A brain atlas of synapse protein lifetime across the mouse lifespan.

Protein turnover is required for synapse maintenance and remodelling and may impact memory duration. We quantified the lifetime of postsynaptic protein PSD95 in individual excitatory synapses across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of protein lifetimes that may extend from a few hours to several months, with distinct spatial distributions in dendrites, neuron types and brain regions. Short protein lifetime (SPL) synapses are enriched in developing animals and in regions controlling innate behaviors, whereas long protein lifetime (LPL) synapses accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. The protein lifetime synaptome architecture is disrupted in an autism model, with synapse protein lifetime increased throughout the brain. These findings add a further layer to synapse diversity in the brain and enrich prevailing concepts in behavior, development, ageing and brain repair.

Cylindromatosis Drives Synapse Pruning and Weakening by Promoting Macroautophagy through Akt-mTOR Signaling

The lysine-63 deubiquitinase cylindromatosis (CYLD) is long recognized as a tumor suppressor in immunity and inflammation and its loss-of-function mutations lead to familial cylindromatosis. However, recent studies reveal that CYLD is enriched in mammalian brain postsynaptic densities, and a gain-of-function mutation causes frontotemporal dementia (FTD), suggesting critical roles at excitatory synapses. Here we report that CYLD drives synapse elimination and weakening by acting on the Akt-mTOR-autophagy axis. Mice lacking CYLD display abnormal sociability, anxiety- and depression-like behaviors, and cognitive inflexibility. These behavioral impairments are accompanied by excessive synapse numbers, increased postsynaptic efficacy, augmented synaptic summation, and impaired NMDA receptor-dependent hippocampal long-term depression (LTD). Exogenous expression of CYLD results in removal of established dendritic spines from mature neurons in a deubiquitinase activity-dependent manner. In search of underlying molecular mechanisms, we find that CYLD knockout mice display marked overactivation of Akt and mTOR and reduced autophagic flux and, conversely, CYLD overexpression potently suppresses Akt and mTOR activity and promotes autophagy. Consequently, abrogating the Akt-mTOR-autophagy signaling pathway abolishes CYLD-induced spine loss, whereas enhancing autophagy in vivo by the mTOR inhibitor rapamycin rescues the synaptic pruning and LTD deficits in mutant mice. Our findings establish CYLD, via Akt-mTOR signaling, as a synaptic autophagy activator that exerts critical modulations on synapse maintenance, function, and plasticity.

Conformational heterogeneity coupled with β-fibril formation of a scaffold protein involved in chronic mental illnesses

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance. It serves as a scaffold hub forming complexes with a variety (~300) of partners that constitute its interactome. Herein, using combinations of structural and biophysical tools, we demonstrate that the C-region of the DISC1 protein is highly polymorphic, with important consequences for its physiological role. Results from solid-state NMR spectroscopy and electron microscopy indicate that the protein not only forms symmetric oligomers but also gives rise to fibrils closely resembling those found in certain established amyloid proteinopathies. Furthermore, its aggregation as studied by isothermal titration calorimetry (ITC) is an exergonic process, involving a negative enthalpy change that drives the formation of oligomeric (presumably tetrameric) species as well as β-fibrils. We have been able to narrow down the β-core region participating in fibrillization to residues 716–761 of full-length human DISC1. This region is absent in the DISC1Δ22aa splice variant, resulting in reduced association with proteins from the dynein motor complex, viz., NDE-like 1 (NDEL1) and lissencephaly 1 (LIS1), which are crucial during mitosis. By employing surface plasmon resonance, we show that the oligomeric DISC1 C-region has an increased affinity and shows cooperativity in binding to LIS1 and NDEL1, in contrast to the noncooperative binding mode exhibited by the monomeric version. Based on the derived structural models, we propose that the association between the binding partners involves two neighboring subunits of DISC1 C-region oligomers. Altogether, our findings highlight the significance of the DISC1 C-region as a crucial factor governing the balance between its physiological role as a multifunctional scaffold protein and aggregation-related aberrations with potential significance for disease.

Long-term Pannexin 1 ablation promotes structural and functional modifications in hippocampal neurons through the regulation of actin cytoskeleton and Rho GTPases activity.

