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.

Deconvolution of spatial sequencing provides accurate characterization of hESC-derived DA transplants in vivo.

Cell therapy for Parkinson's disease has experienced substantial growth in the past decades with several ongoing clinical trials. Despite increasing refinement of differentiation protocols and standardization of the transplanted neural precursors, the transcriptomic analysis of cells in the transplant after its full maturation in vivo has not been thoroughly investigated. Here, we present spatial transcriptomics analysis of fully differentiated grafts in their host tissue. Unlike earlier transcriptomics analyses using single-cell technologies, we observe that cells derived from human embryonic stem cells (hESCs) in the grafts adopt mature dopaminergic signatures. We show that the presence of phenotypic dopaminergic genes, which were found to be differentially expressed in the transplants, is concentrated toward the edges of the grafts, in agreement with the immunohistochemical analyses. Deconvolution shows dopamine neurons being the dominating cell type in many features beneath the graft area. These findings further support the preferred environmental niche of TH-positive cells and confirm their dopaminergic phenotype through the presence of multiple dopaminergic markers.

A novel two-factor monosynaptic TRIO tracing method for assessment of circuit integration of hESC-derived dopamine transplants.

Transplantation in Parkinson's disease using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons is a promising future treatment option. However, many of the mechanisms that govern their differentiation, maturation, and integration into the host circuitry remain elusive. Here, we engrafted hESCs differentiated toward a ventral midbrain DA phenotype into the midbrain of a preclinical rodent model of Parkinson's disease. We then injected a novel DA-neurotropic retrograde MNM008 adeno-associated virus vector capsid, into specific DA target regions to generate starter cells based on their axonal projections. Using monosynaptic rabies-based tracing, we demonstrated for the first time that grafted hESC-derived DA neurons receive distinctly different afferent inputs depending on their projections. The similarities to the host DA system suggest a previously unknown directed circuit integration. By evaluating the differential host-to-graft connectivity based on projection patterns, this novel approach offers a tool to answer outstanding questions regarding the integration of grafted hESC-derived DA neurons.

Next-Generation Gene Therapy for Parkinson's Disease Using Engineered Viral Vectors.

Recent technological and conceptual advances have resulted in a plethora of exciting novel engineered adeno associated viral (AAV) vector variants. They all have unique characteristics and abilities. This review summarizes the development and their potential in treating Parkinson's disease (PD). Clinical trials in PD have shown over the last decade that AAV is a safe and suitable vector for gene therapy but that it also is a vehicle that can benefit significantly from improvement in specificity and potency. This review provides a concise collection of the state-of-the-art for synthetic capsids and their utility in PD. We also summarize what therapeutical strategies may become feasible with novel engineered vectors, including genome editing and neuronal rejuvenation.

FRET-Based Screening Identifies p38 MAPK and PKC Inhibition as Targets for Prevention of Seeded α-Synuclein Aggregation.

Aggregation of α-synuclein is associated with neurodegeneration and a hallmark pathology in synucleinopathies. These aggregates are thought to function as prion-like particles where the conformation of misfolded α-synuclein determines the traits of the induced pathology, similar to prion diseases. Still, little is known about the molecular targets facilitating the conformation-specific biological effects, but their identification could form the basis for new therapeutic interventions. High-throughput screening of annotated compound libraries could facilitate mechanistic investigation by identifying targets with impact on α-synuclein aggregation. To this end, we developed a FRET-based cellular reporter in HEK293T cells, with sensitivity down to 6.5 nM α-synuclein seeds. Using this model system, we identified GF109203X, SB202190, and SB203580 as inhibitors capable of preventing induction of α-synuclein aggregation via inhibition of p38 MAPK and PKC, respectively. We further investigated the mechanisms underlying the protective effects and found alterations in the endo-lysosomal system to be likely candidates of the protection. We found the changes did not stem from a reduction in uptake but rather alteration of lysosomal abundance and degradative capacity. Our findings highlight the value high-throughput screening brings to the mechanistic investigation of α-synuclein aggregation while simultaneously identifying novel therapeutic compounds.

In vivo conversion of dopamine neurons in mouse models of Parkinson's disease - a future approach for regenerative therapy?

Recent advances in cell reprogramming have made it possible to form new therapeutic cells within the body itself via a process called direct conversion or lineage reprogramming. A series of studies have shown that it is possible to reprogram resident glia into new neurons within the brain parenchyma. These studies opened up for the targeted attempts to achieve functional brain repair using in vivo conversion. Because of the relatively focal degeneration, Parkinson's Disease (PD) is an attractive target for both transplantation-based and in vivo conversion-based reparative approaches. Fetal cell transplants have provided proof-of-concept and stem cell-based therapies for PD are now on the verge of entering clinical trials. In the future, in vivo conversion may be an alternative to transplantation-based therapies.

AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In-house AAV Vector Production Based on Chloroform Extraction.

