In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Outside-in regulation of the readily releasable pool of synaptic vesicles by α2δ-1.

The readily releasable pool (RRP) of synaptic vesicles is a key determinant of phasic neurotransmission. Although the size of the RRP is tightly regulated by intracellular factors, there is little evidence for its modification by extracellular signals. By studying the homogeneous population of synapses present in autaptic microcultures, we show that pregabalin, a prototypical gabapentinoid, decreases the effective RRP size. Simultaneous imaging of presynaptic calcium influx and recording of postsynaptic responses shows that the effect is not related to a reduction of calcium entry. The main cause is the impairment of the functional coupling among N-type calcium channels and the RRP, resembling an increase of intracellular mobile calcium buffers. The ectodomain of neurexin-1α shows a similar action to pregabalin, acting as an endogenous ligand of α2δ-1 that reduces the RRP size without affecting presynaptic calcium influx. The regulatory actions described for pregabalin and the ectodomain of neurexin-1α are mutually exclusive. The overexpression of α2δ-1 enhances the effect of pregabalin and the ectodomain of neurexin-1α on neurotransmission by decreasing their effective concentration. In contrast, knockdown of α2δ-1 causes a profound inhibition of synaptic transmission. These observations prompt to consider α2δ-1 as an outside-in signaling platform that binds exogenous and endogenous cues for regulating the coupling of voltage-gated calcium channels to synaptic vesicles.

SPARC triggers a cell-autonomous program of synapse elimination.

Elimination of the excess synaptic contacts established in the early stages of neuronal development is required to refine the function of neuronal circuits. Here we investigate whether secreted protein acidic and rich in cysteine (SPARC), a molecule produced by glial cells, is involved in synapse removal. SPARC production peaks when innervation of the rat superior cervical ganglion and the tail of Xenopus tropicalis tadpoles are remodeled. The formation of new cholinergic synapses in autaptic single-cell microcultures is inhibited by SPARC. The effect resides in the C-terminal domain, which is also responsible for triggering a concentration- and time-dependent disassembly of stable cholinergic synapses. The loss of synaptic contacts is associated with the formation of retracted axon terminals containing multivesicular bodies and secondary lysosomes. The biological relevance of in vitro results was supported by injecting the tail of Xenopus tropicalis tadpoles with peptide 4.2, a 20-aa sequence derived from SPARC that mimics full-length protein effects. Swimming was severely impaired at ∼5 h after peptide application, caused by the massive elimination of neuromuscular junctions and pruning of axonal branches. Effects revert by 6 d after injection, as motor innervation reforms. In conclusion, SPARC triggers a cell-autonomous program of synapse elimination in cholinergic neurons that likely occurs when protein production peaks during normal development.

The epsilon toxin from Clostridium perfringens stimulates calcium-activated chloride channels, generating extracellular vesicles in Xenopus oocytes.

The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.

Multivesicular release favors short term synaptic depression in hippocampal autapses.

Presynaptic terminals of the central nervous system can support univesicular and multivesicular synchronous release of neurotransmitters, however, the functional implications of the prevalence of one mechanism over the other are yet unresolved. Here, we took advantage of the expression of SF-iGluSnFR.S72A in the astrocytic feeder layer of autaptic hippocampal neuronal cultures to associate the liberation of glutamate to excitatory postsynaptic currents. The presence of the glutamate sensor in glial cells avoided any interference with the function of endogenous postsynaptic receptors. It was possible to optically detect changes in neurotransmitter release probability, which was heterogeneous among synaptic boutons studied. For each neuron investigated, the liberation of neurotransmitters occurred through a predominant mechanism. The prevalence of multivesicular over univesicular release increased synaptic strength and enhanced short-term synaptic depression. These results show that the preference of hippocampal boutons to synchronously release one or more vesicles determines the strength and low pass filtering properties of the synapses established.

Axon terminals control endolysosome diffusion to support synaptic remodelling.

Endolysosomes are acidic organelles formed by the fusion of endosomes with lysosomes. In the presynaptic compartment they contribute to protein homeostasis, the maintenance of vesicle pools and synaptic stability. Here, we evaluated the mobility of endolysosomes found in axon terminals of olfactory sensory neurons of Xenopus tropicalis tadpoles. F-actin restricts the motion of these presynaptic acidic organelles which is characterized by a diffusion coefficient of 6.7 × 10-3 µm2·s-1 Local injection of secreted protein acidic and rich in cysteine (SPARC) in the glomerular layer of the olfactory bulb disrupts the structure of synaptic F-actin patches and increases the presence and mobility of endolysosomal organelles found in axon terminals. The increased motion of endolysosomes is localized to the presynaptic compartment and does not promote their access to axonal regions for retrograde transportation to the cell body. Local activation of synaptic degradation mechanisms mediated by SPARC coincides with a loss of the ability of tadpoles to detect waterborne odorants. Together, these observations show that the diffusion of presynaptic endolysosomes increases during conditions of synaptic remodelling to support their local degradative activity.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles.

