Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Intravenous functional gene transfer throughout the brain of non-human primates using AAV.

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.

AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset.

Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood-brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

Unique molecular signature in mucolipidosis type IV microglia.

BACKGROUND: Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease. METHODS: We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1-/-) and Fabry disease (Glay/-) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses. RESULTS: We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1-/- microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1-/- microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer's disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in "disease-associated microglia" pattern among these diseases. CONCLUSIONS: The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01067742, registered on February 12, 2010.

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

Brain Insulin-Like Growth Factor-I Directs the Transition from Stem Cells to Mature Neurons During Postnatal/Adult Hippocampal Neurogenesis.

The specific actions of insulin-like growth factor-I (IGF-I) and the role of brain-derived IGF-I during hippocampal neurogenesis have not been fully defined. To address the influence of IGF-I on the stages of hippocampal neurogenesis, we studied a postnatal/adult global Igf-I knockout (KO) mice (Igf-I(-/-) ) and a nervous system Igf-I conditional KO (Igf-I(Δ/Δ) ). In both KO mice we found an accumulation of Tbr2(+) -intermediate neuronal progenitors, some of which were displaced in the outer granule cell layer (GCL) and the molecular layer (ML) of the dentate gyrus (DG). Similarly, more ectopic Ki67(+) - cycling cells were detected. Thus, the GCL was disorganized with significant numbers of Prox1(+) -granule neurons outside this layer and altered morphology of radial glial cells (RGCs). Dividing progenitors were also generated in greater numbers in clonal hippocampal stem cell (HPSC) cultures from the KO mice. Indeed, higher levels of Hes5 and Ngn2, transcription factors that maintain the stem and progenitor cell state, were expressed in both HPSCs and the GCL-ML from the Igf-I(Δ/Δ) mice. To determine the impact of Igf-I deletion on neuronal generation in vivo, progenitors in Igf-I(-/-) and Igf-I(+/+) mice were labeled with a GFP-expressing vector. This revealed that in the Igf-I(-/-) mice more GFP(+) -immature neurons were formed and they had less complex dendritic trees. These findings indicate that local IGF-I plays critical roles during postnatal/adult hippocampal neurogenesis, regulating the transition from HPSCs and progenitors to mature granule neurons in a cell stage-dependent manner. Stem Cells 2016;34:2194-2209.

Intratracheal exposure of common marmosets to MERS-CoV Jordan-n3/2012 or MERS-CoV EMC/2012 isolates does not result in lethal disease.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) continues to be a threat to human health in the Middle East. Development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. A recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. Based on these data we sought to compare two isolates of MERS-CoV. We followed disease progression in common marmosets after intratracheal exposure with: MERS-CoV-EMC/2012, MERS-CoV-Jordan-n3/2012, media, or inactivated virus. Our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (CT), with limited other clinical signs. Based on CT data, clinical data, and virological data, MERS-CoV inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication.

Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse behavior.

Local translation of nuclear-encoded mitochondrial mRNAs is essential for mitochondrial activity, yet there is little insight into the role that axonal trafficking of these transcripts play in neuronal function and behavior. Previously, we identified a 38 nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the Cytochrome C oxidase IV (COXIV) mRNA that directs the transport of a reporter mRNA to the axon of superior cervical ganglion neurons (SCG). Overexpression of a chimeric reporter mRNA with the COXIV zipcode competed with the axonal trafficking of endogenous COXIV mRNA, and led to attenuated axon growth in SCG neurons. Here, we show that exogenous expression of the COXIV zipcode in cultured SCG neurons also results in the reduction of local ATP levels and increases levels of reactive oxygen species (ROS) in the axon. We took advantage of this "competition" phenotype to investigate the in vivo significance of axonal transport of COXIV mRNA. Toward this end, we generated transgenic mice expressing a fluorescent reporter fused to COXIV zipcode under a forebrain-specific promoter. Immunohistological analyses and RT-PCR analyses of RNA from the transgenic mouse brain showed expression of the reporter in the deep layer neurons in the pre-frontal and frontal cortex. Consistent with the in vitro studies, we observed increased ROS levels in neurons of these transgenic animals. A battery of behavioral tests on transgenic mice expressing the COXIV zipcode revealed an "anxiety-like" behavioral phenotype, suggesting an important role for axonal trafficking of nuclear-encoded mitochondrial mRNAs in neuronal physiology and animal behavior.

Medial prefrontal cortex neuronal activation and synaptic alterations after stress-induced reinstatement of palatable food seeking: a study using c-fos-GFP transgenic female rats.