Recombinant adeno-associated virus (rAAV) is a mammalian virus that has been altered to be used as a gene delivery vehicle. Several changes to the viral genome have made them replication deficient so that this aspect of the viral infection cycle is under full control of the experimenter, while maintaining gene expression machinery. Over the last decades, rAAVs have become the gold standard for studying in vivo gene function and are especially favorable for gene transfer in the central nervous system. AAVs have been proven safe and provide stable gene expression over a long period of time. They are extensively used in preclinical experiments and show great potential for clinical applications. However, the use of AAVs in preclinical settings are often held back due to availability. Waiting lines are long at commercial production facilities, and in-lab production is hindered due to lack of specific laboratory equipment needed. Here we present a novel production method that can be carried out in any molecular biology laboratory using standard laboratory equipment. We provide a simple, fast, and streamlined protocol for production that can result in titers comparable with the more time-consuming iodixanol gradient ultracentrifugation method. The yield using this protocol is high enough for any type of study where AAV is the vector of choice. © 2020 The Authors.

A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism.

Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.

Seeding of protein aggregation causes cognitive impairment in rat model of cortical synucleinopathy.

BACKGROUND: Cortical α-synuclein pathology plays a role in the development of cognitive dysfunction in both Parkinson's disease and dementia with Lewy bodies, although the causative cellular lesions have remained unclear. We aimed to address causal links between α-synuclein-driven pathology in the cerebral cortex and the development of cognitive impairments using new experimental models. METHODS: Neuronal overexpression of human α-synuclein was induced in the rat medial prefrontal cortex using viral vectors. This was combined with inoculations of preformed fibrils of human α-synuclein in some animals. Rats were evaluated with tests probing prefrontal cognitive functions (delayed matching/nonmatching to position and 5-choice serial reaction time task). Patterns of neuropathology were characterized immunohistochemically. RESULTS: Neither α-synuclein overexpression nor the fibril seeds alone yielded any behavioral phenotype. In contrast, combining the 2 approaches produced significant impairments in working memory, attention, and inhibitory control. All animals injected with α-synuclein vectors exhibited high immunoreactivity for human α-synuclein in the medial prefrontal cortex and its primary projection targets. However, only when this overexpression was combined with fibril inoculations did animals exhibit large, proteinase K-resistant and Ser129 -phosphorylated α-synuclein intraneuronal inclusions in the medial prefrontal cortex and its closely interconnected brain regions. The inclusions were associated with distorted dendritic morphologies and partial neuronal loss in the targeted cortical areas. CONCLUSIONS: Cortical overexpression of human α-synuclein is not sufficient to produce cognitive dysfunction, whereas combining this overexpression with fibril seeds yields both cognitive and histopathological phenotypes that are relevant to human Lewy body disease. © 2019 International Parkinson and Movement Disorder Society.

Vector-mediated l-3,4-dihydroxyphenylalanine delivery reverses motor impairments in a primate model of Parkinson's disease.

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.

Practical Considerations for the Use of DREADD and Other Chemogenetic Receptors to Regulate Neuronal Activity in the Mammalian Brain.

Chemogenetics is the process of genetically expressing a macromolecule receptor capable of modulating the activity of the cell in response to selective chemical ligand. This chapter will cover the chemogenetic technologies that are available to date, focusing on the commonly available engineered or otherwise modified ligand-gated ion channels and G-protein-coupled receptors in the context of neuromodulation. First, we will give a brief overview of each chemogenetic approach as well as in vitro/in vivo applications, then we will list their strengths and weaknesses. Finally, we will provide tips for ligand application in each case.Each technology has specific limitations that make them more or less suitable for different applications in neuroscience although we will focus mainly on the most commonly used and versatile family named designer receptors exclusively activated by designer drugs or DREADDs. We here describe the most common cases where these can be implemented and provide tips on how and where these technologies can be applied in the field of neuroscience.

Repairing the Brain: Gene Therapy.

In vivo gene therapy for neurodegenerative disorders has turned out to be a formidable challenge. It is a field not much older than twenty years, but we were many who would have predicted a much easier path towards the clinic using this treatment modality. For Parkinson's disease patients, this has meant a frustrating wait, seeing many promising therapies being forgotten after a few pre-clinical proof-of-concept studies. The reasons for this are both scientific and economical. However, this is slowly but surely changing and over the next two decades we will see a very exciting development in this field. In a foreseeable future, gene therapy will be a very natural component of many clinical therapies, not least in Parkinson's disease.

A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.

Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology.

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.

Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum.

The intricate balance between dopaminergic and cholinergic neurotransmission in the striatum has been thoroughly difficult to characterize. It was initially described as a seesaw with a competing function of dopamine versus acetylcholine. Recent technical advances however, have brought this view into question suggesting that the two systems work rather in concert with the cholinergic interneurons (ChIs) driving dopamine release. In this study, we have utilized two transgenic Cre-driver rat lines, a choline acetyl transferase ChAT-Cre transgenic rat and a novel double-transgenic tyrosine hydroxylase TH-Cre/ChAT-Cre rat to further elucidate the role of striatal ChIs in normal motor function and in Parkinson's disease. Here we show that selective and reversible activation of ChIs using chemogenetic (DREADD) receptors increases locomotor function in intact rats and potentiate the therapeutic effect of L-DOPA in the rats with lesions of the nigral dopamine system. However, the potentiation of the L-DOPA effect is accompanied by an aggravation of L-DOPA induced dyskinesias (LIDs). These LIDs appear to be driven primarily through the indirect striato-pallidal pathway since the same effect can be induced by the D2 agonist Quinpirole. Taken together, the results highlight the intricate regulation of balance between the two output pathways from the striatum orchestrated by the ChIs.

