In vertebrates, the two-step peptide alpha-amidation reaction is catalyzed sequentially by two enzymatic activities contained within one bifunctional enzyme called PAM (peptidylglycine alpha-amidating mono-oxygenase). Drosophila head extracts contained both of these PAM-related enzyme activities: a mono-oxygenase (PHM) and a lyase (PAL). However, no bifunctional PAM protein was detected. We identified cDNAs encoding an active mono-oxygenase that is highly homologous to mammalian PHM. PHM-like immunoreactivity was found within diverse larval tissues, including the CNS, endocrine glands, and gut epithelium. Northern and Western blot analyses demonstrate RNA and protein species corresponding to the cloned PHM, but not to a bifunctional PAM, leading us to predict the existence of separate PHM and PAL genes in Drosophila. The Drosophila PHM gene displays an organization of exons that is highly similar to the PHM-encoding portion of the rat PAM gene. Genetic analysis was consistent with the prediction of separate PHM and PAL gene functions in Drosophila: a P element insertion line containing a transposon within the PHM transcription unit displayed strikingly lower PHM enzyme levels, whereas PAL levels were increased slightly. The lethal phenotype displayed by the dPHM P element insertion indicates a widespread essential function. Reversion analysis indicated that the lethality associated with the insertion chromosome likely is attributable to the P element insertion. These combined data indicate a fundamental evolutionary divergence in the genes coding for critical neurotransmitter biosynthetic enzymes: in Drosophila, the two enzyme activities of PAM are encoded by separate genes.

The amygdala is a key limbic area involved in fear responses and pavlovian conditioning with the potential to directly respond to endocrine signals associated with fear or stress. To gain insights into the molecular mechanisms and subregional specificity of fear conditioning, we disrupted type II glucocorticoid receptors (GRs) in the central nucleus of the amygdala (CeA) by delivering lentiviral vectors containing Cre-recombinase into floxed-GR mice. GR deletion in the CeA (CeAGRKO mice) prevented conditioned fear behavior. In contrast, forebrain disruption of GRs excluding the CeA did not. The conditioned fear deficit in CeAGRKO mice was associated with decreases in cFos and corticotropin-releasing hormone (CRH) expression. Moreover, intracerebroventricular delivery of CRH rescued the conditioned fear deficit in CeAGRKO mice. We conclude that fear conditioning involves a neuroendocrine circuit by using GR activation in the CeA for acute CRH induction and long-lasting behavioral modulation.adrenal ͉ animal behavior ͉ knockout mice ͉ lentivirus ͉ stress

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative lysosomal storage disease (LSD) caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). Studies in Ppt1Ϫ / Ϫ mice demonstrate that glial activation is central to the pathogenesis of INCL. Astrocyte activation precedes neuronal loss, while cytokine upregulation associated with microglial reactivity occurs before and concurrent with neurodegeneration. Therefore, we hypothesized that cytokine cascades associated with neuroinflammation are important therapeutic targets for the treatment of INCL. MW01-2-151SRM (MW151) is a blood-brain barrier penetrant, small-molecule anti-neuroinflammatory that attenuates glial cytokine upregulation in models of neuroinflammation such as traumatic brain injury, Alzheimer's disease, and kainic acid toxicity. Thus, we used MW151, alone and in combination with CNS-directed, AAV-mediated gene therapy, as a possible treatment for INCL. MW151 alone decreased seizure susceptibility. When combined with AAV-mediated gene therapy, treated INCL mice had increased life spans, improved motor performance, and eradication of seizures. Combination-treated INCL mice also had decreased brain atrophy, astrocytosis, and microglial activation, as well as intermediary effects on cytokine upregulation. These data suggest that MW151 can attenuate seizure susceptibility but is most effective when used in conjunction with a therapy that targets the primary genetic defect.

ABSTRACT

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Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by the lack of beta-glucuronidase (GUSB) activity. GUSB deficiency leads to the progressive accumulation of undegraded glycosaminoglycans (GAGs) in cells of most tissues, including the brain, and is associated with mental retardation. Reduction of lysosomal storage in the central nervous system and prevention of cognitive dysfunction may require intracranial delivery of a therapeutic agent during the newborn period that provides a continuous source of GUSB. Therefore, we injected recombinant adeno-associated virus encoding human GUSB into both the anterior cortex and the hippocampus of newborn MPS VII mice. Total GUSB activity in the brain approached normal levels by 18 weeks. Although GUSB activity was concentrated near the injection sites, lysosomal distension was reduced in most areas of the brain. In addition to histopathologic evidence of GAG reduction, the previously undescribed accumulation of GM2 and GM3 gangliosides in the brain was also prevented. Furthermore, GUSB expression and reduced lysosomal distension correlated with improvements in cognitive function as measured in the Morris Water Maze test. These findings indicate that localized overexpression of GUSB has positive effects on the pathology and cognitive function and does not have overt toxicity.

Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and nonphosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated ␤-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, nonphosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the halflives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.

Objective Infantile neuronal ceroid lipofusciniosis (INCL) is an inherited childhood neurodegenerative disorder caused by the loss of palmitoyl protein thioesterase-1 (PPT1) activity. Affected children suffer from blindness, epilepsy, motor dysfunction, cognitive decline, and premature death. The Ppt1−/− mouse shares the histological and clinical features of INCL. Previous single-therapy approaches using small molecule drugs, gene therapy, or neuronal stem cells resulted in partial histological correction, with minimal improvements in motor function or lifespan. Here, we combined CNS-directed AAV2/5-mediated gene therapy with bone marrow transplantation (BMT) in the INCL mouse. Methods At birth, Ppt1−/− and WT mice were given either intracranial injections of AAV2/5-PPT1 or bone marrow transplantation, separately as well as in combination. To assess function, we measured monthly rotorod performance monthly as well as lifespan. At terminal timepoints, we evaluated the therapeutic effects on several INCL specific parameters, such as cortical thickness, autofluorescent accumulation, and glial activation. Finally, we determined levels of PPT1 enzyme activity and bone marrow engraftment in treated mice. Results AAV2/5-mediated gene therapy alone resulted in significant histological correction, improved motor function, and increased life span. Interestingly, the addition of BMT further increased the lifespan of treated mice and led to dramatic, sustained improvements in motor function. These data are truly striking given the fact that BMT alone is ineffective yet it synergizes with CNS-directed gene therapy to dramatically increase efficacy and lifespan. Interpretation AAV2/5-mediated gene therapy in combination with BMT provides an unprecedented increase in lifespan as well as dramatic improvement on functional and histological parameters.