Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling.

The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5' end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.

Aß/Amyloid Precursor Protein-Induced Hyperexcitability and Dysregulation of Homeostatic Synaptic Plasticity in Neuron Models of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly seen as a disease of synapses and diverse evidence has implicated the amyloid-ß peptide (Aß) in synapse damage. The molecular and cellular mechanism(s) by which Aß and/or its precursor protein, the amyloid precursor protein (APP) can affect synapses remains unclear. Interestingly, early hyperexcitability has been described in human AD and mouse models of AD, which precedes later hypoactivity. Here we show that neurons in culture with either elevated levels of Aß or with human APP mutated to prevent Aß generation can both induce hyperactivity as detected by elevated calcium transient frequency and amplitude. Since homeostatic synaptic plasticity (HSP) mechanisms normally maintain a setpoint of activity, we examined whether HSP was altered in AD transgenic neurons. Using methods known to induce HSP, we demonstrate that APP protein levels are regulated by chronic modulation of activity and that AD transgenic neurons have an impaired adaptation of calcium transients to global changes in activity. Further, AD transgenic compared to WT neurons failed to adjust the length of their axon initial segments (AIS), an adaptation known to alter excitability. Thus, we show that both APP and Aß influence neuronal activity and that mechanisms of HSP are disrupted in primary neuron models of AD.

Automated quantification of neuronal swellings in a preclinical rodent model of Parkinson's disease detects region-specific changes in pathology.

BACKGROUND: The development of axonal pathology is a key characteristic of many neurodegenerative disease such as Parkinson's disease and Alzheimer's disease. With advanced disease progression, affected axons do display several signs of pathology such as swelling and fragmentation. In the AAV vector-mediated alpha-synuclein overexpression model of Parkinson's disease, large (> 20 µm2) pathological swellings are prominent characteristics in cortical and subcortical structures. NEW METHOD: This report describes a novel, macro-based workflow to quantify axonal pathology in the form of axonal swellings in the AAV vector-based alpha-synuclein overexpression model. Specifically, the approach is using background correction and thresholding before quantification of structures in 3D throughout a tissue stack. RESULTS: The method was used to quantify TH and aSYN axonal swellings in the prefrontal cortex, striatum, and hippocampus. Regional differences in volume and number of axonal swellings were observed for both in TH and aSYN, with the striatum displaying the greatest signs of pathology. COMPARISON WITH EXISTING METHODS: Existing methods for the quantification of axonal pathology do either rely on proprietary software or are based on manual quantification. The ImageJ workflow described here provides a method to objectively quantify axonal swellings both in volume and number. CONCLUSION: The method described can readily assess axonal pathology in preclinical rodent models of Parkinson's disease and can be easily adapted to other model systems and/or markers.

Development and Validation of CRISPR Activator Systems for Overexpression of CB1 Receptors in Neurons.

Gene therapy approaches using viral vectors for the overexpression of target genes have been for several years the focus of gene therapy research against neurological disorders. These approaches deliver robust expression of therapeutic genes, but are typically limited to the delivery of single genes and often do not manipulate the expression of the endogenous locus. In the last years, the advent of CRISPR-Cas9 technologies have revolutionized many areas of scientific research by providing novel tools that allow simple and efficient manipulation of endogenous genes. One of the applications of CRISPR-Cas9, termed CRISPRa, based on the use of a nuclease-null Cas9 protein (dCas9) fused to transcriptional activators, enables quick and efficient increase in target endogenous gene expression. CRISPRa approaches are varied, and different alternatives exist with regards to the type of Cas9 protein and transcriptional activator used. Several of these approaches have been successfully used in neurons in vitro and in vivo, but have not been so far extensively applied for the overexpression of genes involved in synaptic transmission. Here we describe the development and application of two different CRISPRa systems, based on single or dual Lentiviral and Adeno-Associated viral vectors and VP64 or VPR transcriptional activators, and demonstrate their efficiency in increasing mRNA and protein expression of the Cnr1 gene, coding for neuronal CB1 receptors. Both approaches were similarly efficient in primary neuronal cultures, and achieved a 2-5-fold increase in Cnr1 expression, but the AAV-based approach was more efficient in vivo. Our dual AAV-based VPR system in particular, based on Staphylococcus aureus dCas9, when injected in the hippocampus, displayed almost complete simultaneous expression of both vectors, high levels of dCas9 expression, and good efficiency in increasing Cnr1 mRNA as measured by in situ hybridization. In addition, we also show significant upregulation of CB1 receptor protein in vivo, which is reflected by an increased ability in reducing neurotransmitter release, as measured by electrophysiology. Our results show that CRISPRa techniques could be successfully used in neurons to target overexpression of genes involved in synaptic transmission, and can potentially represent a next-generation gene therapy approach against neurological disorders.