Relapse to maladaptive eating habits during dieting is often provoked by stress and there is evidence for a role of ovarian hormones in stress responses and feeding. We studied the role of these hormones in stress-induced reinstatement of food seeking and medial prefrontal cortex (mPFC) neuronal activation in c-fos-GFP transgenic female rats, which express GFP in strongly activated neurons. Food-restricted ovariectomized or sham-operated c-fos-GFP rats were trained to lever-press for palatable food pellets. Subsequently, lever-pressing was extinguished and reinstatement of food seeking and mPFC neuronal activation was assessed after injections of the pharmacological stressor yohimbine (0.5-2 mg/kg) or pellet priming (1-4 noncontingent pellets). Estrous cycle effects on reinstatement were also assessed in wild-type rats. Yohimbine- and pellet-priming-induced reinstatement was associated with Fos and GFP induction in mPFC; both reinstatement and neuronal activation were minimally affected by ovarian hormones in both c-fos-GFP and wild-type rats. c-fos-GFP transgenic rats were then used to assess glutamatergic synaptic alterations within activated GFP-positive and nonactivated GFP-negative mPFC neurons following yohimbine-induced reinstatement of food seeking. This reinstatement was associated with reduced AMPA receptor/NMDA receptor current ratios and increased paired-pulse facilitation in activated GFP-positive but not GFP-negative neurons. While ovarian hormones do not appear to play a role in stress-induced relapse of food seeking in our rat model, this reinstatement was associated with unique synaptic alterations in strongly activated mPFC neurons. Our paper introduces the c-fos-GFP transgenic rat as a new tool to study unique synaptic changes in activated neurons during behavior.

In vivo role of alternative splicing and serine phosphorylation of the microphthalmia-associated transcription factor.

The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper protein that plays major roles in the development and physiology of vertebrate melanocytes and melanoma cells. It is regulated by post-translational modifications, including phosphorylation at serine 73, which based on in vitro experiments imparts on MITF an increased transcriptional activity paired with a decreased stability. Serine 73 is encoded by the alternatively spliced exon 2B, which is preferentially skipped in mice carrying a targeted serine-73-to-alanine mutation. Here, we measured the relative abundance of exon 2B+ and exon 2B- RNAs in freshly isolated and FACS-sorted wild-type melanoblasts and melanocytes and generated a series of knock-in mice allowing forced incorporation of either alanine, aspartate, or wild-type serine at position 73. None of these knock-in alleles, however, creates a striking pigmentation phenotype on its own, but differences between them can be revealed either by a general reduction of Mitf transcript levels or in heteroallelic combinations with extant Mitf mutations. In fact, compared with straight serine-73 knock-in mice with their relative reduction of 2B+ Mitf, forced incorporation of alanine 73 leads to greater increases in MITF protein levels, melanoblast and melanocyte numbers, and extent of pigmentation in particular allelic combinations. These results underscore, in vivo, the importance of the link between alternative splicing and post-translational modifications and may bear on the recent observation that exon 2B skipping can be found in metastatic melanoma.

Hepatic cannabinoid receptor-1 mediates diet-induced insulin resistance via inhibition of insulin signaling and clearance in mice.

BACKGROUND & AIMS: Obesity-related insulin resistance contributes to cardiovascular disease. Cannabinoid receptor-1 (CB(1)) blockade improves insulin sensitivity in obese animals and people, suggesting endocannabinoid involvement. We explored the role of hepatic CB(1) in insulin resistance and inhibition of insulin signaling pathways. METHODS: Wild-type mice and mice with disruption of CB(1) (CB(1)(-/-) mice) or with hepatocyte-specific deletion or transgenic overexpression of CB(1) were maintained on regular chow or a high-fat diet (HFD) to induce obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp analysis was used to analyze the role of the liver and hepatic CB(1) in HFD-induced insulin resistance. The cellular mechanisms of insulin resistance were analyzed in mouse and human isolated hepatocytes using small interfering or short hairpin RNAs and lentiviral knockdown of gene expression. RESULTS: The HFD induced hepatic insulin resistance in wild-type mice, but not in CB(1)(-/-) mice or mice with hepatocyte-specific deletion of CB(1). CB(1)(-/-) mice that overexpressed CB(1) specifically in hepatocytes became hyperinsulinemic as a result of reduced insulin clearance due to down-regulation of the insulin-degrading enzyme. However, they had increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance; this was further increased by the HFD. In mice with hepatocytes that express CB(1), the HFD or CB(1) activation induced the endoplasmic reticulum stress response via activation of the Bip-PERK-eIF2α protein translation pathway. In hepatocytes isolated from human or mouse liver, CB(1) activation caused endoplasmic reticulum stress-dependent suppression of insulin-induced phosphorylation of akt-2 via phosphorylation of IRS1 at serine-307 and by inducing the expression of the serine and threonine phosphatase Phlpp1. Expression of CB(1) was up-regulated in samples from patients with nonalcoholic fatty liver disease. CONCLUSIONS: Endocannabinoids contribute to diet-induced insulin resistance in mice via hepatic CB(1)-mediated inhibition of insulin signaling and clearance.

