Jimena Baleriola

In neurones, like in any other cell, their function often relies on the fine‐tuning of their protein levels, which is achieved by the balance between protein synthesis and turnover. Defects in protein homeostasis frequently lead to neuronal dysfunction and neurological disorders. Given their extreme morphological complexity and high compartmentalization, neurones highly depend on the asymmetrical distribution of their proteome. The common belief is that proteins that sustain axonal, dendritic and synaptic functions are synthesized in the soma and then transported to distal neuronal compartments. However, there is a complementary mechanism by which the mRNAs, and not the proteins, are transported to distal subneuronal domains, and once they reach their destination, they are locally translated. Although once considered heretical, local translation (or local protein synthesis) is now widely accepted by the scientific community. Nonetheless, there is one question that remains largely unexplored in the field, and that is whether local translation in dendrites, axons and synapses is fully regulated by the neurone itself or if non‐neuronal cells (e.g., glia) can modulate this mechanism in a non‐cell‐autonomous manner. Here, we combined primary neuronal cultures, astrocyte‐derived extracellular vesicle (EVs) isolation, and proteomics to investigate whether astroglial EVs modulate local translation in axons. We show that EVs released by astrocytes exposed to amyloid‐β peptide (Aβ) enhance protein synthesis specifically in distal axons and increase synaptic integrity. Proteomics analysis and western blotting identified the ribosomal protein Rps6 as an astroglial Aβ‐EV cargo delivered to axons. Interestingly, genetic downregulation revealed the contribution of vesicular Rps6 to translation regulation in axons and synaptic integrity. To our knowledge, this is the first report that directly demonstrates glial control of local translation in neurones through EVs, revealing a novel glia‐to‐neurone communication mechanism in an experimental model of Alzheimer's disease (AD).

Polarized cells in the brain, such as neurons and glia, rely on the asymmetric distribution of their proteins compartmentalizing the function of dendrites, axons, glial projections, and endfeet. Subcellular proteomes can be assembled either by the transport of proteins synthesized in the cell soma or by the delivery of mRNAs to target compartments where they are locally translated into proteins. This latter mechanism is known as local protein synthesis or local translation, and it has been best studied in neurons. Increasing evidence suggests it is also required to maintain local protein homeostasis in glial cells; however, in microglia, local translation remains largely unexplored. Given the scant evidence, we aimed at exploring the existence of local translation in peripheral microglial processes (PeMPs) and unraveling its functional significance. We report that local translation indeed happens in PeMPs, and it is enhanced by triggering a microglial inflammatory response with bacterial lipopolysaccharides (LPS) suggesting a functional relevance of this molecular mechanism in response to inflammation. We found that Actb mRNA polarizes to PeMPs and is locally translated upon LPS exposure. Interestingly, downregulation of the Actb -binding protein IMP1/ZBP1 impaired Actb mRNA polarization and its localized translation, and led to defects in filopodia distribution, PeMP motility, lamellar directed migration, and phagocytosis in microglia. Thus, our work contributes to recent findings that mRNA localization and localized translation occur in microglia and gives a mechanistic insight into the relevance of this molecular mechanism in fundamental microglial functions in response to inflammation.

Oligodendrocyte dysfunction, myelin degeneration, and white matter changes are critical events in the cognitive decline of Alzheimer’s disease (AD). Amyloid-β peptide (Aβ), a hallmark of AD, disrupts oligodendrocyte and myelin homeostasis, through mechanisms that remain poorly understood. Here, transcriptomic profiling of Aβ-exposed oligodendrocytes revealed widespread gene expression changes, particularly in RNA-related processes. Among these, hnRNP A2, a key regulator of RNA transport and myelin protein regulation, was aberrantly upregulated in hippocampal oligodendrocytess from AD patients with high Aβ levels, from AD mouse models, and in Aβ-treated oligodendrocytes. RNA-immunoprecipitation sequencing of the hnRNP A2 interactome revealed Aβ-induced changes in mRNA interactions, particularly enriched binding to Mbp and Mobp, indicating impaired RNA metabolism of myelin components. Furthermore, Aβ, through hnRNP A2 disruption, increased the number, cargo and dynamics of Mbp- and Mobp-containing granules, enhanced MBP and MOBP synthesis, and decreased oligodendroglial voltage-gated Ca²⁺ influx in an MBP-dependent manner. These findings suggest that Aβ-induced dysregulation of hnRNP A2 impairs RNA metabolism and myelin protein synthesis, altering the intracellular Ca²⁺ homeostasis critical for oligodendrocyte function. Graphical abstract

