
    (2017-03-14) Ghorpade, Anuja Ph.D.; Pandey, Richa Ph.D.
    Background and Objective: Over the past few decades, ~25 million people died with human immunodeficiency virus (HIV)-1 disease. About 70% of HIV patients suffer from HIV-associated neurocognitive disorders (HAND). The prevalence of alcohol abuse among HIV-1+ve individuals is estimated to be 2-3 times that of the general population in the USA. HIV patient brains harbor up to 20% HIV-infected astrocytes, thus making them critical players in HAND. Previously, we reported that HIV-1 and/or alcohol (EtOH) activated astrocytes induced inflammation via cytosolic phospholipase A2 (cPLA2) activation. How EtOH regulates HIV-1-mediated inflammatory episodes initiated at the cell surface level is still unclear. Toll-like receptor (TLR) signaling in immune cells, astrocytes, microglia and neurons may play roles in pathogenesis of multiple diseases including HIV-1. Hypothesis: We propose that TLR4 may serve as critical regulator of alcohol-mediated inflammatory responses in HAND directly or by controlling cPLA2 signaling. Materials and Methods: To investigate the temporal order of events, primary human astrocytes were cultured and treated with HIV-1 (10ng/ml) and/or EtOH (50mM). TLR4, COX2 and CCL2 mRNA levels were measured by RT2PCR at 8h whereas protein levels were analyzed by ELISA, western blot and immunocytochemistry on 24h. We also explored the phosphorylation studies of cPLA2 and TLR4 downstream molecules such as IRAK4 and NF-kB by western blot and immuno-staining analysis on 30 min. TLR4-RNAi and cPLA2-specific inhibitor AACOCF3 were employed to carried out TLR4 and cPLA2-specific responses. Results: We showed EtOH, HIV-1, IL-1b and anti-retroviral (ARV) drugs significantly upregulated TLR4 in human astrocytes. Our results established that EtOH+/- HIV-1 activated TLR4 signaling leads to IRAK4 phosphorylation followed by NF-kB activation, ultimately leading to excessive production of inflammatory mediators such as COX2 & CCL2. EtOH and/or HIV-1 increased inflammatory molecules in MyD88-dependent manner. TLR4-RNAi studies reversed EtOH and/or HIV-1-regulated effects. Moreover, on silencing TLR4, the increase in EtOH+/-HIV-1-induced cPLA2 phosphorylation was not observed. Conclusions: Our study demonstrated that TLR4 regulates inflammatory responses in primary human astrocytes directly or by controlling cPLA2 cascade in HAND. Hence, TLR4 could be the critical regulator of alcohol-induced astrocyte inflammation with HIV-1.
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    A High Throughput and Integrative Approach to Evaluating the Functional Significance of a Glycosidase NGLY1 in Human Brain Development
    (2017-03-14) Shakhbazau, Antos; Zolekar, Ashwini; Wang, Jack; Lin, Victor
    Background: Mutations of the NGLY1 gene, leading to NGLY1 deficiency and associated neurodysfunction in pediatric patients, have been identified as the cause of a previously undiagnosed congenital disorder of deglycosylation. Despite the identification of the causal mutations, how NGLY1 deficiency disturbs normal cerebral development and causes neurological abnormalities is unknown. Purpose: Our desire is to unravel the mystery behind this novel disease and how it influences the assembly and function of the human cerebral landscape. Further, our hope is to develop mid-to-high throughput platforms that can be applied to discover and test druggable targets for this disease and adapted for associated neurocognitive or neurodegenerative disorders. Methods: Using human induced pluripotent stem cells (hiPSCs) and the state-of-the-art gene editing technology, CRISPR-Cas9, NGLY1 deficient human pluripotent stem cells (hPSCs) were created and used to elucidate the disease pathophysiology. In succession, middle-to-high throughput platforms were applied to recapitulate the disease in 2D and 3D, used in tandem with systems biology and novel imaging capabilities to discover new understandings and the importance glycosylation states for cerebral development and function. Results: The CRISPR-Cas9 mediated knockout of NGLY1 was confirmed by DNA sequencing and a biochemical test. Our optimized two-dimensional and three-dimensional differentiation protocols for neurogenesis in the control and NGLY1-deficient hESCs and hiPSCs showed that the loss of NGLY1 appears to have a negligible impact on the viability and cellular pluripotency in undifferentiated hPSCs. Neuroepithelial differentiation can be successfully generated in both control and NGLY1-deficient hPSCs, suggesting that the commitment of hPSCs to the neural lineage is not profoundly hindered by the loss of NGLY1 activity. However, compared with the differentiated derivatives of control hPSCs, the derivatives from neural differentiation in NGLY1-deficient hPSCs showed noticeably increased apoptosis, suggesting that NGLY1 activity may play a critical role in the viability of neural progenitor cells, as well as, play a role in the success of their subsequent differentiation into neuronal or astroglial lineages. Conclusions: We have built a new and unique model that can recapitulate the early-stage neurodevelopment patterns associated with NGLY1 deficiency. Using systems biology and imaging approaches, we are uncovering unprecedented insights into this newly identified disease. With the hiPSC and CRISPR-Cas9 gene editing, we demonstrate how regenerative medicine and genetic engineering approaches can be applied to studying the pathogenesis of human hereditary disease, applied in like to understand other brain pathologies, and possibly assist in the discovery of new therapeutics.
