We examined the feedback between the major protein degradation pathway, the ubiquitin-proteasome system (UPS), and protein synthesis in rat and mouse neurons. When protein degradation was inhibited, we observed a coordinate dramatic reduction in nascent protein synthesis in neuronal cell bodies and dendrites. The mechanism for translation inhibition involved the phosphorylation of eIF2α, surprisingly mediated by eIF2α kinase 1, or heme-regulated kinase inhibitor (HRI). Under basal conditions, neuronal expression of HRI is barely detectable. Following proteasome inhibition, HRI protein levels increase owing to stabilization of HRI and enhanced translation, likely via the increased availability of tRNAs for its rare codons. Once expressed, HRI is constitutively active in neurons because endogenous heme levels are so low; HRI activity results in eIF2α phosphorylation and the resulting inhibition of translation. These data demonstrate a novel role for neuronal HRI that senses and responds to compromised function of the proteasome to restore proteostasis.

Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered ∼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

Photoreceptor ribbon synapses translate light-dependent changes of membrane potential into graded transmitter release over several orders of magnitude in intensity. A specialized organelle at the active zone--the synaptic ribbon--is a key player in this process, and it is well known that the ribbon undergoes illumination and thus activity-dependent structural changes. However, the molecular basis for these changes is unknown. The aim of this study was to correlate the known ultrastructural ribbon changes to the distribution of proteins of the presynaptic ribbon complex.

To form synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of messenger RNAs (mRNAs) near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally. It is not generally known, however, if, how, and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons (neuropil). Thousands of transcripts were differentially translated between somatic and synaptic regions, with many scaffold and signaling molecules displaying increased translation levels in the neuropil. Most translational changes between compartments could be accounted for by differences in RNA abundance. Pervasive translational regulation was observed in both somata and neuropil influenced by specific mRNA features (e.g., untranslated region [UTR] length, RNA-binding protein [RBP] motifs, and upstream open reading frames [uORFs]). For over 800 mRNAs, the dominant source of translation was the neuropil. We constructed a searchable and interactive database for exploring mRNA transcripts and their translation levels in the somata and neuropil [MPI Brain Research, The mRNA translation landscape in the synaptic neuropil. https://public.brain.mpg.de/dashapps/localseq/ Accessed 5 October 2021]. Overall, our findings emphasize the substantial contribution of local translation to maintaining synaptic protein levels and indicate that on-site translational control is an important mechanism to control synaptic strength.

The retinal photoreceptor ribbon synapse is a chemical synapse structurally and functionally specialized for the tonic release of neurotransmitter. It is characterized by the presynaptic ribbon, an electron-dense organelle at the active zone covered by hundreds of synaptic vesicles. In conventional synapses, dense-core transport vesicles carrying a set of active zone proteins are implicated in early steps of synapse formation. In photoreceptor ribbon synapses, synaptic spheres are suggested to be involved in ribbon synapse assembly, but nothing is known about the molecular composition of these organelles. With light, electron, and stimulated emission depletion microscopy and immunocytochemistry, we investigated a series of presynaptic proteins during photoreceptor synaptogenesis. The cytomatrix proteins Bassoon, Piccolo, RIBEYE, and RIM1 appear early in synaptogenesis. They are transported in nonmembranous, electron-dense, spherical transport units, which we called precursor spheres, to the future presynaptic site. Other presynaptic proteins, i.e., Munc13, CAST1, RIM2, and an L-type Ca(2+) channel alpha1 subunit are not associated with the precursor spheres. They cluster directly at the active zone some time after the first set of cytomatrix proteins has arrived. By quantitative electron microscopy, we found an inverse correlation between the numbers of spheres and synaptic ribbons in the postnatally developing photoreceptor synaptic terminals. From these results, we suggest that the precursor spheres are the transport units for proteins of the photoreceptor ribbon compartment and are involved in the assembly of mature synaptic ribbons.