In neurons, like in any other cell, their function often relies on the fine tuning of their protein levels, which is achieved by the balance between protein synthesis and turnover. Defects in protein homeostasis frequently leads to neuronal dysfunction and neurological disorders. Given their extreme morphological complexity and high compartmentalization, neurons highly depend on the asymmetrical distribution of their proteome. The common belief is that proteins that sustain axonal, dendritic and synaptic functions are synthesized in the soma and then transported to distal neuronal compartments. However, there is a complementary mechanism by which the mRNAs, and not the proteins, are transported to distal subneuronal domains, and once they reach their destination they are locally translated. Although once considered heretical, local translation (or local protein synthesis) is now widely accepted by the scientific community. Nonetheless there is one question that remains largely unexplored in the field and that is whether local translation in dendrites, axons and synapses is fully regulated by the neuron itself or if non-neuronal cells (e.g. glia) can modulate this mechanism in a non-cell-autonomous manner. Here we show that astroglia regulates local protein synthesis and enhances synaptic markers by releasing extracellular vesicles (EVs) containing ribosomal protein Rps6. To our knowledge this is the first report that directly demonstrates glial control of local translation in neurons through EV-mediated glia-to-neuron communication.

The synaptic proteome can be shaped by proteins transported from the neuronal soma and/or by mRNAs that are delivered to synapses where proteins are locally synthesized. This last mechanism is known as local translation. Local translation has been extensively studied in neurons in physiological conditions and, more recently, in neurological disorders, in which local transcriptomes and translatomes become dysregulated. It is widely believed that in neurons, the main source of localized transcripts is the neuronal soma and that localized translation is primarily regulated by the neuron itself. However, we wondered whether glial cells, especially astrocytes, could contribute to the modulation of synaptic local protein synthesis. To address this question, we compared levels of proteins produced in synaptic compartments in neuronal and neuron–astrocyte co-cultures using modified Boyden chambers or astrocyte-conditioned medium. We developed a methodology to measure local protein synthesis by puromycin labeling of isolated synaptosomes devoid of somatic input. Our results show that synaptic local translation is enhanced or retained when neurons are cultured in the presence of astrocytes and in response to astrocyte-conditioned medium. Puromycin labeling coupled with proximity ligation identified Rpl26 as one of the proteins whose local synthesis is regulated by astrocyte-secreted factors. Our results thus unravel the contribution of glia to synaptic protein synthesis and point to a previously unexplored extra layer of complexity in the regulation of local translation in neurons.

Oligodendrocyte dysfunction, myelin degeneration, and white matter structural alterations are critical events in Alzheimer’s disease (AD) that contribute to cognitive decline. A key hallmark of AD, Aβ oligomers, disrupt oligodendrocyte and myelin homeostasis, but a comprehensive global analysis of the mechanisms involved is lacking. Here, transcriptomic profiling of Aβ-exposed oligodendrocytes revealed widespread gene expression changes, particularly affecting pathways related to RNA localisation. Among the genes identified, we focused on Hnrnpa2/b1, the gene encoding the hnRNP A2 protein, which is essential for RNA transport and translation of myelin proteins. We confirmed aberrant upregulation of hnRNP A2 in hippocampal oligodendrocytes from post-mortem human brains of early-stage AD patients, Aβ-injected mouse hippocampi and Aβ-treated disrupting cells in vitro. RIP-seq analysis of the hnRNP A2 interactome revealed attenuated interactions with Hnrnpk and Hnrnpa2/b1, while interactions with Mbp and Mobp were enriched, suggesting changes in RNA metabolism of molecules associated with mRNA transport of myelin proteins. Aβ increased the total number and dynamics of mRNA-containing granules, facilitating local translation of the myelin proteins MBP and MOBP and attenuating Ca²⁺ signalling. These findings suggest that Aβ oligomers disrupt RNA metabolism mechanisms crucial for oligodendrocyte myelination through dysregulation of hnRNP A2 and myelin protein levels, potentially affecting oligodendroglia Ca²⁺ homeostasis.