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    Identification of stable individual variation in learning of drug-associated cues in mice
    (2017-03-14) Shetty, Ritu; Forster, Michael; Wagner, Alison
    Hypothesis/Purpose/Objective: Conditioned place preference (CPP) is a behavioral assay used to assess learning of drug-associated cues and drug reward. Though the reliability of the assay is established, considerable variability exists when examining the outcome of conditioning in individual mice. Indeed, at doses expected to produce robust CPP, some mice exhibit weak preference, or even aversion. The present study characterized the reliability and stability of these CPP phenotypes (i.e. robust, weak, or averse) to determine if they represent true individual differences. These phenotypes were investigated using the psychostimulants d-amphetamine and methylenedioxypyrovalerone (MDPV), as recent studies and trends in drug use suggest that synthetic cathinones, such as MDPV, have a high potential for abuse. Materials/Methods: The CPP phenotypes were examined in subsets of two hundred fifty-two male Swiss-Webster mice used in dose-response studies, in which separate groups received either saline, MDPV, or d-amphetamine. These groups were subsequently assessed for conditioned place preference. One post-test was conducted at 24 hours after the initial test in a group receiving 2.5 mg/kg d-amphetamine. Three post-tests were conducted at 24, 48, and 72 hours after the initial test in groups receiving 10 mg/kg MDPV and 0.5 mg/kg d-amphetamine. At the initial test session, outcome of conditioning was examined by calculating a preference score; higher preference score indicated greater learning. Pearson’s r was used to analyze the relationship between place preference during the test and the post-tests. An Analysis of Variance was used to ensure the partitioning criteria were appropriate and the phenotypes were, in fact, separate groups. Results: When examining preference scores, three distinct phenotypes emerge. Data from groups receiving 10 mg/kg MDPV and 0.5 and 2.5 mg/kg d-amphetamine demonstrate a strong correlation between the initial test and the post-test(s). Conclusions: Outbred mice exhibit differential conditioning to psychostimulants, a phenomenon that can be qualified by distinct phenotypes. These phenotypes are stable over additional post-tests, as mice seem to persist in the phenotype they exhibit during the initial test. Taken together, these results suggest robust individual differences in the development of place preference.
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    Mild CIH Does Not Induce Cell Loss in the Substantia Nigra
    (2017-03-14) Snyder, Brina; Cunningham, Rebecca; Davis, William
    Purpose: Sleep apnea severity has been associated with Parkinson’s disease (PD) severity in men. Chronic intermittent hypoxia (CIH) is an animal model for sleep apnea. Mild CIH increases oxidative stress (OS) and inflammation in substantia nigral dopamine neurons, a neuron type lost in PD. Currently there is no model for early stage PD, wherein cell loss is not evident. Clinical symptoms of PD are not observed until about 80% of the substantia nigra (SN) is lost. It is unknown what causes PD, nor is there a cure for PD. The purpose of this study was to determine if CIH impacted neuronal viability in the SN in order to establish an early stage PD model. Methods: Gonadally intact male Sprague Dawley rats were exposed to either room air (normoxia) or six-minute chronic intermittent hypoxia (CIH) cycles, during which oxygen levels were rapidly decreased from 21% to 10% then returned to normal room air levels, eight hours a day during the light phase for seven days. Animals were perfused and brain tissue containing the SN was prepared for 8-OhDg (OS damage marker) and DAPI (cell nuclear marker) immunohistochemical staining. Afterwards, tissue sections were mounted and imaged to analyze the specific effects of CIH on OS damage, cell nuclear size, and cell number. Specifically, 8-OhDg and DAPI expression within the SN were summed and averaged across multiple sections of the SN. Comparisons were made between normoxia and CIH groups. In addition to OS damage and cell number, cell nuclear sizes were quantified and averaged across sections. 8-OhDg and DAPI staining were visualized using a digital camera on fluorescent microscope. Results: CIH increased OS, as shown by increased 8-OhDg expression, in the SN compared to normoxia. No significant differences in cell number or cell nuclear size were found between CIH and normoxia. Conclusions: This is the first study to show that mild CIH does not alter SN cell number or nuclear size, even though CIH increases OS damage in cells. These results support the use of CIH as an early stage animal model for PD. Data generated from this model aid in the understanding of the PD and its pathophysiology.