Adeno-associated virus vector-mediated expression and constitutive secretion of galanin suppresses limbic seizure activity

Theoretically, gene therapy techniques offer an attractive alternative treatment option for intractable, focal epilepsies. Although logical gene therapy targets include excitatory and inhibitory receptors, variable viral vector tropism interjects an uncertainty as to the direction of change, seizure suppression, or seizure sensitization. To circumvent this therapeutic liability, adeno-associated virus (AAV) vectors have been constructed where the gene product is constitutively secreted from the transduced cell. Using AAV vectors, the fibronectin secretory signal sequence (FIB) was placed in front of the coding sequence for green fluorescent protein or the active portion of the neuroactive peptide galanin (GAL). Subsequent studies showed that these vectors supported expression and constitutive secretion of these gene products from transfected cells in vitro. More importantly, upon transduction in vivo, AAV-FIB-GAL vectors significantly attenuated focal seizure sensitivity, and this seizure attenuation could be controlled in vivo by using a tetracycline-regulated promoter. The expression and constitutive secretion of green fluorescent protein, or the expression of GAL alone, exerted no effect on focal seizure sensitivity. Moreover, unilateral infusion of the AAV-FIB-GAL vectors into the hippocampus prevented kainic acid-induced hilar cell death. With regard to limbic seizures, bilateral infusion of AAV-FIB-GAL vectors into the piriform cortex prevented both behavioral and localized electrographic seizure activity after the peripheral administration of kainic acid. Also, when rats were electrically kindled to class V seizure activity, subsequent infusion of AAV-FIB-GAL proved capable of significantly elevating the seizure initiation threshold. Thus, these studies clearly demonstrate the anti-seizure effectiveness of AAV vector-mediated expression and constitutive secretion of galanin

Current prospects and challenges for epilepsy gene therapy

This review addresses the state of gene therapy research for the treatment of epilepsy. Preclinical studies have demonstrated the anti-seizure efficacy of viral vector-based gene transfer through the use of a variety of strategies – from modulating classic neurotransmitter systems to targeting or overexpressing of neuropeptide receptors in seizure-specific brain regions. While these studies provide substantive proof of principle for viral vector gene therapy, future studies must address the challenges of vector immunity, cellular specificity and effective global delivery. As these issues are resolved, viral vector gene therapy should significantly impact the treatment of intractable epilepsy

Mechanistic and Functional Divergence Between Thyrotropin-releasing Hormone and RO 15-4513 Interactions with Ethanol

Both thyrotropin-releasing hormone (TRH) and RO 15-4513 antagonize ethanol-induced depression, but this common property does not infer that both compounds share similar mechanisms of action. In the present studies, both TRH (30 mg/kg, i.p.) and RO 15-4513 (10 mg/kg, i.p.) reversed ethanol-induced depression of locomotor activity, in accord with previous reports. However, the benzodiazepine antagonist, RO 15-1788, blocked this action of RO 15-4513, while exerting no effect on the analeptic action of TRH. Using a model of seizure activity electrically elicited from the inferior colliculus, ethanol exerted a dose-related attenuation of seizure activity. This anticonvulsant action of ethanol was not altered by TRH (30 mg/kg, i.p.), but RO 15-4513 (3 mg/kg) reversed the effect of the 0.5, but not the 1.0 g/kg, dose of ethanol. In addition, pretreatment with RO 15-4513 (1 or 3 mg/kg, i.p.), but not TRH (30 mg/kg, i.p.), caused seizure generalization into the forebrain following inferior collicular stimulation, further verifying the proconvulsant properties of RO 15-4513. In conclusion, the analeptic action of TRH appears independent of benzodiazepine activity, and in contrast to RO 15-4513, TRH does not exhibit proconvulsant properties. Furthermore, because TRH did not antagonize both depressant actions of ethanol studied, it appears unlikely that TRH directly interacts with the molecular basis of ethanol action

