Plectin dysfunction in neurons leads to tau accumulation on microtubules affecting neuritogenesis, organelle trafficking, pain sensitivity and memory.

AIMS: Plectin, a universally expressed multi-functional cytolinker protein, is crucial for intermediate filament networking, including crosstalk with actomyosin and microtubules. In addition to its involvement in a number of diseases affecting skin, skeletal muscle, heart, and other stress-exposed tissues, indications for a neuropathological role of plectin have emerged. Having identified P1c as the major isoform expressed in neural tissues in previous studies, our aim for the present work was to investigate whether, and by which mechanism(s), the targeted deletion of this isoform affects neuritogenesis and proper nerve cell functioning. METHODS: For ex vivo phenotyping, we used dorsal root ganglion and hippocampal neurons derived from isoform P1c-deficient and plectin-null mice, complemented by in vitro experiments using purified proteins and cell fractions. To assess the physiological significance of the phenotypic alterations observed in P1c-deficient neurons, P1c-deficient and wild-type littermate mice were subjected to standard behavioural tests. RESULTS: We demonstrate that P1c affects axonal microtubule dynamics by isoform-specific interaction with tubulin. P1c deficiency in neurons leads to altered dynamics of microtubules and excessive association with tau protein, affecting neuritogenesis, neurite branching, growth cone morphology, and translocation and directionality of movement of vesicles and mitochondria. On the organismal level, we found P1c deficiency manifesting as impaired pain sensitivity, diminished learning capabilities and reduced long-term memory of mice. CONCLUSIONS: Revealing a regulatory role of plectin scaffolds in microtubule-dependent nerve cell functions, our results have potential implications for cytoskeleton-related neuropathies.

Astroglial vesicular network: evolutionary trends, physiology and pathophysiology.

Intracellular organelles, including secretory vesicles, emerged when eukaryotic cells evolved some 3 billion years ago. The primordial organelles that evolved in Archaea were similar to endolysosomes, which developed, arguably, for specific metabolic tasks, including uptake, metabolic processing, storage and disposal of molecules. In comparison with prokaryotes, cell volume of eukaryotes increased by several orders of magnitude and vesicle traffic emerged to allow for communication between distant intracellular locations. Lysosomes, first described in 1955, a prominent intermediate of endo- and exocytotic pathways, operate virtually in all eukaryotic cells including astroglia, the most heterogeneous type of homeostatic glia in the central nervous system. Astrocytes support neuronal network activity in particular through elaborated secretion, based on a complex intracellular vesicle network dynamics. Deranged homeostasis underlies disease and astroglial vesicle traffic contributes to the pathophysiology of neurodegenerative (Alzheimer's disease, Huntington's disease), neurodevelopmental diseases (intellectual deficiency, Rett's disease) and neuroinfectious (Zika virus) disorders. This review addresses astroglial cell-autonomous vesicular traffic network, as well as its into primary and secondary vesicular network defects in diseases, and considers this network as a target for developing new therapies for neurological conditions.

Presenilin PS1∆E9 disrupts mobility of secretory organelles in rat astrocytes.

AIM: Alzheimer's disease (AD) is largely considered a neuron-derived insult, but also involves failure of astroglia. A recent study indicated that mutated presenilin 1 (PS1M146V), a putative endoplasmic reticulum (ER) Ca2+ channel with decreased Ca2+ conductance, impairs the traffic of astroglial peptidergic vesicles. Whether other pathogenically relevant PS1 mutants, such as PS1ΔE9, which code for ER channel with putative increased Ca2+ conductance, similarly affect vesicle traffic, is unknown. METHODS: Here, we cotransfected rat astrocytes with plasmids encoding mutant PS1ΔE9 and atrial natriuretic peptide or vesicular glutamate transporter 1 tagged with fluorescent proteins (pANP.emd or pVGLUT1-EGFP respectively), to microscopically examine whether alterations in vesicle mobility and Ca2+ -regulated release of gliosignalling molecules manifest as a general vesicle-based defect; control cells were transfected to co-express exogenous or native wild-type PS1 and pANP.emd or pVGLUT1-EGFP. The vesicle mobility was analysed at rest and after ATP stimulation that increased intracellular calcium activity. RESULTS: In PS1ΔE9 astrocytes, spontaneous mobility of both vesicle types was reduced (P < .001) when compared to controls. Post-stimulatory recovery of fast vesicle mobility was hampered in PS1ΔE9 astrocytes. The ATP-evoked peptide release was less efficient in PS1ΔE9 astrocytes than in the controls (P < .05), as was the pre-stimulatory mobility of these vesicles. CONCLUSION: Although the PS1 mutants PS1M146V and PS1ΔE9 differently affect ER Ca2+ conductance, our results revealed a common, vesicle-type indiscriminate trafficking defect in PS1ΔE9 astrocytes, indicating that reduced secretory vesicle-based signalling is a general deficit in AD astrocytes.