Optimization of production and transgene expression of a retrogradely transported pseudotyped lentiviral vector.

BACKGROUND: To target specific neuronal populations by gene transfer is challenging. A complicating fact is that populations of neurons may have opposing roles despite being found adjacent to each other. One example is the medium spiny neurons of the striatum. These cells have different projection patterns, a trait used in this study to specifically target one population. NEW METHOD: Here we present a way of labeling and further studying neurons based on their projections. This was achieved by pseudotyping lentiviral vectors with a chimeric glycoprotein allowing for retrograde transport in combination with optimizing the promoter element used. RESULTS: We transduced on average 4000 neurons of the direct pathway in the striatum, with the viral vector allowing for microscopy and miRNA immunoprecipitation. In addition, we were able to optimize vector production, reducing the time and material used. COMPARISON WITH EXISTING METHOD: The optimized protocol is more reproducible compared to previously published protocols. Alternative methods to study specific populations of neurons are transgenic animals or, if available, specific promoter elements. However, very specific promoter elements are rarely available and often large, limiting the usefulness in viral vectors. Our optimized retrograde vectors allow for selection based on neuronal projections and are therefore independent of such elements. CONCLUSION: We have developed a method that allows for specific analysis of neuronal subpopulations in the brain either by microscopy or by biochemical methods e.g. immunoprecipitation. This method is simple to use and can be combined with transgenic animals for studying disease models.

GDNF-mediated rescue of the nigrostriatal system depends on the degree of degeneration.

Glial cell-line derived neurotrophic factor (GDNF) is a promising therapeutic molecule to treat Parkinson's disease. Despite an excellent profile in experimental settings, clinical trials testing GDNF have failed. One of the theories to explain these negative outcomes is that the clinical trials were done in late-stage patients that have advanced nigrostriatal degeneration and may therefore not respond to a neurotrophic factor therapy. Based on this idea, we tested if the stage of nigrostriatal degeneration is important for GDNF-based therapies. Lentiviral vectors expressing regulated GDNF were delivered to the striatum of rats to allow GDNF expression to be turned on either while the nigrostriatal system was degenerating or after the nigrostriatal system had been fully lesioned by 6-OHDA. In the group of animals where GDNF expression was on during degeneration, neurons were rescued and there was a reversal of motor deficits. Turning GDNF expression on after the nigrostriatal system was lesioned did not rescue neurons or reverse motor deficits. In fact, these animals were indistinguishable from the control groups. Our results suggest that GDNF can reverse motor deficits and nigrostriatal pathology despite an ongoing nigrostriatal degeneration, if there is still a sufficient number of remaining neurons to respond to therapy.

Destabilizing Domains Enable Long-Term and Inert Regulation of GDNF Expression in the Brain.

Regulation of therapeutic transgene expression can increase the safety of gene therapy interventions, especially when targeting critical organs such as the brain. Although several gene expression systems have been described, none of the current systems has the required safety profile for clinical applications. Our group has previously adapted a system for novel gene regulation based on the destabilizing domain degron technology to successfully regulate glial cell-line derived neurotrophic factor in the brain (GDNF-F-DD). In the present study, we used GDNF-F-DD as a proof-of-principle molecule to fully characterize DD regulation in the brain. Our results indicate that DD could be regulated in a dose-dependent manner. In addition, GDNF-F-DD could also be induced in vivo repeatedly, without loss of activity or efficacy in vivo. Finally, DD regulation was able to be sustained for 24 weeks without loss of expression or any overt toxicity. The present study shows that DD has great potential to regulate gene expression in the brain.

Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing.

Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease dependency. Furthermore, de novo sequences can be inserted without the requirement of homologous recombination. Such reprogramming would enable efficient editing in quiescent cells (e.g., neurons) with an attractive safety profile for translational therapies. In this study, we applied a novel molecular-barcoded screening assay to investigate RNA trans-splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determining trans-splicing are reproducible regardless of the screening system. With this screening, we have located the most permissive trans-splicing sequences targeting an intron in the Synapsin I gene. Using viral vectors, we were able to splice full-length fluorophores into the mRNA while retaining very low off-target expression. Furthermore, this approach also showed evidence of functionality in the mouse striatum. However, in its current form, the trans-splicing events are stochastic and the overall activity lower than would be required for therapies targeting loss-of-function mutations. Nevertheless, the herein described barcode-based screening assay provides a unique possibility to screen and map large libraries in single animals or cell assays with very high precision.

Regulated Gene Therapy.

Gene therapy represents a promising approach for the treatment of monogenic and multifactorial neurological disorders. It can be used to replace a missing gene and mutated gene or downregulate a causal gene. Despite the versatility of gene therapy, one of the main limitations lies in the irreversibility of the process: once delivered to target cells, the gene of interest is constitutively expressed and cannot be removed. Therefore, efficient, safe and long-term gene modification requires a system allowing fine control of transgene expression.Different systems have been developed over the past decades to regulate transgene expression after in vivo delivery, either at transcriptional or post-translational levels. The purpose of this chapter is to give an overview on current regulatory system used in the context of gene therapy for neurological disorders. Systems using external regulation of transgenes using antibiotics are commonly used to control either gene expression using tetracycline-controlled transcription or protein levels using destabilizing domain technology. Alternatively, specific promoters of genes that are regulated by disease mechanisms, increasing expression as the disease progresses or decreasing expression as disease regresses, are also examined. Overall, this chapter discusses advantages and drawbacks of current molecular methods for regulated gene therapy in the central nervous system.

Functional neuroprotection and efficient regulation of GDNF using destabilizing domains in a rodent model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) has great potential to treat Parkinson's disease (PD). However, constitutive expression of GDNF can over time lead to side effects. Therefore, it would be useful to regulate GDNF expression. Recently, a new gene inducible system using destabilizing domains (DD) from E. coli dihydrofolate reductase (DHFR) has been developed and characterized. The advantage of this novel DD is that it is regulated by trimethoprim (TMP), a well-characterized drug that crosses the blood-brain barrier and can therefore be used to regulate gene expression in the brain. We have adapted this system to regulate expression of GDNF. A C-terminal fusion of GDNF and a DD with an additional furin cleavage site was able to be efficiently regulated in vitro, properly processed and was able to bind to canonical GDNF receptors, inducing a signaling cascade response in target cells. In vivo characterization of the protein showed that it could be efficiently induced by TMP and it was only functional when gene expression was turned on. Further characterization in a rodent model of PD showed that the regulated GDNF protected neurons, improved motor behavior of animals and was efficiently regulated in a pathological setting.

FACS binding assay for analysing GDNF interactions.

Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9.

Functional studies of resident microglia require molecular tools for their genetic manipulation. Here we show that microRNA-9-regulated lentiviral vectors can be used for the targeted genetic modification of resident microglia in the rodent brain. Using transgenic reporter mice, we demonstrate that murine microglia lack microRNA-9 activity, whereas most other cells in the brain express microRNA-9. Injection of microRNA-9-regulated vectors into the adult rat brain induces transgene expression specifically in cells with morphological features typical of ramified microglia. The majority of transgene-expressing cells colabels with the microglia marker Iba1. We use this approach to visualize and isolate activated resident microglia without affecting circulating and infiltrating monocytes or macrophages in an excitotoxic lesion model in rat striatum. The microRNA-9-regulated vectors described here are a straightforward and powerful tool that facilitates functional studies of resident microglia.