Polarized cells in the brain, such as neurons and glia, rely on the asymmetric distribution of their proteins compartmentalizing the function of dendrites, axons, glial projections and endfeet. Subcellular proteomes can be assembled either by the transport of proteins synthesized in the cell soma or by the delivery of mRNAs to target compartments where they are locally translated into protein. This latter mechanism is known as local protein synthesis or local translation, and it has been best studied in neurons. Increasing evidence suggest it is also required to maintain local protein homeostasis in glial cells, however, in microglia, local translation remains largely unexplored. Given the scant evidence, we aimed at exploring the existence of local translation in microglial peripheral processes (MPPs) and unravel its functional significance. We report that local translation indeed happens in MPPs and it is enhanced by triggering a microglial inflammatory response with bacterial lipopolysaccharides (LPS) suggesting a functional relevance of this molecular mechanism in response to inflammation. We found that Actb and Par3 mRNAs polarize to MMPs and are locally translated upon LPS exposure. Interestingly, downregulation of the Actb binding protein IMP1/ZBP1 impaired Actb mRNA polarization and its localized translation, leading to defects in filopodia distribution, lamellar directed migration and phagocytosis in microglia. Thus, our work contributes to recent findings that localized translation occurs in microglia and gives a mechanistic insight into the relevance of this molecular mechanism in fundamental microglial functions in response to LPS-induced inflammation.

Polarized cells in the brain, such as neurons and glia, rely on the asymmetric distribution of their proteins compartmentalizing the function of dendrites, axons, glial projections and endfeet. Subcellular proteomes can be assembled either by the transport of proteins synthesized in the cell soma or by the delivery of mRNAs to target compartments where they are locally translated into protein. This latter mechanism is known as local protein synthesis or local translation, and it has been best studied in neurons. Increasing evidence suggest it is also required to maintain local protein homeostasis in glial cells, however, in microglia, local translation remains largely unexplored. Given the scant evidence, we aimed at exploring the existence of local translation in peripheral microglial processes (PeMPs) and unravel its functional significance in response to inflammation, a major hallmark of neurodegenerative diseases. We report that local translation in PeMPs is enhanced by triggering a microglial inflammatory response with bacterial lipopolysaccharides (LPS). We found that Actb mRNA polarizes to PeMPs and is locally translated upon LPS exposure. mRNA localization in eukaryotic cells is driven by RNA binding proteins. Interestingly, downregulation of the Actb binding protein IMP1/ZBP1 impaired Actb mRNA polarization and its localized translation, and led to defects in filopodia distribution, PeMP motility, lamellar directed migration and phagocytosis in microglia. Thus, our work contributes to recent findings that mRNA localization and localized translation occur in microglia and gives a mechanistic insight into the relevance of this molecular mechanism in fundamental microglial functions in response to inflammation.

Gene expression is changed by disease, but how these molecular responses arise and contribute to pathophysiology remains less understood. We discover that β-amyloid, a trigger of Alzheimer’s disease (AD), promotes the formation of pathological CREB3L2-ATF4 transcription factor heterodimers in neurons. Through a multilevel approach based on AD datasets and a novel chemogenetic method that resolves the genomic binding profile of dimeric transcription factors (ChIPmera), we find that CREB3L2-ATF4 activates a transcription network that interacts with roughly half of the genes differentially expressed in AD, including subsets associated with β-amyloid and tau neuropathologies. CREB3L2-ATF4 activation drives tau hyperphosphorylation and secretion in neurons, in addition to misregulating the retromer, an endosomal complex linked to AD pathogenesis. We further provide evidence for increased heterodimer signaling in AD brain and identify dovitinib as a candidate molecule for normalizing β-amyloid–mediated transcriptional responses. The findings overall reveal differential transcription factor dimerization as a mechanism linking disease stimuli to the development of pathogenic cellular states.

Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson’s disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chamber microfluidic platform and established a cortico-striato-nigral microcircuit recapitulating the striatal presynaptic triad in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, although cortical glutamatergic projections facilitated MSN synaptic activity, dopaminergic transmission was essential for excitability maturation of MSNs in vitro . Replacement of wild-type iPSC-dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation induced early hyperexcitability in iPSC-MSNs through reduction of voltage-gated sodium and potassium intrinsic currents. Such deficits were resolved in aged cultures or with antagonism of protein kinase A activity in nigrostriatal iPSC-DaNs. Hence, our results highlight the unique utility of modelling striatal neurons in a modular and highly physiological circuit which is essential to reveal mechanistic insights of the loss of electrical functional integrity in the striata of GBA1 PD patients.