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EMBO Workshop: Proteostasis shaping health span: from protein folding to failure
This EMBO Workshop examines how protein synthesis, folding, trafficking, and degradation shape health across the lifespan, connecting fundamental proteostasis mechanisms with ageing and degenerative disease.
Translation at the Synapse
The complex morphology of neurons, with synapses located hundreds of microns from the cell body, necessitates the localization of important cell biological machines, including ribosomes, within dendrites and axons. Local translation of mRNAs is important for the function and plasticity of synapses. Using advanced sequencing and imaging techniques we have updated our understanding of the local transcriptome and identified the local translatome- identifying over 800 transcripts for which local translation is the dominant source of protein. In addition, we have explored the unique mechanisms neurons use to meet protein demands at synapses, identifying surprising features of neuronal and synaptic protein synthesis.
Mechanisms to medicines in neurodegeneration
Dysregulation of protein synthesis both globally and locally in neurons and astrocytes is a key feature of neurodegenerative diseases. Aberrant signalling through the Unfolded Protein Response (UPR) and related Integrated Stress Response (ISR) have become major targets for neuroprotection in these disorders. In addition, other homeostatic mechanisms and stress responses, including the cold shock response, appear to regulate local translation and RNA splicing to control synapse maintenance and regeneration and can also be targeted therapeutically for neuroprotection. We have defined the role of UPR/ISR and the cold-shock response in neurodegenerative disorders and have developed translational strategies targeting them for new treatments for dementia.
Translational upregulation of STXBP1 by non-coding RNAs as an innovative treatment for STXBP1 encephalopathy
Developmental and epileptic encephalopathies (DEEs) are a broad spectrum of genetic epilepsies associated with impaired neurological development as a direct consequence of a genetic mutation, in addition to the effect of the frequent epileptic activity on brain. Compelling genetic studies indicate that heterozygous de novo mutations represent the most common underlying genetic mechanism, in accordance with the sporadic presentation of DEE. De novo mutations may exert a loss-of-function (LOF) on the protein by decrementing expression level and/or activity, leading to functional haploinsufficiency. These diseases share several features: severe and frequent refractory seizures, diffusely abnormal background activity on EEG, intellectual disability often profound, and severe consequences on global development. One of major causes of early onset DEE are de novo heterozygous mutations in syntaxin-binding-protein-1 gene STXBP1, which encodes a membrane trafficking protein playing critical role in vesicular docking and fusion. LOF STXBP1 mutations lead to a failure of neurotransmitter secretion from synaptic vesicles. Core clinical features of STXBP1 encephalopathy include early-onset epilepsy with hypsarrhythmic EEG, or burst-suppression pattern, or multifocal epileptiform activity. Seizures are often resistant to standard treatments and patients typically show intellectual disability, mostly severe to profound. Additional neurologic features may include autistic traits, movement disorders (dyskinesia, dystonia, tremor), axial hypotonia, and ataxia, indicating a broader neurologic impairment. Patients with severe neuro-cognitive features but without epilepsy have been reported. Recently, a new class of natural and synthetic non-coding RNAs have been identified, enabling upregulation of protein translation in a gene-specific way (SINEUPs), without any increase in mRNA of the target gene. SINEUPs are translational activators composed by a Binding Domain (BD) that overlaps, in antisense orientation, to the sense protein-coding mRNA, and determines target selection; and an Effector Domain (ED), that is essential for protein synthesis up regulation. SINEUPs have been shown to restore the physiological expression of a protein in case of haploinsufficiency, without driving excessive overexpression out of the physiological range. This technology brings many advantages, as it mainly acts on endogenous target mRNAs produced in situ by the wild-type allele; this action is limited to mRNA under physiological regulation, therefore no off-site effects can be expected in cells and tissues that do not express the target transcript; by acting only on a posttranscriptional level, SINEUPs do not trigger hereditable genome editing. After bioinformatic analysis of the promoter region of interest, we designed SINEUPs with 3 different BD for STXBP1. Human neurons from iPSCs were treated and STXBP1 levels showed a 1.5-fold increase compared to the Negative control. RNA levels of STXBP1 after the administration of SINEUPs remained stable as expected. These preliminary results proved the SINEUPs potential to specifically increase the protein levels without impacting on the genome. This is an extremely flexible approach to target many developmental and epileptic encephalopathies caused by haploinsufficiency, and therefore to address these diseases in a more tailored and radical way.
Protein Synthesis at Neuronal Synapses
The complex morphology of neurons, with synapses located 100’s of microns from the cell body, necessitates the localization of important cell biological machines and processes within dendrites and axons. Using expansion microscopy together with metabolic labeling we have discovered that both postsynaptic spines and presynaptic terminals exhibit rapid translation, which exhibits differential sensitivity to different neurotransmitters and neuromodulators. In addition, we have explored the unique mechanisms neurons use to meet protein demands at synapses, identifying the transcriptome and translatome in the neuropil.