Gene Electrotransfer via Conductivity-Clamped Electric Field Focusing Pivots Sensori-Motor DNA Therapeutics: "A Spoonful of Sugar Helps the Medicine Go Down".

Viral vectors and lipofection-based gene therapies have dispersion-dependent transduction/transfection profiles that thwart precise targeting. The study describes the development of focused close-field gene electrotransfer (GET) technology, refining spatial control of gene expression. Integration of fluidics for precise delivery of "naked" plasmid deoxyribonucleic acid (DNA) in sucrose carrier within the focused electric field enables negative biasing of near-field conductivity ("conductivity-clamping"-CC), increasing the efficiency of plasma membrane molecular translocation. This enables titratable gene delivery with unprecedently low charge transfer. The clinic-ready bionics-derived CC-GET device achieved neurotrophin-encoding miniplasmid DNA delivery to the cochlea to promote auditory nerve regeneration; validated in deafened guinea pig and cat models, leading to improved central auditory tuning with bionics-based hearing. The performance of CC-GET is evaluated in the brain, an organ problematic for pulsed electric field-based plasmid DNA delivery, due to high required currents causing Joule-heating and damaging electroporation. Here CC-GET enables safe precision targeting of gene expression. In the guinea pig, reporter expression is enabled in physiologically critical brainstem regions, and in the striatum (globus pallidus region) delivery of a red-shifted channelrhodopsin and a genetically-encoded Ca2+ sensor, achieved photoactivated neuromodulation relevant to the treatment of Parkinson's Disease and other focal brain disorders.

TRPC Channels Activated by G Protein-Coupled Receptors Drive Ca2+ Dysregulation Leading to Secondary Brain Injury in the Mouse Model.

Canonical transient receptor potential (TRPC) non-selective cation channels, particularly those assembled with TRPC3, TRPC6, and TRPC7 subunits, are coupled to Gαq-type G protein-coupled receptors for the major classes of excitatory neurotransmitters. Sustained activation of this TRPC channel-based pathophysiological signaling hub in neurons and glia likely contributes to prodigious excitotoxicity-driven secondary brain injury expansion. This was investigated in mouse models with selective Trpc gene knockout (KO). In adult cerebellar brain slices, application of glutamate and the class I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine to Purkinje neurons expressing the GCaMP5g Ca2+ reporter demonstrated that the majority of the Ca2+ loading in the molecular layer dendritic arbors was attributable to the TRPC3 effector channels (Trpc3KO compared with wildtype (WT)). This Ca2+ dysregulation was associated with glutamate excitotoxicity causing progressive disruption of the Purkinje cell dendrites (significantly abated in a GAD67-GFP-Trpc3KO reporter brain slice model). Contribution of the Gαq-coupled TRPC channels to secondary brain injury was evaluated in a dual photothrombotic focal ischemic injury model targeting cerebellar and cerebral cortex regions, comparing day 4 post-injury in WT mice, Trpc3KO, and Trpc1/3/6/7 quadruple knockout (TrpcQKO), with immediate 2-h (primary) brain injury. Neuroprotection to secondary brain injury was afforded in both brain regions by Trpc3KO and TrpcQKO models, with the TrpcQKO showing greatest neuroprotection. These findings demonstrate the contribution of the Gαq-coupled TRPC effector mechanism to excitotoxicity-based secondary brain injury expansion, which is a primary driver for mortality and morbidity in stroke, traumatic brain injury, and epilepsy.

Dual-function AAV gene therapy reverses late-stage Canavan disease pathology in mice.

