Local mitochondrial replication in the periphery of neurons requires the eEF1A1 protein and thetranslation of nuclear-encoded proteins.

In neurons, it is commonly assumed that mitochondrial replication only occurs in the cell body, after which the mitochondria must travel to the neuron's periphery. However, while mitochondrial DNA replication has been observed to occur away from the cell body, the specific mechanisms involved remain elusive. Using EdU-labelling in mouse primary neurons, we developed a tool to determine the mitochondrial replication rate. Taking of advantage of microfluidic devices, we confirmed that mitochondrial replication also occurs locally in the periphery of neurons. To achieve this, mitochondria require de novo nuclear-encoded, but not mitochondrial-encoded protein translation. Following a proteomic screen comparing synaptic with non-synaptic mitochondria, we identified two elongation factors - eEF1A1 and TUFM - that were upregulated in synaptic mitochondria. We found that mitochondrial replication is impaired upon the downregulation of eEF1A1, and this is particularly relevant in the periphery of neurons.

Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan.

The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues.

Cochlear ribbon synapse maturation requires Nlgn1 and Nlgn3.

Hearing depends on precise synaptic transmission between cochlear inner hair cells and spiral ganglion neurons through afferent ribbon synapses. Neuroligins (Nlgns) facilitate synapse maturation in the brain, but they have gone unstudied in the cochlea. We report Nlgn3 and Nlgn1 knockout (KO) cochleae have fewer ribbon synapses and have impaired hearing. Nlgn3 KO is more vulnerable to noise trauma with limited activity at high frequencies one day after noise. Furthermore, Nlgn3 KO cochleae have a 5-fold reduction in synapse number compared to wild type after two weeks of recovery. Double KO cochlear phenotypes are more prominent than the KOs, for example, 5-fold smaller synapses, 25% reduction in synapse density, and 30% less synaptic output. These observations indicate Nlgn3 and Nlgn1 are essential to cochlear ribbon synapse maturation and function.

Long-lived mitochondrial proteins and why they exist.

Intracellular long-lived proteins (LLPs) provide structural support for several highly stable protein complexes and assemblies that play essential roles in ensuring cellular homeostasis and function. Recently, mitochondrial long-lived proteins (mt-LLPs) were discovered within inner mitochondria membranes (IMMs) and cristae invagination in tissues with old postmitotic cells. This observation is at odds with the fact that mitochondria are highly dynamic organelles that are continually remodeled through processes of fission, fusion, biogenesis, and multiple quality control pathways. In this opinion article, we propose that a subset of the mitochondrial proteome persists over long time frames and these mt-LLPs provide key structural support for the lifelong maintenance of mitochondrial structure.

Herpesviruses assimilate kinesin to produce motorized viral particles.

Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established1. Recurrent infections arise sporadically from the peripheral nervous system throughout the life of the host, and invasion of the central nervous system may occur, with severe outcomes2. These viruses directly recruit cellular motors for transport along microtubules in nerve axons, but how the motors are manipulated to deliver the virus to neuronal nuclei is not understood. Here, using herpes simplex virus type I and pseudorabies virus as model alphaherpesviruses, we show that a cellular kinesin motor is captured by virions in epithelial cells, carried between cells, and subsequently used in neurons to traffic to nuclei. Viruses assembled in the absence of kinesin are not neuroinvasive. The findings explain a critical component of the alphaherpesvirus neuroinvasive mechanism and demonstrate that these viruses assimilate a cellular protein as an essential proviral structural component. This principle of viral assimilation may prove relevant to other virus families and offers new strategies to combat infection.

Long-lived mitochondrial cristae proteins in mouse heart and brain.