Enhanced activity and overexpression of Pannexin 1 (PANX1) channels contribute to neuronal pathologies, such as epilepsy and Alzheimers disease (AD). In the hippocampus, the PANX1 channels ablation alters glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, PANX1-knockout (KO) mice still preserve the ability to learn, suggesting that compensatory mechanisms work to stabilize neuronal activity. Here, we show that the absence of PANX1 in the adult brain promotes a series of structural and functional modifications in KO hippocampal synapses, preserving spontaneous activity. Adult CA1 neurons of KO mice exhibit enhanced excitability, complex dendritic branching, spine maturation, and multiple synaptic contacts compared to the WT condition. These modifications seem to rely on the actin-cytoskeleton dynamics as an increase in actin polymerization and an imbalance between Rac1 and RhoA GTPase activity is observed in the absence of PANX1. Our findings highlight a novel interaction between PANX1, actin, and small Rho GTPases that appear to be relevant for synapse maintenance as a long-term compensatory mechanism for PANX1 deficiency.

Heat Shock Factor 1 Directly Regulates Postsynaptic Scaffolding PSD-95 in Aging and Huntington’s Disease and Influences Striatal Synaptic Density

PSD-95 (Dlg4) is an ionotropic glutamate receptor scaffolding protein essential in synapse stability and neurotransmission. PSD-95 levels are reduced during aging and in neurodegenerative diseases like Huntington’s disease (HD), and it is believed to contribute to synaptic dysfunction and behavioral deficits. However, the mechanism responsible for PSD-95 dysregulation under these conditions is unknown. The Heat Shock transcription Factor 1 (HSF1), canonically known for its role in protein homeostasis, is also depleted in both aging and HD. Synaptic protein levels, including PSD-95, are influenced by alterations in HSF1 levels and activity, but the direct regulatory relationship between PSD-95 and HSF1 has yet to be determined. Here, we showed that HSF1 chronic or acute depletion in cell lines and mice decreased PSD-95 expression. Furthermore, HSF1(+/-) mice had reduced PSD-95 synaptic puncta that paralleled a loss in thalamo-striatal excitatory synapses, an important circuit disrupted early in HD. We demonstrated that HSF1 binds to regulatory elements present in the PSD-95 gene and directly regulates PSD-95 expression. HSF1 DNA-binding on the PSD-95 gene was disrupted in an age-dependent manner in WT mice and worsened in HD cells and mice, leading to reduced PSD-95 levels. These results demonstrate a direct role of HSF1 in synaptic gene regulation that has important implications in synapse maintenance in basal and pathological conditions.

Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons

In neurons, endoplasmic reticulum forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance. However, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high resolution microscopy and live cell imaging to investigate rapid movements of endoplasmic reticulum and ribosomes in axons of cultured motoneurons after stimulation with Brain-derived neurotrophic factor. Our results indicate that the endoplasmic reticulum extends into axonal growth cone filopodia where its integrity and dynamic remodeling are regulated mainly by actin and its motor protein myosin VI. Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with ER in response to extracellular stimuli which describes a novel function of axonal ER in dynamic regulation of local translation.

Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons

In neurons, endoplasmic reticulum forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance. However, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high resolution microscopy and live cell imaging to investigate rapid movements of endoplasmic reticulum and ribosomes in axons of cultured motoneurons after stimulation with Brain-derived neurotrophic factor. Our results indicate that the endoplasmic reticulum extends into axonal growth cone filopodia where its integrity and dynamic remodeling are regulated mainly by actin and its motor protein myosin VI. Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with ER in response to extracellular stimuli which describes a novel function of axonal ER in dynamic regulation of local translation.

Running and Swimming Differently Adapt the BDNF/TrkB Pathway to a Slow Molecular Pattern at the NMJ

Physical exercise improves motor control and related cognitive abilities and reinforces neuroprotective mechanisms in the nervous system. As peripheral nerves interact with skeletal muscles at the neuromuscular junction, modifications of this bidirectional communication by physical activity are positive to preserve this synapse as it increases quantal content and resistance to fatigue, acetylcholine receptors expansion, and myocytes’ fast-to-slow functional transition. Here, we provide the intermediate step between physical activity and functional and morphological changes by analyzing the molecular adaptations in the skeletal muscle of the full BDNF/TrkB downstream signaling pathway, directly involved in acetylcholine release and synapse maintenance. After 45 days of training at different intensities, the BDNF/TrkB molecular phenotype of trained muscles from male B6SJLF1/J mice undergo a fast-to-slow transition without affecting motor neuron size. We provide further knowledge to understand how exercise induces muscle molecular adaptations towards a slower phenotype, resistant to prolonged trains of stimulation or activity that can be useful as therapeutic tools.