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Role of neuron-glia interactions in developmental synapse elimination.

During the embryonic development of the nervous system there is a massive formation of synapses. However, the exuberant connectivity present after birth must be pruned during postnatal growth to optimize the function of neuronal circuits. Whilst glial cells play a fundamental role in the formation of early synaptic contacts, their contribution to developmental modifications of established synapses is not well understood. The present review aims to highlight the various roles of glia in the developmental refinement of embryonic synaptic connectivity. We summarize recent evidences linking secretory abilities of glial cells to the disassembly of synaptic contacts that are complementary of a well-established phagocytic role. Considering a theoretical framework, it is discussed how release of glial molecules could be relevant to the developmental refinement of synaptic connectivity. Finally, we propose a three-stage model of synapse elimination in which neurons and glia are functionally associated to timely eliminate synapses.

Tight temporal coupling between synaptic rewiring of olfactory glomeruli and the emergence of odor-guided behavior in Xenopus tadpoles.

Olfactory sensory neurons (OSNs) are chemoreceptors that establish excitatory synapses within glomeruli of the olfactory bulb. OSNs undergo continuous turnover throughout life, causing the constant replacement of their synaptic contacts. Using Xenopus tadpoles as an experimental system to investigate rewiring of glomerular connectivity, we show that novel OSN synapses can transfer information immediately after formation, mediating olfactory-guided behavior. Tadpoles recover the ability to detect amino acids 4 days after bilateral olfactory nerve transection. Restoration of olfactory-guided behavior depends on the efficient reinsertion of OSNs to the olfactory bulb. Presynaptic terminals of incipient synaptic contacts generate calcium transients in response to odors, triggering long lasting depolarization of olfactory glomeruli. The functionality of reconnected terminals relies on well-defined readily releasable and cytoplasmic vesicle pools. The continuous growth of non-compartmentalized axonal processes provides a vesicle reservoir to nascent release sites, which contrasts to the gradual development of cytoplasmic vesicle pools in conventional excitatory synapses. The immediate availability of fully functional synapses upon formation supports an age-independent contribution of OSNs to the generation of odor maps.

Acute Effect of Pore-Forming Clostridium perfringens ε-Toxin on Compound Action Potentials of Optic Nerve of Mouse.

ε-Toxin is a pore forming toxin produced by Clostridium perfringens types B and D. It is synthesized as a less active prototoxin form that becomes fully active upon proteolytic activation. The toxin produces highly lethal enterotoxaemia in ruminants, has the ability to cross the blood-brain barrier (BBB) and specifically binds to myelinated fibers. We discovered that the toxin induced a release of ATP from isolated mice optic nerves, which are composed of myelinated fibers that are extended from the central nervous system. We also investigated the effect of the toxin on compound action potentials (CAPs) in isolated mice optic nerves. When nerves were stimulated at 100 Hz during 200 ms, the decrease of the amplitude and the area of the CAPs was attenuated in the presence of ε-toxin. The computational modelling of myelinated fibers of mouse optic nerve revealed that the experimental results can be mimicked by an increase of the conductance of myelin and agrees with the pore forming activity of the toxin which binds to myelin and could drill it by making pores. The intimate ultrastructure of myelin was not modified during the periods of time investigated. In summary, the acute action of the toxin produces a subtle functional impact on the propagation of the nerve action potential in myelinated fibers of the central nervous system with an eventual desynchronization of the information. These results may agree with the hypothesis that the toxin could be an environmental trigger of multiple sclerosis (MS).

Abnormal Expression and Distribution of MMP2 at Initial Stages of Alzheimer's Disease-Related Pathology.

Previous studies have shown that metalloproteinases (MMPs) participate in the clearance of amyloid-ß (Aß) in Alzheimer's disease (AD); MMP2 and MMP3 cleave soluble Aß, and both MMP9 and MT1-MMP are able to degrade soluble and fibrillar forms of Aß. The present study shows increased expression levels of active MMP2 in the entorhinal cortex at early stages of AD-related pathology (Braak and Braak stages I/II-0 and III/IV-A) as revealed by western blotting and gelatin zymography. Confocal microscopy discloses co-localization of MMP2 and phospho-tau in neurofibrillary tangles and dystrophic neurites. MMP2 has the capacity to cleave recombinant tau in vitro in a dose-dependent manner, consistent with a physiological function of MMP2 in normal tau proteolysis. However, MMP2 does not cleave hyperphosphorylated and dephosphorylated tau from enriched paired helical filament fractions. These observations raise the possibility that accumulation of MMP2 in neurofibrillary tangles and concomitant loss of proteolytic capacity on tau protein is a response geared to eliminating production of toxic truncated tau species in AD brains.