Rab3 proteins: key players in the control of exocytosis.

Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic.

Astrocytic vesicles and gliotransmitters: Slowness of vesicular release and synaptobrevin2-laden vesicle nanoarchitecture.

Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent merger of the vesicle membrane with the plasmalemma. Up to 25 molecules of synaptobrevin 2 (Sb2), a SNARE complex protein, reside at a single astroglial vesicle; an individual neuronal, i.e. synaptic, vesicle contains ∼70 Sb2 molecules. It is proposed that this paucity of Sb2 molecules in astrocytic vesicles may determine the slow secretion. In the present essay we shall overview multiple aspects of vesicular architecture and types of vesicles based on their cargo and dynamics in astroglial cells.

Astrocytes in physiological aging and Alzheimer's disease.

Astrocytes are fundamental for homoeostasis, defence and regeneration of the central nervous system. Loss of astroglial function and astroglial reactivity contributes to the aging of the brain and to neurodegenerative diseases. Changes in astroglia in aging and neurodegeneration are highly heterogeneous and region-specific. In animal models of Alzheimer's disease (AD) astrocytes undergo degeneration and atrophy at the early stages of pathological progression, which possibly may alter the homeostatic reserve of the brain and contribute to early cognitive deficits. At later stages of AD reactive astrocytes are associated with neurite plaques, the feature commonly found in animal models and in human diseased tissue. In animal models of the AD reactive astrogliosis develops in some (e.g. in the hippocampus) but not in all regions of the brain. For instance, in entorhinal and prefrontal cortices astrocytes do not mount gliotic response to emerging ß-amyloid deposits. These deficits in reactivity coincide with higher vulnerability of these regions to AD-type pathology. Astroglial morphology and function can be regulated through environmental stimulation and/or medication suggesting that astrocytes can be regarded as a target for therapies aimed at the prevention and cure of neurodegenerative disorders.

FM1-43 measurements of local exocytotic events in rat melanotrophs.

We have explored the existence of fusion- and secretion-competent sites on the plasma membrane of peptide secreting rat pituitary melanotrophs at rest, and following stimulation with glutamate. We monitored changes in fluorescence of FM1-43, a styryl dye which labels plasma membrane. The results show spontaneous local increases in FM1-43 reporting changes in membrane surface area due to cumulative exocytosis. Addition of glutamate, further increased the occurrence of these events. Statistical analysis of local FM1-43 fluorescence changes suggests that this is due to the recruitment of inactive exocytotic domains and due to the stimulation of already active exocytotic domains.

Increased cytosolic calcium stimulates exocytosis in bovine lactotrophs. Direct evidence from changes in membrane capacitance.

The patch-clamp technique has been used to measure changes in membrane capacitance (Cm) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo- and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2(+)-containing solutions. The resting Cm of 6.05 +/- 1.68 pF was found to correlate well with squared cell radius, suggesting a specific Cm of 0.8 microF/cm2. Discrete Cm steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 microns in diameter). High Ca2+ resulted in a Cm increase of 20-50% from the resting value, demonstrating a role for [Ca2+]i in stimulus-secretion coupling. Spontaneous Cm changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca2+ through the membrane. This is supported by the following findings: addition of Co2+ diminished or reversed the spontaneous Cm changes and decreased resting [Ca2+]i; and membrane depolarization increased Cm, indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by Cm.

Calcium signaling and secretion in pituitary cells.

An important trigger of hormone secretion from pituitary cells is a rise in cytosolic Ca(2+) ([Ca(2+)](i)). Pituitary cells may modulate [Ca(2+)](i) by an increased membrane flux from the extracellular space and/or by a release from intracellular stores. Both mechanisms can support exocytosis, although in different pituitary cell types one or the other mechanism may predominate. Molecular events transducing a rise in [Ca(2+)](i) into hormone secretion are still poorly understood. Here, the exocytotic machinery in pituitary cells is briefly reviewed in terms of the spatial organization of [Ca(2+)](i) elevation relative to the Ca(2+) sensor(s).

Fura-2 imaging of thyrotropin-releasing hormone and dopamine effects on calcium homeostasis of bovine lactotrophs.