The leukodystrophy Canavan disease is a fatal white matter disorder caused by loss-of-function mutations of the aspartoacylase-encoding ASPA gene. There are no effective treatments available and experimental gene therapy trials have failed to provide sufficient amelioration from Canavan disease symptoms. Preclinical studies suggest that Canavan disease-like pathology can be addressed by either ASPA gene replacement therapy or by lowering the expression of the N-acetyl-L-aspartate synthesizing enzyme NAT8L. Both approaches individually prevent or even reverse pathological aspects in Canavan disease mice. Here, we combined both strategies and assessed whether intracranial adeno-associated virus-mediated gene delivery to a Canavan disease mouse model at 12 weeks allows for reversal of existing pathology. This was enabled by a single vector dual-function approach. In vitro and in vivo biopotency assessment revealed significant knockdown of neuronal Nat8l paired with robust ectopic aspartoacylase expression. Following nomination of the most efficient cassette designs, we performed proof-of-concept studies in post-symptomatic Aspa-null mice. Late-stage gene therapy resulted in a decrease of brain vacuoles and long-term reversal of all pathological hallmarks, including loss of body weight, locomotor impairments, elevated N-acetyl-L-aspartate levels, astrogliosis, and demyelination. These data suggest feasibility of a dual-function vector combination therapy, directed at replacing aspartoacylase with concomitantly suppressing N-acetyl-L-aspartate production, which holds potential to permanently alleviate Canavan disease symptoms and expands the therapeutic window towards a treatment option for adult subjects.

Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice.

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the 'cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, "contralateral suppression" of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24-32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.

Emerging Concepts in Vector Development for Glial Gene Therapy: Implications for Leukodystrophies.

Central Nervous System (CNS) homeostasis and function rely on intercellular synchronization of metabolic pathways. Developmental and neurochemical imbalances arising from mutations are frequently associated with devastating and often intractable neurological dysfunction. In the absence of pharmacological treatment options, but with knowledge of the genetic cause underlying the pathophysiology, gene therapy holds promise for disease control. Consideration of leukodystrophies provide a case in point; we review cell type - specific expression pattern of the disease - causing genes and reflect on genetic and cellular treatment approaches including ex vivo hematopoietic stem cell gene therapies and in vivo approaches using adeno-associated virus (AAV) vectors. We link recent advances in vectorology to glial targeting directed towards gene therapies for specific leukodystrophies and related developmental or neurometabolic disorders affecting the CNS white matter and frame strategies for therapy development in future.

Transgenic Analyses of Homer2 Function Within Nucleus Accumbens Subregions in the Regulation of Methamphetamine Reward and Reinforcement in Mice.

Problems associated with the abuse of amphetamine-type stimulants, including methamphetamine (MA), pose serious health and socioeconomic issues world-wide. While it is well-established that MA's psychopharmacological effects involve interactions with monoamine neurotransmission, accumulating evidence from animal models implicates dysregulated glutamate in MA addiction vulnerability and use disorder. Recently, we discovered an association between genetic vulnerability to MA-taking and increased expression of the glutamate receptor scaffolding protein Homer2 within both the shell and core subregions of the nucleus accumbens (NAC) and demonstrated a necessary role for Homer2 within the shell subregion in MA reward and reinforcement in mice. This report extends our earlier work by interrogating the functional relevance of Homer2 within the NAC core for the conditioned rewarding and reinforcing properties of MA. C57BL/6J mice with a virus-mediated knockdown of Homer2b expression within the NAC core were first tested for the development and expression of a MA-induced conditioned place-preference/CPP (four pairings of 2 mg/kg MA) and then were trained to self-administer oral MA under operant-conditioning procedures (5-80 mg/L). Homer2b knockdown in the NAC core augmented a MA-CPP and shifted the dose-response function for MA-reinforced responding, above control levels. To determine whether Homer2b within NAC subregions played an active role in regulating MA reward and reinforcement, we characterized the MA phenotype of constitutive Homer2 knockout (KO) mice and then assayed the effects of virus-mediated overexpression of Homer2b within the NAC shell and core of wild-type and KO mice. In line with the results of NAC core knockdown, Homer2 deletion potentiated MA-induced CPP, MA-reinforced responding and intake, as well as both cue- and MA-primed reinstatement of MA-seeking following extinction. However, there was no effect of Homer2b overexpression within the NAC core or the shell on the KO phenotype. These data provide new evidence indicating a globally suppressive role for Homer2 in MA-seeking and MA-taking but argue against specific NAC subregions as the neural loci through which Homer2 actively regulates MA addiction-related behaviors.

