Mitochondrial-derived vesicles (MDVs) are implicated in diverse physiological processes-for example, mitochondrial quality control-and are linked to various neurodegenerative diseases. However, their ...specific cargo composition and complex molecular biogenesis are still unknown. Here we report the proteome and lipidome of steady-state TOMM20
MDVs. We identified 107 high-confidence MDV cargoes, which include all β-barrel proteins and the TOM import complex. MDV cargoes are delivered as fully assembled complexes to lysosomes, thus representing a selective mitochondrial quality control mechanism for multi-subunit complexes, including the TOM machinery. Moreover, we define key biogenesis steps of phosphatidic acid-enriched MDVs starting with the MIRO1/2-dependent formation of thin membrane protrusions pulled along microtubule filaments, followed by MID49/MID51/MFF-dependent recruitment of the dynamin family GTPase DRP1 and finally DRP1-dependent scission. In summary, we define the function of MDVs in mitochondrial quality control and present a mechanistic model for global GTPase-driven MDV biogenesis.
Low-voltage fast (LVF) and hypersynchronous (HYP) - onset seizures occur in the EEG obtained with depth electrodes from mesial temporal lobe epilepsy (MTLE) patients and animal models. In epileptic ...rats analyzed up to approximately two weeks after pilocarpine-induced status epilepticus (SE), these patterns are associated with specific high-frequency oscillation (HFO) content: ripples (80–200Hz) or fast-ripples (250–500Hz) predominate in LVF or HYP seizures, respectively. To establish whether these features change over the course of the disease, we recorded the EEG from the hippocampal CA3 subfield, subiculum, entorhinal cortex and dentate gyrus in two groups of pilocarpine-treated rats: the “early stage group” (n=8) was analyzed from day 3 to 20 post-SE while the “late stage group” (n=7) was studied from day 27 to 53 post-SE. We found that: (i) HYP and LVF seizures prevail in the early and late stage group, respectively; (ii) HYP seizures mainly originate from CA3 in the early stage group only; (iii) LVF seizures in both early and late stage group originate from a diffuse network; (iv) LVF and HYP seizures in the early stage group are mainly associated with ripples and fast ripples, respectively; but (v) fast ripples predominate in the late stage group, regardless of seizure onset pattern. Finally, extensive neuronal loss occurred in the hippocampus of the late stage group. Our results reveal that significant changes in ictogenesis and HFO occurrence, which are associated with the manifestation of severe hippocampal damage, occur over time in this MTLE model.
•Hypersynchronous-onset seizures prevail from 3 to 20days after status epilepticus.•Low-voltage fast-onset seizures prevail from 27 to 53days after status epilepticus.•Fast ripples predominate in the late stage group, during both seizure onset patterns.•Hippocampal damage could explain these changes over time.
While leptin is a well-known regulator of body fat mass, it remains unclear how circulating leptin is sensed centrally to maintain energy homeostasis. Here we show that genetic and pharmacological ...ablation of adult NG2-glia (also known as oligodendrocyte precursors), but not microglia, leads to primary leptin resistance and obesity in mice. We reveal that NG2-glia contact the dendritic processes of arcuate nucleus leptin receptor (LepR) neurons in the median eminence (ME) and that these processes degenerate upon NG2-glia elimination, which explains the consequential attenuation of these neurons’ molecular and electrical responses to leptin. Our data therefore indicate that LepR dendrites in the ME represent the principal conduits of leptin’s anorexigenic action and that NG2-glia are essential for their maintenance. Given that ME-directed X-irradiation confirmed the pharmacological and genetically mediated ablation effects on body weight, our findings provide a rationale for the known obesity risk associated with cranial radiation therapy.