Dual wavelength digital imaging microscopy to detect fura-2 has been employed to characterize in normal bovine PRL-secreting cells the effects of TRH and dopamine on the intracellular ionized calcium concentration [( Ca2+]i). Concentrations of TRH greater than 10 nM caused a rapid but transient increase in [Ca2+]i, arising mainly from intracellular calcium stores, since it was unaffected by lowering extracellular calcium with EGTA or blocking calcium channels with Co2+. The threshold for TRH action was close to 0.1 nM. TRH action was dose dependent, with lower concentrations (less than 1-10 nM) slowing the time to peak [Ca2+]i response. The TRH-induced [Ca2+]i rise had a Q10 of about 2. TRH caused multiple transient increases in [Ca2+]i, but a recovery time of 10-15 min was required for full restoration of the TRH-induced response. In some cells the [Ca2+]i response to TRH was polarized to one region of the cell, suggesting the following possibilities, none of them exclusive: 1) Ca2+ release sites may be localized within the cell; or 2) an efficient local mechanism exists for lowering Ca2+ once it is liberated inside the cells; or 3) barriers may exist to diffusion of Ca2+ released within the cell. Extracellular application of Co2+, Mn2+, and EGTA under basal conditions resulted in lowering of [Ca2+]i within seconds, consistent with tonic Ca2+ influx under resting conditions which could contribute to the basal release of hormone. Dopamine, a PRL release-inhibiting factor, also lowered [Ca2+]i under basal conditions. However, the [Ca2+]i response of lactotrophs to TRH was unaffected by dopamine. This suggests that dopamine and TRH act via separate intracellular pathways to modulate hormone secretion. Applications of forskolin preceding the TRH-induced transient rise in [Ca2+]i resulted in a prolonged plateau rise in [Ca2+]i. This was mainly due to increased influx of Ca2+ since addition of Co2+ or EGTA-containing or Ca(2+)-free medium during this phase of response lowered the plateau concentration of [Ca2+]i.

Cytosolic chloride ions stimulate Ca(2+)-induced exocytosis in melanotrophs.

We used the whole-cell patch-clamp technique to study the secretory activity of single cells by monitoring changes in membrane capacitance [Neher, E. and Marty, A. (1982) Proc. Natl. Acad. Sci. USA 79, 523-535] in anterior pituitary cells. Unexpectedly we have observed that increasing intracellular chloride ions stimulate Ca(2+)-induced exocytosis in a dose-dependent fashion (Kd = 12 mM). These results demonstrate a role of cytosolic chloride ions in the regulation of exocytotic secretion in anterior pituitary cells. It is suggest that chloride channels, in addition to playing a part in regulating membrane electrical activity [Korn, S.J., Bolden, A. and Horn, A. (1991) J. Physiol. 439, 423-437; Penner, R., Matthews, G. and Horn, A. (1988) Nature 334, 499-504] and cytosolic pH [Kaila, K. and Voipio, J. (1987) Nature 330, 163-165], are also involved in the modulation of cytosolic chloride concentration and thus in the control of exocytosis.

Rapid pressure driven exocytosis-endocytosis cycle in a single plant cell. Capacitance measurements in aleurone protoplasts.

The whole-cell patch-clamp technique has been used to increase intracellular pressure via the pipette while monitoring changes in membrane capacitance (related to membrane area) in single aleurone protoplasts. Increased pressure increased membrane capacitance and diameter, upon the release of pressure both parameters returned to resting levels. Pressure also reversibly increased membrane conductance. Comparison between diameter and capacitance shows that the capacitance changes are due to changes in cell surface area. These results show that pressure can rapidly alter the balance between exo- and endocytosis. Pressure-induced changes in conductance are suggested to be due to insertion/withdrawal of channels with the exo-/endocytosed membrane.

cAMP directly facilitates Ca-induced exocytosis in bovine lactotrophs.

We have used the whole cell patch clamp technique on single prolactin-secreting bovine lactotrophs to measure plasma membrane capacitance (Cm), an index of membrane surface area, under voltage-clamp during cytosol dialysis with Ca and cAMP. cAMP increased the magnitude and rate of Ca-induced exocytosis (Cm increase) without affecting membrane conductance; however, cAMP had no detectable effect on Cm when intracellular Ca was low. We thus report new evidence that cAMP can facilitate Ca-induced secretion in a synergistic fashion, by acting directly on the secretory apparatus, independently of membrane conductance activation.

Mastoparan and Rab3AL peptide potentiation of calcium-independent secretory activity in rat melanotrophs is inhibited by GDPbetaS.

