Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau In Vivo

Article Number: e1001026Peer reviewe

In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment

Treating the placenta to prevent adverse effects of gestational hypoxia on fetal brain development.

Some neuropsychiatric disease, including schizophrenia, may originate during prenatal development, following periods of gestational hypoxia and placental oxidative stress. Here we investigated if gestational hypoxia promotes damaging secretions from the placenta that affect fetal development and whether a mitochondria-targeted antioxidant MitoQ might prevent this. Gestational hypoxia caused low birth-weight and changes in young adult offspring brain, mimicking those in human neuropsychiatric disease. Exposure of cultured neurons to fetal plasma or to secretions from the placenta or from model trophoblast barriers that had been exposed to altered oxygenation caused similar morphological changes. The secretions and plasma contained altered microRNAs whose targets were linked with changes in gene expression in the fetal brain and with human schizophrenia loci. Molecular and morphological changes in vivo and in vitro were prevented by a single dose of MitoQ bound to nanoparticles, which were shown to localise and prevent oxidative stress in the placenta but not in the fetus. We suggest the possibility of developing preventative treatments that target the placenta and not the fetus to reduce risk of psychiatric disease in later life

A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs

Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes

miR-132 Enhances Dendritic Morphogenesis, Spine Density, Synaptic Integration, and Survival of Newborn Olfactory Bulb Neurons

An array of signals regulating the early stages of postnatal subventricular zone (SVZ) neurogenesis has been identified, but much less is known regarding the molecules controlling late stages. Here, we investigated the function of the activity-dependent and morphogenic microRNA miR-132 on the synaptic integration and survival of olfactory bulb (OB) neurons born in the neonatal SVZ. In situ hybridization revealed that miR-132 expression occurs at the onset of synaptic integration in the OB. Using in vivo electroporation we found that sequestration of miR-132 using a sponge-based strategy led to a reduced dendritic complexity and spine density while overexpression had the opposite effects. These effects were mirrored with respective changes in the frequency of GABAergic and glutamatergic synaptic inputs reflecting altered synaptic integration. In addition, timely directed overexpression of miR-132 at the onset of synaptic integration using an inducible approach led to a significant increase in the survival of newborn neurons. These data suggest that miR-132 forms the basis of a structural plasticity program seen in SVZ-OB postnatal neurogenesis. miR-132 overexpression in transplanted neurons may thus hold promise for enhancing neuronal survival and improving the outcome of transplant therapies

Modeling Spinal Muscular Atrophy in Drosophila

Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets

Understanding neuronal connectivity through the post-transcriptional toolkit

Post-transcriptional regulatory mechanisms have emerged as a critical component underlying the diversification and spatiotemporal control of the proteome during the establishment of precise neuronal connectivity. These mechanisms have been shown to be important for virtually all stages of assembling a neural network, from neurite guidance, branching, and growth to synapse morphogenesis and function. From the moment a gene is transcribed, it undergoes a series of post-transcriptional regulatory modifications in the nucleus and cytoplasm until its final deployment as a functional protein. Initially, a message is subjected to extensive structural regulation through alternative splicing, which is capable of greatly expanding the protein repertoire by generating, in some cases, thousands of functionally distinct isoforms from a single gene locus. Then, RNA packaging into neuronal transport granules and recognition by RNA-binding proteins and/or microRNAs is capable of restricting protein synthesis to selective locations and under specific input conditions. This ability of the post-transcriptional apparatus to expand the informational content of a cell and control the deployment of proteins in both spatial and temporal dimensions is a feature well adapted for the extreme morphological properties of neural cells. In this review, we describe recent advances in understanding how post-transcriptional regulatory mechanisms refine the proteomic complexity required for the assembly of intricate and specific neural networks

SRβ coordinates signal sequence release from SRP with ribosome binding to the translocon

Protein targeting to the endoplasmic reticulum (ER) membrane is regulated by three GTPases, the 54 kDa subunit of the signal recognition particle (SRP) and the α- and β-subunits of the SRP receptor (SR). Using a soluble form of SR and an XTP-binding mutant of SRβ, we show that SRβ is essential for protein translocation across the ER membrane. SRβ can be cross-linked to a 21 kDa ribosomal protein in its empty and GDP-bound state, but not when GTP is bound. GTP binding to SRβ is required to induce signal sequence release from SRP. This is achieved by the presence of the translocon, which changes the interaction between the 21 kDa ribosomal protein and SRβ and thereby allows SRβ to bind GTP. We conclude that SRβ coordinates the release of the signal sequence from SRP with the presence of the translocon

Oxidative stress mediates tau-induced neurodegeneration in Drosophila

Markers of oxidative damage have been detected in brain tissue from patients with Alzheimer disease (AD) and other neurodegenerative disorders. These findings implicate oxidative injury in the neurodegenerative process, but whether oxidative stress is a cause or a consequence of neurotoxicity remains unclear. We used a Drosophila model of human tauopathies to investigate the role of oxidative stress in neurodegeneration. Genetic and pharmacological manipulation of antioxidant defense mechanisms significantly modified neurodegeneration in our model, suggesting that oxidative stress plays a causal role in neurotoxicity. We demonstrate that the JNK signaling pathway is activated in our model, which is in agreement with previous findings in AD tissue. Furthermore, we show that the extent of JNK activation correlates with the degree of tau-induced neurodegeneration. Finally, our findings suggest that oxidative stress acts not to promote tau phosphorylation, but to enhance tau-induced cell cycle activation. In summary, our study identifies oxidative stress as a causal factor in tau-induced neurodegeneration in Drosophila