Carine Cleren

Functional engraftment of human ES cell–derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes

To direct human embryonic stem (HES) cells to a dopaminergic neuronal fate, we cocultured HES cells that were exposed to both sonic hedgehog and fibroblast growth factor 8 with telomerase-immortalized human fetal midbrain astrocytes. These astrocytes substantially potentiated dopaminergic neurogenesis by both WA09 and WA01 HES cells, biasing them to the A9 nigrostriatal phenotype. When transplanted into the neostriata of 6-hydroxydopamine–lesioned parkinsonian rats, the dopaminergic implants yielded a significant, substantial and long-lasting restitution of motor function. However, although rich in donor-derived tyrosine hydroxylase–expressing neurons, the grafts exhibited expanding cores of undifferentiated mitotic neuroepithelial cells, which can be tumorigenic. These results show the utility of recreating the cellular environment of the developing human midbrain while driving dopaminergic neurogenesis from HES cells, and they demonstrate the potential of the resultant cells to mediate substantial functional recovery in a model of Parkinson disease. Yet these data also mandate caution in the clinical application of HES cell–derived grafts, given their potential for phenotypic instability and undifferentiated expansion.

Expression profiling of Huntington's disease models suggests that brain-derived neurotrophic factor depletion plays a major role in striatal degeneration.

Many pathways have been proposed as contributing to Huntingtons disease (HD) pathogenesis, but generally the in vivo effects of their perturbation have not been compared with reference data from human patients. Here we examine how accurately mechanistically motivated and genetic HD models recapitulate the striatal gene expression phenotype of human HD. The representative genetic model was the R6/2 transgenic mouse, which expresses a fragment of the huntingtin protein containing a long CAG repeat. Pathogenic mechanisms examined include mitochondrial dysfunction; profiled in 3-nitropropionic acid-treated rats, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, and PGC-1α knock-out mice; and depletion of brain-derived neurotrophic factor (BDNF) using heterozygous and forebrain-specific BDNF-knock-out mice (BDNF HET , Emx-BDNF KO ). Based on striatal gene expression, we find the BDNF models, both heterozygous and homozygous knock-outs, to be more like human HD than the other HD models. This implicates reduced trophic support as a major pathway contributing to striatal degeneration in HD. Because the majority of striatal BDNF is synthesized by cortical neurons, the data also imply that cortical dysfunction contributes to HDs hallmark effects on the basal ganglia. Finally, the results suggest that striatal lesions caused by mitochondrial toxins may arise via pathways different from those that drive neurodegeneration in HD. Based on these findings, we present a testable model of HD pathogenesis that, unlike most models, begins to account for regional specificity in human HD and the absence of such specificity in genetic mouse models of HD.

Celastrol protects against MPTP- and 3-nitropropionic acid-induced neurotoxicity.

Oxidative stress and inflammation are implicated in neurodegenerative diseases including Parkinsons disease (PD) and Huntingtons disease (HD). Celastrol is a potent anti‐inflammatory and antioxidant compound extracted from a perennial creeping plant belonging to the Celastraceae family. Celastrol is known to prevent the production of proinflammatory cytokines, inducible nitric oxide synthase and lipid peroxidation. Mice were treated with celastrol before and after injections of MPTP, a dopaminergic neurotoxin, which produces a model of PD. A 48% loss of dopaminergic neurons induced by MPTP in the substantia nigra pars compacta was significantly attenuated by celastrol treatment. Moreover, celastrol treatment significantly reduced the depletion in dopamine concentration induced by MPTP. Similarly, celastrol significantly decreased the striatal lesion volume induced by 3‐nitropropionic acid, a neurotoxin used to model HD in rats. Celastrol induced heat shock protein 70 within dopaminergic neurons and decreased tumor necrosis factor‐α and nuclear factor κ B immunostainings as well as astrogliosis. Celastrol is therefore a promising neuroprotective agent for the treatment of PD and HD.