The whole-cell patch-clamp membrane capacitance measurement was used to monitor secretory activity in rat melanotrophs, while rab3AL, putative effector domain peptides of Rab3 small GTPases (20-30 kDa), were introduced into cytosol. In melanotrophs dialyzed with calcium free solutions membrane capacitance tends to decrease slightly. This decrease is further potentiated with GDPbetaS (500 microM). We found that rab3AL (100 microM) stimulated secretory activity in the absence of calcium. The rab3AL response was qualitatively comparable to the response to mastoparan (1 microM), an activator of certain heterotrimeric GTP-binding proteins. Interestingly, inclusion of GDPbetaS (500 microM) resulted in a blockade of both rab3AL and mastoparan induced responses. We conclude that rab3AL and mastoparan induce calcium-independent stimulation of secretory activity in rat melanotrophs by activation of a downstream heterotrimeric GTP-binding protein.

Dual effects of G-protein activation on Ca-dependent exocytosis in bovine lactotrophs.

The whole-cell patch-clamp technique was used to measure cell membrane capacitance (Cm) to monitor exocytosis in single-cultured bovine prolactin-secreting cells (lactotrophs) of the anterior pituitary. The cells were dialyzed with solutions containing different concentrations of ionised Ca and non-hydrolyzable GTP analogues (GTP-gamma-S and GMP-PNP) to activate G-proteins. We have identified two distinct effects of G-protein activation on Ca-induced exocytosis: (i) the maximum Cm increase due to intracellular Ca-dependent exocytosis was diminished, suggesting an inhibitory role of G-proteins close to the site of granule fusion, while (ii) the rate of Cm increase (delta Cm/delta t) was facilitated, revealing conversely a stimulatory role of G-proteins in the translocation of secretory granules to the fusion sites.

Apoptosis triggered redistribution of caspase-9 from cytoplasm to mitochondria.

Caspase-9 is an apoptosis initiator protease activated as a response to the mitochondrial damage in the cytoplasmic complex apoptosome. By fluorescence labelling of proteins, confocal microscopy and subcellular fractionations we demonstrate that caspase-9 is in the cytoplasm of non-apoptotic pituitary cells. The activation of apoptosis with rotenone triggers the redistribution of caspase-9 to mitochondria. Experiments using the general caspase inhibitor z-VAD.fmk and the specific caspase-9 inhibitor z-LEHD.fmk show that the caspase-9 redistribution is a regulated process and requires the activity of a caspase other than the caspase-9. We propose that this spatial regulation is required to control the activity of caspase-9.

Dopaminergic regulation of synaptotagmin I and IV mRNAs in hemiparkinsonian rats.

Synaptotagmins (Syts) I and IV are synaptic proteins involved in the regulation of neurosecretion. Dopaminergic drugs have been shown to modulate their expression. Here we investigate whether dopaminergic regulation of syt I and syt IV expression could play a role in the hypersensitive striatum of rats with unilateral lesions of dopaminergic nigrostriatal neurons with 6-hydroxydopamine. We show that chronic dopaminergic denervation resulted in a small down-regulation of striatal syt I mRNA, whereas acute treatment with SKF-82958, a dopamine D1 receptor agonist, induced a massive syt IV mRNA upregulation in the striatum on the lesioned side. We conclude that chronic lack of dopamine and treatment with dopamine D1 receptor agonists alter the synaptic plasticity in dopamine depleted basal ganglia.

Brefeldin A and a synthetic peptide to ADP-ribosylation factor (ARF) inhibit regulated exocytosis in melanotrophs.

We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.

The voltage-clamp apparatus assisted by a 'current pump'.

A current pump is described, which is attached to the last stage of the 2-microelectrode voltage-clamp apparatus. Thus a voltage-to-current conversion independent of the impedance of the current microelectrode is obtained. In this way it is possible to pass relatively large currents through the current microelectrode and in the same time to have a stable negative feed-back system.

Nicardipine enantiomers inhibit calcium and outward currents in rat pars intermedia cells.

The whole-cell patch clamp technique was used to investigate the effect of the dihydropyridine antagonist nicardipine and its two enantiomers on calcium and outward currents in rat pars intermedia cells. Inward voltage-activated calcium currents as well as outward currents were blocked by nicardipines (enantiomers and racemic substance). We found a highly significant correlation between the blockage of voltage-activated currents and outward currents, indicating an association between recorded currents. Therefore, the inhibitory effect of nicardipines was measured as outward current inhibition. The effects of (-) and (+) enantiomers as well as the racemic nicardipine were concentration-dependent. The (+)-enantiomer was the least potent with an ED50 of 3.8 +/- 1.2 microM, while the (-)-enantiomer inhibited currents with an ED50 of 0.7 +/- 0.1 microM. Racemic nicardipine blocked currents with an ED50 of 2.2 +/- 0.6 microM.