DKC1 is a transcriptional target of GATA1 and drives upregulation of telomerase activity in normal human erythroblasts.

Telomerase is a ribonucleoprotein complex that maintains the length and integrity of telomeres, and thereby enables cellular proliferation. Understanding the regulation of telomerase in hematopoietic cells is relevant to the pathogenesis of leukemia, in which telomerase is constitutively activated, as well as bone marrow failure syndromes that feature telomerase insufficiency. Past studies showing high levels of telomerase in human erythroblasts and a prevalence of anemia in disorders of telomerase insufficiency provide the rationale for investigating telomerase regulation in erythroid cells. Here it is shown for the first time that the telomerase RNA-binding protein dyskerin (encoded by DKC1) is dramatically upregulated as human hematopoietic stem and progenitor cells commit to the erythroid lineage, driving an increase in telomerase activity in the presence of limiting amounts of TERT mRNA. It is also shown that upregulation of DKC1 was necessary for expansion of glycophorin A+ erythroblasts and sufficient to extend telomeres in erythroleukemia cells. Chromatin immunoprecipitation and reporter assays implicated GATA1-mediated transcriptional regulation of DKC1 in the modulation of telomerase in erythroid lineage cells. Together these results describe a novel mechanism of telomerase regulation in erythroid cells which contrasts with mechanisms centered on transcriptional regulation of TERT that are known to operate in other cell types. This is the first study to reveal a biological context in which telomerase is upregulated by DKC1 and to implicate GATA1 in telomerase regulation. The results from this study are relevant to hematopoietic disorders involving DKC1 mutations, GATA1 deregulation and/or telomerase insufficiency.

Neurotrophin gene augmentation by electrotransfer to improve cochlear implant hearing outcomes.

This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed 'close-field electroporation'. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4; plasmid Free of Antibiotic Resistance v.4) which could be used to package a 'humanized' neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies.

Human Brain Region-Specific Alternative Splicing of TRPC3, the Type 3 Canonical Transient Receptor Potential Non-Selective Cation Channel.

Canonical transient receptor potential (TRPC) non-selective cation channels are broadly expressed by neurons, glia and the microvasculature of the brain. In neurons and astrocytes, these ion channels are coupled to group I metabotropic glutamate receptors via Gαq-phospholipase C signal transduction. In the mouse cerebellar Purkinje neurons, TRPC channels assembled as tetramers of TRPC3 subunits exclusively mediate this glutamatergic signalling mechanism and regulation of alternative splicing results in dominance of a high Ca2+ conducting TRPC3c isoform. This regional control of TRPC3 transcript type likely has physiological and pathophysiological sequelae. The current study provides a quantitative comparison of the TRPC3c splice variant and the TRPC3b full-length isoform expression across seven regions of the human brain. This shows that the cerebellum has the highest expression level of both isoforms and that regulation of alternative splicing results in a higher propensity of the TRPC3c isoform in the cerebellum relative to the TRPC3b isoform (in a 1:3 ratio). This compares with the other regions (motor cortex, hippocampus, midbrain subregions, pons and medulla) where the prevalence of TRPC3c relative to TRPC3b is typically less than half as abundant. The finding here of a bias in the high-conductance TRPC3c isoform in the cerebellum is consistent with the enhanced vulnerability of the cerebellum to ischaemic injury.

Increased Alcohol-Drinking Induced by Manipulations of mGlu5 Phosphorylation within the Bed Nucleus of the Stria Terminalis.

