Modifying the m6A brain methylome by ALKBH5-mediated demethylation: a new contender for synaptic tagging

Synaptic plasticity processes, which underlie learning and memory formation, require RNA to be translated local to synapses. The synaptic tagging hypothesis has previously been proposed to explain how mRNAs are available at specific activated synapses. However how RNA is regulated, and which transcripts are silenced or processed as part of the tagging process is still unknown. Modification of RNA by N6-methyladenosine (m6A/m) influences the cellular fate of mRNA. Here, by advanced microscopy, we showed that m6A demethylation by the eraser protein ALKBH5 occurs at active synaptic ribosomes and at synapses during short term plasticity. We demonstrated that at activated glutamatergic post-synaptic sites, both the YTHDF1 and YTHDF3 reader and the ALKBH5 eraser proteins increase in co-localisation to m6A-modified RNAs; but only the readers showed high co-localisation to modified RNAs during late-stage plasticity. The YTHDF1 and YTHFDF3 readers also exhibited differential roles during synaptic maturation suggesting that temporal and subcellular abundance may determine specific function. m6A-sequencing of human parahippocampus brain tissue revealed distinct white and grey matter m6A methylome profiles indicating that cellular context is a fundamental factor dictating regulated pathways. However, in both neuronal and glial cell-rich tissue, m6A effector proteins are themselves modified and m6A epitranscriptional and posttranslational modification processes coregulate protein cascades. We hypothesise that the availability m6A effector protein machinery in conjunction with RNA modification, may be important in the formation of condensed synaptic nanodomain assemblies through liquid-liquid phase separation. Our findings support that m6A demethylation by ALKBH5 is an intrinsic component of the synaptic tagging hypothesis and a molecular switch which leads to alterations in the RNA methylome, synaptic dysfunction and potentially reversible disease states.

Neuroligin-1 mediates presynaptic maturation through brain-derived neurotrophic factor signaling

Background Maturation is a process that allows synapses to acquire full functionality, optimizing their activity to diverse neural circuits, and defects in synaptic maturation may contribute to neurodevelopmental disorders. Neuroligin-1 (NL1) is a postsynaptic cell adhesion molecule essential for synapse maturation, a role typically attributed to binding to pre-synaptic ligands, the neurexins. However, the pathways underlying the action of NL1 in synaptic maturation are incompletely understood, and some of its previously observed effects seem reminiscent of those described for the neurotrophin brain-derived neurotrophic factor (BDNF). Here, we show that maturational increases in active zone stability and synaptic vesicle recycling rely on the joint action of NL1 and brain-derived neurotrophic factor (BDNF). Results Applying BDNF to hippocampal neurons in primary cultures or organotypical slice cultures mimicked the effects of overexpressing NL1 on both structural and functional maturation. Overexpressing a NL1 mutant deficient in neurexin binding still induced presynaptic maturation. Like NL1, BDNF increased synaptic vesicle recycling and the augmentation of transmitter release by phorbol esters, both hallmarks of presynaptic maturation. Mimicking the effects of NL1, BDNF also increased the half-life of the active zone marker bassoon at synapses, reflecting increased active zone stability. Overexpressing NL1 increased the expression and synaptic accumulation of BDNF. Inhibiting BDNF signaling pharmacologically or genetically prevented the effects of NL1 on presynaptic maturation. Applying BDNF to NL1-knockout mouse cultures rescued defective presynaptic maturation, indicating that BDNF acts downstream of NL1 and can restore presynaptic maturation at late stages of network development. Conclusions Our data introduce BDNF as a novel and essential component in a transsynaptic pathway linking NL1-mediated cell adhesion, neurotrophin action, and presynaptic maturation. Our findings connect synaptic cell adhesion and neurotrophin signaling and may provide a therapeutic approach to neurodevelopmental disorders by targeting synapse maturation.

Developmental shift to mitochondrial respiration for energetic support of sustained transmission during maturation at the calyx of Held

A considerable amount of energy is expended following presynaptic activity to regenerate electrical polarization and maintain efficient release and recycling of neurotransmitter. Mitochondria are the major suppliers of neuronal energy, generating ATP via oxidative phosphorylation. However, the specific utilization of energy from cytosolic glycolysis rather than mitochondrial respiration at the presynaptic terminal during synaptic activity remains unclear and controversial. We use a synapse specialized for high frequency transmission in mice, the calyx of Held, to test the sources of energy utilized to maintain energy during short activity bursts (<1 sec) and sustained neurotransmission (30-150 sec). We dissect the role of presynaptic glycolysis versus mitochondrial respiration by acutely and selectively blocking these ATP-generating pathways in a synaptic preparation where mitochondria and synaptic vesicles are prolific, under near-physiological conditions. Surprisingly, if either glycolysis or mitochondrial ATP production is intact, transmission during repetitive short bursts of activity is not affected. In slices from young animals prior to the onset of hearing, where the synapse is not yet fully specialized, both glycolytic and mitochondrial ATP production are required to support sustained, high frequency neurotransmission. In mature synapses, sustained transmission relies exclusively on mitochondrial ATP production supported by bath lactate, but not glycolysis. At both ages, we observe that action potential propagation begins to fail prior to defects in synaptic vesicle recycling. Our data describe a specific metabolic profile to support high-frequency information transmission at the mature calyx of Held, shifting during postnatal synaptic maturation from glycolysis to rely on monocarboxylates as a fuel source.