Therapeutic effects of coenzyme Q10 (CoQ10) and reduced CoQ10 in the MPTP model of Parkinsonism

J. Neurochem. (2008) 104, 1613–1621.

Neuroprotective effects of phenylbutyrate against MPTP neurotoxicity

There is increasing evidence that administration of histone deacetylase (HDAC) inhibitors can exert neuroprotective effects by a variety of mechanisms. Phenylbutyrate is a well-known HDAC inhibitor, which increases gene transcription of a number of genes, and also exerts neuroprotective effects. These include several antioxidant enzymes, chaperones, and genes involved in cell survival. We examined whether administration of phenylbutyrate could exert significant neuroprotective effects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which has been used to model Parkinson’s disease. Administration of phenylbutyrate significantly attenuated MPTP-induced depletion of striatal dopamine and loss of tyrosine hydroxylase-positive neurons in the substantia nigra. These findings provide further evidence that administration of phenylbutyrate may be a useful approach for the treatment of neurodegenerative diseases.

Promethazine protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity.

Promethazine (PMZ) is an FDA-approved antihistaminergic drug that was identified as a potentially neuroprotective compound in the NINDS screening program. PMZ accumulates in brain mitochondria in vivo and inhibits Ca2+-induced mitochondrial permeability transition pore (PTP) in rat liver mitochondria in vitro. We hypothesized that PMZ may have a protective effect in a mitochondrial toxin model of Parkinsons disease (PD). Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) sustained a significant loss of dopaminergic neurons within the SNpc that was strongly attenuated by PMZ treatment. However, neither striatal MPP+ concentrations nor MPTP-induced inhibition of mitochondrial complex I were affected by PMZ treatment. In isolated mouse brain mitochondria, PMZ partially prevented and reversed MPP+-induced depolarization of membrane potential and inhibited the Ca2+-induced PTP in brain mitochondria. The sum of data indicates that PMZ is a strong neuroprotective agent capable of protecting dopaminergic neurons against MPTP toxicity in vivo.

Molecular and electrophysiological changes in the prefrontal cortex-amygdala-dorsal periaqueductal grey pathway during persistent pain state and fear-conditioned analgesia.

Fear-conditioned analgesia (FCA) is the reduction in pain responding which is expressed upon re-exposure to a context previously paired with an aversive stimulus. Projections along the prefrontal cortex (PFC)-amygdala-dorsal periaqueductal grey (dPAG) pathway may mediate FCA. However, there is a paucity of studies measuring both molecular and electrophysiological changes in this pathway in rats expressing persistent pain-related behaviour or FCA. Male Lister-hooded rats, with stimulating and recording electrodes implanted in the amygdala and dPAG, respectively, either received or did not receive footshock (0.4 mA) paired with context, followed 23.5 h later by an intraplantar injection of saline or formalin (50 μL, 2.5%) into the right hindpaw. Thirty minutes post-formalin/saline, rats were re-exposed to the context for 15 min, during which pain-related behaviours were assessed in addition to evoked field potential recordings in the amygdala-dPAG pathway. Immediately after the 15-minute trial, PFC tissue was isolated for measurement of total and phosphorylated extracellular-signal regulated kinase (ERK) by western blotting. Formalin-evoked nociceptive behaviour in non-fear-conditioned rats was associated with increased field potential amplitude in the dPAG and increased relative expression of phospho-ERK in the PFC. These effects were abolished in rats expressing FCA. Fear conditioning in non-formalin treated rats was associated with increased phospho-ERK in the PFC but no change in field potential amplitude in the dPAG. Together, these data suggest differential, state-dependent alterations in electrophysiological activity and ERK phosphorylation along the PFC-amygdala-dPAG pathway during pain, conditioned fear, and FCA.

Prefrontal tetanic stimulation, following fear reconditioning, facilitates expression of previously acquired extinction.

