GABAergic interneurons contribute to the fatal seizure phenotype of CLN2 disease mice.

GABAergic interneuron deficits have been implicated in the epileptogenesis of multiple neurological diseases. While epileptic seizures are a key clinical hallmark of CLN2 disease, a childhood-onset neurodegenerative lysosomal storage disorder caused by a deficiency of tripeptidyl peptidase 1 (TPP1), the etiology of these seizures remains elusive. Given that Cln2 R207X/R207X mice display fatal spontaneous seizures and an early loss of several cortical interneuron populations, we hypothesized that those two events might be causally related. To address this hypothesis, we first generated an inducible transgenic mouse expressing lysosomal membrane-tethered TPP1 (TPP1LAMP1) on the Cln2 R207X/R207X genetic background to study the cell-autonomous effects of cell-type-specific TPP1 deficiency. We crossed the TPP1LAMP1 mice with Vgat-Cre mice to introduce interneuron-specific TPP1 deficiency. Vgat-Cre ; TPP1LAMP1 mice displayed storage material accumulation in several interneuron populations both in cortex and striatum, and increased susceptibility to die after PTZ-induced seizures. Secondly, to test the role of GABAergic interneuron activity in seizure progression, we selectively activated these cells in Cln2 R207X/R207X mice using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) in in Vgat-Cre : Cln2 R207X/R207X mice. EEG monitoring revealed that DREADD-mediated activation of interneurons via chronic deschloroclozapine administration accelerated the onset of spontaneous seizures and seizure-associated death in Vgat-Cre : Cln2 R207X/R207X mice, suggesting that modulating interneuron activity can exert influence over epileptiform abnormalities in CLN2 disease. Taken together, these results provide new mechanistic insights into the underlying etiology of seizures and premature death that characterize CLN2 disease.

An anti-neuroinflammatory that targets dysregulated glia enhances the efficacy of CNS-directed gene therapy in murine infantile neuronal ceroid lipofuscinosis.

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 Ppt1(-/-) 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.

Synergistic effects of central nervous system-directed gene therapy and bone marrow transplantation in the murine model of infantile neuronal ceroid lipofuscinosis.

OBJECTIVE: Infantile neuronal ceroid lipofuscinosis (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 central nervous system (CNS)-directed adeno-associated virus (AAV)2/5-mediated gene therapy with bone marrow transplantation (BMT) in the INCL mouse. METHODS: At birth, Ppt1(-/-) and wild-type 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 rotorod performance monthly as well as lifespan. At terminal time points, 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 lifespan. 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 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.

Combination small molecule PPT1 mimetic and CNS-directed gene therapy as a treatment for infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a profoundly neurodegenerative disease of children caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). There is currently no effective therapy for this invariably fatal disease. To date, preclinical experiments using single treatments have resulted in incremental clinical improvements. Therefore, we determined the efficacy of CNS-directed AAV2/5-mediated gene therapy alone and in combination with the systemic delivery of the lysosomotropic PPT1 mimetic phosphocysteamine. Since CNS-directed gene therapy provides relatively high levels of PPT1 activity to specific regions of the brain, we hypothesized that phosphocysteamine would complement that activity in regions expressing subtherapeutic levels of the enzyme. Results indicate that CNS-directed gene therapy alone provided the greatest improvements in biochemical and histological measures as well as motor function and life span. Phosphocysteamine alone resulted in only minor improvements in motor function and no increase in lifespan. Interestingly, phosphocysteamine did not increase the biochemical and histological response when combined with AAV2/5-mediated gene therapy, but it did result in an additional improvement in motor function. These data suggest that a CNS-directed gene therapy approach provides significant clinical benefit, and the addition of the small molecule PPT1 mimetic can further increase that response.

Central amygdala glucocorticoid receptor action promotes fear-associated CRH activation and conditioning.

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.