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

Long-term memory has been associated with morphological changes in the brain, which in turn tightly correlate with changes in synaptic efficacy. Such plasticity is proposed to rely on dendritic spines as a neuronal canvas on which these changes can occur. Given the key role of actin cytoskeleton dynamics in spine morphology, major regulating factors of this process such as Cofilin 1 (Cfl1) and LIM kinase (LIMK), an inhibitor of Cfl1 activity, are prime molecular targets that may regulate dendritic plasticity. Using a contextual fear conditioning paradigm in mice, we found that pharmacological induction of depolymerization of actin filaments through the inhibition of LIMK causes an impairment in memory reconsolidation, as well as in memory consolidation. On top of that, Cfl1 activity is inhibited and its mRNA is downregulated in CA1 neuropil after re-exposure to the training context. Moreover, by pharmacological disruption of actin cytoskeleton dynamics, the process of memory extinction can either be facilitated or impaired. Our results lead to a better understanding of the role of LIMK, Cfl1 and actin cytoskeleton dynamics in the morphological and functional changes underlying the synaptic plasticity of the memory trace.

Immunocytochemical discrimination of distinct bipolar cell types in the mouse retina is a prerequisite for analyzing retinal circuitry in wild-type and transgenic mice. Here we demonstrate that among the more than 10 anatomically defined mouse bipolar cell types, type 4 bipolar cells are specifically recognized by anti-calsenilin antibodies. Axon terminals in the inner plexiform layer are not readily identifiable because calsenilin is also expressed in a subset of amacrine and ganglion cells. In contrast, in the outer plexiform layer calsenilin immunoreactivity allows the analysis of photoreceptor to type 4 bipolar cell contacts. A dense plexus of calsenilin-positive dendrites makes several basal contacts at cone pedicles. An individual calsenilin-positive bipolar cell contacts five to seven cones. In addition, some calsenilin-positive dendrites contact rod photoreceptors. On average we counted 10 rod spherule contacts per type 4 bipolar cell, and approximately 10% of rods contacted type 4 bipolar cells. We suggest that type 4 bipolar cells, together with the recently described type 3a and b cells, provide an alternative and direct route from rods to OFF cone bipolar cells. In the Bassoon DeltaEx4/5 mouse, a mouse mutant that shows extensive remodeling of the rod system including sprouting of horizontal and rod bipolar cells into the outer nuclear layer due to impaired synaptic transmission, we found that in addition mixed-input (type 3 and 4) OFF bipolar cells sprout to ectopic sites. In contrast, true cone-selective type 1 and 2 OFF cone bipolar cells did not show sprouting in the Bassoon mouse mutant.

MeCP2-a chromatin-binding protein associated with Rett syndrome-has two main isoforms, MeCP2-E1 and MeCP2-E2, differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest that they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored.

Although mRNAs are localized in the processes of excitatory neurons, it is still unclear whether interneurons also localize a large population of mRNAs. In addition, the variability in the localized mRNA population within and between cell types is unknown. Here we describe the unbiased transcriptomic characterization of the subcellular compartments of hundreds of single neurons. We separately profiled the dendritic and somatic transcriptomes of individual rat hippocampal neurons and investigated mRNA abundances in the soma and dendrites of single glutamatergic and GABAergic neurons. We found that, like their excitatory counterparts, interneurons contain a rich repertoire of ~4000 mRNAs. We observed more cell type-specific features among somatic transcriptomes than their associated dendritic transcriptomes. Finally, using celltype-specific metabolic labeling of isolated neurites, we demonstrated that the processes of glutamatergic and, notably, GABAergic neurons were capable of local translation, suggesting mRNA localization and local translation are general properties of neurons.

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization. While brain transcriptome, connectome and global proteome maps are being generated, system-wide analysis of temporal protein dynamics at the subcellular level are currently lacking. Here, we perform a temporally-resolved surfaceome analysis of primary neuron cultures and reveal dynamic surface protein clusters that reflect the functional requirements during distinct stages of neuronal development. Direct comparison of surface and total protein pools during development and homeostatic synaptic scaling demonstrates system-wide proteostasis-independent remodeling of the neuronal surface, illustrating widespread regulation on the level of surface trafficking. Finally, quantitative analysis of the neuronal surface during chemical long-term potentiation (cLTP) reveals fast externalization of diverse classes of surface proteins beyond the AMPA receptor, providing avenues to investigate the requirement of exocytosis for LTP. Our resource (neurosurfaceome.ethz.ch) highlights the importance of subcellular resolution for systems-level understanding of cellular processes.