Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model.

Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. FXS results from the lack of the RNA-binding protein FMRP and is associated with the deregulation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk), a main mRNA target of FMRP in cortical neurons and a master regulator of lipid signaling, is downregulated in the absence of FMRP in the brain of Fmr1-KO mouse model. Here we show that adeno-associated viral vector delivery of a modified and FMRP-independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS-relevant behavioral phenotypes in the Fmr1-KO mouse. Our data suggest that DGKk is an important factor in FXS pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in FXS.

Disruption of RNA Metabolism in Neurological Diseases and Emerging Therapeutic Interventions.

RNA binding proteins are critical to the maintenance of the transcriptome via controlled regulation of RNA processing and transport. Alterations of these proteins impact multiple steps of the RNA life cycle resulting in various molecular phenotypes such as aberrant RNA splicing, transport, and stability. Disruption of RNA binding proteins and widespread RNA processing defects are increasingly recognized as critical determinants of neurological diseases. Here, we describe distinct mechanisms by which the homeostasis of RNA binding proteins is compromised in neurological disorders through their reduced expression level, increased propensity to aggregate or sequestration by abnormal RNAs. These mechanisms all converge toward altered neuronal function highlighting the susceptibility of neurons to deleterious changes in RNA expression and the central role of RNA binding proteins in preserving neuronal integrity. Emerging therapeutic approaches to mitigate or reverse alterations of RNA binding proteins in neurological diseases are discussed.

CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts.

Expansion of G4C2 repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded transcripts undergo repeat-associated non-AUG (RAN) translation producing dipeptide repeat proteins from all reading frames. We determined cis-factors and trans-factors influencing translation of the human C9ORF72 transcripts. G4C2 translation operates through a 5'-3' cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNAMeti. Production of poly-GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG start codon supporting a frameshifting mechanism. RAN translation is also regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5'-flanking G4C2 sequence block ribosomal scanning and prevent translation. Finally, we identified an unexpected affinity of expanded transcripts for the ribosomal subunits independently from translation.

Polyglutamine-Expanded Huntingtin Exacerbates Age-Related Disruption of Nuclear Integrity and Nucleocytoplasmic Transport.

Onset of neurodegenerative disorders, including Huntington's disease, is strongly influenced by aging. Hallmarks of aged cells include compromised nuclear envelope integrity, impaired nucleocytoplasmic transport, and accumulation of DNA double-strand breaks. We show that mutant huntingtin markedly accelerates all of these cellular phenotypes in a dose- and age-dependent manner in cortex and striatum of mice. Huntingtin-linked polyglutamine initially accumulates in nuclei, leading to disruption of nuclear envelope architecture, partial sequestration of factors essential for nucleocytoplasmic transport (Gle1 and RanGAP1), and intranuclear accumulation of mRNA. In aged mice, accumulation of RanGAP1 together with polyglutamine is shifted to perinuclear and cytoplasmic areas. Consistent with findings in mice, marked alterations in nuclear envelope morphology, abnormal localization of RanGAP1, and nuclear accumulation of mRNA were found in cortex of Huntington's disease patients. Overall, our findings identify polyglutamine-dependent inhibition of nucleocytoplasmic transport and alteration of nuclear integrity as a central component of Huntington's disease.

Fragile X syndrome: Are signaling lipids the missing culprits?

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability and autism. FXS results from the absence of FMRP, an RNA binding protein associated to ribosomes that influences the translation of specific mRNAs in post-synaptic compartments of neurons. The main molecular consequence of the absence of FMRP is an excessive translation of neuronal protein in several areas of the brain. This local protein synthesis deregulation is proposed to underlie the defect in synaptic plasticity responsible for FXS. Recent findings in neurons of the fragile X mouse model (Fmr1-KO) uncovered another consequence of the lack of FMRP: a deregulation of the diacylglycerol (DAG)/phosphatidic acid (PA) homeostasis. DAG and PA are two interconvertible lipids that influence membrane architecture and that act as essential signaling molecules that activate various downstream effectors, including master regulators of local protein synthesis and actin polymerization. As a consequence, DAG and PA govern a variety of cellular processes, including cell proliferation, vesicle/membrane trafficking and cytoskeletal organization. At the synapse, the level of these lipids is proposed to influence the synaptic activation status. FMRP appears as a master regulator of this neuronal process by controlling the translation of a diacylglycerol kinase enzyme that converts DAG into PA. The deregulated levels of DAG and PA caused by the absence of FMRP could represent a novel therapeutic target for the treatment of FXS.

Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons.

Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

Sequestration of DROSHA and DGCR8 by expanded CGG RNA repeats alters microRNA processing in fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55-200 CGG repeats in the 5' UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery.

G-quadruplex RNA structure as a signal for neurite mRNA targeting.

Targeting of messenger RNAs (mRNAs) in neuron processes relies on cis-acting regulatory elements, the nature of which is poorly understood. Here, we report that approximately 30% of the best-known dendritic mRNAs contain a guanine (G)-quadruplex consensus in their 3'-untranslated region. Among these mRNAs, we show by using RNA structure probing that a G-quadruplex is present in the mRNAs of two key postsynaptic proteins: PSD-95 and CaMKIIa. The G-quadruplex structure is necessary and sufficient for the potent and fast localization of mRNAs in cortical neurites and this occurs in a metabotropic glutamate receptor-responsive manner. Thus, G-quadruplex seems to be a common neurite localization signal.