The bed nucleus of the stria terminalis (BNST) is part of the limbic-hypothalamic system important for behavioral responses to stress, and glutamate transmission within this region has been implicated in the neurobiology of alcoholism. Herein, we used a combination of immunoblotting, neuropharmacological and transgenic procedures to investigate the role for metabotropic glutamate receptor 5 (mGlu5) signaling within the BNST in excessive drinking. We discovered that mGlu5 signaling in the BNST is linked to excessive alcohol consumption in a manner distinct from behavioral or neuropharmacological endophenotypes that have been previously implicated as triggers for heavy drinking. Our studies demonstrate that, in male mice, a history of chronic binge alcohol-drinking elevates BNST levels of the mGlu5-scaffolding protein Homer2 and activated extracellular signal-regulated kinase (ERK) in an adaptive response to limit alcohol consumption. Male and female transgenic mice expressing a point mutation of mGlu5 that cannot be phosphorylated by ERK exhibit excessive alcohol-drinking, despite greater behavioral signs of alcohol intoxication and reduced anxiety, and are insensitive to local manipulations of signaling in the BNST. These transgenic mice also show selective insensitivity to alcohol-aversion and increased novelty-seeking, which may be relevant to excessive drinking. Further, the insensitivity to alcohol-aversion exhibited by male mice can be mimicked by the local inhibition of ERK signaling within the BNST. Our findings elucidate a novel mGluR5-linked signaling state within BNST that plays a central and unanticipated role in excessive alcohol consumption.SIGNIFICANCE STATEMENT The bed nucleus of the stria terminalis (BNST) is part of the limbic-hypothalamic system important for behavioral responses to stress and alcohol, and glutamate transmission within BNST is implicated in the neurobiology of alcoholism. The present study provides evidence that a history of excessive alcohol drinking increases signaling through the metabotropic glutamate receptor 5 (mGlu5) receptor within the BNST in an adaptive response to limit alcohol consumption. In particular, disruption of mGlu5 phosphorylation by extracellular signal-regulated kinase within this brain region induces excessive alcohol-drinking, which reflects a selective insensitivity to the aversive properties of alcohol intoxication. These data indicate that a specific signaling state of mGlu5 within BNST plays a central and unanticipated role in excessive alcohol consumption.

Expression Pattern of the Aspartyl-tRNA Synthetase DARS in the Human Brain.

Translation of mRNA into protein is an evolutionarily conserved, fundamental process of life. A prerequisite for translation is the accurate charging of tRNAs with their cognate amino acids, a reaction catalyzed by specific aminoacyl-tRNA synthetases. One of these enzymes is the aspartyl-tRNA synthetase DARS, which pairs aspartate with its corresponding tRNA. Missense mutations of the gene encoding DARS result in the leukodystrophy hypomyelination with brainstem and spinal cord involvement and leg spasticity (HBSL) with a distinct pattern of hypomyelination, motor abnormalities, and cognitive impairment. A thorough understanding of the DARS expression domains in the central nervous system is essential for the development of targeted therapies to treat HBSL. Here, we analyzed endogenous DARS expression on the mRNA and protein level in different brain regions and cell types of human post mortem brain tissue as well as in human stem cell derived neurons, oligodendrocytes, and astrocytes. DARS expression is significantly enriched in the cerebellum, a region affected in HBSL patients and important for motor control. Although obligatorily expressed in all cells, DARS shows a distinct expression pattern with enrichment in neurons but only low abundance in oligodendrocytes, astrocytes, and microglia. Our results reveal little homogeneity across the different cell types, largely matching previously published data in the murine brain. This human gene expression study will significantly contribute to the understanding of DARS gene function and HBSL pathology and will be instrumental for future development of animal models and targeted therapies. In particular, we anticipate high benefit from a gene replacement approach in neurons of HBSL mouse models, given the abundant endogenous DARS expression in this lineage cell.

Gene therapy mediated seizure suppression in Genetic Generalised Epilepsy: Neuropeptide Y overexpression in a rat model.

