Intrathymic AAV delivery results in therapeutic site-specific integration at TCR loci in mice.

Adeno-associated virus (AAV) vectors have been successfully exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector, but it has been shown to integrate within the host cell genome after the generation of double-strand DNA breaks or nicks. Although AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T-cell progenitors and long-term T-cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T cells and compared these with liver and brain tissue from treated mice. Notably, we found that although AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T cells were clustered within the T-cell receptor α, ß, and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAGs) during variable, diversity, and joining recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.

In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice.

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.

Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery.

P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

The Tbr2 Molecular Network Controls Cortical Neuronal Differentiation Through Complementary Genetic and Epigenetic Pathways.

The T-box containing Tbr2 gene encodes for a transcription factor essential for the specification of the intermediate neural progenitors (INPs) originating the excitatory neurons of the cerebral cortex. However, its overall mechanism of action, direct target genes and cofactors remain unknown. Herein, we carried out global gene expression profiling combined with genome-wide binding site identification to determine the molecular pathways regulated by TBR2 in INPs. This analysis led to the identification of novel protein-protein interactions that control multiple features of INPs including cell-type identity, morphology, proliferation and migration dynamics. In particular, NEUROG2 and JMJD3 were found to associate with TBR2 revealing unexplored TBR2-dependent mechanisms. These interactions can explain, at least in part, the role of this transcription factor in the implementation of the molecular program controlling developmental milestones during corticogenesis. These data identify TBR2 as a major determinant of the INP-specific traits by regulating both genetic and epigenetic pathways.

AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy.

The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.

COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons.

COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, we investigated COUP-TFI expression and function in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. Here, we show that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). We found that odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, we demonstrate that conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. Our study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons.

New AAV9 engineered variants with enhanced neurotropism and reduced liver off-targeting in mice and marmosets.

Although adeno-associated virus 9 (AAV9) has been highly exploited as delivery platform for gene-based therapies, its efficacy is hampered by low efficiency in crossing the adult blood-brain barrier (BBB) and pronounced targeting to the liver upon intravenous delivery. We generated a new galactose binding-deficient AAV9 peptide display library and selected two new AAV9 engineered capsids with enhanced targeting in mouse and marmoset brains after intravenous delivery. Interestingly, the loss of galactose binding greatly reduced undesired targeting to peripheral organs, particularly the liver, while not compromising transduction of the brain vasculature. However, the galactose binding was necessary to efficiently infect non-endothelial brain cells. Thus, the combinatorial actions of the galactose-binding domain and the incorporated displayed peptide are crucial to enhance BBB crossing along with brain cell transduction. This study describes two novel capsids with high brain endothelial infectivity and extremely low liver targeting based on manipulating the AAV9 galactose-binding domain.

Altered Brain Cholesterol Machinery in a Down Syndrome Mouse Model: A Possible Common Feature with Alzheimer's Disease.

Down syndrome (DS) is a complex chromosomal disorder considered as a genetically determined form of Alzheimer's disease (AD). Maintenance of brain cholesterol homeostasis is essential for brain functioning and development, and its dysregulation is associated with AD neuroinflammation and oxidative damage. Brain cholesterol imbalances also likely occur in DS, concurring with the precocious AD-like neurodegeneration. In this pilot study, we analyzed, in the brain of the Ts2Cje (Ts2) mouse model of DS, the expression of genes encoding key enzymes involved in cholesterol metabolism and of the levels of cholesterol and its main precursors and products of its metabolism (i.e., oxysterols). The results showed, in Ts2 mice compared to euploid mice, the downregulation of the transcription of the genes encoding the enzymes 3-hydroxy-3-methylglutaryl-CoA reductase and 24-dehydrocholesterol reductase, the latter originally recognized as an indicator of AD, and the consequent reduction in total cholesterol levels. Moreover, the expression of genes encoding enzymes responsible for brain cholesterol oxidation and the amounts of the resulting oxysterols were modified in Ts2 mouse brains, and the levels of cholesterol autoxidation products were increased, suggesting an exacerbation of cerebral oxidative stress. We also observed an enhanced inflammatory response in Ts2 mice, underlined by the upregulation of the transcription of the genes encoding for α-interferon and interleukin-6, two cytokines whose synthesis is increased in the brains of AD patients. Overall, these results suggest that DS and AD brains share cholesterol cycle derangements and altered oxysterol levels, which may contribute to the oxidative and inflammatory events involved in both diseases.

