Circular RNAs (circRNAs) are formed in all domains of life and via different mechanisms. There has been an explosion in the number of circRNA papers in recent years; however, as a relatively young ...field, circRNA biology has an urgent need for common experimental standards for isolating, analyzing, expressing and depleting circRNAs. Here we propose a set of guidelines for circRNA studies based on the authors' experience. This Perspective will specifically address the major class of circRNAs in Eukarya that are generated by a spliceosome-catalyzed back-splicing event. We hope that the implementation of best practice principles for circRNA research will help move the field forward and allow a better functional understanding of this fascinating group of RNAs.
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains ...unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.
The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell ...perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively induce actin disassembly in eukaryotic cells. We demonstrate that DeActs are universal tools for studying the actin cytoskeleton in single cells in culture, tissues, and multicellular organisms including various neurodevelopmental model systems.
Monoaminergic neurons serotonergic (5-HT) and dopaminergic (mdDA) in the brainstem project axons along the anterior-posterior axis. Despite their important physiological functions and implication in ...disease, the molecular mechanisms that dictate the formation of these projections along the anterior-posterior axis remain unknown. Here we reveal a novel requirement for Wnt/planar cell polarity signaling in the anterior-posterior organization of the monoaminergic system. We find that 5-HT and mdDA axons express the core planar cell polarity components Frizzled3, Celsr3, and Vangl2. In addition, monoaminergic projections show anterior-posterior guidance defects in Frizzled3, Celsr3, and Vangl2 mutant mice. The only known ligands for planar cell polarity signaling are Wnt proteins. In culture, Wnt5a attracts 5-HT but repels mdDA axons, and Wnt7b attracts mdDA axons. However, mdDA axons from Frizzled3 mutant mice are unresponsive to Wnt5a and Wnt7b. Both Wnts are expressed in gradients along the anterior-posterior axis, consistent with their role as directional cues. Finally, Wnt5a mutants show transient anterior-posterior guidance defects in mdDA projections. Furthermore, we observe during development that the cell bodies of migrating descending 5-HT neurons eventually reorient along the direction of their axons. In Frizzled3 mutants, many 5-HT and mdDA neuron cell bodies are oriented abnormally along the direction of their aberrant axon projections. Overall, our data suggest that Wnt/planar cell polarity signaling may be a global anterior-posterior guidance mechanism that controls axonal and cellular organization beyond the spinal cord.
Axons Navigate Noise with 190RhoGAP Zelina, Pavol; Pasterkamp, R. Jeroen
Neuron (Cambridge, Mass.),
05/2019, Volume:
102, Issue:
3
Journal Article
Peer reviewed
Open access
In this issue of Neuron, Bonanomi et al. (2019) investigate how navigating axons ignore irrelevant guidance signals. They show that the binding of p190RhoGAP to DCC suppresses inappropriate responses ...to Netrin-1, allowing motor axons to exit the embryonic spinal cord.
In this issue of Neuron, Bonanomi et al. (2019) investigate how navigating axons ignore irrelevant guidance signals. They show that the binding of p190RhoGAP to DCC suppresses inappropriate responses to Netrin-1, allowing motor axons to exit the embryonic spinal cord.
Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disorder characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE may involve defects in the post-transcriptional ...regulation of gene expression. MicroRNAs (miRNAs) are non-coding RNAs that control the expression of genes at the post-transcriptional level. Here, we performed a genome-wide miRNA profiling study to examine whether miRNA-mediated mechanisms are affected in human mTLE. miRNA profiles of the hippocampus of autopsy control patients and two mTLE patient groups were compared. This revealed segregated miRNA signatures for the three different patient groups and 165 miRNAs with up- or down-regulated expression in mTLE. miRNA in situ hybridization detected cell type-specific changes in miRNA expression and an abnormal nuclear localization of select miRNAs in neurons and glial cells of mTLE patients. Of several cellular processes implicated in mTLE, the immune response was most prominently targeted by deregulated miRNAs. Enhanced expression of inflammatory mediators was paralleled by a reduction in miRNAs that were found to target the 3′-untranslated regions of these genes in reporter assays. miR-221 and miR-222 were shown to regulate endogenous ICAM1 expression and were selectively co-expressed with ICAM1 in astrocytes in mTLE patients. Our findings suggest that miRNA changes in mTLE affect the expression of immunomodulatory proteins thereby further facilitating the immune response. This mechanism may have broad implications given the central role of astrocytes and the immune system in human neurological disease. Overall, this work extends the current concepts of human mTLE pathogenesis to the level of miRNA-mediated gene regulation.
Objective
How hexanucleotide (GGGGCC) repeat expansions in C9ORF72 cause amyotrophic lateral sclerosis (ALS) remains poorly understood. Both gain‐ and loss‐of‐function mechanisms have been proposed. ...Evidence supporting these mechanisms in vivo is, however, incomplete. Here we determined the effect of C9orf72 loss‐of‐function in mice.