Neuropeptide Y (NPY) is an important 36 amino acid peptide that is abundantly expressed in the mammalian CNS and is known to be an endogenous modulator of seizure activity, including in rat models of Genetic Generalised Epilepsy (GGE) with absence seizures. Studies have shown that viral-mediated "gene therapy" with overexpression of NPY in the hippocampus can suppress seizures in acquired epilepsy animal models. This study investigated whether NPY gene delivery to the thalamus or somatosensory cortex, using recombinant adeno-associated viral vector (rAAV), could produce sustained seizure suppression in the GAERS model of GGE with absence seizures. Three cohorts of GAERS were injected bilaterally into the thalamus (short term n = 14 and long term n = 8) or the somatosensory cortex (n = 26) with rAAV-NPY or rAAV-empty. EEG recordings were acquired weekly post-treatment and seizure expression was quantified. Anxiety levels were tested using elevated plus maze and open field test. NPY and NPY receptor mRNA and protein expression were evaluated using quantitative PCR, immunohistochemistry and immunofluorescence. Viral overexpression of human NPY in the thalamus and somatosensory cortex in GAERS significantly reduced the time spent in seizure activity and number of seizures, whereas seizure duration was only reduced after thalamic NPY overexpression. Human and rat NPY and rat Y2 receptor mRNA expression was significantly increased in the somatosensory cortex. NPY overexpression in the thalamus was observed in rAAV-NPY treated rats compared to controls in the long term cohort. No effect was observed on anxiety behaviour. We conclude that virally-mediated human NPY overexpression in the thalamus or somatosensory cortex produces sustained anti-epileptic effects in GAERS. NPY gene therapy may represent a novel approach for the treatment of patients with genetic generalised epilepsies.

Uncoupling N-acetylaspartate from brain pathology: implications for Canavan disease gene therapy.

N-Acetylaspartate (NAA) is the second most abundant organic metabolite in the brain, but its physiological significance remains enigmatic. Toxic NAA accumulation appears to be the key factor for neurological decline in Canavan disease-a fatal neurometabolic disorder caused by deficiency in the NAA-degrading enzyme aspartoacylase. To date clinical outcome of gene replacement therapy for this spongiform leukodystrophy has not met expectations. To identify the target tissue and cells for maximum anticipated treatment benefit, we employed comprehensive phenotyping of novel mouse models to assess cell type-specific consequences of NAA depletion or elevation. We show that NAA-deficiency causes neurological deficits affecting unconscious defensive reactions aimed at protecting the body from external threat. This finding suggests, while NAA reduction is pivotal to treat Canavan disease, abrogating NAA synthesis should be avoided. At the other end of the spectrum, while predicting pathological severity in Canavan disease mice, increased brain NAA levels are not neurotoxic per se. In fact, in transgenic mice overexpressing the NAA synthesising enzyme Nat8l in neurons, supra-physiological NAA levels were uncoupled from neurological deficits. In contrast, elimination of aspartoacylase expression exclusively in oligodendrocytes elicited Canavan disease like pathology. Although conditional aspartoacylase deletion in oligodendrocytes abolished expression in the entire CNS, the remaining aspartoacylase in peripheral organs was sufficient to lower NAA levels, delay disease onset and ameliorate histopathology. However, comparable endpoints of the conditional and complete aspartoacylase knockout indicate that optimal Canavan disease gene replacement therapies should restore aspartoacylase expression in oligodendrocytes. On the basis of these findings we executed an ASPA gene replacement therapy targeting oligodendrocytes in Canavan disease mice resulting in reversal of pre-existing CNS pathology and lasting neurological benefits. This finding signifies the first successful post-symptomatic treatment of a white matter disorder using an adeno-associated virus vector tailored towards oligodendroglial-restricted transgene expression.

Behavioral and Neurochemical Phenotyping of Mice Incapable of Homer1a Induction.

