Amar K. Pani

Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson's disease: Critical role for the astrocyte

Oxidative stress has been implicated in the etiology of Parkinsons disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. It is known that under conditions of oxidative stress, the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes. To investigate the role of Nrf2 in the process of MPTP-induced toxicity, mice expressing the human placental alkaline phosphatase (hPAP) gene driven by a promoter containing a core ARE sequence (ARE-hPAP) were used. ARE-hPAP mice were injected (30 mg/kg) once per day for 5 days and killed 7 days after the last MPTP injection. In response to this design, ARE-dependent gene expression was decreased in striatum whereas it was increased in substantia nigra. The same MPTP protocol was applied in Nrf2+/+ and Nrf2−/− mice; Nrf2 deficiency increases MPTP sensitivity. Furthermore, we evaluated the potential for astrocytic Nrf2 overexpression to protect from MPTP toxicity. Transgenic mice with Nrf2 under control of the astrocyte-specific promoter for the glial fribillary acidic protein (GFAP-Nrf2) on both a Nrf2+/+ and Nrf2−/− background were administered MPTP. In the latter case, only the astrocytes expressed Nrf2. Independent of background, MPTP-mediated toxicity was abolished in GFAP-Nrf2 mice. These striking results indicate that Nrf2 expression restricted to astrocytes is sufficient to protect against MPTP and astrocytic modulation of the Nrf2-ARE pathway is a promising target for therapeutics aimed at reducing or preventing neuronal death in PD.

Extensive enteric nervous system abnormalities in mice transgenic for artificial chromosomes containing Parkinson disease-associated α-synuclein gene mutations precede central nervous system changes

Parkinson disease (PD) is a neurodegenerative disease with motor as well as non-motor signs in the gastrointestinal tract that include dysphagia, gastroparesis, prolonged gastrointestinal transit time, constipation and difficulty with defecation. The gastrointestinal dysfunction commonly precedes the motor symptoms by decades. Most PD is sporadic and of unknown etiology, but a fraction is familial. Among familial forms of PD, a small fraction is caused by missense (A53T, A30P and E46K) and copy number mutations in SNCA which encodes alpha-synuclein, a primary protein constituent of Lewy bodies, the pathognomonic protein aggregates found in neurons in PD. We set out to develop transgenic mice expressing mutant alpha-synuclein (either A53T or A30P) from insertions of an entire human SNCA gene as models for the familial disease. Both the A53T and A30P lines show robust abnormalities in enteric nervous system (ENS) function and synuclein-immunoreactive aggregates in ENS ganglia by 3 months of age. The A53T line also has abnormal motor behavior but neither demonstrates cardiac autonomic abnormalities, olfactory dysfunction, dopaminergic neurotransmitter deficits, Lewy body inclusions or neurodegeneration. These animals recapitulate the early gastrointestinal abnormalities seen in human PD. The animals also serve as an in vivo system in which to investigate therapies for reversing the neurological dysfunction that target alpha-synuclein toxicity at its earliest stages.

Exercise protects against MPTP-induced neurotoxicity in mice.

Exercise has been shown to be potently neuroprotective in several neurodegenerative models, including 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) model of Parkinsons disease (PD). In order to determine the critical duration of exercise necessary for DA neuroprotection, mice were allowed to run for either 1, 2 or 3months prior to treatment with saline or MPTP. Quantification of DA neurons in the SNpc show that mice allowed to run unrestricted for 1 or 2months lost significant numbers of neurons following MPTP administration as compared to saline treated mice; however, 3months of exercise provided complete protection against MPTP-induced neurotoxicity. To determine the critical intensity of exercise for DA neuroprotection, mice were restricted in their running to either 1/3 or 2/3 that of the full running group for 3months prior to treatment with saline or MPTP. Quantification of DA neurons in the SNpc show that mice whose running was restricted lost significant numbers of DA neurons due to MPTP toxicity; however, the 2/3 running group demonstrated partial protection. Neurochemical analyses of DA and its metabolites DOPAC and HVA show that exercise also functionally protects neurons from MPTP-induced neurotoxicity. Proteomic analysis of SN and STR tissues indicates that 3months of exercise induces changes in proteins related to energy regulation, cellular metabolism, the cytoskeleton, and intracellular signaling events. Taken together, these data indicate that exercise potently protects DA neurons from acute MPTP toxicity, suggesting that this simple lifestyle element may also confer significant protection against developing PD in humans.

