Neuronal Secretory Pathway Research Articles

Human cytomegalovirus induces neuronal gene expression for viral maturation.

Viral invasion of the host cell causes some of the most dramatic changes in biology. Human cytomegalovirus (HCMV) extensively remodels host cells, altering nuclear shape and generating a cytoplasmic viral-induced assembly compartment (vIAC). How these striking morphology changes take place in the context of host gene regulation is still emerging. Here, we discovered that histone variant macroH2A1 is essential for producing infectious progeny. Because virion maturation and cellular remodeling are closely linked processes, we investigated structural changes in the host cell upon HCMV infection. We discovered that macroH2A1 is necessary for HCMV-induced reorganization of the host nucleus, cytoskeleton, and endoplasmic reticulum. Furthermore, using RNA-seq we found that while all viral genes were highly expressed in the absence of macroH2A1, many HCMV-induced host genes were not. Remarkably, hundreds of these HCMV-induced macroH2A1-dependent host genes are associated with neuronal synapse formation and vesicle trafficking. Knock-down of these HCMV-induced neuronal genes during infection resulted in malformed vIACs and smaller plaques, establishing their importance to HCMV infection. Together, our findings demonstrate that HCMV manipulates host gene expression by hijacking a dormant neuronal secretory pathway for efficient virion maturation.

Amyloid β production along the neuronal secretory pathway: Dangerous liaisons in the Golgi?

β-amyloid peptides (Aβ) are generated in intracellular compartments of neurons and secreted to form cytotoxic fibrils and plaques. Dysfunctional membrane trafficking contributes to aberrant Aβ production and Alzheimer's disease. Endosomes represent one of the major sites for Aβ production and recently the Golgi has re-emerged also as a major location for amyloid precursor protein (APP) processing and Aβ production. Based on recent findings, here we propose that APP processing in the Golgi is finely tuned by segregating newly-synthesised APP and the β-secretase BACE1 within the Golgi and into distinct trans-Golgi network transport pathways. We hypothesise that there are multiple mechanisms responsible for segregating APP and BACE1 during transit through the Golgi, and that perturbation in Golgi morphology associated with Alzheimer's disease, and or changes in cholesterol metabolism associated with Alzheimer's disease risk factors, may lead to a loss of partitioning and enhanced Aβ production.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Synaptic activity and strength are reflected by changes in the post-synaptic secretory pathway

Neurons are highly asymmetric cells that span long distances and need to react promptly to local demands. Consequently, neuronal secretory pathway elements are distributed throughout neurites, specifically in post-synaptic compartments, to enable local protein synthesis and delivery. Whether and how changes in local synaptic activity correlate to post-synaptic secretory elements is still unclear. To assess this, we used STED nanoscopy and automated quantitative image analysis of post-synaptic markers of the endoplasmic reticulum, ER-Golgi intermediate compartment, trans-Golgi network, and spine apparatus. We found that the distribution of these proteins was dependent on pre-synaptic activity, measured as the amount of recycling vesicles. Moreover, their abundance correlated to both pre- and post-synaptic markers of synaptic strength. Overall, the results suggest that in small, low-activity synapses the secretory pathway components are tightly clustered in the synaptic area, presumably to enable rapid local responses, while bigger synapses utilise secretory machinery components from larger, more diffuse areas.

Increased expression and retention of the secretory chaperone proSAAS following cell stress

The secretory pathway of neurons and endocrine cells contains a variety of mechanisms designed to combat cellular stress. These include not only the unfolded protein response pathways but also diverse chaperone proteins that collectively work to ensure proteostatic control of secreted and membrane-bound molecules. One of the least studied of these chaperones is the neural- and endocrine-specific molecule known as proSAAS. This small chaperone protein acts as a potent anti-aggregant both in vitro and in cellulo and also represents a cerebrospinal fluid biomarker in Alzheimer’s disease. In the present study, we have examined the idea that proSAAS, like other secretory chaperones, might represent a stress-responsive protein. We find that exposure of neural and endocrine cells to the cell stressors tunicamycin and thapsigargin increases cellular proSAAS mRNA and protein in Neuro2A cells. Paradoxically, proSAAS secretion is inhibited by these same drugs. Exposure of Neuro2A cells to low concentrations of the hypoxic stress inducer cobalt chloride, or to sodium arsenite, an oxidative stressor, also increases cellular proSAAS content and reduces its secretion. We conclude that the cellular levels of the small secretory chaperone proSAAS are positively modulated by cell stress.