24-Hydroxycholesterol Induces Tau Proteasome-Dependent Degradation via the SIRT1/PGC1α/Nrf2 Pathway: A Potential Mechanism to Counteract Alzheimer's Disease.

Considerable evidence indicates that cholesterol oxidation products, named oxysterols, play a key role in several events involved in Alzheimer's disease (AD) pathogenesis. Although the majority of oxysterols causes neuron dysfunction and degeneration, 24-hydroxycholesterol (24-OHC) has recently been thought to be neuroprotective also. The present study aimed at supporting this concept by exploring, in SK-N-BE neuroblastoma cells, whether 24-OHC affected the neuroprotective SIRT1/PGC1α/Nrf2 axis. We demonstrated that 24-OHC, through the up-regulation of the deacetylase SIRT1, was able to increase both PGC1α and Nrf2 expression and protein levels, as well as Nrf2 nuclear translocation. By acting on this neuroprotective pathway, 24-OHC favors tau protein clearance by triggering tau ubiquitination and subsequently its degradation through the ubiquitin-proteasome system. We also observed a modulation of SIRT1, PGC1α, and Nrf2 expression and synthesis in the brain of AD patients with the progression of the disease, suggesting their potential role in neuroprotection. These findings suggest that 24-OHC contributes to tau degradation through the up-regulation of the SIRT1/PGC1α/Nrf2 axis. Overall, the evidence points out the importance of avoiding 24-OHC loss, which can occur in the AD brain, and of limiting SIRT1, PGC1α, and Nrf2 deregulation in order to prevent the neurotoxic accumulation of hyperphosphorylated tau and counteract neurodegeneration.

MCT1-dependent energetic failure and neuroinflammation underlie optic nerve degeneration in Wolfram syndrome mice.

Wolfram syndrome 1 (WS1) is a rare genetic disorder caused by mutations in the WFS1 gene leading to a wide spectrum of clinical dysfunctions, among which blindness, diabetes, and neurological deficits are the most prominent. WFS1 encodes for the endoplasmic reticulum (ER) resident transmembrane protein wolframin with multiple functions in ER processes. However, the WFS1-dependent etiopathology in retinal cells is unknown. Herein, we showed that Wfs1 mutant mice developed early retinal electrophysiological impairments followed by marked visual loss. Interestingly, axons and myelin disruption in the optic nerve preceded the degeneration of the retinal ganglion cell bodies in the retina. Transcriptomics at pre-degenerative stage revealed the STAT3-dependent activation of proinflammatory glial markers with reduction of the homeostatic and pro-survival factors glutamine synthetase and BDNF. Furthermore, label-free comparative proteomics identified a significant reduction of the monocarboxylate transport isoform 1 (MCT1) and its partner basigin that are highly enriched on retinal glia and myelin-forming oligodendrocytes in optic nerve together with wolframin. Loss of MCT1 caused a failure in lactate transfer from glial to neuronal cell bodies and axons leading to a chronic hypometabolic state. Thus, this bioenergetic impairment is occurring concurrently both within the axonal regions and cell bodies of the retinal ganglion cells, selectively endangering their survival while impacting less on other retinal cells. This metabolic dysfunction occurs months before the frank RGC degeneration suggesting an extended time-window for intervening with new therapeutic strategies focused on boosting retinal and optic nerve bioenergetics in WS1.

Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells.

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.

Genome-wide screening in pluripotent cells identifies Mtf1 as a suppressor of mutant huntingtin toxicity.

Huntington's disease (HD) is a neurodegenerative disorder caused by CAG-repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT (mHTT) protein induces toxicity and cell death via multiple mechanisms and no effective therapy is available. Here, we employ a genome-wide screening in pluripotent mouse embryonic stem cells (ESCs) to identify suppressors of mHTT toxicity. Among the identified suppressors, linked to HD-associated processes, we focus on Metal response element binding transcription factor 1 (Mtf1). Forced expression of Mtf1 counteracts cell death and oxidative stress caused by mHTT in mouse ESCs and in human neuronal precursor cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mHTT. In R6/2 mice, Mtf1 ablates motor defects and reduces mHTT aggregates and oxidative stress. Our screening strategy enables a quick in vitro identification of promising suppressor genes and their validation in vivo, and it can be applied to other monogenic diseases.

Adult human Müller glia cells are a highly efficient source of rod photoreceptors.

There is growing evidence that Müller glia cells (MGCs) might act as regenerative elements in injured retinas of fishes and amniotes. However, their differentiation potential in humans is yet unknown. We isolated Müller glia from adult human retinas and propagated them in vitro revealing for the first time their ability to differentiate into rod photoreceptors. These results were also confirmed with mice retinas. Here, we describe conditions by which human MGCs adopt a rod photoreceptor commitment with a surprising efficiency as high as 54%. Functional characterization of Müller glia-derived photoreceptors by patch-clamp recordings revealed that their electrical properties are comparable to those of adult rods. Interestingly, our procedure allowed efficient derivation of MGC cultures starting from both injured and degenerating and postmortem human retinas. Human transplanted Müller glia-derived photoreceptors integrate and survive within immunodeficient mouse retinas. These data provide evidence that Müller glia retains an unpredicted plasticity and multipotent potential into adulthood, and it is therefore a promising source of novel therapeutic applications in retinal repair.

Macrophage polarization by potential nutraceutical compounds: A strategic approach to counteract inflammation in atherosclerosis.

Chronic inflammation represents a main event in the onset and progression of atherosclerosis and is closely associated with oxidative stress in a sort of vicious circle that amplifies and sustains all stages of the disease. Key players of atherosclerosis are monocytes/macrophages. According to their pro- or anti-inflammatory phenotype and biological functions, lesional macrophages can release various mediators and enzymes, which in turn contribute to plaque progression and destabilization or, alternatively, lead to its resolution. Among the factors connected to atherosclerotic disease, lipid species carried by low density lipoproteins and pro-oxidant stimuli strongly promote inflammatory events in the vasculature, also by modulating the macrophage phenotyping. Therapies specifically aimed to balance macrophage inflammatory state are increasingly considered as powerful tools to counteract plaque formation and destabilization. In this connection, several molecules of natural origin have been recognized to be active mediators of diverse metabolic and signaling pathways regulating lipid homeostasis, redox state, and inflammation; they are, thus, considered as promising candidates to modulate macrophage responsiveness to pro-atherogenic stimuli. The current knowledge of the capability of nutraceuticals to target macrophage polarization and to counteract atherosclerotic lesion progression, based mainly on in vitro investigation, is summarized in the present review.

ApoE3 vs. ApoE4 Astrocytes: A Detailed Analysis Provides New Insights into Differences in Cholesterol Homeostasis.