Destabilizing domains mediate reversible transgene expression in the brain.

Regulating transgene expression in vivo by delivering oral drugs has been a long-time goal for the gene therapy field. A novel gene regulating system based on targeted proteasomal degradation has been recently developed. The system is based on a destabilizing domain (DD) of the Escherichia coli dihydrofolate reductase (DHFR) that directs fused proteins to proteasomal destruction. Creating YFP proteins fused to destabilizing domains enabled TMP based induction of YFP expression in the brain, whereas omission of TMP resulted in loss of YFP expression. Moreover, induction of YFP expression was dose dependent and at higher TMP dosages, induced YFP reached levels comparable to expression of unregulated transgene., Transgene expression could be reversibly regulated using the DD system. Importantly, no adverse effects of TMP treatment or expression of DD-fusion proteins in the brain were observed. To show proof of concept that destabilizing domains derived from DHFR could be used with a biologically active molecule, DD were fused to GDNF, which is a potent neurotrophic factor of dopamine neurons. N-terminal placement of the DD resulted in TMP-regulated release of biologically active GDNF. Our findings suggest that TMP-regulated destabilizing domains can afford transgene regulation in the brain. The fact that GDNF could be regulated is very promising for developing future gene therapies (e.g. for Parkinson's disease) and should be further investigated.

Novel disease-specific promoters for use in gene therapy for Parkinson's disease.

Gene therapy is a promising therapeutic tool for Parkinson's disease (PD), but there is a lack of evaluated cell specific promoters that are relevant for the disease. We have chosen PD relevant promoter candidates for gene therapy vectors based on either previous studies; Drd1a, Drd2 and pDyn, or from a microarray study on parkinsonian patients; ACE, DNAJC3, GALNS, MAP1a and RNF25. These candidates have been evaluated in rat striatum to determine their suitability for use in cell specific vectors. The promoters had a neuronal specificity of 91-100%. The efficiency of the promoters was variable, but RNF25, DNAJC3 and MAP1a were comparable to widely used ubiquitous promoters. MAP1a was also affected by dopamine depletion.

A model of GDNF gene therapy in mice with 6-Hydroxydopamine lesions: time course of Neurorestorative effects and ERK1/2 activation.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is the most promising neurotrophin for restorative treatments in Parkinson's disease, but its biological effects are not completely understood. OBJECTIVE: To define a model of GDNF gene therapy in the mouse, we studied the long-term effects of lentiviral GDNF delivery in mice with striatal 6-hydroxydopamine (6-OHDA) lesions. METHODS: Lentiviral vectors coding for GDNF or green fluorescent protein (GFP) were injected unilaterally in the striatum two weeks prior to the 6-OHDA lesion. Mice were monitored on tests of spontaneous activity and amphetamine-induced rotation at 1, 4, 10 and 35 weeks post-lesion. Brains were processed immunohistochemically for tyrosine hydroxylase (TH) and markers of extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation at the same time points. RESULTS: Lentiviral GDNF significantly inhibited both spontaneous and amphetamine-induced rotation. Compared to the control vector, lentiviral GDNF resulted in a partial protection of TH-positive cells in the substantia nigra, and in a nearly total restoration of striatal TH immunostaining by 35 weeks. A progressive sprouting of TH-positive neurites occurred in both the globus pallidus and the substantia nigra, reaching a 4-5 fold increase above controls by 35 weeks. This effect was paralleled by a long-term supranormal activation of ERK1/2 and its downstream target, phospho-Ser31 TH. CONCLUSIONS: Lentiviral GDNF delivery produced robust long-term signaling responses and neurorestoration. This experimental model of GDNF gene therapy will be particularly suitable to study the molecular mechanisms of dopaminergic fiber sprouting, a long-term response to GDNF delivery that also occurs in Parkinson's disease patients.