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.

Normal Midbrain Dopaminergic Neuron Development and Function in miR-133b Mutant Mice

Midbrain dopaminergic (mDA) neurons control movement and emotion, and their degeneration leads to motor and cognitive defects in Parkinsons disease (PD). miR-133b is a conserved microRNA that is thought to regulate mDA neuron differentiation by targeting Pitx3, a transcription factor required for appropriate development of mDA substantia nigra neurons. Moreover, miR-133b has been found to be depleted in the midbrain of PD patients. However, the function of miR-133b in the intact midbrain has not been determined. Here we show that miR-133b null mice have normal numbers of mDA neurons during development and aging. Dopamine levels are unchanged in the striatum, while expression of dopaminergic genes, including Pitx3, is also unaffected. Finally, motor coordination and both spontaneous and psychostimulant-induced locomotion are unaltered in miR-133b null mice, suggesting that miR-133b does not play a significant role in mDA neuron development and maintenance in vivo.

Specific disruption of thalamic inputs to the auditory cortex in schizophrenia models.

Genes, synapses, and hallucinations In a schizophrenia mouse model, Chun et al. found that an abnormal increase of dopamine D2 receptors in the brains thalamic nuclei caused thalamocortical synapse deficits owing to reduced glutamate release. Antipsychotic agents or a dopamine receptor antagonist reversed this down-regulation. The defect was associated with the loss of a component of the microRNA processing machinery encoded by the dgcr8 gene. Science, this issue p. 1178 In mice, dopamine D2 receptor up-regulation in the thalamus is responsible for auditory hallucinations. Auditory hallucinations in schizophrenia are alleviated by antipsychotic agents that inhibit D2 dopamine receptors (Drd2s). The defective neural circuits and mechanisms of their sensitivity to antipsychotics are unknown. We identified a specific disruption of synaptic transmission at thalamocortical glutamatergic projections in the auditory cortex in murine models of schizophrenia-associated 22q11 deletion syndrome (22q11DS). This deficit is caused by an aberrant elevation of Drd2 in the thalamus, which renders 22q11DS thalamocortical projections sensitive to antipsychotics and causes a deficient acoustic startle response similar to that observed in schizophrenic patients. Haploinsufficiency of the microRNA-processing gene Dgcr8 is responsible for the Drd2 elevation and hypersensitivity of auditory thalamocortical projections to antipsychotics. This suggests that Dgcr8-microRNA-Drd2–dependent thalamocortical disruption is a pathogenic event underlying schizophrenia-associated psychosis.

Inflammatory Effects of Highly Pathogenic H5N1 Influenza Virus Infection in the CNS of Mice

The A/VN/1203/04 strain of the H5N1 influenza virus is capable of infecting the CNS of mice and inducing a number of neurodegenerative pathologies. Here, we examined the effects of H5N1 on several pathological aspects affected in parkinsonism, including loss of the phenotype of dopaminergic neurons located in the substantia nigra pars compacta (SNpc), expression of monoamines and indolamines in brain, alterations in SNpc microglia number and morphology, and expression of cytokines, chemokines, and growth factors. We find that H5N1 induces a transient loss of the dopaminergic phenotype in SNpc and now report that this loss recovers by 90 d after infection. A similar pattern of loss and recovery was seen in monoamine levels of the basal ganglia. The inflammatory response in lung and different regions of the brain known to be targets of the H5N1 virus (brainstem, substantia nigra, striatum, and cortex) were examined at 3, 10, 21, 60, and 90 d after infection. In each of these brain regions, we found a significant increase in the number of activated microglia that lasted at least 90 d. We also quantified expression of IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, eotaxin, interferon-inducible protein 10, cytokine-induced neutrophil chemoattractant, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP) 1α, MIP-1β, and VEGF, and found that the pattern and levels of expression are dependent on both brain region and time after infection. We conclude that H5N1 infection in mice induces a long-lasting inflammatory response in brain and may play a contributing factor in the development of pathologies in neurodegenerative disorders.