Autophagy-Mediated Secretory Pathway is Responsible for Both Normal and Pathological Tau in Neurons.

Increased levels of total tau (t-tau) and hyperphosphorylated tau (p-tau) proteins in the cerebrospinal fluid of Alzheimer's disease (AD) patients are well documented and strongly correlate with AD pathology. Recent studies have further shown that human tau can be released into the extracellular space and transferred to nascent neurons. However, because the tau protein has no signal peptide identity, the mechanisms underlying its secretion remain poorly understood. In the present study, we confirmed that tau protein secretion was promoted by autophagy inducers and downregulated by beclin1 knockdown or autophagy inhibitors derived from human wild type tau (wt-tau)-overexpressing SH-SY5Y cells. Moreover, both t-tau and p-tau secretion were increased by autophagy activation. Furthermore, we identified that six isoforms of tau protein are secreted in an autophagy-dependent manner. These results indicate that both normal and pathological tau are secreted via an autophagy-mediated secretory pathway in neurons. Understanding this new pathway for tau secretion may provide critical future insights into tau pathologies, such as AD.

A NOVEL DROSOPHILA MODEL OF ALZHEIMER’S DISEASE

Alzheimer's disease (AD) is a progressive neurodegenerative disease and is the main cause of senile dementia. AD is characterized by two pathological hallmarks, senile plaques composed by amyloid β peptides (Aβ) and neurofibrillary tangles composed by hyper-phosphorylated tau proteins. The accumulation of Aβ42 in the brain is thought to be the primary event in AD pathogenesis. To understand pathological roles of Aβ42 in vivo, we previously established a Drosophila model of AD expressing Aβ42 peptides in the secretory pathway of neurons (Iijima, K. et al. 2004, PNAS). This fly model developed several key pathological phenotypes in an age-dependent manner. However, substantial amount of Aβ42 was accumulated intracellularly and caused chronic ER stress, which may not be common pathologies in AD patients. To overcome this issue, we generated novel transgenic flies that efficiently secrete Aβ42 peptides from neurons to extracellular space in the brains. To examine the effects of and metabolism of Aβ42 peptides in the extracellular space of fly brains, we generated transgenic fly lines carrying engineered Aβ42 construct that efficiently secrete Aβ42 from neurons in the brains. We then systematically characterized their phenotypes including Aβ42 accumulation, locomotor functions, neurodegeneration and life span during aging and compared those in our previous Aβ42 fly model. As reported, our previous Aβ42 fly model showed age-dependent Aβ42 accumulation, locomotor defects, neurodegeneration and premature death. In contrast, despite high expression levels of Aβ42 in the brains, the new Aβ42 fly model did not show any significant phenotypes including Aβ42 deposits, suggesting that secreted Aβ42 did not accumulate and aggregate in the extracellular space even in aged fly brains. Our data indicate that overexpression of Aβ42 in the extracellular space of fly brain is not sufficient to produce Aβ42 deposits and neurotoxicity in flies. This result might be similar to recent observation in App knock-in mouse of AD carrying only Swedish mutation (Salas, IH. et al. 2018, J Alzheimers Dis. & Sakakibara, Y. et al. 2019, BMC Neurosci.). Our new Aβ42 fly model can be used as an efficient genetic model system to provide significant insights into pathomechanisms of an early phase of AD.

A novel function for the ER retention signals in the C-terminus of kainate receptor subunit, GluK5

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons. SignificanceWe present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways.

