Genome-wide, integrative analysis of circular RNA dysregulation and the corresponding circular RNA-microRNA-mRNA regulatory axes in autism.

Circular RNAs (circRNAs), a class of long noncoding RNAs, are known to be enriched in mammalian neural tissues. Although a wide range of dysregulation of gene expression in autism spectrum disorder (ASD) have been reported, the role of circRNAs in ASD remains largely unknown. Here, we performed genome-wide circRNA expression profiling in postmortem brains from individuals with ASD and controls and identified 60 circRNAs and three coregulated modules that were perturbed in ASD. By integrating circRNA, microRNA, and mRNA dysregulation data derived from the same cortex samples, we identified 8170 ASD-associated circRNA-microRNA-mRNA interactions. Putative targets of the axes were enriched for ASD risk genes and genes encoding inhibitory postsynaptic density (PSD) proteins, but not for genes implicated in monogenetic forms of other brain disorders or genes encoding excitatory PSD proteins. This reflects the previous observation that ASD-derived organoids show overproduction of inhibitory neurons. We further confirmed that some ASD risk genes (NLGN1, STAG1, HSD11B1, VIP, and UBA6) were regulated by an up-regulated circRNA (circARID1A) via sponging a down-regulated microRNA (miR-204-3p) in human neuronal cells. Particularly, alteration of NLGN1 expression is known to affect the dynamic processes of memory consolidation and strengthening. To the best of our knowledge, this is the first systems-level view of circRNA regulatory networks in ASD cortex samples. We provided a rich set of ASD-associated circRNA candidates and the corresponding circRNA-microRNA-mRNA axes, particularly those involving ASD risk genes. Our findings thus support a role for circRNA dysregulation and the corresponding circRNA-microRNA-mRNA axes in ASD pathophysiology.

BDNF elevates the axonal levels of hnRNPs Q and R in cultured rat cortical neurons.

Local translation plays important roles in the maintenance and various functions of axons, and dysfunctions of local translation in axons are implicated in various neurological diseases. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA binding proteins with multiple functions in RNA metabolism. Here, we identified 20 hnRNPs in the axons of cultured rat cortical neurons by interrogating published axon mass spectrometric databases with rat protein databases. Among those identified in axons are highly related hnRNPs Q and R. RT-PCR analysis indicated that axons also contained low levels of hnRNPs Q and R mRNAs. We further found that BDNF treatments raised the levels of hnRNPs Q and R proteins in whole neurons and axons. BDNF also increased the level of poly(A) RNA as well as the proportion of poly(A) RNA granules containing hnRNPs Q and R in the axon. However, following severing the connection between the cell bodies and axons, BDNF did not affect the levels of hnRNPs Q and R, the content of poly(A) RNA, or the colocalization of poly(A) RNA and hnRNPs Q and R in the axon any more, although BDNF still stimulated the local translation in severed axons as it did in intact axons. The results are consistent with that BDNF enhances the axonal transport of RNA granules. The results further suggest that hnRNPs Q and R play a role in the mechanism underlying the enhancement of axonal RNA transport by BDNF.

Unbiased Proteomic Study of the Axons of Cultured Rat Cortical Neurons.

The axon is a long projection connecting a neuron to its targets. Here, the axons of cultured rat cortical neurons were isolated with micropatterned chips that enable the separation of axons from their cell bodies. Proteins extracted from isolated axons and whole neurons were subjected to analyses using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) analyses without and with stable isotope dimethyl labeling, resulting in the identification of >2500 axonal proteins and 103 axon-enriched proteins. A strong correlation exists between the abundances of axonal proteins and their counterparts in whole neurons. The proteomic results confirm the axonal protein constituents of the subcellular structures documented in earlier electron microscopic studies. Cortical axons have proteins that are components of machineries for protein degradation and the synthesis of soluble, membrane, and secretory proteins, although axons lack conventional Golgi apparatus. Despite the fact that axons lack nucleus, nuclear proteins were identified, and 67 of them were found enriched in axons. Some of the results obtained by the MS-based studies were validated by quantitative Western blotting and immunofluorescence staining analyses. The results represent the first comprehensive description of the axonal protein landscape. The MS proteomics data are available via ProteomeXchange with identifier PXD005527.

A compartmentalized culture device for studying the axons of CNS neurons.

We report here the development of a compartmentalized culture device that allows the spatial separation of the somatodendrites and axons of central nervous system (CNS) neurons. The device consists of two compartments separated by a septum constructed by attaching a porous polycarbonate track etch (PCTE) filter on top of a microchannel-filled polydimethylsiloxane (PDMS) membrane. The surface and microchannels of the septum are coated and filled, respectively, with materials that support neuron growth and neurite migration. When rat hippocampal neurons are cultured in the top compartment, axons are the only processes that can migrate through the septum to the bottom compartment. The axons in the bottom compartment can be studied directly in real-time or through immunofluorescence staining after fixation. Axons containing ∼3 µg protein can be isolated from each device for biochemical analyses. In addition, the septum also impedes the movement of small molecules between the top and bottom compartments. This feature allows the somatodendrites and axons of neurons, which occupy the top and bottom compartments of the device, respectively, to be manipulated independently. The potential applications of the device as a tool in diverse studies concerning neuronal axons and in screening reagents that regulate axonal functions have also been discussed.