Structure activity relationship (SAR) studies of neurotoxin quinoline-derivatives

Structure activity relationship (SAR) studies are performed in order to identify the core structure that is responsible for the biological activity of an organic molecule. Recently, we have synthesized a drug prototype which contains several functional groups, such as an alcohol, an ester, a fluorine, and an aromatic ring. While studying in vivo toxicity of this molecule in zebrafish (Danio rerio) embryo, we observed that it has a unique biological activity that causes a sudden inactivity in embryo movement. Continued investigation revealed that this molecule blocks sodium channels in neurons causing a temporary anesthesia in Danio rerio embryo. The biological activity in zebrafish was performed with Dr. Sittaramane. We have also observed that after transferring the embryo to fresh water, the embryo resumed normal behavior. As our next step, we would like to synthesize a variety of structural analogs and determine their activity. The ultimate goal of this project was to develop effective methods of synthesizing various molecules that have one of the functional groups removed in order to identify its role in biological activity. All products and intermediates will be fully analyzed using NMR, IR and Mass spectrometer analysis. Further structural modifications may be required depending on the activity findings. After successful synthesis of the proposed molecules, Dr. Sittaramane and I performed in vivo activity determination

Neuroanatomical and neurochemical effects of prolonged social isolation in adult mice

IntroductionAs social animals, our health depends in part on interactions with other human beings. Yet millions suffer from chronic social isolation, including those in nursing/assisted living facilities, people experiencing chronic loneliness as well as those in enforced isolation within our criminal justice system. While many historical studies have examined the effects of early isolation on the brain, few have examined its effects when this condition begins in adulthood. Here, we developed a model of adult isolation using mice (C57BL/6J) born and raised in an enriched environment.MethodsFrom birth until 4 months of age C57BL/6J mice were raised in an enriched environment and then maintained in that environment or moved to social isolation for 1 or 3 months. We then examined neuronal structure and catecholamine and brain derived neurotrophic factor (BDNF) levels from different regions of the brain, comparing animals from social isolation to enriched environment controls.ResultsWe found significant changes in neuronal volume, dendritic length, neuronal complexity, and spine density that were dependent on brain region, sex, and duration of the isolation. Isolation also altered dopamine in the striatum and serotonin levels in the forebrain in a sex-dependent manner, and also reduced levels of BDNF in the motor cortex and hippocampus of male but not female mice.ConclusionThese studies show that isolation that begins in adulthood imparts a significant change on the homeostasis of brain structure and chemistry

Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice

Background: Methylphenidate (MPH) is a psychostimulant that exerts its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. Clinically, methylphenidate is prescribed for the symptomatic treatment of ADHD and narcolepsy; although lately, there has been an increased incidence of its use in individuals not meeting the criteria for these disorders. MPH has also been misused as a cognitive enhancer and as an alternative to other psychostimulants. Here, we investigate whether chronic or acute administration of MPH in mice at either 1 mg/kg or 10 mg/kg, affects cell number and gene expression in the basal ganglia. Methodology/Principal Findings: Through the use of stereological counting methods, we observed a significant reduction (~20%) in dopamine neuron numbers in the substantia nigra pars compacta (SNpc) following chronic administration of 10 mg/kg MPH. This dosage of MPH also induced a significant increase in the number of activated microglia in the SNpc. Additionally, exposure to either 1 mg/kg or 10 mg/kg MPH increased the sensitivity of SNpc dopaminergic neurons to the parkinsonian agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Unbiased gene screening employing Affymetrix GeneChip® HT MG-430 PM revealed changes in 115 and 54 genes in the substantia nigra (SN) of mice exposed to 1 mg/kg and 10 mg/kg MPH doses, respectively. Decreases in the mRNA levels of gdnf, dat1, vmat2, and th in the substantia nigra (SN) were observed with both acute and chronic dosing of 10 mg/kg MPH. We also found an increase in mRNA levels of the pro-inflammatory genes il-6 and tnf-α in the striatum, although these were seen only at an acute dose of 10 mg/kg and not following chronic dosing. Conclusion: Collectively, our results suggest that chronic MPH usage in mice at doses spanning the therapeutic range in humans, especially at prolonged higher doses, has long-term neurodegenerative consequences

Murine MPDZ-linked hydrocephalus is caused by hyperpermeability of the choroid plexus.

Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus

Neuroanatomical and Neurochemical Effects of Prolonged Social Isolation in Adult Mice

INTRODUCTION: As social animals, our health depends in part on interactions with other human beings. Yet millions suffer from chronic social isolation, including those in nursing/assisted living facilities, people experiencing chronic loneliness as well as those in enforced isolation within our criminal justice system. While many historical studies have examined the effects of early isolation on the brain, few have examined its effects when this condition begins in adulthood. Here, we developed a model of adult isolation using mice (C57BL/6J) born and raised in an enriched environment. METHODS: From birth until 4 months of age C57BL/6J mice were raised in an enriched environment and then maintained in that environment or moved to social isolation for 1 or 3 months. We then examined neuronal structure and catecholamine and brain derived neurotrophic factor (BDNF) levels from different regions of the brain, comparing animals from social isolation to enriched environment controls. RESULTS: We found significant changes in neuronal volume, dendritic length, neuronal complexity, and spine density that were dependent on brain region, sex, and duration of the isolation. Isolation also altered dopamine in the striatum and serotonin levels in the forebrain in a sex-dependent manner, and also reduced levels of BDNF in the motor cortex and hippocampus of male but not female mice. CONCLUSION: These studies show that isolation that begins in adulthood imparts a significant change on the homeostasis of brain structure and chemistry

Mutant LRRK2 in lymphocytes regulates neurodegeneration via IL-6 in an inflammatory model of Parkinson\u27s disease

Mutations in a number of genes contribute to development of Parkinson\u27s disease (PD), including several within the LRRK2 gene. However, little is known about the signals that underlie LRRK2-mediated neuronal loss. One clue resides in the finding that the neurodegenerative cascades emanate from signals arising from the peripheral immune system. Here, using two chimeric mouse models, we demonstrate that: 1) the replacement of mutant LRRK2 with wt form of the protein in T- and B-lymphocytes diminishes LPS-mediated inflammation and rescues the SNpc DA neuron loss in the mutant LRRK2 brain; 2) the presence of G2019S or R1441G LRRK2 mutation in lymphocytes alone is sufficient for LPS-induced DA neuron loss in the genotypically wt brain; and 3) neutralization of peripheral IL-6 overproduction prevents the SNpc DA neuron loss in LPS-treated mutant LRRK2 mice. These results represent a major paradigm shift in our understanding of PD pathogenesis and suggest that immune dysfunction in some forms of familial PD may have primacy over the CNS as the initiating site of the disorder

Daun02 inactivation of behaviorally-activated Fos-expressing neuronal ensembles

Learned associations about salient experiences (e.g. drug exposure, stress) and their associated environmental stimuli are mediated by a minority of sparsely distributed, behaviorally activated neurons coined ‘neuronal ensembles’. For many years, it was not known whether these neuronal ensembles played causal roles in mediating learned behaviors. However, in the last several years the ‘Daun02 inactivation technique’ in Fos-lacZ transgenic rats has proved very useful in establishing causal links between neuronal ensembles that express the activity-regulated protein ‘Fos’ and learned behaviors. Fosexpressing neurons in these rats also express the bacterial protein b-galactosidase (b-gal) in strongly activated neurons. When the prodrug Daun02 is injected into the brains of these rats 90 min after a behavior (e.g. drug-seeking) or cue exposure, then the Daun02 is converted into daunorubicin by b-gal, which selectively inactivates the Fos and b-galexpressing neurons that were activated 90 min before the Daun02 injection. This unit presents protocols for breeding the Fos-lacZ rats and conducting appropriate Daun02 inactivation experiments

Manganese exposure in juvenile C57BL/6 mice increases glial inflammatory responses in the substantia nigra following infection with H1N1 influenza virus.

