Tau aggregation is central to the pathogenesis of a large group of neurodegenerative diseases termed tauopathies, but it is still unclear in which way neurons respond to tau pathology and how tau ...accumulation leads to neurodegeneration. A striking neuron-specific response to tau pathology is presented by granulovacuolar degeneration bodies (GVBs), lysosomal structures that accumulate specific cargo in a dense core. Here we employed different tau aggregation models in primary neurons to investigate which properties of pathological tau assemblies affect GVB accumulation using a combination of confocal microscopy, transmission electron microscopy, and quantitative automated high-content microscopy. Employing GFP-tagged and untagged tau variants that spontaneously form intraneuronal aggregates, we induced pathological tau assemblies with a distinct subcellular localization, morphology, and ultrastructure depending on the presence or absence of the GFP tag. The quantification of the GVB load in the different models showed that an increased GVB accumulation is associated with the untagged tau aggregation model, characterized by shorter and more randomly distributed tau filaments in the neuronal soma. Our data indicate that tau aggregate structure and/or subcellular localization may be key determinants of GVB accumulation.
Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless ...(Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.
Secretion principles are conserved from yeast to humans, and many yeast orthologs have established roles in synaptic vesicle exocytosis in the mammalian brain. Surprisingly, SEC4 orthologs and their ...effectors, the exocyst, are dispensable for synaptic vesicle exocytosis. Here, we identify the SEC4 ortholog RAB3 and its neuronal effector, RIM1, as essential molecules for neuropeptide and neurotrophin release from dense-core vesicles (DCVs) in mammalian neurons. Inactivation of all four RAB3 genes nearly ablated DCV exocytosis, and re-expression of RAB3A restored this deficit. In RIM1/2-deficient neurons, DCV exocytosis was undetectable. Full-length RIM1, but not mutants that lack RAB3 or MUNC13 binding, restored release. Strikingly, a short N-terminal RIM1 fragment only harboring RAB3- and MUNC13-interacting domains was sufficient to support DCV exocytosis. We propose that RIM and MUNC13 emerged as mammalian alternatives to the yeast exocyst complex as essential RAB3/SEC4 effectors and organizers of DCV fusion sites by recruiting DCVs via RAB3.
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•RAB3 and RIM1/2 are essential for neuromodulator release from dense-core vesicles•RIM’s N-terminal RAB3- and MUNC13-binding domains are sufficient to support release•The indispensable role of RAB3 marks a main difference between secretory pathways•RIM/MUNC13 may provide a mammalian substitute for the yeast exocyst complex
Neurons secrete neuromodulators/neuropeptides from dense-core vesicles (DCVs) by a largely unknown mechanism. Persoon et al. identify RAB3 and RIM1/2 as essential factors. RAB3’s indispensable role is the first distinct feature of DCV secretion as compared to synaptic vesicle secretion.
Neuropeptides are essential signaling molecules transported and secreted by dense‐core vesicles (DCVs), but the number of DCVs available for secretion, their subcellular distribution, and release ...probability are unknown. Here, we quantified DCV pool sizes in three types of mammalian CNS neurons in vitro and in vivo. Super‐resolution and electron microscopy reveal a total pool of 1,400–18,000 DCVs, correlating with neurite length. Excitatory hippocampal and inhibitory striatal neurons in vitro have a similar DCV density, and thalamo‐cortical axons in vivo have a slightly higher density. Synapses contain on average two to three DCVs, at the periphery of synaptic vesicle clusters. DCVs distribute equally in axons and dendrites, but the vast majority (80%) of DCV fusion events occur at axons. The release probability of DCVs is 1–6%, depending on the stimulation. Thus, mammalian CNS neurons contain a large pool of DCVs of which only a small fraction can fuse, preferentially at axons.
Synopsis
This study characterized the total and release pool of dense‐core vesicles (DCVs) in mammalian CNS neurons, showing a total pool of 1,400–18,000 DCVs which distribute equally in axons and dendrites. Depending on the stimulation 1–6% of the DCVs can fuse, which occurs predominantly at axons.
Mammalian neurons contain a total pool of 1,400‐18,000 dense‐core vesicles, correlating with neurite length.
Dense‐core vesicles distribute equally in axons and dendrites, but the vast majority (80%) of fusion events occurs at axons.
Different stimulations release up to 6% of the total pool of dense‐core vesicles.