Mitochondrial ATP-synthase in the entorhinal cortex is a target of oxidative stress at stages I/II of Alzheimer's disease pathology.

Oxidative stress has been implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD). Several proteins have been identified as targets of oxidative damage in AD dementia (usually stages V/VI of Braak) and in subjects with mild cognitive impairment associated with middle stages of AD pathology (stage IV of Braak). In this study, we investigate whether brain proteins are locally modified by oxidative stress at the first stages of AD-related pathology when morphological lesions are restricted to the entorhinal and transentorhinal cortices of neurofibrillary pathology (stages I/II of Braak). Using a proteomic approach, we show that the alpha subunit of the mitochondrial adenosine triphosphate (ATP)-synthase is distinctly lipoxidized in the entorhinal cortex at Braak stages I/II compared with age-matched controls. In addition, ATP-synthase activity is significantly lower in Braak stages I/II than age-matched control, while electron transport chain, expressed by the mitochondrial complex I activity, remains not affected. This is the first study showing oxidative damage in the first stage, and clinically silent period, of AD-related pathology characterized by entorhinal and transentorhinal tauopathy.

Frontotemporal dementia with tau pathology.

Tau is a microtubule-associated protein involved in microtubule assembly and stabilization. Filamentous deposits made of tau constitute a major defining characteristic of several neurodegenerative diseases known as tauopathies including Alzheimer's disease. The involvement of tau in neurodegeneration has been clarified by the identification of genetic mutations in the tau gene in cases with familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Although the mechanism through which tau mutations lead to neuronal death is still unresolved, it is clear that tau mutations lead to formation of tau filaments that have a different morphology, contain different types of tau isoforms and produce distinct tau deposits. The range of tau pathology identified in FTDP-17 recapitulates the tau pathology present in sporadic tauopathies and indicates that tau dysfunction plays a major role also in these diseases.

Mutant ubiquitin and p62 immunoreactivity in cases of combined multiple system atrophy and Alzheimer's disease.

Recent studies have shown the co-existence of alpha-synuclein and phosphorylated tau (pTau) in several neurodegenerative diseases. Here, we report two autopsy cases of combined multiple system atrophy (MSA) and Alzheimer's disease (AD). In both cases, abundant alpha-synuclein-positive glial and neuronal cytoplasmic inclusions were found in the brainstem, amygdala and hippocampal formation. pTau-positive neurofibrillary tangles (NFTs) were widely distributed in case 1 (Braak stage VI) and moderate in case 2 (Braak stage III). Although alpha-synuclein and pTau pathology co-occurred in the hippocampus and entorhinal cortex, only a few neurons showed co-existence of these two proteins. Immunoreactivity for p62, a ubiquitin proteasome system related protein, was found in the majority of NFTs, but in only a small proportion of neuronal alpha-synuclein inclusions. In addition, UBB+1, a mutant form of ubiquitin and a marker for proteasomal dysfunction, was present in the majority of NFTs, whereas co-existence of alpha-synuclein and UBB+1 was found in only a few neurons. These findings indicate that alpha-synuclein and phosphorylated tau co-occur in certain brain regions in cases of combined MSA and AD and that the proteasomal pathways differ between alpha-synuclein- and pTau-bearing neurons.

Conformational variability of matrix metalloproteinases: beyond a single 3D structure.

The structures of the catalytic domain of matrix metalloproteinase 12 in the presence of acetohydroxamic acid and N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid have been solved by x-ray diffraction in the crystalline state at 1.0 and 1.3-A resolution, respectively, and compared with the previously published x-ray structure at 1.2-A resolution of the adduct with batimastat. The structure of the N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid adduct has been solved by NMR in solution. The three x-ray structures and the solution structure are similar but not identical to one another, the differences being sizably higher in the loops. We propose that many of the loops show a dynamical behavior in solution on a variety of time scales. Different conformations of some flexible regions of the protein can be observed as "frozen" in different crystalline environments. The mobility in solution studied by NMR reveals conformational equilibria in accessible time scales, i.e., from 10(-5) s to ms and more. Averaging of some residual dipolar couplings is consistent with further motions down to 10(-9) s. Finally, local thermal motions of each frozen conformation in the crystalline state at 100 K correlate well with local motions on the picosecond time scale. Flexibility/conformational heterogeneity in crucial parts of the catalytic domain is a rule rather than an exception in matrix metalloproteinases, and its extent may be underestimated by inspection of one x-ray structure. Backbone flexibility may play a role in the difficulties encountered in the design of selective inhibitors, whereas it may be a requisite for substrate binding and broad substrate specificity.