Long-lived proteins (LLPs) have recently emerged as vital components of intracellular structures whose function is coupled to long-term stability. Mitochondria are multifaceted organelles, and their function hinges on efficient proteome renewal and replacement. Here, using metabolic stable isotope labeling of mice combined with mass spectrometry (MS)-based proteomic analysis, we demonstrate remarkable longevity for a subset of the mitochondrial proteome. We discovered that mitochondrial LLPs (mt-LLPs) can persist for months in tissues harboring long-lived cells, such as brain and heart. Our analysis revealed enrichment of mt-LLPs within the inner mitochondrial membrane, specifically in the cristae subcompartment, and demonstrates that the mitochondrial proteome is not turned over in bulk. Pioneering cross-linking experiments revealed that mt-LLPs are spatially restricted and copreserved within protein OXPHOS complexes, with limited subunit exchange throughout their lifetimes. This study provides an explanation for the exceptional mitochondrial protein lifetimes and supports the concept that LLPs provide key structural stability to multiple large and dynamic intracellular structures.

Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer's Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals.

Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aß) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aß accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aß. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper's transparent peer review process is included in the Supplemental Information.

Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways.

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.

Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons.

Recent reports suggest that botulinum neurotoxin (BoNT) A, which is widely used clinically to inhibit neurotransmission, can spread within networks of neurons to have distal effects, but this remains controversial. Moreover, it is not known whether other members of this toxin family are transferred between neurons. Here, we investigate the potential distal effects of BoNT/A, BoNT/D, and tetanus toxin (TeNT), using central neurons grown in microfluidic devices. Toxins acted upon the neurons that mediated initial entry, but all three toxins were also taken up, via an alternative pathway, into non-acidified organelles that mediated retrograde transport to the somato-dendritic compartment. Toxins were then released into the media, where they entered and exerted their effects upon upstream neurons. These findings directly demonstrate that these agents undergo transcytosis and interneuronal transfer in an active form, resulting in long-distance effects.

Increased expression of AT-1/SLC33A1 causes an autistic-like phenotype in mice by affecting dendritic branching and spine formation.

The import of acetyl-CoA into the lumen of the endoplasmic reticulum (ER) by AT-1/SLC33A1 regulates Nε-lysine acetylation of ER-resident and -transiting proteins. Specifically, lysine acetylation within the ER appears to influence the efficiency of the secretory pathway by affecting ER-mediated quality control. Mutations or duplications in AT-1/SLC33A1 have been linked to diseases such as familial spastic paraplegia, developmental delay with premature death, and autism spectrum disorder with intellectual disability. In this study, we generated an AT-1 Tg mouse model that selectively overexpresses human AT-1 in neurons. These animals demonstrate cognitive deficits, autistic-like social behavior, aberrations in synaptic plasticity, an increased number of dendritic spines and branches, and widespread proteomic changes. We also found that AT-1 activity regulates acetyl-CoA flux, causing epigenetic modulation of the histone epitope H3K27 and mitochondrial adaptation. In conclusion, our results indicate that increased expression of AT-1 can cause an autistic-like phenotype by affecting key neuronal metabolic pathways.

Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure.

Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of gait disorders. Their pathological hallmark is a length-dependent distal axonopathy of nerve fibers in the corticospinal tract. Involvement of other neurons can cause additional neurological symptoms, which define a diverse set of complex hereditary spastic paraplegias. We present two siblings who have the unusual combination of early-onset spastic paraplegia, optic atrophy, and neuropathy. Genome-wide SNP-typing, linkage analysis, and exome sequencing revealed a homozygous c.316C>T (p.R106C) variant in the Trk-fused gene (TFG) as the only plausible mutation. Biochemical characterization of the mutant protein demonstrated a defect in its ability to self-assemble into an oligomeric complex, which is critical for normal TFG function. In cell lines, TFG inhibition slows protein secretion from the endoplasmic reticulum (ER) and alters ER morphology, disrupting organization of peripheral ER tubules and causing collapse of the ER network onto the underlying microtubule cytoskeleton. The present study provides a unique link between altered ER architecture and neurodegeneration.

Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen.

The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function.