A decay of the adaptive capacity of the unfolded protein response exacerbates Alzheimer's disease

Alterations in the buffering capacity of the proteostasis network are a salient feature of Alzheimer's disease, associated with the occurrence of chronic endoplasmic reticulum (ER) stress. To cope with ER stress, cells activate the unfolded protein response (UPR), a signal transduction pathway that enforces adaptive programs through the induction of transcription factors such as X-box binding protein 1 (XBP1). A new study by Marcora et al used a fly model to study amyloid β pathogenesis in the secretory pathway of neurons. Through genetic manipulation, authors identified a new role of XBP1s in the clearance of amyloid β and the improvement of neuronal function. However, although the activation of the UPR signaling was sustained over time, the transcriptional upregulation of XBP1-target genes was attenuated during aging. This study suggests that aging has a negative impact in the ability of the UPR to manage proteostasis alterations in Alzheimer's disease.

Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains.

The secretory pathway in neurons requires efficient targeting of cargos and regulatory proteins to their release sites. Tomosyn contributes to synapse function by regulating synaptic vesicle (SV) and dense-core vesicle (DCV) secretion. While there is large support for the presynaptic accumulation of tomosyn in fixed preparations, alternative subcellular locations have been suggested. Here we studied the dynamic distribution of tomosyn-1 (Stxbp5) and tomosyn-2 (Stxbp5l) in mouse hippocampal neurons and observed a mixed diffuse and punctate localization pattern of both isoforms. Tomosyn-1 accumulations were present in axons and dendrites. As expected, tomosyn-1 was expressed in about 75% of the presynaptic terminals. Interestingly, also bidirectional moving tomosyn-1 and -2 puncta were observed. Despite the lack of a membrane anchor these puncta co-migrated with synapsin and neuropeptide Y, markers for respectively SVs and DCVs. Genetic blockade of two known tomosyn interactions with synaptotagmin-1 and its cognate SNAREs did not abolish its vesicular co-migration, suggesting an interplay of protein interactions mediated by the WD40 and SNARE domains. We hypothesize that the vesicle-binding properties of tomosyns may control the delivery, pan-synaptic sharing and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane.

Epigenetic modifications reveal an effective use of the protein quality control system to suppress accumulation and toxicity of beta-amyloid 42 in the secretory pathway in neurons

Epigenetic modifications reveal an effective use of the protein quality control system to suppress accumulation and toxicity of beta-amyloid 42 in the secretory pathway in neurons

Structure-dependent Impairment of Intracellular Apolipoprotein E4 Trafficking and Its Detrimental Effects Are Rescued by Small-molecule Structure Correctors

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

Processing-dependent trafficking of Sonic hedgehog to the regulated secretory pathway in neurons

Neurons are an important source of the secreted morphogen Sonic hedgehog (Shh), however, little is known about neuron-specific regulation of Shh transport and secretion. To study this process, we investigated the subcellular distribution of Shh in primary neurons and differentiated cells of a neuroendocrine cell line by fluorescence microscopy and biochemical fractionation. In retinal ganglion cells, endogenous Shh was distributed as intra- and extracellular puncta at the soma, dendrites, axons and neurite terminals. Shh + puncta move bidirectionally and colocalize with markers of synaptic vesicles (SVs) and dense core granules. Lipid modification and proteolysis were required for Shh sorting to SVs and cell surface association. Finally, consistent with its association with regulated secretory vesicles, Shh secretion could be induced under depolarizing conditions. Taken together, these observations suggest that long-range Shh transport and signalling in neurons involves trafficking to the regulated secretory pathway and cell surface accumulation of Shh on axons and suggests a link between neuronal activity and Shh release.