Expression of the metabotropic glutamate receptor 5 (mGluR5) induces melanoma in transgenic mice.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS and mediates fast synaptic transmission upon activation of glutamate-gated ion channels. In addition, glutamate modulates a variety of other synaptic responses and intracellular signaling by activating metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors. The mGluRs are also expressed in nonneuronal tissues and are implicated in a variety of normal biological functions as well as diseases. To study mGluR-activated calcium signaling in neurons, we generated mGluR5 transgenic animals using a Thy1 promoter to drive expression in the forebrain, and one founder unexpectedly developed melanoma. To directly investigate the role of mGluR5 in melanoma formation, we generated mGluR5 transgenic lines under a melanocyte-specific promoter, tyrosinase-related protein 1. A majority of the founders showed a severe phenotype with early onset. Hyperpigmentation of the pinnae and tail could be detected as early as 3-5 d after birth for most of the mGluR5 transgene-positive mice. There was 100% penetrance in the progeny from the tyrosinase-related protein 1-mGluR5 lines generated from founders that developed melanoma. Expression of mGluR5 was detected in melanoma samples by RT-PCR, immunoblotting, and immunohistochemistry. We evaluated the expression of several cancer-related proteins in tumor samples and observed a dramatic increase in the phosphorylation of ERK, implicating ERK as a downstream effector of mGluR5 signaling in tumors. Our findings show that mGluR5-mediated glutamatergic signaling can trigger melanoma in vivo. The aggressive growth and severe phenotype make these mouse lines unique and a potentially powerful tool for therapeutic studies.

Adult hippocampal neurogenesis buffers stress responses and depressive behaviour.

Glucocorticoids are released in response to stressful experiences and serve many beneficial homeostatic functions. However, dysregulation of glucocorticoids is associated with cognitive impairments and depressive illness. In the hippocampus, a brain region densely populated with receptors for stress hormones, stress and glucocorticoids strongly inhibit adult neurogenesis. Decreased neurogenesis has been implicated in the pathogenesis of anxiety and depression, but direct evidence for this role is lacking. Here we show that adult-born hippocampal neurons are required for normal expression of the endocrine and behavioural components of the stress response. Using either transgenic or radiation methods to inhibit adult neurogenesis specifically, we find that glucocorticoid levels are slower to recover after moderate stress and are less suppressed by dexamethasone in neurogenesis-deficient mice than intact mice, consistent with a role for the hippocampus in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Relative to controls, neurogenesis-deficient mice also showed increased food avoidance in a novel environment after acute stress, increased behavioural despair in the forced swim test, and decreased sucrose preference, a measure of anhedonia. These findings identify a small subset of neurons within the dentate gyrus that are critical for hippocampal negative control of the HPA axis and support a direct role for adult neurogenesis in depressive illness.

eIF2alpha Phosphorylation-dependent translation in CA1 pyramidal cells impairs hippocampal memory consolidation without affecting general translation.

Protein synthesis inhibitor antibiotics are widely used to produce amnesia, and have been recognized to inhibit general or global mRNA translation in the basic translational machinery. For instance, anisomycin interferes with protein synthesis by inhibiting peptidyl transferase or the 80S ribosomal function. Therefore, de novo general or global protein synthesis has been thought to be necessary for long-term memory formation. However, it is unclear which mode of translation-gene-specific translation or general/global translation-is actually crucial for the memory consolidation process in mammalian brains. Here, we generated a conditional transgenic mouse strain in which double-strand RNA-dependent protein kinase (PKR)-mediated phosphorylation of eIF2alpha, a key translation initiation protein, was specifically increased in hippocampal CA1 pyramidal cells by the chemical inducer AP20187. Administration of AP20187 significantly increased activating transcription factor 4 (ATF4) translation and concomitantly suppressed CREB-dependent pathways in CA1 cells; this led to impaired hippocampal late-phase LTP and memory consolidation, with no obvious reduction in general translation. Conversely, inhibition of general translation by low-dose anisomycin failed to block hippocampal-dependent memory consolidation. Together, these results indicated that CA1-restricted genetic manipulation of particular mRNA translations is sufficient to impair the consolidation and that consolidation of memories in CA1 pyramidal cells through eIF2alpha dephosphorylation depends more on transcription/translation of particular genes than on overall levels of general translation. The present study sheds light on the critical importance of gene-specific translations for hippocampal memory consolidation.

CD45-positive blood cells give rise to uterine epithelial cells in mice.

The uterine endometrium is composed of epithelial and stromal cells, which undergo extensive degeneration and regeneration in every estrous cycle, and dramatic changes occur during pregnancy. The high turnover of cells requires a correspondingly high level of cell division by progenitor cells in the uterus, but the character and source of these cells remain obscure. In the present study, using a novel transgenic mouse, we showed that CD45-positive hematopoietic progenitor cells colonize the uterine epithelium and that in pregnancy more than 80% of the epithelium can derive from these cells. Since we also found green fluorescent protein (GFP)-positive uterine endothelial cells in long-term GFP bone marrow-transplanted mice, we conclude that circulating CD45+ cells play an important role in regenerating the uterine epithelium.