Allopregnanolone Reinstates Tyrosine Hydroxylase Immunoreactive Neurons and Motor Performance in an MPTP-Lesioned Mouse Model of Parkinson's Disease

Restorative/protective therapies to restore dopamine neurons in the substantia nigra pars compacta (SNpc) are greatly needed to effectively change the debilitating course of Parkinsons disease. In this study, we tested the therapeutic potential of a neurogenic neurosteroid, allopregnanolone, in the restoration of the components of the nigrostriatal pathway in MPTP-lesioned mice by measuring striatal dopamine levels, total and tyrosine hydroxylase immunoreactive neuron numbers and BrdU-positive cells in the SNpc. An acute treatment (once/week for two weeks) with allopregnanolone restored the number of tyrosine hydroxylase-positive and total cell numbers in the SNpc of MPTP-lesioned mice, even though this did not increase striatal dopamine. It was also noted that MPTP treated mice to which allopregnanolone was administered had an increase in BrdU-positive cells in the SNpc. The effects of allopregnanolone in MPTP-lesioned mice were more apparent in mice that underwent behavioral tests. Interestingly, mice treated with allopregnanolone after MPTP lesion were able to perform at levels similar to that of non-lesioned control mice in a rotarod test. These data demonstrate that allopregnanolone promotes the restoration of tyrosine hydroxylase immunoreactive neurons and total cells in the nigrostriatal tract, improves the motor performance in MPTP-treated mice, and may serve as a therapeutic strategy for Parkinsons disease.

Neurochemical Measurement of Adenosine in Discrete Brain Regions of Five Strains of Inbred Mice

Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models.

Restoring auditory cortex plasticity in adult mice by restricting thalamic adenosine signaling

Reopening a critical period Young brains, compared with adult brains, are plastic. This phenomenon has given rise to the concept of critical periods, during which acquisition of certain skills is optimal. In mice, an auditory critical period is only open in early postnatal days. The youthful brain tunes circuits to sounds in its environment in a way that the adult brain does not. This facility may form the basis for childhood language acquisition in humans. Blundon et al. show that by manipulating adenosine signaling in mice, some plasticity of the adult auditory cortex can be regained (see the Perspective by Kehayas and Holmaat). Disruption of adenosine production or adenosine receptor signaling in adult mice leads to improved tone discrimination abilities. Science, this issue p. 1352; see also p. 1335 The auditory cortex of adult mice acquires juvenile flexibility if adenosine signaling is disrupted. Circuits in the auditory cortex are highly susceptible to acoustic influences during an early postnatal critical period. The auditory cortex selectively expands neural representations of enriched acoustic stimuli, a process important for human language acquisition. Adults lack this plasticity. Here we show in the murine auditory cortex that juvenile plasticity can be reestablished in adulthood if acoustic stimuli are paired with disruption of ecto-5′-nucleotidase–dependent adenosine production or A1–adenosine receptor signaling in the auditory thalamus. This plasticity occurs at the level of cortical maps and individual neurons in the auditory cortex of awake adult mice and is associated with long-term improvement of tone-discrimination abilities. We conclude that, in adult mice, disrupting adenosine signaling in the thalamus rejuvenates plasticity in the auditory cortex and improves auditory perception.