Immediate early and constitutively expressed products of the Homer1 gene regulate the functional assembly of post-synaptic density proteins at glutamatergic synapses to influence excitatory neurotransmission and synaptic plasticity. Earlier studies of Homer1 gene knock-out (KO) mice indicated active, but distinct, roles for IEG and constitutively expressed Homer1 gene products in regulating cognitive, emotional, motivational and sensorimotor processing, as well as behavioral and neurochemical sensitivity to cocaine. More recent characterization of transgenic mice engineered to prevent generation of the IEG form (a.k.a Homer1a KO) pose a critical role for Homer1a in cocaine-induced behavioral and neurochemical sensitization of relevance to drug addiction and related neuropsychiatric disorders. Here, we extend our characterization of the Homer1a KO mouse and report a modest pro-depressant phenotype, but no deleterious effects of the KO upon spatial learning/memory, prepulse inhibition, or cocaine-induced place-conditioning. As we reported previously, Homer1a KO mice did not develop cocaine-induced behavioral or neurochemical sensitization within the nucleus accumbens; however, virus-mediated Homer1a over-expression within the nucleus accumbens reversed the sensitization phenotype of KO mice. We also report several neurochemical abnormalities within the nucleus accumbens of Homer1a KO mice that include: elevated basal dopamine and reduced basal glutamate content, Group1 mGluR agonist-induced glutamate release and high K+-stimulated release of dopamine and glutamate within this region. Many of the neurochemical anomalies exhibited by Homer1a KO mice are recapitulated upon deletion of the entire Homer1 gene; however, Homer1 deletion did not affect NAC dopamine or alter K+-stimulated neurotransmitter release within this region. These data show that the selective deletion of Homer1a produces a behavioral and neurochemical phenotype that is distinguishable from that produced by deletion of the entire Homer1 gene. Moreover, the data indicate a specific role for Homer1a in regulating cocaine-induced behavioral and neurochemical sensitization of potential relevance to the psychotogenic properties of this drug.

Gene therapy targeting oligodendrocytes provides therapeutic benefit in a leukodystrophy model.

Pelizaeus-Merzbacher-like disease or hypomyelinating leukodystrophy-2 is an autosomal recessively inherited leukodystrophy with childhood onset resulting from mutations in the gene encoding the gap junction protein connexin 47 (Cx47, encoded by GJC2). Cx47 is expressed specifically in oligodendrocytes and is crucial for gap junctional communication throughout the central nervous system. Previous studies confirmed that a cell autonomous loss-of-function mechanism underlies hypomyelinating leukodystrophy-2 and that transgenic oligodendrocyte-specific expression of another connexin, Cx32 (GJB1), can restore gap junctions in oligodendrocytes to achieve correction of the pathology in a disease model. To develop an oligodendrocyte-targeted gene therapy, we cloned the GJC2/Cx47 gene under the myelin basic protein promoter and used an adeno-associated viral vector (AAV.MBP.Cx47myc) to deliver the gene to postnatal Day 10 mice via a single intracerebral injection in the internal capsule area. Lasting Cx47 expression specifically in oligodendrocytes was detected in Cx47 single knockout and Cx32/Cx47 double knockout mice up to 12 weeks post-injection, including the corpus callosum and the internal capsule but also in more distant areas of the cerebrum and in the spinal cord. Application of this oligodendrocyte-targeted somatic gene therapy at postnatal Day 10 in groups of double knockout mice, a well characterized model of hypomyelinating leukodystrophy-2, resulted in significant improvement in motor performance and coordination at 1 month of age in treated compared to mock-treated mice, as well as prolonged survival. Furthermore, immunofluorescence and morphological analysis revealed improvement in demyelination, oligodendrocyte apoptosis, inflammation, and astrogliosis, all typical features of this leukodystrophy model in both brain and spinal cord. Functional dye transfer analysis confirmed the re-establishment of oligodendrocyte gap junctional connectivity in treated as opposed to untreated mice. These results provide a significant advance in the development of oligodendrocyte-cell specific gene therapy. Adeno-associated viral vectors can be used to target therapeutic expression of a myelin gene to oligodendrocytes. We show evidence for the first somatic gene therapy approach to treat hypomyelinating leukodystrophy-2 preclinically, providing a potential treatment for this and similar forms of leukodystrophies.