Integrated analysis of clinical signs and literature data for the diagnosis and therapy of a previously undescribed 6p21.3 deletion syndrome

A de novo 0.3 Mb deletion on 6p21.3 was detected by array-comparative genomic hybridization in a girl with mental retardation, drug-resistant seizures, facial dysmorphisms, gut malrotation and abnormal pancreas segmentation. Consistent with phenotypic manifestations is haploinsufficiency of SYNGAP1, which was recently demonstrated to cause non-syndromic mental retardation, and of the flanking genes CuTA, a likely modulator of the processing and trafficking of secretory proteins in the human brain, and hPHF1, involved in HOX gene silencing. Mutations of both CuTA and hPHF1 were never reported as causative of human diseases. Similarly, the present syndromic condition was not previously described and it can be regarded as a human model confirming the suggested biological properties of the genes included in the deletion interval. In addition, experimental evidence that SYNGAP1 and CuTA are involved in the secretory pathway in neurons, through glutamate and acetylcholinesterase signalling, prompted us to consider modulation of the glutamate pathway as target of a therapeutic strategy for seizure control.

Gamma‐protocadherins are enriched and transported in specialized vesicles associated with the secretory pathway in neurons

Gamma protocadherins (Pcdh-γs) resemble classical cadherins and have the potential to engage in cell-cell interactions with homophilic properties. Emerging evidence suggests non-conventional roles for some protocadherins in neural development. We sought to determine whether Pcdh-γ trafficking in neurons is consistent with an intracellular role for these molecules. Here we show that, in contrast to the largely surface localization of classical cadherins, endogenous Pcdh-γs are primarily intracellular in rat neurons in vivo and are equally distributed within organelles of subsynaptic dendritic and axonal compartments. A strikingly higher proportion of Pcdh-γ-containing organelles in synaptic compartments was observed at postnatal day 16. To determine the origin of Pcdh-γ-trafficking organelles, we isolated organelles with Pcdh-γ antibody-coupled magnetic beads from brain organelle suspensions. Vesicles with high levels of COPII and endoplasmic reticulum-Golgi intermediate compartment (ERGIC) components were isolated with the Pcdh-γ antibody but not with the classical cadherin antibody. In cultured hippocampal neurons, Pcdh-γ immunolabeling partially overlapped with calnexin- and COPII-positive puncta in dendrites. Mobile Pcdh-γ-GFP profiles dynamically codistributed with a DsRed construct coupled to ER retention signals by live imaging. Pcdh-γ expression correlated with accumulations of tubulovesicular and ER-like organelles in dendrites. Our results are consistent with the possibility that Pcdh-γs could have a unique function within the secretory pathway in addition to their documented surface roles.

Resolving the Assembly State of Herpes Simplex Virus during Axon Transport by Live-Cell Imaging

Neurotropic herpesviruses depend on long-distance axon transport for the initial establishment of latency in peripheral ganglia (retrograde transport) and for viral spread in axons to exposed body surfaces following reactivation (anterograde transport). Images of neurons infected with herpes simplex virus type 1 (HSV-1), acquired using electron microscopy, have led to a debate regarding why different types of viral structures are seen in axons and which of these particles are relevant to the axon transport process. In this study, we applied time-lapse fluorescence microscopy to image HSV-1 virion components actively translocating to distal axons in primary neurons and neuronal cell lines. Key to these findings, only a small fraction of viral particles were engaged in anterograde transport during the egress phase of infection at any given time. By selective analysis of the composition of the subpopulation of actively transporting capsids, a link between transport of fully assembled HSV-1 virions and the neuronal secretory pathway was identified. Last, we have evaluated the seemingly opposing findings made in previous studies of HSV-1 axon transport in fixed cells and demonstrate a limitation to assessing the composition of individual HSV-1 particles using antibody detection methods.