A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing.

Detailed characterization and mapping of oligonucleotide function in vivo is generally a very time consuming effort that only allows for hypothesis driven subsampling of the full sequence to be analysed. Recent advances in deep sequencing together with highly efficient parallel oligonucleotide synthesis and cloning techniques have, however, opened up for entirely new ways to map genetic function in vivo. Here we present a novel, optimized protocol for the generation of universally applicable, barcode labelled, plasmid libraries. The libraries are designed to enable the production of viral vector preparations assessing coding or non-coding RNA function in vivo. When generating high diversity libraries, it is a challenge to achieve efficient cloning, unambiguous barcoding and detailed characterization using low-cost sequencing technologies. With the presented protocol, diversity of above 3 million uniquely barcoded adeno-associated viral (AAV) plasmids can be achieved in a single reaction through a process achievable in any molecular biology laboratory. This approach opens up for a multitude of in vivo assessments from the evaluation of enhancer and promoter regions to the optimization of genome editing. The generated plasmid libraries are also useful for validation of sequencing clustering algorithms and we here validate the newly presented message passing clustering process named Starcode.

Identification of Multiple QTLs Linked to Neuropathology in the Engrailed-1 Heterozygous Mouse Model of Parkinson's Disease.

Motor symptoms in Parkinson's disease are attributed to degeneration of midbrain dopaminergic neurons (DNs). Heterozygosity for Engrailed-1 (En1), one of the key factors for programming and maintenance of DNs, results in a parkinsonian phenotype featuring progressive degeneration of DNs in substantia nigra pars compacta (SNpc), decreased striatal dopamine levels and swellings of nigro-striatal axons in the SwissOF1-En1+/- mouse strain. In contrast, C57Bl/6-En1+/- mice do not display this neurodegenerative phenotype, suggesting that susceptibility to En1 heterozygosity is genetically regulated. Our goal was to identify quantitative trait loci (QTLs) that regulate the susceptibility to PD-like neurodegenerative changes in response to loss of one En1 allele. We intercrossed SwissOF1-En1+/- and C57Bl/6 mice to obtain F2 mice with mixed genomes and analyzed number of DNs in SNpc and striatal axonal swellings in 120 F2-En1+/- 17 week-old male mice. Linkage analyses revealed 8 QTLs linked to number of DNs (p = 2.4e-09, variance explained = 74%), 7 QTLs linked to load of axonal swellings (p = 1.7e-12, variance explained = 80%) and 8 QTLs linked to size of axonal swellings (p = 7.0e-11, variance explained = 74%). These loci should be of prime interest for studies of susceptibility to Parkinson's disease-like damage in rodent disease models and considered in clinical association studies in PD.

Cellular Barcoding Links B-1a B Cell Potential to a Fetal Hematopoietic Stem Cell State at the Single-Cell Level.

Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigated whether the developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. Using cellular barcoding for in vivo single-cell fate analyses, we found that fetal liver definitive HSCs gave rise to both B-1a and B-2 cells. Whereas B-1a potential diminished in all HSCs with time, B-2 output was maintained. B-1a and B-2 plasticity could be reinitiated in a subset of adult HSCs by ectopic expression of the RNA binding protein LIN28B, a key regulator of fetal hematopoiesis, and this coincided with the clonal reversal to fetal-like elevated self-renewal and repopulation potential. These results anchor the attenuation of B-1a cell output to fetal HSC behavior and demonstrate that the developmental decline in regenerative potential represents a reversible HSC state.

DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor.

Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson's disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still unknown. Here we have used a new model where the activity of the transplanted DA neurons can be selectively modulated using a bimodal chemogenetic (DREADD) approach, allowing either enhancement or reduction of the therapeutic effect. We show that exclusive activation of a cAMP-linked (Gs-coupled) DREADD or serotonin 5-HT6 receptor, located on the grafted DA neurons, is sufficient to induce GIDs. These findings establish a mechanistic link between the 5-HT6 receptor, intracellular cAMP, and GIDs in transplanted PD patients. This effect is thought to be mediated through counteraction of the D2 autoreceptor feedback inhibition, resulting in a dysplastic DA release from the transplant.

Expression of Multiple Functional RNAs or Proteins from One Viral Vector.

In this chapter, we will cover the available design choices for enabling expression of two functional protein or RNA sequences from a single viral vector. Such vectors are very useful in the neuroscience-related field of neuronal control and modulation, e.g., using optogenetics or DREADDs, but are also desirable in applications of CRISPR/Cas9 in situ genome editing and more refined therapeutic approaches. Each approach to achieving this combined expression has its own strengths and limitations, which makes them more or less suitable for different applications. In this chapter, we describe the available alternatives and provide tips on how they can be implemented.