Opposing actions of hippocampus TNFα receptors on limbic seizure susceptibility

Resected epileptic tissues exhibit elements of chronic neuroinflammation that include elevated TNFα and increased TNFα receptor activation, but the seizure related consequences of chronic TNFα expression remain unknown. Twenty four hours after acute limbic seizures the rat hippocampus exhibited a rapid upregulation of TNFR1, but a simultaneous downregulation of TNFR2. These limbic seizures also evoked significant increases in measures of neuroinflammation and caused significant neuronal cell death in both the hilus and CA3 of the hippocampus. In order to mimic a state of chronic TNFα exposure, adeno-associated viral vectors were packaged with a TNF receptor 1 (TNFR1) specific agonist, human TNFα, or a TNF receptor 1/2 agonist, rat TNFα. Subsequently, chronic hippocampal overexpression of either TNFR ligand caused microglial activation and blood-brain barrier compromise, a pattern similar to limbic seizure-induced neuroinflammation. However, no evidence was found for neuronal cell death or spontaneous seizure activity. Thus, chronic, in vivo TNFα expression and the subsequent neuroinflammation alone did not cause cell death or elicit seizure activity. In contrast, chronic hippocampal activation of TNFR1 alone significantly increased limbic seizure sensitivity in both amygdala kainic acid and electrical amygdala kindling models, while chronic activation of both TNFR1 and TNFR2 significantly attenuated the amygdala kindling rate. With regard to endogenous TNFα, chronic hippocampal expression of a TNFα decoy receptor significantly reduced seizure-induced cell death in the hippocampus, but did not alter seizure susceptibility. These findings suggest that blockade of endogenous TNFα could attenuate seizure related neuropathology, while selective activation of TNFR2 could exert beneficial therapeutic effects on in vivo seizure sensitivity

Interactions between TRH and ethanol in the medial septum

When rats were pretreated with ethanol (3.0 g/kg, IP), subsequent microinjection of thyrotropin-releasing hormone (TRH) (500 or 1000 ng) into the medial septum, 30 minutes later, significantly shortened the time necessary for the rats to regain their righting reflex. Conversely, microinjection of TRH into the nucleus accumbens (1000 ng/side) or the area of the raphe obscurus (1000 ng) had no effect on ethanol-induced depression, although both of these structures mediate specific TRH effects in the CNS. In order to determine if this antagonism was due to a specific TRH interaction, TRH Fab fragments were microinjected into the medial septum just prior to the microinjection of TRH. Under these conditions, TRH did not alter ethanol’s depressant actions. Finally, this TRH antagonism of ethanol-induced depression appears attributable to a net increase in neuronal activity, because electrical stimulation (160 μA, 120 Hz, 1.5 msec duration) of the medial septum antagonized ethanol’s impairment of the righting reflex. These results are discussed in relationship to a potential CNS site for the action of ethanol

Comparison of the CNS effects induced by TRH and bicuculline after microinjection into medial septum, substantia nigra and inferior colliculus: Absence of support for a GABA antagonist action for TRH

Antagonism of ethanol-induced depression of locomotion was observed after intracisternal injection of thyrotropin releasing hormone (TRH) and bicuculline methiodide (BICM), as well as after microinjection of these drugs into the medial septum. The present investigation compared the behavioral and physiological consequence of administering TRH and BICM into the medial septum, inferior colliculus and substantia nigra to quantitate the similarities between these compounds. BICM produced a major increase in locomotor activity when injected into the medial septum and stereotypies when injected into the substantia nigra, suggesting that GABA-containing neurons have widespread influences on motor function. The wild running and seizure activity observed after BICM injection into the inferior colliculus was also consistent with this latter view. The marked increase in rectal temperature observed when BICM was injected into the medial septum may also implicate GABAergic mechanisms in temperature control at this brain site. TRH produced no such behavioral or physiological changes when administered into these three sites. Thus, this work strongly suggests that TRH does not exert a widespread action as a GABA antagonist because TRH did not produce the same changes induced by BICM. The actions of BICM and TRH to antagonize ethanol-induced depression when microinjected into the medial septum suggests that this brain area may be a critical site for the depressant action of ethanol

Neurological Correction of Lysosomal Storage in a Mucopolysaccharidosis IIIB Mouse Model by Adeno-associated Virus-Mediated Gene Delivery