Infection with Influenza A virus can lead to the development of encephalitis and subsequent neurological deficits ranging from headaches to neurodegeneration. Post-encephalitic parkinsonism has been reported in surviving patients of H1N1 infections, but not all cases of encephalitic H1N1 infection present with these neurological symptoms, suggesting that interactions with an environmental neurotoxin could promote more severe neurological damage. The heavy metal, manganese (Mn), is a potential interacting factor with H1N1 because excessive exposure early in life can induce long-lasting effects on neurological function through inflammatory activation of glial cells. In the current study, we used a two-hit model of neurotoxin-pathogen exposure to examine whether exposure to Mn during juvenile development would induce a more severe neuropathological response following infection with H1N1 in adulthood. To test this hypothesis, C57BL/6 mice were exposed to MnCl2 in drinking water (50 mg/kg/day) for 30 days from days 21-51 postnatal, then infected intranasally with H1N1 three weeks later. Analyses of dopaminergic neurons, microglia and astrocytes in basal ganglia indicated that although there was no significant loss of dopaminergic neurons within the substantia nigra pars compacta, there was more pronounced activation of microglia and astrocytes in animals sequentially exposed to Mn and H1N1, as well as altered patterns of histone acetylation. Whole transcriptome Next Generation Sequencing (RNASeq) analysis was performed on the substantia nigra and revealed unique patterns of gene expression in the dual-exposed group, including genes involved in antioxidant activation, mitophagy and neurodegeneration. Taken together, these results suggest that exposure to elevated levels of Mn during juvenile development could sensitize glial cells to more severe neuro-immune responses to influenza infection later in life through persistent epigenetic changes

Acute Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or paraquat on core temperature in C57BL/6J mice

Background: MPTP and paraquat are two compounds that have been used to model Parkinson’s disease in mice. Previous studies in two non-traditional strains of mice have shown that a single dose of MPTP can induce changes in body temperature, while the effects of paraquat have not been examined. Examination of body temperature is important since small fluctuations in an animal’s core temperature can significantly affect drug metabolism, and if significant enough can even culminate in an animal’s death. Objective: To determine how external heating can alter the survival of C57BL/6J mice following MPTP administration. Methods: In this study, we examine the effects of MPTP (4×20 mg/kg, 2 hours apart) and paraquat (2×10 mg/kg/week for 3 weeks) on core temperature of C57BL/6J mice. Correlations of purine and catecholamine levels were also done in mice treated with MPTP. Results: We find that MPTP induces a significant hypothermia in C57BL/6J mice that reduces their core temperature below the limit of fatal hypothermia. Unlike MPTP, paraquat did not induce a significant hypothermia. Placement of animals on heating pads significantly abrogates the loss of core temperature. In both heated and non-heated conditions, mice treated with MPTP showed a significant depletion of ATP within 2 hours of administration in both striatum and SN that started to recover 2 hours after MPTP administration was complete. Striatal DA and DOPAC are significantly reduced starting 4–6 hours after MPTP. Conclusions: The fatal hypothermic effects of MPTP can be abrogated through use of external heating

Infection and Risk of Parkinson\u27s Disease

Parkinson\u27s disease (PD) is thought to be caused by a combination of genetic and environmental factors. Bacterial or viral infection has been proposed as a potential risk factor, and there is supporting although not entirely consistent epidemiologic and basic science evidence to support its role. Encephalitis caused by influenza has included parkinsonian features. Epidemiological evidence is most compelling for an association between PD and hepatitis C virus. Infection with Helicobacter pylori may be associated not only with PD risk but also response to levodopa. Rapidly evolving knowledge regarding the role of the microbiome also suggests a role of resident bacteria in PD risk. Biological plausibility for the role for infectious agents is supported by the known neurotropic effects of specific viruses, particular vulnerability of the substantia nigra and even the promotion of aggregation of alpha-synuclein. A common feature of implicated viruses appears to be production of high levels of cytokines and chemokines that can cross the blood-brain barrier leading to microglial activation and inflammation and ultimately neuronal cell death. Based on multiple avenues of evidence it appears likely that specific bacterial and particularly viral infections may increase vulnerability to PD. The implications of this for PD prevention requires attention and may be most relevant once preventive treatments for at-risk populations are developed