• Bimodal DREADDs and transgenic rats enable remote control of transplant function • Gs-coupled increase of cAMP using DREADDs leads to graft-induced dyskinesias (GIDs) • Serotonin 5-HT6 receptor stimulation gives GIDs through dysplastic DA release • DA neurons grafted to the human brain have high levels of the 5-HT6 receptor

Synucleinopathies, characterized by intracellular aggregation of α-synuclein protein, share a number of features in pathology and disease progression. However, the vulnerable cell population differs significantly between the disorders, despite being caused by the same protein. While the vulnerability of dopamine cells in the substantia nigra to α-synuclein over-expression, and its link to Parkinson's disease, is well studied, animal models recapitulating the cortical degeneration in dementia with Lewy-bodies (DLB) are much less mature. The aim of this study was to develop a first rat model of widespread progressive synucleinopathy throughout the forebrain using adeno-associated viral (AAV) vector mediated gene delivery. Through bilateral injection of an AAV6 vector expressing human wild-type α-synuclein into the forebrain of neonatal rats, we were able to achieve widespread, robust α-synuclein expression with preferential expression in the frontal cortex. These animals displayed a progressive emergence of hyper-locomotion and dysregulated response to the dopaminergic agonist apomorphine. The animals receiving the α-synuclein vector displayed significant α-synuclein pathology including intra-cellular inclusion bodies, axonal pathology and elevated levels of phosphorylated α-synuclein, accompanied by significant loss of cortical neurons and a progressive reduction in both cortical and striatal ChAT positive interneurons. Furthermore, we found evidence of α-synuclein sequestered by IBA-1 positive microglia, which was coupled with a distinct change in morphology. In areas of most prominent pathology, the total α-synuclein levels were increased to, on average, two-fold, which is similar to the levels observed in patients with SNCA gene triplication, associated with cortical Lewy body pathology. This study provides a novel rat model of progressive cortical synucleinopathy, showing for the first time that cholinergic interneurons are vulnerable to α-synuclein over-expression. This animal model provides a powerful new tool for studies of neuronal degeneration in conditions of widespread cortical α-synuclein pathology, such as DLB, as well an attractive model for the exploration of novel biomarkers.

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 RNAtrans-splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determiningtrans-splicing are reproducible regardless of the screening system. With this screening, we have located the most permissivetrans-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, thetrans-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.

Engineering of Adeno-associated viral (AAV) vector capsids through directed evolution has been used to generate novel capsids with altered tropism and function _1-9 . This approach, however, involves a selection process that requires multiple generations of screenings to identify real functional capsids _2-4 . Due to the random nature of this process, it is also inherently unreproducible, and the resulting capsid variants provide little mechanistic insights into the molecular targets engaged. To overcome this, we have developed a novel method for rational capsid evolution named Barcoded Rational AAV Vector Evolution (BRAVE). The key to this method is a novel viral production approach where each virus particle displays a protein-derived peptide on the surface which is linked to a unique barcode in the packaged genome ₁₀ . Through hidden Markov model-based clustering ₁₁ , we were able to identify novel consensus motifs for cell-type specific retrograde transport in neurons in vivo in the brain. The BRAVE approach enables the selection of novel capsid structures using only a single-generation screening. Furthermore, it can be used to map, with high resolution, the putative binding sequences of large protein libraries.