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•Pharmacological and genetic ablation of NG2-glia, but not microglia, leads to obesity•NG2-glial ablation causes LepR processes in the median eminence to degenerate•Arcuate nucleus LepR neurons lose responsiveness to leptin after NG2-glia ablation•X-irradiation aimed at the median eminence is sufficient for weight gain induction
Djogo et al. show that ablation of NG2-glia, but not microglia, in the median eminence (ME) leads to selective degeneration of LepR dendrites in the arcuate nucleus, causing primary leptin resistance and obesity. These effects are reproduced with ME-directed X-irradiation, possibly explaining the obesity risk associated with cranial radiation therapy.
Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among ...biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.
Puberty is a critical period in mesocorticolimbic dopamine (DA) system development, particularly for the medial prefrontal cortex (mPFC) projection which achieves maturity in early adulthood. The ...guidance cue netrin-1 organizes neuronal networks by attracting or repelling cellular processes through DCC (deleted in colorectal cancer) and UNC-5 homologue (UNC5H) receptors, respectively. We have shown that variations in netrin-1 receptor levels lead to selective reorganization of mPFC DA circuitry, and changes in DA-related behaviors, in transgenic mice and in rats. Significantly, these effects are only observed after puberty, suggesting that netrin-1 mediated effects on DA systems vary across development. Here we report on the normal expression of DCC and UNC5H in the ventral tegmental area (VTA) by DA neurons from embryonic life to adulthood, in both mice and rats. We show a dramatic and enduring pubertal change in the ratio of DCC:UNC5H receptors, reflecting a shift toward predominant UNC5H function. This shift in DCC:UNC5H ratio coincides with the pubertal emergence of UNC5H expression by VTA DA neurons. Although the distribution of DCC and UNC5H by VTA DA neurons changes during puberty, the pattern of netrin-1 immunoreactivity in these cells does not. Together, our findings suggest that DCC:UNC5H ratios in DA neurons at critical periods may have important consequences for the organization and function of mesocorticolimbic DA systems.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neuroendocrine circuits are orchestrated by the pituitary gland in response to hypothalamic hormone-releasing and inhibiting factors to generate an ultradian and/or circadian rhythm of hormone ...secretion. However, mechanisms that govern this rhythmicity are not fully understood. It has been shown that synaptic transmission in the rodent hypothalamus undergoes cyclical changes in parallel with rhythmic hormone secretion and a growing body of evidence suggests that rapid rewiring of hypothalamic neurons may be the source of these changes. For decades, structural synaptic studies have been utilizing electron microscopy, which provides the resolution suitable for visualizing synapses. However, the small field of view, limited specificity and manual analysis susceptible to bias fuel the search for a more quantitative approach. Here, we apply the fluorescence super-resolution microscopy approach
Stochastic Optical Reconstruction Microscopy (
STORM) to quantify and structurally characterize excitatory and inhibitory synapses that contact growth hormone-releasing-hormone (GHRH) neurons during peak and trough values of growth hormone (GH) concentration in mice. This approach relies on a three-color immunofluorescence staining of GHRH and pre- and post-synaptic markers, and a quantitative analysis with a Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm. With this method we confirm our previous findings, using electron microscopy, of increased excitatory synaptic input to GHRH neurons during peak levels of GH. Additionally, we find a shift in synapse numbers during low GH levels, where more inhibitory synaptic inputs are detected. Lastly, we utilize
STORM to study novel aspects of synaptic structure. We show that more excitatory (but not inhibitory) pre-synaptic clusters associate with excitatory post-synaptic clusters during peaks of GH secretion and that the numbers of post-synaptic clusters increase during high hormone levels. The results presented here provide an opportunity to highlight
STORM as a valuable quantitative approach to study synaptic structure in the neuroendocrine circuit. Importantly, our analysis of GH circuitry sheds light on the potential mechanism that drives ultradian changes in synaptic transmission and possibly aids in GH pulse generation in mice.
