Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.

TDP-43 protein is dysregulated in several neurodegenerative diseases, which often have a multifactorial nature and may have extrinsic stressors as a "second hit." TDP-43 undergoes reversible nuclear condensation in stressed cells including neurons. Here, we demonstrate that stress-inducible nuclear TDP-43 condensates are RNA-depleted, non-liquid assemblies distinct from the known nuclear bodies. Their formation requires TDP-43 oligomerization and ATP and is inhibited by RNA. Using a confocal nanoscanning assay, we find that amyotrophic lateral sclerosis (ALS)-linked mutations alter stress-induced TDP-43 condensation by changing its affinity to liquid-like ribonucleoprotein assemblies. Stress-induced nuclear condensation transiently inactivates TDP-43, leading to loss of interaction with its protein binding partners and loss of function in splicing. Splicing changes are especially prominent and persisting for STMN2 RNA, and STMN2 protein becomes rapidly depleted early during stress. Our results point to early pathological changes to TDP-43 in the nucleus and support therapeutic modulation of stress response in ALS.

Bringing synapses into focus: Recent advances in synaptic imaging and mass-spectrometry for studying synaptopathy.

Synapses are integral for healthy brain function and are becoming increasingly recognized as key structures in the early stages of brain disease. Understanding the pathological processes driving synaptic dysfunction will unlock new therapeutic opportunities for some of the most devastating diseases of our time. To achieve this we need a solid repertoire of imaging and molecular tools to interrogate synaptic biology at greater resolution. Synapses have historically been examined in small numbers, using highly technical imaging modalities, or in bulk, using crude molecular approaches. However, recent advances in imaging techniques are allowing us to analyze large numbers of synapses, at single-synapse resolution. Furthermore, multiplexing is now achievable with some of these approaches, meaning we can examine multiple proteins at individual synapses in intact tissue. New molecular techniques now allow accurate quantification of proteins from isolated synapses. The development of increasingly sensitive mass-spectrometry equipment means we can now scan the synaptic molecular landscape almost in totality and see how this changes in disease. As we embrace these new technical developments, synapses will be viewed with clearer focus, and the field of synaptopathy will become richer with insightful and high-quality data. Here, we will discuss some of the ways in which synaptic interrogation is being facilitated by methodological advances, focusing on imaging, and mass spectrometry.

Array tomography: 15 years of synaptic analysis.

Synapses are minuscule, intricate structures crucial for the correct communication between neurons. In the 125 years since the term synapse was first coined, we have advanced a long way when it comes to our understanding of how they work and what they do. Most of the fundamental discoveries have been invariably linked to advances in technology. However, due to their size, delicate structural integrity and their sheer number, our knowledge of synaptic biology has remained somewhat elusive and their role in neurodegenerative diseases still remains largely unknown. Here, we briefly discuss some of the imaging technologies used to study synapses and focus on the utility of the high-resolution imaging technique array tomography (AT). We introduce the AT technique and highlight some of the ways it is utilised with a particular focus on its power for analysing synaptic composition and pathology in human post-mortem tissue. We also discuss some of the benefits and drawbacks of techniques for imaging synapses and highlight some recent advances in the study of form and function by combining physiology and high-resolution synaptic imaging.

Synaptic proteomics reveal distinct molecular signatures of cognitive change and C9ORF72 repeat expansion in the human ALS cortex.

Increasing evidence suggests synaptic dysfunction is a central and possibly triggering factor in Amyotrophic Lateral Sclerosis (ALS). Despite this, we still know very little about the molecular profile of an ALS synapse. To address this gap, we designed a synaptic proteomics experiment to perform an unbiased assessment of the synaptic proteome in the ALS brain. We isolated synaptoneurosomes from fresh-frozen post-mortem human cortex (11 controls and 18 ALS) and stratified the ALS group based on cognitive profile (Edinburgh Cognitive and Behavioural ALS Screen (ECAS score)) and presence of a C9ORF72 hexanucleotide repeat expansion (C9ORF72-RE). This allowed us to assess regional differences and the impact of phenotype and genotype on the synaptic proteome, using Tandem Mass Tagging-based proteomics. We identified over 6000 proteins in our synaptoneurosomes and using robust bioinformatics analysis we validated the strong enrichment of synapses. We found more than 30 ALS-associated proteins in synaptoneurosomes, including TDP-43, FUS, SOD1 and C9ORF72. We identified almost 500 proteins with altered expression levels in ALS, with region-specific changes highlighting proteins and pathways with intriguing links to neurophysiology and pathology. Stratifying the ALS cohort by cognitive status revealed almost 150 specific alterations in cognitively impaired ALS synaptic preparations. Stratifying by C9ORF72-RE status revealed 330 protein alterations in the C9ORF72-RE +ve group, with KEGG pathway analysis highlighting strong enrichment for postsynaptic dysfunction, related to glutamatergic receptor signalling. We have validated some of these changes by western blot and at a single synapse level using array tomography imaging. In summary, we have generated the first unbiased map of the human ALS synaptic proteome, revealing novel insight into this key compartment in ALS pathophysiology and highlighting the influence of cognitive decline and C9ORF72-RE on synaptic composition.