The strongest genetic risk factor for sporadic Alzheimer's disease (AD) is the presence of the ε4 allele of the apolipoprotein E (ApoE) gene, the major apolipoprotein involved in brain cholesterol homeostasis. Being astrocytes the main producers of cholesterol and ApoE in the brain, we investigated the impact of the ApoE genotype on astrocyte cholesterol homeostasis. Two mouse astrocytic cell lines expressing the human ApoE3 or ApoE4 isoform were employed. Gas chromatography-mass spectrometry (GC-MS) analysis pointed out that the levels of total cholesterol, cholesterol precursors, and various oxysterols are altered in ApoE4 astrocytes. Moreover, the gene expression analysis of more than 40 lipid-related genes by qRT-PCR showed that certain genes are up-regulated (e.g., CYP27A1) and others down-regulated (e.g., PPARγ, LXRα) in ApoE4, compared to ApoE3 astrocytes. Beyond confirming the significant reduction in the levels of PPARγ, a key transcription factor involved in the maintenance of lipid homeostasis, Western blotting showed that both intracellular and secreted ApoE levels are altered in ApoE4 astrocytes, as well as the levels of receptors and transporters involved in lipid uptake/efflux (ABCA1, LDLR, LRP1, and ApoER2). Data showed that the ApoE genotype clearly affects astrocytic cholesterol homeostasis; however, further investigation is needed to clarify the mechanisms underlying these differences and the consequences on neighboring cells. Indeed, drug development aimed at restoring cholesterol homeostasis could be a potential strategy to counteract AD.

The epilepsy-associated protein PCDH19 undergoes NMDA receptor-dependent proteolytic cleavage and regulates the expression of immediate-early genes.

Protocadherin-19 (PCDH19) is a synaptic cell-adhesion molecule encoded by X-linked PCDH19, a gene linked with epilepsy. Here, we report a synapse-to-nucleus signaling pathway through which PCDH19 bridges neuronal activity with gene expression. In particular, we describe the NMDA receptor (NMDAR)-dependent proteolytic cleavage of PCDH19, which leads to the generation of a PCDH19 C-terminal fragment (CTF) able to enter the nucleus. We demonstrate that PCDH19 CTF associates with chromatin and with the chromatin remodeler lysine-specific demethylase 1 (LSD1) and regulates expression of immediate-early genes (IEGs). Our results are consistent with a model whereby PCDH19 favors maintenance of neuronal homeostasis via negative feedback regulation of IEG expression and provide a key to interpreting PCDH19-related hyperexcitability.

Scn1a gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome.

Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Nav1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells.

Modeling native and seeded Synuclein aggregation and related cellular dysfunctions in dopaminergic neurons derived by a new set of isogenic iPSC lines with SNCA multiplications.

Triplication of the SNCA gene, encoding the protein alpha-Synuclein (αSyn), is a rare cause of aggressive and early-onset parkinsonism. Herein, we generated iPSCs from two siblings with a recently described compact SNCA gene triplication and suffering from severe motor impairments, psychiatric symptoms, and cognitive deterioration. Using CRISPR/Cas9 gene editing, each SNCA copy was inactivated by targeted indel mutations generating a panel of isogenic iPSCs with a decremental number from 4 down to none of functional SNCA gene alleles. We differentiated these iPSC lines in midbrain dopaminergic (DA) neuronal cultures to characterize αSyn aggregation in native and seeded conditions and evaluate its associated cellular dysfunctions. Utilizing a new nanobody-based biosensor combined with super-resolved imaging, we were able to visualize and measure αSyn aggregates in early DA neurons in unstimulated conditions. Calcium dysregulation and mitochondrial alterations were the first pathological signs detectable in early differentiated DA neuronal cultures. Accelerated αSyn aggregation was induced by exposing neurons to structurally well-characterized synthetic αSyn fibrils. 4xSNCA DA neurons showed the highest vulnerability, which was associated with high levels of oxidized DA and amplified by TAX1BP1 gene disruption. Seeded DA neurons developed large αSyn deposits whose morphology and internal constituents resembled Lewy bodies commonly observed in Parkinson's disease (PD) patient brain tissues. These findings provide strong evidence that this isogenic panel of iPSCs with SNCA multiplications offers a remarkable cellular platform to investigate mechanisms of PD and validate candidate inhibitors of native and seeded αSyn aggregation.