Reduced PTPRD expression differentially alters brain phosphotyrosine phosphoproteomic profiles of 2 and 12 month-old mice

The receptor type protein tyrosine phosphatase PTPRD is implicated in maturation of synapses of expressing neurons, vulnerability to addictions, reward from addictive substances, vulnerability to restless leg syndrome and densities of neurofibrillary pathology in Alzheimer’s disease brains by a variety of evidence. However, PTPRD’s physiological substrates and adaptations to differences in levels of PTPRD expression in brains of young and aging animals have not been explored in depth. We report phosphoproteomic studies of brains of young and aged mice with different levels of PTPRD expression, gene ontology studies of genes identified in this way and validation of several candidate PTPRD substrates with in vitro assays using recombinant PTPRD phosphatase. PTPRD is well positioned to modulate the extent of phosphorylation of phosphotyrosine phosphoprotein substrates, including those involved in synaptic maturation and adaptation.

PKN1 Is a Novel Regulator of Hippocampal GluA1 Levels

Alterations in the processes that control α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression, assembly and trafficking are closely linked to psychiatric and neurodegenerative disorders. We have recently shown that the serine/threonine kinase Protein kinase N1 (PKN1) is a developmentally active regulator of cerebellar synaptic maturation by inhibiting AKT and the neurogenic transcription factor neurogenic differentiation factor-2 (NeuroD2). NeuroD2 is involved in glutamatergic synaptic maturation by regulating expression levels of various synaptic proteins. Here we aimed to study the effect of Pkn1 knockout on AKT phosphorylation and NeuroD2 levels in the hippocampus and the subsequent expression levels of the NeuroD2 targets and AMPAR subunits: glutamate receptor 1 (GluA1) and GluA2/3. We show that PKN1 is expressed throughout the hippocampus. Interestingly, not only postnatal but also adult hippocampal phospho-AKT and NeuroD2 levels were significantly elevated upon Pkn1 knockout. Postnatal and adult Pkn1–/– hippocampi showed enhanced expression of the AMPAR subunit GluA1, particularly in area CA1. Surprisingly, GluA2/3 levels were not different between both genotypes. In addition to higher protein levels, we also found an enhanced GluA1 content in the membrane fraction of postnatal and adult Pkn1–/– animals, while GluA2/3 levels remained unchanged. This points toward a very specific regulation of GluA1 expression and/or trafficking by the novel PKN1-AKT-NeuroD2 axis. Considering the important role of GluA1 in hippocampal development as well as the pathophysiology of several disorders, ranging from Alzheimer’s, to depression and schizophrenia, our results validate PKN1 for future studies into neurological disorders related to altered AMPAR subunit expression in the hippocampus.

Human Astrocytes Model Derived from Induced Pluripotent Stem Cells

Induced pluripotent stem cell (iPSC)-based disease modeling has a great potential for uncovering the mechanisms of pathogenesis, especially in the case of neurodegenerative diseases where disease-susceptible cells can usually not be obtained from patients. So far, the iPSC-based modeling of neurodegenerative diseases has mainly focused on neurons because the protocols for generating astrocytes from iPSCs have not been fully established. The growing evidence of astrocytes’ contribution to neurodegenerative diseases has underscored the lack of iPSC-derived astrocyte models. In the present study, we established a protocol to efficiently generate iPSC-derived astrocytes (iPasts), which were further characterized by RNA and protein expression profiles as well as functional assays. iPasts exhibited calcium dynamics and glutamate uptake activity comparable to human primary astrocytes. Moreover, when co-cultured with neurons, iPasts enhanced neuronal synaptic maturation. Our protocol can be used for modeling astrocyte-related disease phenotypes in vitro and further exploring the contribution of astrocytes to neurodegenerative diseases.

γ-secretase promotes postsynaptic maturation through the cleavage of a Wnt receptor

An emerging feature of neurodegenerative disease is synaptic dysfunction and loss, leading to the suggestion that mechanisms required for synaptic maturation may be linked to disease. Synaptic maturation requires the transmission of signals between nascent synaptic sites and the nucleus, but how these signals are generated is not well understood. We posit that proteolytic cleavage of receptors, which enables their translocation to the nucleus, may be a shared molecular mechanism between the events that promote synaptic maturation and those linked to later-onset disorders of the nervous system, including neurodegenerative disease. Here we show during synaptic development, that cleavage of synaptic maturation molecules requires γ-secretase, a protein complex linked to Alzheimer’s Disease, a devastating neurodegenerative condition, is required for postsynaptic maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to appropriately recruit postsynaptic scaffolding and cytoskeletal proteins, and mutant larvae display behavioral deficits. At the NMJ, γ-secretase promotes synaptic maturation through the cleavage of the Wnt receptor Fz2, and the subsequent entry of its C-terminus into the nucleus. A developmental synaptic role for γ-secretase is also conserved in both the Drosophila central nervous system and mammalian cortical neuron dendrites. Finally, we found that similar maturation defects are evident in fly models for ALS, Alzheimer’s, Huntington’s, and Parkinson’s Diseases. The previously unknown, but conserved, role for γ-secretase coupled with its well-known role in neurodegenerative disease suggest that neurodevelopmental defects may be common to diverse neurodegenerative disease models.