Transcriptional Orchestration of the Regulated Secretory Pathway in Neurons by the bHLH protein DIMM

The Drosophila basic helix-loop-helix (bHLH) gene dimmed (dimm) promotes a neurosecretory/neuroendocrine phenotype in cells but is not associated with specific neuropeptides or neurohormones. Rather, it is expressed by those peptidergic neurons that project long axons and appear to produce large amounts of secretory peptides. Here, we genetically transform nonpeptidergic neurons in Drosophila to study DIMM's action mechanisms. Nonpeptidergic neurons normally fail to accumulate ectopic neuropeptides. We now show that they will do so when they are also forced to express ectopic DIMM. Furthermore, mass spectrometry shows that photoreceptors, which are normally nonpeptidergic, fail to process an ectopic neuropeptide precursor to make bioactive peptides but will do so efficiently when DIMM is co-misexpressed. Likewise, photoreceptors, which normally package the fast neurotransmitter histamine within small clear synaptic vesicles, produce numerous large dense-core vesicles (LDCVs) when they misexpress DIMM. These novel LDCVs accumulate ectopic neuropeptide when photoreceptors co-misexpress a neuropeptide transgene. DIMM-expressing photoreceptors no longer accumulate histamine and lose synaptic organelles critical to their normal physiology. These findings indicate that DIMM suppresses conventional fast neurotransmission and promotes peptidergic neurosecretory properties. We conclude that DIMM normally provides a comprehensive transcriptional control to direct the differentiation of dedicated neuroendocrine neurons.

Heterogeneity of a Fluorescent Tegument Component in Single Pseudorabies Virus Virions and Enveloped Axonal Assemblies

The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.

Impaired extracellular secretion of mutant superoxide dismutase 1 associates with neurotoxicity in familial amyotrophic lateral sclerosis.

Mutations in the intracellular metalloenzyme superoxide dismutase 1 (SOD1) are linked to neurotoxicity in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism. Golgi fragmentation and endoplasmic reticulum stress are early hallmarks of spinal motor neuron pathology in transgenic mice overexpressing mutant SOD1, suggesting that dysfunction of the neuronal secretory pathway may contribute to ALS pathogenesis. We therefore proposed that mutant SOD1 directly engages and modulates the secretory pathway based on recent evidence of SOD1 secretion in diverse human cell lines. Here, we demonstrate that a fraction of active endogenous SOD1 is secreted by NSC-34 motor neuron-like cells via a brefeldin-A (BFA)-sensitive pathway. Expression of enhanced green fluorescent protein-tagged mutant human SOD1 (hSOD1-EGFP) in NSC-34 cells induced frequent cytoplasmic inclusions and protein insolubility that correlated with toxicity. In contrast, transfection of non-neuronal COS-7 cells resulted in mutant hSOD1-EGFP cytoplasmic inclusions, oligomerization, and fragmentation without detectable toxicity. Importantly, impaired secretion of hSOD1-EGFP was common to all 10 SOD1 mutants tested relative to wild-type protein in NSC-34 cells. Treatment with BFA inhibited hSOD1-EGFP secretion with pronounced BFA-induced toxicity in mutant cells. Extracellular targeting of mutant hSOD1-EGFP via SOD3 signal peptide fusion attenuated cytoplasmic inclusion formation and toxicity. The effect of elevated extracellular SOD1 was then evaluated in a transgenic rat model of ALS. Chronic intraspinal infusion of exogenous wild-type hSOD1 significantly delayed disease progression and endpoint in transgenic SOD1(G93A) rats. Collectively, these results suggest novel extracellular roles for SOD1 in ALS and support a causal relationship between mutant SOD1 secretion and intraneuronal toxicity.

Secretory trafficking in neuronal dendrites.

The neuronal secretory pathway represents the intracellular route for proteins involved in synaptic transmission and plasticity, as well as lipids required for outgrowth and remodelling of dendrites and axons. Although neurons use the same secretory compartments as other eukaryotic cells, the enormous distances involved, as well as the unique morphology of the neuron and its signalling requirements, challenge canonical models of secretory pathway organization. Here, we review evidence for a distributed secretory pathway in neurons, suggest mechanisms that may regulate secretory compartment distribution, and discuss the implications of a distributed secretory pathway for neuronal morphogenesis and neural-circuit plasticity.