In vivocharacterization of the aspartyl-tRNA synthetase DARS: Homing in on the leukodystrophy HBSL.

BACKGROUND: The recently diagnosed leukodystrophy Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity (HBSL) is caused by mutations of the cytoplasmic aspartyl-tRNA synthetase geneDARS. The physiological role of DARS in translation is to accurately pair aspartate with its cognate tRNA. Clinically, HBSL subjects show a distinct pattern of hypomyelination and develop progressive leg spasticity, variable cognitive impairment and epilepsy. To elucidate the underlying pathomechanism, we comprehensively assessed endogenous DARS expression in mice. Additionally, aiming at creating the first mammalian HBSL model, we genetically engineered and phenotyped mutant mice with a targetedDarslocus. RESULTS: DARS, although expressed in all organs, shows a distinct expression pattern in the adult brain with little immunoreactivity in macroglia but enrichment in neuronal subpopulations of the hippocampus, cerebellum, and cortex. Within neurons, DARS is mainly located in the cell soma where it co-localizes with other components of the translation machinery. Intriguingly, DARS is also present along neurites and at synapses, where it potentially contributes to local protein synthesis.Dars-null mice are not viable and die before embryonic day 11. Heterozygous mice with only one functionalDarsallele display substantially reduced DARS levels in the brain; yet these mutants show no gross abnormalities, including unchanged motor performance. However, we detected reduced pre-pulse inhibition of the acoustic startle response indicating dysfunction of attentional processing inDars+/-mice. CONCLUSIONS: Our results, for the first time, show an in-depth characterization of the DARS tissue distribution in mice, revealing surprisingly little uniformity across brain regions or between the major neural cell types. The complete loss of DARS function is not tolerated in mice suggesting that the identified HBSL mutations in humans retain some residual enzyme activity. The mild phenotype of heterozygousDars-null carriers indicates that even partial restoration of DARS levels would be therapeutically relevant. Despite the fact that they do not resemble the full spectrum of clinical symptoms, the robust pre-pulse inhibition phenotype ofDars+/-mice will be instrumental for future preclinical therapeutic efficacy studies. In summary, our data is an important contribution to a better understanding of DARS function and HBSL pathology.

Methamphetamine Addiction Vulnerability: The Glutamate, the Bad, and the Ugly.

BACKGROUND: The high prevalence and severity of methamphetamine (MA) abuse demands greater neurobiological understanding of its etiology. METHODS: We conducted immunoblotting and in vivo microdialysis procedures in MA high/low drinking mice, as well as in isogenic C57BL/6J mice that varied in their MA preference/taking, to examine the glutamate underpinnings of MA abuse vulnerability. Neuropharmacological and Homer2 knockdown approaches were also used in C57BL/6J mice to confirm the role for nucleus accumbens (NAC) glutamate/Homer2 expression in MA preference/aversion. RESULTS: We identified a hyperglutamatergic state within the NAC as a biochemical trait corresponding with both genetic and idiopathic vulnerability for high MA preference and taking. We also confirmed that subchronic subtoxic MA experience elicits a hyperglutamatergic state within the NAC during protracted withdrawal, characterized by elevated metabotropic glutamate 1/5 receptor function and Homer2 receptor-scaffolding protein expression. A high MA-preferring phenotype was recapitulated by elevating endogenous glutamate within the NAC shell of mice and we reversed MA preference/taking by lowering endogenous glutamate and/or Homer2 expression within this subregion. CONCLUSIONS: Our data point to an idiopathic, genetic, or drug-induced hyperglutamatergic state within the NAC as a mediator of MA addiction vulnerability.

MYC-Driven Neuroblastomas Are Addicted to a Telomerase-Independent Function of Dyskerin.

