Visible light-activatable Q-dye molecular beacons for long-term mRNA monitoring in neurons.

Herein, we present a new class of Q-dye molecular beacons (MBs) that can be locally activated with visible light in hippocampal neurons. Our novel architecture increases the available monitoring time for neuronal mRNA from several minutes to 14 hours, since a lower light-sampling rate is required for tracking.

Correction of amygdalar dysfunction in a rat model of fragile X syndrome.

Fragile X syndrome (FXS), a commonly inherited form of autism and intellectual disability, is associated with emotional symptoms that implicate dysfunction of the amygdala. However, current understanding of the pathogenesis of the disease is based primarily on studies in the hippocampus and neocortex, where FXS defects have been corrected by inhibiting group I metabotropic glutamate receptors (mGluRs). Here, we observe that activation, rather than inhibition, of mGluRs in the basolateral amygdala reverses impairments in a rat model of FXS. FXS rats exhibit deficient recall of auditory conditioned fear, which is accompanied by a range of in vitro and in vivo deficits in synaptic transmission and plasticity. We find presynaptic mGluR5 in the amygdala, activation of which reverses deficient synaptic transmission and plasticity, thereby restoring normal fear learning in FXS rats. This highlights the importance of modifying the prevailing mGluR-based framework for therapeutic strategies to include circuit-specific differences in FXS pathophysiology.

Differential regulation of local mRNA dynamics and translation following long-term potentiation and depression.

Decades of work have demonstrated that messenger RNAs (mRNAs) are localized and translated within neuronal dendrites and axons to provide proteins for remodeling and maintaining growth cones or synapses. It remains unknown, however, whether specific forms of plasticity differentially regulate the dynamics and translation of individual mRNA species. To address this, we targeted three individual synaptically localized mRNAs, CamkIIa, ß-actin, Psd95, and used molecular beacons to track endogenous mRNA movements. We used reporters and CRISPR/Cas9 gene editing to track mRNA translation in cultured neurons. We found alterations in mRNA dynamic properties occurred during two forms of synaptic plasticity, long-term potentiation (cLTP) and depression (mGluR-LTD). Changes in mRNA dynamics following either form of plasticity resulted in an enrichment of mRNA in the vicinity of dendritic spines. Both the reporters and tagging of endogenous proteins revealed the transcript-specific stimulation of protein synthesis following cLTP or mGluR-LTD. As such, the plasticity-induced enrichment of mRNA near synapses could be uncoupled from its translational status. The enrichment of mRNA in the proximity of spines allows for localized signaling pathways to decode plasticity milieus and stimulate a specific translational profile, resulting in a customized remodeling of the synaptic proteome.

The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition.

We examined the feedback between the major protein degradation pathway, the ubiquitin-proteasome system (UPS), and protein synthesis in rat and mouse neurons. When protein degradation was inhibited, we observed a coordinate dramatic reduction in nascent protein synthesis in neuronal cell bodies and dendrites. The mechanism for translation inhibition involved the phosphorylation of eIF2α, surprisingly mediated by eIF2α kinase 1, or heme-regulated kinase inhibitor (HRI). Under basal conditions, neuronal expression of HRI is barely detectable. Following proteasome inhibition, HRI protein levels increase owing to stabilization of HRI and enhanced translation, likely via the increased availability of tRNAs for its rare codons. Once expressed, HRI is constitutively active in neurons because endogenous heme levels are so low; HRI activity results in eIF2α phosphorylation and the resulting inhibition of translation. These data demonstrate a novel role for neuronal HRI that senses and responds to compromised function of the proteasome to restore proteostasis.

Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments.

There is ample evidence for localization of messenger RNAs (mRNAs) and protein synthesis in neuronal dendrites; however, demonstrations of these processes in presynaptic terminals are limited. We used expansion microscopy to resolve pre- and postsynaptic compartments in rodent neurons. Most presynaptic terminals in the hippocampus and forebrain contained mRNA and ribosomes. We sorted fluorescently labeled mouse brain synaptosomes and then sequenced hundreds of mRNA species present within excitatory boutons. After brief metabolic labeling, >30% of all presynaptic terminals exhibited a signal, providing evidence for ongoing protein synthesis. We tested different classic plasticity paradigms and observed distinct patterns of rapid pre- and/or postsynaptic translation. Thus, presynaptic terminals are translationally competent, and local protein synthesis is differentially recruited to drive compartment-specific phenotypes that underlie different forms of plasticity.

Local translation in neuronal processes.