We conclude that mouse CNS neurons contain a large pool of dense‐core vesicles of which only a small fraction fuse, preferentially at axons.
A quantitative analysis of neuropeptide‐secreting dense‐core vesicles (DCVs) using super‐resolution and electron microscopy provides insight into DCV distribution, abundance and release probability.
Tomosyns are widely thought to attenuate membrane fusion by competing with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Here, we present evidence against this scenario. In a novel mouse model, ...tomosyn-1/2 deficiency lowered the fusion barrier and enhanced the probability that synaptic vesicles fuse, resulting in stronger synapses with faster depression and slower recovery. While wild-type tomosyn-1m rescued these phenotypes, substitution of its SNARE motif with that of synaptobrevin-2/VAMP2 did not. Single-molecule force measurements indeed revealed that tomosyn's SNARE motif cannot substitute synaptobrevin-2/VAMP2 to form template complexes with Munc18-1 and syntaxin-1, an essential intermediate for SNARE assembly. Instead, tomosyns extensively bind synaptobrevin-2/VAMP2-containing template complexes and prevent SNAP-25 association. Structure-function analyses indicate that the C-terminal polybasic region contributes to tomosyn's inhibitory function. These results reveal that tomosyns regulate synaptic transmission by cooperating with synaptobrevin-2/VAMP2 to prevent SNAP-25 binding during SNARE assembly, thereby limiting initial synaptic strength and equalizing it during repetitive stimulation.
Extracellular vesicles (EVs) are present in human cerebrospinal fluid (CSF), yet little is known about their protein composition. The aim of this study is to provide a comprehensive analysis of the ...proteome of CSF EVs by electron microscopy and high resolution tandem mass spectrometry (MS/MS) in conjunction with bioinformatics. We report an extensive catalog of 1315 proteins identified in EVs isolated from two different CSF pools by ultracentrifugation, including 230 novel EV proteins. Out of 1315 proteins, 760 were identified in both CSF pools and about 30% of those were also quantitatively enriched in the EV fraction versus the soluble CSF fraction. The proteome of CSF EVs was enriched in exosomal markers such as alix and syntenin-1, heat shock proteins and tetraspanins and contained a high proportion of brain-derived proteins (n=373). Interestingly, several known biomarkers for neurodegenerative diseases such as the amyloid precursor protein, the prion protein and DJ-1 were identified in the EV fractions. Our dataset represents the first comprehensive inventory of the EV proteome in CSF, underscoring the biomarker potential of this organelle. Further comparative studies on CSF EVs isolated from patients diagnosed with neurological disorders are warranted. Data are available via ProteomeXchange with identifier PXD000608.
Biological significance
In this study we analyzed the protein composition of extracellular vesicles isolated from pooled samples of human cerebrospinal fluid (CSF). CSF is a colorless fluid surrounding the brain and the spinal cord, important for the physiology of the central nervous system, ensuing mechanical protection, regulation of brain blood flow and elimination of byproducts of the brain. Since brain (patho)physiology is reflected in CSF, this biological fluid represents an ideal source of soluble and vesicle-based biomarkers for neurological diseases. Here we confirm the presence of exosome-like extracellular vesicles in CSF, underscoring a potential role in the physiology of the brain. These extracellular vesicles provide a rich source of candidate biomarkers, representing a brain “fluid biopsy”. Most interestingly, the involvement of extracellular vesicles in transferring toxic proteins such as α-synuclein and β-amyloid has been postulated as one of the mechanisms involved in the spreading of neurodegeneration to different brain areas. In line with this, we show that human CSF extracellular vesicles contain prionogenic proteins such as the amyloid precursor protein and the prion protein. Delineating the protein composition of extracellular vesicles in CSF is a first and crucial step to comprehend their origin and their function in the central nervous system and to establish their biomarker potential.
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•First in-depth proteomic inventory of CSF extracellular vesicles (EVs).•Different types of EVs are present in CSF, including exosomes.•The proteome of CSF EVs is composed of a high proportion of brain-derived proteins.•Prionogenic and neurodegeneration-related proteins were identified in CSF EVs.
Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP) and dense‐core vesicle (DCV) biogenesis. To test this, we analyzed the ...release of neuropeptide Y (NPY)‐pHluorin, a live RSP reporter, and the distribution, number, and appearance of DCVs, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT‐PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B−/− neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP, we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B−/− neurons. Moreover, the number of DCVs outside the soma, stained with endogenous marker Secretogranin II, the number of NPY‐pHluorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH‐sensitive NPY‐pHluorin fluorescence, were all normal in CgA/B−/− neurons. Electron microscopy revealed that synapses contained a normal number of DCVs, with a normal diameter, in CgA/B−/− neurons. In contrast, CgA/B−/− chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live‐cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B−/− neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.
Chromogranin A and B (Cgs) have been implicated in the biogenesis and release of Dense‐Core Vesicles (DCVs) in chromaffin cells. Here, we demonstrate that despite the reduction in the number and size of DCVs in chromaffin cells, this phenotype is not reproduced in hippocampal neurons. We conclude that Cgs are not essential for DCVs biogenesis, cargo sorting, and release in hippocampal neurons.
Loss of the exocytic Sec1/MUNC18 protein MUNC18‐1 or its target‐SNARE partners SNAP25 and syntaxin‐1 results in rapid, cell‐autonomous and unexplained neurodegeneration, which is independent of their ...known role in synaptic vesicle exocytosis. cis‐Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18‐1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18‐1 expression results in a smaller cis‐Golgi. In addition, we now show that medial‐Golgi and the trans‐Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum‐to‐Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18‐1 caused reduced retrograde Cholera Toxin B‐subunit transport from the plasma membrane to the Golgi. In addition, MUNC18‐1‐deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18‐1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18‐1 in endosomal SNARE‐dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18‐1 deficient neurons.
Loss of MUNC18‐1 or the t‐SNAREs SNAP25 and syntaxin‐1 leads to massive, yet unexplained, neurodegeneration. Previous research showed that Golgi abnormalities are the earliest, common phenotype. In this issue, Van Berkel et al. provide an explanation for the mechanism underlying the Golgi abnormalities upon loss of MUNC18‐1. By systematically assessing transport routes to and from the Golgi, they show that retrograde endosome‐to‐Golgi, but not anterograde transport from the Golgi, is disturbed. This imbalance in transport routes provides a plausible explanation for the Golgi abnormalities, and may explain neurodegeneration.
Objective
Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 ...function causes MLC. MLC1 is involved in ion–water homeostasis, but its exact role is unknown. We generated Mlc1‐null mice for further studies.
Methods
We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1‐null mice.
Results
Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid–brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1‐null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid–brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1‐null astrocytes showed slowed regulatory volume decrease and reduced volume‐regulated anion currents, which increased upon MLC1 re‐expression. Mlc1‐null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC‐2, but no substantial changes in other known MLC1‐interacting proteins.
Interpretation
Mlc1‐null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC‐2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC. ANN NEUROL 2015;77:114–131
Loss of function of the astrocyte membrane protein MLC1 is the primary genetic cause of the rare white matter disease Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC), which is ...characterized by disrupted brain ion and water homeostasis. MLC1 is prominently present around fluid barriers in the brain, such as in astrocyte endfeet contacting blood vessels and in processes contacting the meninges. Whether the protein plays a role in other astrocyte domains is unknown. Here, we show that MLC1 is present in distal astrocyte processes, also known as perisynaptic astrocyte processes (PAPs) or astrocyte leaflets, which closely interact with excitatory synapses in the CA1 region of the hippocampus. We find that the PAP tip extending toward excitatory synapses is shortened in Mlc1‐null mice. This affects glutamatergic synaptic transmission, resulting in a reduced rate of spontaneous release events and slower glutamate re‐uptake under challenging conditions. Moreover, while PAPs in wildtype mice retract from the synapse upon fear conditioning, we reveal that this structural plasticity is disturbed in Mlc1‐null mice, where PAPs are already shorter. Finally, Mlc1‐null mice show reduced contextual fear memory. In conclusion, our study uncovers an unexpected role for the astrocyte protein MLC1 in regulating the structure of PAPs. Loss of MLC1 alters excitatory synaptic transmission, prevents normal PAP remodeling induced by fear conditioning and disrupts contextual fear memory expression. Thus, MLC1 is a new player in the regulation of astrocyte‐synapse interactions.
Main points
MLC1 is present in perisynaptic astrocyte processes (PAPs).
Loss of MLC1 leads to shorter PAP tips.
PAPs lacking MLC1 do not display learning‐induced structural remodeling.
Mlc1‐null mice show impaired contextual fear memory expression.
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