Background Autism is a heterogeneous collection of disorders with a complex molecular underpinning. Evidence from postmortem brain studies have indicated that early prenatal development may be altered in autism. Induced pluripotent stem cells (iPSCs) generated from individuals with autism with macrocephaly also indicate prenatal development as a critical period for this condition. But little is known about early altered cellular events during prenatal stages in autism. Methods iPSCs were generated from 9 unrelated individuals with autism without macrocephaly and with heterogeneous genetic backgrounds, and 6 typically developing control individuals. iPSCs were differentiated toward either cortical or midbrain fates. Gene expression and high throughput cellular phenotyping was used to characterize iPSCs at different stages of differentiation. Results A subset of autism-iPSC cortical neurons were RNA-sequenced to reveal autism-specific signatures similar to postmortem brain studies, indicating a potential common biological mechanism. Autism-iPSCs differentiated toward a cortical fate displayed impairments in the ability to self-form into neural rosettes. In addition, autism-iPSCs demonstrated significant differences in rate of cell type assignment of cortical precursors and dorsal and ventral forebrain precursors. These cellular phenotypes occurred in the absence of alterations in cell proliferation during cortical differentiation, differing from previous studies. Acquisition of cell fate during midbrain differentiation was not different between control- and autism-iPSCs. Conclusions Taken together, our data indicate that autism-iPSCs diverge from control-iPSCs at a cellular level during early stage of neurodevelopment. This suggests that unique developmental differences associated with autism may be established at early prenatal stages.

30Autism is a heterogenous collection of disorders with a complex molecular underpinning. 31Evidence from post mortem studies using adult brains have identified atypical co-expression 32 of genes which may occur during the prenatal period. Recent studies using induced pluripotent 33 stem cells (iPSCs) generated from autistic individuals have suggested that prenatal 34 development is a critical period for the emergence of pathophysiology associated with this 35 condition. However, how early during development these differences emerge and whether such 36 alterations can be seen across the development of multiple brain regions is unclear. In this study 37 we investigated whether early prenatal stages of neurodevelopment differ between iPSCs 38 generated from typically developing and autistic individuals. We specifically selected autistic 39 individuals unrelated to each other with a heterogeneous genetic background and no known 40 comorbidities, to probe for common molecular phenotypes. Differentiation of iPSCs towards a 41 cortical lineage revealed abnormal cell fate acquisition from an early stage of development. 42Interestingly, abnormal differentiation occurred in the absence of alteration in cell proliferation 43 during cortical differentiation, differing from previous studies. Moreover, these effects 44 appeared specific for the acquisition of a cortical fate, as differentiation of iPSCs towards a 45 midbrain lineage was not accompanied by differences in neurogenesis between typically 46 developing and autism iPSC lines. RNA-sequencing on a subset of our cohort further revealed 47 autism-specific signatures during cortical differentiation similar to that observed in post 48 mortem studies, indicating a potential common biological mechanism. Together, these data 49 suggest unique developmental differences associated with autism may establish at an early 50 prenatal stage. 51 52 53 4 54 Recent methodological advances in the field of induced pluripotent stem cell (iPSC) 75technology [15][16][17][18][19][20] has made it possible to study prenatal cellular behaviour in autism in detail, 76 something that was not possible using post mortem brains. IPSCs have similar abilities to 77 5 embryonic stem cells to generate any tissue of the body. These can then be differentiated into 78 neurons of various lineages 15-17, 19, 20 . As the neurons contain the same genetic information as 79 the individual from whom it was derived, typical or autistic, its cellular behaviours is 80 influenced by its genetic background. Using these methods, studies have shown significant 81 anomalies in cellular/molecular behaviour during prenatal-equivalent periods of development 82 in autistic individuals [21][22][23] . One such study generated iPSCs from autistic individuals who were 83 comorbid for macrocephaly, and demonstrated: (1) atypical cortical differentiation and 84 increased cell proliferation of cortical neural precursor cells (NPCs) from iPSCs, and (2) an 85 imbalance in excitatory (glutamate-producing) and inhibitory (GABA-produ...

Background The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. Results Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. Conclusion We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson’s disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.

The degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson’s Disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons results in locomotor impairments and mitochondrial abnormality in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modelling and treating PD.