Neurons exhibit a unique degree of spatial compartmentalization and are able to maintain and remodel their proteomes independently from the cell body. While much effort has been devoted to understanding the capacity and role for local protein synthesis in dendrites and spines, local mRNA translation in mature axons, projecting over distances up to a meter, has received much less attention. Also, little is known about the spatio-temporal dynamics of axonal and dendritic gene expression as function of mRNA abundance, protein synthesis and degradation. Here, we summarize key recent findings that have shaped our knowledge of the precise location of local protein production and discuss unique strategies used by neurons to shape presynaptic and postsynaptic proteomes.

TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD.

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.

KDM1A/LSD1 regulates the differentiation and maintenance of spermatogonia in mice.

The proper regulation of spermatogenesis is crucial to ensure the continued production of sperm and fertility. Here, we investigated the function of the H3K4me2 demethylase KDM1A/LSD1 during spermatogenesis in developing and adult mice. Conditional deletion of Kdm1a in the testis just prior to birth leads to fewer spermatogonia and germ cell loss before 3 weeks of age. These results demonstrate that KDM1A is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis. In addition, inducible deletion of Kdm1a in the adult testis results in the abnormal accumulation of meiotic spermatocytes, as well as apoptosis and progressive germ cell loss. These results demonstrate that KDM1A is also required during adult spermatogenesis. Furthermore, without KDM1A, the stem cell factor OCT4 is ectopically maintained in differentiating germ cells. This requirement for KDM1A is similar to what has been observed in other stem cell populations, suggesting a common function. Taken together, we propose that KDM1A is a key regulator of spermatogenesis and germ cell maintenance in the mouse.

Internal modifications in the CENP-A nucleosome modulate centromeric dynamics.

BACKGROUND: Posttranslational modifications of core histones are correlated with changes in transcriptional status, chromatin fiber folding, and nucleosome dynamics. However, within the centromere-specific histone H3 variant CENP-A, few modifications have been reported, and their functions remain largely unexplored. In this multidisciplinary report, we utilize in silico computational and in vivo approaches to dissect lysine 124 of human CENP-A, which was previously reported to be acetylated in advance of replication. RESULTS: Computational modeling demonstrates that acetylation of K124 causes tightening of the histone core and hinders accessibility to its C-terminus, which in turn diminishes CENP-C binding. Additionally, CENP-A K124ac/H4 K79ac containing nucleosomes are prone to DNA sliding. In vivo experiments using a CENP-A acetyl or unacetylatable mimic (K124Q and K124A, respectively) reveal alterations in CENP-C levels and a modest increase in mitotic errors. Furthermore, mutation of K124 results in alterations in centromeric replication timing. Purification of native CENP-A proteins followed by mass spectrometry analysis reveals that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early S and mid-S phases. Finally, we provide evidence implicating the histone acetyltransferase (HAT) p300 in this cycle. CONCLUSIONS: Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome contribute to the binding of key kinetochore proteins, the integrity of mitosis, and centromeric replication. These data support the paradigm that modifications in histone variants can influence key biological processes.

Spatially and temporally regulating translation via mRNA-binding proteins in cellular and neuronal function.

Coordinated regulation of mRNA localization and local translation are essential steps in cellular asymmetry and function. It is increasingly evident that mRNA-binding proteins play critical functions in controlling the fate of mRNA, including when and where translation occurs. In this review, we discuss the robust and complex roles that mRNA-binding proteins play in the regulation of local translation that impact cellular function in vertebrates. First, we discuss the role of local translation in cellular polarity and possible links to vertebrate development and patterning. Next, we discuss the expanding role for local protein synthesis in neuronal development and function, with special focus on how a number of neurological diseases have given us insight into the importance of translational regulation. Finally, we discuss the ever-increasing set of tools to study regulated translation and how these tools will be vital in pushing forward and addressing the outstanding questions in the field.

The Survival of Motor Neuron Protein Acts as a Molecular Chaperone for mRNP Assembly.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival of motor neuron (SMN) protein. SMN is part of a multiprotein complex that facilitates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN has also been found to associate with mRNA-binding proteins, but the nature of this association was unknown. Here, we have employed a combination of biochemical and advanced imaging methods to demonstrate that SMN promotes the molecular interaction between IMP1 protein and the 3' UTR zipcode region of ß-actin mRNA, leading to assembly of messenger ribonucleoprotein (mRNP) complexes that associate with the cytoskeleton to facilitate trafficking. We have identified defects in mRNP assembly in cells and tissues from SMA disease models and patients that depend on the SMN Tudor domain and explain the observed deficiency in mRNA localization and local translation, providing insight into SMA pathogenesis as a ribonucleoprotein (RNP)-assembly disorder.

A role for the survival of motor neuron protein in mRNP assembly and transport.

Localization and local translation of mRNA plays a key role in neuronal development and function. While studies in various systems have provided insights into molecular mechanisms of mRNA transport and local protein synthesis, the factors that control the assembly of mRNAs and mRNA binding proteins into messenger ribonucleoprotein (mRNP) transport granules remain largely unknown. In this review we will discuss how insights on a motor neuron disease, spinal muscular atrophy (SMA), is advancing our understanding of regulated assembly of transport competent mRNPs and how defects in their assembly and delivery may contribute to the degeneration of motor neurons observed in SMA and other neurological disorders.

Deficiency of the Survival of Motor Neuron Protein Impairs mRNA Localization and Local Translation in the Growth Cone of Motor Neurons.

Spinal muscular atrophy (SMA) is a neurodegenerative disease primarily affecting spinal motor neurons. It is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays an essential role in the biogenesis of spliceosomal small nuclear ribonucleoproteins in all tissues. The etiology of the specific defects in the motor circuitry in SMA is still unclear, but SMN has also been implicated in mediating the axonal localization of mRNA-protein complexes, which may contribute to the axonal degeneration observed in SMA. Here, we report that SMN deficiency severely disrupts local protein synthesis within neuronal growth cones. We also identify the cytoskeleton-associated growth-associated protein 43 (GAP43) mRNA as a new target of SMN and show that motor neurons from SMA mouse models have reduced levels ofGAP43mRNA and protein in axons and growth cones. Importantly, overexpression of two mRNA-binding proteins, HuD and IMP1, restoresGAP43mRNA and protein levels in growth cones and rescues axon outgrowth defects in SMA neurons. These findings demonstrate that SMN plays an important role in the localization and local translation of mRNAs with important axonal functions and suggest that disruption of this function may contribute to the axonal defects observed in SMA. SIGNIFICANCE STATEMENT: The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays a key role in assembling RNA/protein complexes that are essential for mRNA splicing. It remains unclear whether defects in this well characterized housekeeping function cause the specific degeneration of spinal motor neurons observed in SMA. Here, we describe an additional role of SMN in regulating the axonal localization and local translation of the mRNA encoding growth-associated protein 43 (GAP43). This study supports a model whereby SMN deficiency impedes transport and local translation of mRNAs important for neurite outgrowth and stabilization, thus contributing to axon degeneration, muscle denervation, and motor neuron cell death in SMA.

Dynamics of survival of motor neuron (SMN) protein interaction with the mRNA-binding protein IMP1 facilitates its trafficking into motor neuron axons.

Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the ß-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the ß-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1.

Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle.

DREF was first characterized for its role in the regulation of transcription of genes encoding proteins involved in DNA replication and found to interact with sequences similar to the DNA recognition motif of the BEAF-32 insulator protein. Insulators are DNA-protein complexes that mediate intra- and inter-chromosome interactions. Several DNA-binding insulator proteins have been described in Drosophila, including BEAF-32, dCTCF and Su(Hw). Here we find that DREF and BEAF-32 co-localize at the same genomic sites, but their enrichment shows an inverse correlation. Furthermore, DREF co-localizes in the genome with other insulator proteins, suggesting that the function of this protein may require components of Drosophila insulators. This is supported by the finding that mutations in insulator proteins modulate DREF-induced cell proliferation. DREF persists bound to chromatin during mitosis at a subset of sites where it also co-localizes with dCTCF, BEAF-32 and CP190. These sites are highly enriched for sites where Orc2 and Mcm2 are present during interphase and at the borders of topological domains of chromosomes defined by Hi-C. The results suggest that DREF and insulator proteins may help maintain chromosome organization during the cell cycle and mark a subset of genomic sites for the assembly of pre-replication complexes and gene bookmarking during the M/G1 transition.

A subset of Drosophila Myc sites remain associated with mitotic chromosomes colocalized with insulator proteins.

Myc has been characterized as a transcription factor that activates expression of genes involved in pluripotency and cancer, and as a component of the replication complex. Here we find that Myc is present at promoters and enhancers of Drosophila melanogaster genes during interphase. Myc colocalizes with Orc2, which is part of the prereplication complex, during G1. As is the case in mammals, Myc associates preferentially with paused genes, suggesting that it may also be involved in the release of RNA polymerase II from the promoter-proximal pausing in Drosophila. Interestingly, about 40% of Myc sites present in interphase persists during mitosis. None of the Myc mitotic sites correspond to enhancers, and only some correspond to promoters. The rest of the mitotic Myc sites overlap with binding sites for multiple insulator proteins that are also maintained in mitosis. These results suggest alternative mechanisms to explain the role of Myc in pluripotency and cancer.