The RNA-binding protein dyskerin, encoded by the DKC1 gene, functions as a core component of the telomerase holoenzyme as well as ribonuclear protein complexes involved in RNA processing and ribosome biogenesis. The diverse roles of dyskerin across many facets of RNA biology implicate its potential contribution to malignancy. In this study, we examined the expression and function of dyskerin in neuroblastoma. We show that DKC1 mRNA levels were elevated relative to normal cells across a panel of 15 neuroblastoma cell lines, where both N-Myc and c-Myc directly targeted the DKC1 promoter. Upregulation of MYCN was shown to dramatically increase DKC1 expression. In two independent neuroblastoma patient cohorts, high DKC1 expression correlated strongly with poor event-free and overall survival (P < 0.0001), independently of established prognostic factors. RNAi-mediated depletion of dyskerin inhibited neuroblastoma cell proliferation, including cells immortalized via the telomerase-independent ALT mechanism. Furthermore, dyskerin attenuation impaired anchorage-independent proliferation and tumor growth. Overexpression of the telomerase RNA component, hTR, demonstrated that this proliferative impairment was not a consequence of telomerase suppression. Instead, ribosomal stress, evidenced by depletion of small nucleolar RNAs and nuclear dispersal of ribosomal proteins, was the likely cause of the proliferative impairment in dyskerin-depleted cells. Accordingly, dyskerin suppression caused p53-dependent G1 cell-cycle arrest in p53 wild-type cells, and a p53-independent pathway impaired proliferation in cells with p53 dysfunction. Together, our findings highlight dyskerin as a new therapeutic target in neuroblastoma with crucial telomerase-independent functions and broader implications for the spectrum of malignancies driven by MYC family oncogenes. Cancer Res; 76(12); 3604-17. ©2016 AACR.

Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes.

Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene expression (>90%) in vivo. Moreover, cy5-MAG2.2-GFP delivery to the neonate CNS resulted in selective GFP expression in oligodendrocytes for at least 8 months. Broadly, the characterization of the extremely short yet oligodendrocyte-specific human MAG promoter may facilitate modeling neurological diseases caused by oligodendrocyte pathology and has translational relevance for leukodystrophy gene therapy.

Septal Glucagon-Like Peptide 1 Receptor Expression Determines Suppression of Cocaine-Induced Behavior.

Glucagon-like peptide 1 (GLP-1) and its receptor GLP-1R are a key component of the satiety signaling system, and long-acting GLP-1 analogs have been approved for the treatment of type-2 diabetes mellitus. Previous reports demonstrate that GLP-1 regulates glucose homeostasis alongside the rewarding effects of food. Both palatable food and illicit drugs activate brain reward circuitries, and pharmacological studies suggest that central nervous system GLP-1 signaling holds potential for the treatment of addiction. However, the role of endogenous GLP-1 in the attenuation of reward-oriented behavior, and the essential domains of the mesolimbic system mediating these beneficial effects, are largely unknown. We hypothesized that the central regions of highest Glp-1r gene activity are essential in mediating responses to drugs of abuse. Here, we show that Glp-1r-deficient (Glp-1r(-/-)) mice have greatly augmented cocaine-induced locomotor responses and enhanced conditional place preference compared with wild-type (Glp-1r(+/+)) controls. Employing mRNA in situ hybridization we located peak Glp-1r mRNA expression in GABAergic neurons of the dorsal lateral septum, an anatomical site with a crucial function in reward perception. Whole-cell patch-clamp recordings of dorsal lateral septum neurons revealed that genetic Glp-1r ablation leads to increased excitability of these cells. Viral vector-mediated Glp-1r gene delivery to the dorsal lateral septum of Glp-1r(-/-) animals reduced cocaine-induced locomotion and conditional place preference to wild-type levels. This site-specific genetic complementation did not affect the anxiogenic phenotype observed in Glp-1r(-/-) controls. These data reveal a novel role of GLP-1R in dorsal lateral septum function driving behavioral responses to cocaine.