We have recently shown that post-extinction retraining of rats, with a shock intensity that is too weak to induce by itself significant fear acquisition, impairs the recall of fear extinction memory. Tetanic stimulation (TS) of the medial prefrontal cortex (mPFC), applied before or following this retraining, facilitates extinction recall. Here we investigated whether mPFC TS can also facilitate expression of fear extinction when rats are retrained with the same shock intensity as during the initial fear acquisition. Rats were implanted with stimulating electrodes in the mPFC and were trained to acquire freezing to a conditioning chamber, in which they had to enter freely. In Experiment 1, extinction of this response was followed by reconditioning and then another extinction training. Acquired freezing was extinguished successfully, while reacquired freezing, which was associated with increased chamber entry latencies, was resistant to subsequent extinction. Both reacquired freezing and increased chamber entry latencies were absent in rats that received post-reconditioning mPFC TS. In Experiment 2, post-conditioning mPFC TS had no effect on initially acquired freezing. In Experiment 3, rats were submitted to reconditioning without experiencing extinction training. In this condition, both reacquired freezing and increased chamber entry latencies were still present in rats that received post-reconditioning mPFC TS. These findings provide additional evidence for the fundamental role of the mPFC in maintaining expression of fear extinction.

Promethazine protects against 3-nitropropionic acid-induced neurotoxicity

Promethazine (PMZ), an FDA-approved antihistaminergic drug, was identified as a potentially neuroprotective compound in a NINDS screening program. It was shown to protect against ischemia in mice, to delay disease onset in a mouse model of amyotrophic lateral sclerosis and to inhibit Ca(2+)-induced mitochondrial permeability transition in rat liver mitochondria. We investigated whether PMZ could protect against the neurotoxic effects induced by 3-nitropropionic acid (3-NP), an inhibitor of the succinate dehydrogenase, used to model Huntingtons disease (HD) in rats. Lewis rats receiving chronic subcutaneous infusion of 3-NP were treated with PMZ. The findings indicate that chronic PMZ treatment significantly reduced 3-NP-induced striatal lesion volume, loss of GABAergic neurons and number of apoptotic cells in the striatum. PMZ showed a strong neuroprotective effect against 3-NP toxicity in vivo.

Time- and sex-dependent efficacy of magnesium sulfate to prevent behavioral impairments and cerebral damage in a mouse model of cerebral palsy

Cerebral lesions acquired in the perinatal period can induce cerebral palsy (CP), a multifactorial pathology leading to lifelong motor and cognitive deficits. Several risk factors, including perinatal hypoxia-ischemia (HI), can contribute to the emergence of CP in preterm infants. Currently, there is no international consensus on treatment strategies to reduce the risk of developing CP. A meta-analysis showed that magnesium sulfate (MgSO4) administration to mothers at risk of preterm delivery reduces the risk of developing CP (Crowther et al., 2017). However, only a few studies have investigated the long-term effects of MgSO4 and it is not known whether sex would influence MgSO4 efficacy. In addition, the search for potential deleterious effects is essential to enable broad use of MgSO4 in maternity wards. We used a mouse model of perinatal HI to study MgSO4 effects until adolescence, focusing on cognitive and motor functions, and on some apoptosis and inflammation markers. Perinatal HI at postnatal day 5 (P(5)) induced (1) sensorimotor deficits in pups; (2) increase in caspase-3 activity 24 h after injury; (3) production of proinflammatory cytokines from 6 h to 5 days after injury; (4) behavioral and histological alterations in adolescent mice with considerable interindividual variability. MgSO4 prevented sensorimotor alterations in pups, with the same efficacy in males and females. MgSO4 displayed anti-apoptotic and anti-inflammatory effects without deleterious side effects. Perinatal HI led to motor coordination impairments in female adolescent mice and cognitive deficits in both sexes. MgSO4 tended to prevent these motor and cognitive deficits only in females, while it prevented global brain tissue damage in both sexes. Moreover, interindividual and intersexual differences appeared regarding the lesion size and neuroprotection by MgSO4 in a region-specific manner. These differences, the partial prevention of disorders, as well as the mismatch between histological and behavioral observations mimic clinical observations. This underlines that this perinatal HI model is suitable to further analyze the mechanisms of sex-dependent perinatal lesion susceptibility and MgSO4 efficacy.