Methods
We generated and analyzed a conditional C9orf72 knockout mouse model. C9orf72fl/fl mice were crossed with Nestin‐Cre mice to selectively remove C9orf72 from neurons and glial cells. Immunohistochemistry was performed to study motor neurons and neuromuscular integrity, as well as several pathological hallmarks of ALS, such as gliosis and TDP‐43 mislocalization. In addition, motor function and survival were assessed.
Results
Neural‐specific ablation of C9orf72 in conditional C9orf72 knockout mice resulted in significantly reduced body weight but did not induce motor neuron degeneration, defects in motor function, or altered survival.
Interpretation
Our data suggest that C9orf72 loss‐of‐function, by itself, is insufficient to cause motor neuron disease. These results may have important implications for the development of therapeutic strategies for C9orf72‐associated ALS. Ann Neurol 2015;78:426–438
Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease affecting upper and lower motor neurons with no cure available. Clinical and animal studies reveal that the ...neuromuscular junction (NMJ), a synaptic connection between motor neurons and skeletal muscle fibers, is highly vulnerable in ALS and suggest that NMJ defects may occur at the early stages of the disease. However, mechanistic insight into how NMJ dysfunction relates to the onset and progression of ALS is incomplete, which hampers therapy development. This is, in part, caused by a lack of robust in vitro models. The ability to combine microfluidic and induced pluripotent stem cell (iPSC) technologies has opened up new avenues for studying molecular and cellular ALS phenotypes in vitro. Microfluidic devices offer several advantages over traditional culture approaches when modeling the NMJ, such as the spatial separation of different cell types and increased control over the cellular microenvironment. Moreover, they are compatible with 3D cell culture, which enhances NMJ functionality and maturity. Here, we review how microfluidic technology is currently being employed to develop more reliable in vitro NMJ models. To validate and phenotype such models, various morphological and functional read‐outs have been developed. We describe and discuss the relevance of these read‐outs and specifically illustrate how these read‐outs have enhanced our understanding of NMJ pathology in ALS. Finally, we share our view on potential future directions and challenges.
The neuromuscular junction (NMJ) is highly vulnerable and affected at the early stages of the pathogenic process leading to ALS. Advances in microfluidic technology and stem cell biology have led to the development of ALS‐on‐a‐chip models. This has provided new avenues for studying ALS disease mechanisms and for drug development. One important advantage of these models is their high spatial control over individual cell types, enabling studies on the contribution of each cell type to NMJ dysfunction. Furthermore, in recent years a variety of morphological and functional assays has been developed to facilitate analysis of NMJ (patho)physiology in microfluidic platforms.
Midbrain dopaminergic (DA) axons make long longitudinal projections towards the striatum. Despite the importance of DA striatal innervation, processes involved in establishment of DA axonal ...connectivity remain largely unknown. Here we demonstrate a striatal-specific requirement of transcriptional regulator Nolz1 in establishing DA circuitry formation. DA projections are misguided and fail to innervate the striatum in both constitutive and striatal-specific Nolz1 mutant embryos. The lack of striatal Nolz1 expression results in nigral to pallidal lineage conversion of striatal projection neuron subtypes. This lineage switch alters the composition of secreted factors influencing DA axonal tract formation and renders the striatum non-permissive for dopaminergic and other forebrain tracts. Furthermore, transcriptomic analysis of wild-type and Nolz1
mutant striatal tissue led to the identification of several secreted factors that underlie the observed guidance defects and proteins that promote DA axonal outgrowth. Together, our data demonstrate the involvement of the striatum in orchestrating dopaminergic circuitry formation.
Long non‐coding RNAs (lncRNAs) are RNAs that exceed 200 nucleotides in length and that are not translated into proteins. Thousands of lncRNAs have been identified with functions in processes such as ...transcription and translation regulation, RNA processing, and RNA and protein sponging. LncRNAs show prominent expression in the nervous system and have been implicated in neural development, function and disease. Recent work has begun to report on the expression and roles of lncRNAs in motor neurons (MNs). The cell bodies of MNs are located in cortex, brainstem or spinal cord and their axons project into the brainstem, spinal cord or towards peripheral muscles, thereby controlling important functions such as movement, breathing and swallowing. Degeneration of MNs is a pathological hallmark of diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. LncRNAs influence several aspects of MN development and disruptions in these lncRNA‐mediated effects are proposed to contribute to the pathogenic mechanisms underlying MN diseases (MNDs). Accumulating evidence suggests that lncRNAs may comprise valuable therapeutic targets for different MNDs. In this review, we discuss the role of lncRNAs (including circular RNAs circRNAs) in the development of MNs, discuss how lncRNAs may contribute to MNDs and provide directions for future research.
During development, distinct classes of long non‐coding RNAs (lncRNAs) display specific spatiotemporal patterns to drive motor neuron (MN) differentiation and maturation leading to the generation of functional subsets of MNs. MN diseases such as ALS are characterized by changes in the expression of specific classes of lncRNAs and mutant antisense (AS) RNAs, resulting in disease phenotypes. These include the formation of aberrant RNA accumulations (foci) that sequester and interfere with RNA‐binding protein (RBP) function and the production of toxic peptides (DPRs). These changes ultimately lead to MN degeneration and muscle atrophy.
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