Scy1-like 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, was recently identified as the gene product altered in muscle-deficient mice, which suffer from motor ...neuron degeneration and cerebellar atrophy. To determine the function of Scyl1, we have now used a mass spectrometry-based screen to search for Scyl1-binding partners and identified components of coatomer I (COPI) coats. The interaction was confirmed in pull-down assays, and Scyl1 co-immunoprecipitates with βCOP from brain lysates. Interestingly, and unique for a non-transmembrane domain protein, Scyl1 binds COPI coats using a C-terminal RKLD-COO- sequence, similar to the KKXX-COO- COPI-binding motif found in transmembrane endoplasmic reticulum (ER) proteins. Scyl1 co-localizes with βCOP and is localized, in an Arf1-independent manner, to the ER-Golgi intermediate compartment and the cis-Golgi, sites of COPI-mediated membrane budding. The localization and binding properties of Scyl1 strongly suggest a function in COPI transport, and inhibitory RNA-mediated knock down of the protein disrupts COPI-mediated retrograde traffic of the KDEL receptor to the ER without affecting anterograde traffic from the ER. Our data demonstrate a function for Scyl1 as an accessory factor in COPI trafficking and suggest for the first time that alterations in the COPI pathway result in neurodegenerative disease.
The netrin-1 receptor Deleted in Colorectal Cancer (DCC) is required for the formation of major axonal projections by embryonic cortical neurons, including the corpus callosum, hippocampal ...commissure, and cortico-thalamic tracts. The presentation of DCC by axonal growth cones is tightly regulated, but the mechanisms regulating DCC trafficking within neurons are not well understood. Here, we investigated the mechanisms regulating DCC recruitment to the plasma membrane of embryonic cortical neurons. In embryonic spinal commissural neurons, protein kinase A (PKA) activation recruits DCC to the plasma membrane and enhances axon chemoattraction to netrin-1. We demonstrate that PKA activation similarly recruits DCC and increases embryonic cortical neuron axon extension, which, like spinal commissural neurons, respond to netrin-1 as a chemoattractant. We then determined if depolarization might recruit DCC to the plasma membrane. Neither netrin-1 induced axon extension, nor levels of plasma membrane DCC, were altered by depolarizing embryonic spinal commissural neurons with elevated levels of KCl. In contrast, depolarizing embryonic cortical neurons increased the amount of plasma membrane DCC, including at the growth cone, and increased axon outgrowth evoked by netrin-1. Inhibition of PKA, phosphatidylinositol-3-kinase, protein kinase C, or exocytosis blocked the depolarization-induced recruitment of DCC and suppressed axon outgrowth. Inhibiting protein synthesis did not affect DCC recruitment, nor were the distributions of trkB or neural cell adhesion molecule (NCAM) influenced by depolarization, consistent with selective mobilization of DCC. These findings identify a role for membrane depolarization modulating the response of axons to netrin-1 by regulating DCC recruitment to the plasma membrane.
By yeast two-hybrid screening we have identified interaction partners for the intracellular C-terminal tail of the human and rodent somatostatin receptor subtype 5 (SSTR5). Interactions with the PDZ ...domain-containing proteins PIST and PDZK1 are mediated by the PDZ ligand motif at the C terminus of the receptor; in case of the human and mouse (but not the rat) receptors, a slight sequence variation of this motif also allows for binding of the peroxisomal receptor PEX5. PIST is Golgi-associated and retains SSTR5 in the Golgi apparatus when coexpressed with the receptor; PDZK1 on the other hand associates with the SSTR5 at the plasma membrane. Endogenous SSTR5 in the neuroendocrine AtT-20 tumor cell line is colocalized with PIST in the Golgi apparatus. On a functional level, removal of the PDZ ligand motif of the receptor does not interfere with agonist-dependent internalization of the receptor or its targeting to a Golgi-associated compartment; however, recycling of the receptor to the plasma membrane after washout of the agonist is inhibited, suggesting that the PDZ-mediated interaction of SSTR5 is required for postendocytic sorting.
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