AbstractMucopolysaccharidosis (MPS) IIIB is characterized by mild somatic features and severe neurological diseases leading to premature death. No definite treatment is available for MPS IIIB patients. We constructed two recombinant adeno-associated virus (rAAV) vectors containing the human α-N-acetylglucosaminidase (NaGlu) cDNA driven by either a CMV or a neuron-specific enolase (NSE) promoter. In vitro, these rAAV vectors mediated efficient expression of recombinant NaGlu in human MPS IIIB fibroblasts and mouse MPS IIIB somatic and brain primary cell cultures. The secreted rNaGlu was taken up by both human and mouse MPS IIIB cells in culture and degraded the accumulated glycosaminoglycans (GAG). A direct microinjection (107 viral particles, 1 μl/10 minutes per injection) of vectors containing the NSE promoter resulted in long-term (6 months, the duration of the experiments) expression of rNaGlu in multiple brain structures/areas of adult MPS IIIB mice. Consistent with previous studies, the main target cells were neurons. However, while vector typically transduced an area of 400–500 μm surrounding the infusion sites, the correction of GAG storage involved neurons of a much broader area (1.5 mm) in a 6-month duration of experiments. These results provide a basis for the development of a treatment for neurological disease in MPS IIIB patients using AAV vectors

Treatment Strategies Targeting Excess Hippocampal Activity Benefit Aged Rats with Cognitive Impairment

Excess neural activity in the CA3 region of the hippocampus has been linked to memory impairment in aged rats. We tested whether interventions aimed at reducing this excess activity would improve memory performance. Aged (24 to 28 months old) male Long–Evans rats were characterized in a spatial memory task known to depend on the functional integrity of the hippocampus, such that aged rats with identified memory impairment were used in a series of experiments. Overexpression of the inhibitory neuropeptide Y 13–36 in the CA3 via adeno-associated viral transduction was found to improve hippocampal-dependent long-term memory in aged rats, which had been characterized with impairment. Subsequent experiments with two commonly used antiepileptic agents, sodium valproate and levetiracetam, similarly produced dose-dependent memory improvement in such aged rats. Improved spatial memory with low doses of these agents was observed in both appetitve and aversive spatial tasks. The benefits of these different modalities of treatment are consistent with the concept that excess activity in the CA3 region of the hippocampus is a dysfunctional condition that may have a key role underlying age-related impairment in hippocampal-dependent memory processes. Because increased hippocampal activation occurs in age-related memory impairment in humans as observed in functional neuroimaging, the current findings also suggest that low doses of certain antiepileptic drugs in cognitively impaired elderly humans may have therapeutic potential and point to novel targets for this indication

Changes in apical dendritic structure correlate with sustained ERK1/2 phosphorylation in medial prefrontal cortex of a rat model of dopamine D 1 receptor agonist sensitization

Rats lesioned with 6-hydroxydopamine (6-OHDA) as neonates exhibit behavioral and neurochemical abnormalities in adulthood that mimic Lesch-Nyhan disease, schizophrenia and other developmental disorders of frontostriatal circuit dysfunction. In these animals, a latent sensitivity to D1 agonists is maximally exposed by repeated administration of dopamine agonists in the post-pubertal period (D1 priming). In neonate-lesioned, adult rats primed with SKF-38393, we found selective, persistent alterations in the morphology of pyramidal neuron apical dendrites in the prelimbic area of the medial prefrontal cortex (mPFC). In these animals, dendrite bundling patterns and the typically straight trajectories of primary dendritic shafts were disrupted, whereas the diameter of higher-order oblique branches was increased. Although not present in neonate-lesioned rats treated with saline, these morphological changes persisted at least 21 days after repeated dosing with SKF-38393, and were not accompanied by markers of neurodegenerative change. A sustained increase in phospho-ERK immunoreactivity in wavy dendritic shafts over the same period suggested a relationship between prolonged ERK phosphorylation and dendritic remodeling in D1-primed rats. In support of this hypothesis, pretreatment with the MEK1/2-ERK1/2 pathway inhibitors PD98059 or SL327, prior to each priming dose of SKF-38393, prevented the morphological changes associated with D1 priming. Together, these findings demonstrate that repeated stimulation of D1 receptors in adulthood interacts with the developmental loss of dopamine to profoundly and persistently modify neuronal signaling and dendrite morphology in the mature prefrontal cortex. Furthermore, sustained elevation of ERK activity in mPFC pyramidal neurons may play a role in guiding these morphological changes in vivo

N-acetylaspartate supports the energetic demands of developmental myelination via oligodendroglial aspartoacylase

Breakdown of neuro-glial N-acetyl-aspartate (NAA) metabolism results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathological progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation “oligotropic” adeno-associated virus vector (AAV-Olig001) to quantitatively assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathology and deteriorating motor function. Long-term effects included increased oligodendrocyte numbers, a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy observed following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile observed in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase