Impact of differential and time-dependent autophagy activation on therapeutic efficacy in a model of Huntington disease.

Activation of macroautophagy/autophagy, a key mechanism involved in the degradation and removal of aggregated proteins, can successfully reverse Huntington disease phenotypes in various model systems. How neuronal autophagy impairments need to be considered in Huntington disease progression to achieve a therapeutic effect is currently not known. In this study, we used a mouse model of HTT (huntingtin) protein aggregation to investigate how different methods and timing of autophagy activation influence the efficacy of autophagy-activating treatment in vivo. We found that overexpression of human TFEB, a master regulator of autophagy, did not decrease mutant HTT aggregation. On the other hand, Becn1 overexpression, an autophagic regulator that plays a key role in autophagosome formation, partially cleared mutant HTT aggregates and restored neuronal pathology, but only when administered early in the disease progression. When Becn1 was administered at a later stage, when prominent mutant HTT accumulation and autophagy impairments have occurred, Becn1 overexpression did not rescue the mutant HTT-associated phenotypes. Together, these results demonstrate that the targets used to activate autophagy, as well as the timing of autophagy activation, are crucial for achieving efficient therapeutic effects.Abbreviations: AAV: adeno-associated viral vectors; ACTB: actin beta; BECN1: beclin 1, autophagy related; DAPI: 4',6-diamidino-2-phenylindole; GO: gene ontology; HD: Huntington disease; HTT: huntingtin; ICQ: Li's intensity correlation quotient; IHC: immunohistochemistry; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mHTT: mutant huntingtin; PCA: principal component analysis; PPP1R1B/DARPP-32: protein phosphatase 1 regulatory inhibitor subunit 1B; SQSTM1: sequestosome 1; TFEB: transcription factor EB; WB: western blot; WT: wild-type.

Driving Neuronal Differentiation through Reversal of an ERK1/2-miR-124-SOX9 Axis Abrogates Glioblastoma Aggressiveness.

Identifying cellular programs that drive cancers to be stem-like and treatment resistant is critical to improving outcomes in patients. Here, we demonstrate that constitutive extracellular signal-regulated kinase 1/2 (ERK1/2) activation sustains a stem-like state in glioblastoma (GBM), the most common primary malignant brain tumor. Pharmacological inhibition of ERK1/2 activation restores neurogenesis during murine astrocytoma formation, inducing neuronal differentiation in tumorspheres. Constitutive ERK1/2 activation globally regulates miRNA expression in murine and human GBMs, while neuronal differentiation of GBM tumorspheres following the inhibition of ERK1/2 activation requires the functional expression of miR-124 and the depletion of its target gene SOX9. Overexpression of miR124 depletes SOX9 in vivo and promotes a stem-like-to-neuronal transition, with reduced tumorigenicity and increased radiation sensitivity. Providing a rationale for reports demonstrating miR-124-induced abrogation of GBM aggressiveness, we conclude that reversal of an ERK1/2-miR-124-SOX9 axis induces a neuronal phenotype and that enforcing neuronal differentiation represents a therapeutic strategy to improve outcomes in GBM.

LINE-2 transposable elements are a source of functional human microRNAs and target sites.

Transposable elements (TEs) are dynamically expressed at high levels in multiple human tissues, but the function of TE-derived transcripts remains largely unknown. In this study, we identify numerous TE-derived microRNAs (miRNAs) by conducting Argonaute2 RNA immunoprecipitation followed by small RNA sequencing (AGO2 RIP-seq) on human brain tissue. Many of these miRNAs originated from LINE-2 (L2) elements, which entered the human genome around 100-300 million years ago. L2-miRNAs derived from the 3' end of the L2 consensus sequence and thus shared very similar sequences, indicating that L2-miRNAs could target transcripts with L2s in their 3'UTR. In line with this, many protein-coding genes carried fragments of L2-derived sequences in their 3'UTR: these sequences served as target sites for L2-miRNAs. L2-miRNAs and their targets were generally ubiquitously expressed at low levels in multiple human tissues, suggesting a role for this network in buffering transcriptional levels of housekeeping genes. In addition, we also found evidence that this network is perturbed in glioblastoma. In summary, our findings uncover a TE-based post-transcriptional network that shapes transcriptional regulation in human cells.

Distinct cognitive effects and underlying transcriptome changes upon inhibition of individual miRNAs in hippocampal neurons.

MicroRNAs (miRNA) are small, non-coding RNAs mediating post-transcriptional regulation of gene expression. miRNAs have recently been implicated in hippocampus-dependent functions such as learning and memory, although the roles of individual miRNAs in these processes remain largely unknown. Here, we achieved stable inhibition using AAV-delivered miRNA sponges of individual, highly expressed and brain-enriched miRNAs; miR-124, miR-9 and miR-34, in hippocampal neurons. Molecular and cognitive studies revealed a role for miR-124 in learning and memory. Inhibition of miR-124 resulted in an enhanced spatial learning and working memory capacity, potentially through altered levels of genes linked to synaptic plasticity and neuronal transmission. In contrast, inhibition of miR-9 or miR-34 led to a decreased capacity of spatial learning and of reference memory, respectively. On a molecular level, miR-9 inhibition resulted in altered expression of genes related to cell adhesion, endocytosis and cell death, while miR-34 inhibition caused transcriptome changes linked to neuroactive ligand-receptor transduction and cell communication. In summary, this study establishes distinct roles for individual miRNAs in hippocampal function.

Identification of the miRNA targetome in hippocampal neurons using RIP-seq.

MicroRNAs (miRNAs) are key players in the regulation of neuronal processes by targeting a large network of target messenger RNAs (mRNAs). However, the identity and function of mRNAs targeted by miRNAs in specific cells of the brain are largely unknown. Here, we established an adeno-associated viral vector (AAV)-based neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high-throughput sequencing to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this approach, we identified more than two thousand miRNA targets in hippocampal neurons, regulating essential neuronal features such as cell signalling, transcription and axon guidance. Furthermore, we found that stable inhibition of the highly expressed miR-124 and miR-125 in hippocampal neurons led to significant but distinct changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. These findings greatly enhance our understanding of the miRNA targetome in hippocampal neurons.