Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly.

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.

In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) characterized by loss of myelin accompanied by infiltration of T-lymphocytes and monocytes. Although it has been shown that these infiltrates are important for the progression of MS, the role of microglia, the resident macrophages of the CNS, remains ambiguous. Therefore, we have compared the phenotypes of microglia and macrophages in a mouse model for MS, experimental autoimmune encephalomyelitis (EAE). In order to properly discriminate between these two cell types, microglia were defined as CD11b(pos) CD45(int) Ly-6C(neg) , and infiltrated macrophages as CD11b(pos) CD45(high) Ly-6C(pos) . During clinical EAE, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecules (CD80, CD86, and CD40) and proinflammatory genes [interleukin-1ß (IL-1ß) and tumour necrosis factor- α (TNF-α)]. In contrast, CD11b(pos) CD45(high) Ly-6C(pos) infiltrated macrophages were strongly activated and could be divided into two populations Ly-6C(int) and Ly-6C(high) , respectively. Ly-6C(high) macrophages contained less myelin than Ly-6C(int) macrophages and expression levels of the proinflammatory cytokines IL-1ß and TNF-α were higher in Ly-6C(int) macrophages. Together, our data show that during clinical EAE, microglia are only weakly activated whereas infiltrated macrophages are highly immune reactive.

Pathophysiogenesis of mesial temporal lobe epilepsy: is prevention of damage antiepileptogenic?

Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused by a primary brain injury that occurred a long time before the appearance of neurological symptoms. This type of epilepsy is characterized by refractoriness to drug treatment, so to require surgical resection of mesial temporal regions involved in seizure onset. Even this last therapeutic approach may fail in giving relief to patients. Although prevention of hippocampal damage and epileptogenesis after a primary event could be a key innovative approach to TLE, the lack of clear data on the pathophysiological mechanisms leading to TLE does not allow any rational therapy. Here we address the current knowledge on mechanisms supposed to be involved in epileptogenesis, as well as on the possible innovative treatments that may lead to a preventive approach. Besides loss of principal neurons and of specific interneurons, network rearrangement caused by axonal sprouting and neurogenesis are well known phenomena that are integrated by changes in receptor and channel functioning and modifications in other cellular components. In particular, a growing body of evidence from the study of animal models suggests that disruption of vascular and astrocytic components of the blood-brain barrier takes place in injured brain regions such as the hippocampus and piriform cortex. These events may be counteracted by drugs able to prevent damage to the vascular component, as in the case of the growth hormone secretagogue ghrelin and its analogues. A thoroughly investigation on these new pharmacological tools may lead to design effective preventive therapies.

Neurosteroids and epileptogenesis.

Epileptogenesis is defined as the latent period at the end of which spontaneous recurrent seizures occur. This concept has been recently re-evaluated to include exacerbation of clinically-manifested epilepsy. Thus, in patients affected by pharmacoresistant seizures, the progression toward a worse condition may be viewed as the result of a durable epileptogenic process. However, the mechanism potentially responsible for this progression remains unclear. Neuroinflammation has been consistently detected both in the latent period and in the chronic phase of epilepsy, especially when brain damage is present. This phenomenon is accompanied by glial cell reaction, leading to gliosis. We have previously described rats presenting an increased expression of the cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, during the latent period, in glial cells of the hippocampus. The P450scc enzyme is critically involved in the synthesis of neurosteroids and its up-regulation is associated with a delayed appearance of spontaneous recurrent seizures in rats that experienced status epilepticus induced by pilocarpine. Moreover, by decreasing the synthesis of neurosteroids able to promote inhibition, such as allopregnanolone, through the administration of the 5α-reductase blocker finasteride, it is possible to terminate the latent period in pilocarpine-treated rats. Finasteride was also found to promote seizures in the chronic period of epileptic rats, suggesting that neurosteroids are continuously produced to counteract seizures. In humans, exacerbation of epilepsy has been also described in patients occasionally exposed to finasteride. Overall, these findings suggest a major role of neurosteroids in the progression of epilepsy and a possible antiepileptogenic role of allopregnanolone and cognate molecules.

Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR.

BACKGROUND AND PURPOSE: Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor-like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine-like receptor proteins D6 and DARC as well as CCX-CKR. Here, we provide evidence for an additional biological function of human (h)CCX-CKR. EXPERIMENTAL APPROACH: We used transfection strategies in HEK293 and human T cells. KEY RESULTS: Co-expression of hCCX-CKR completely inhibits hCXCR3-induced chemotaxis. We found that hCCX-CKR forms complexes with hCXCR3, suggesting a relationship between CCX-CKR heteromerization and inhibition of chemotaxis. Moreover, negative binding cooperativity induced by ligands both for hCXCR3 and hCCX-CKR was observed in cells expressing both receptors. This negative cooperativity may also explain the hCCX-CKR-induced inhibition of chemotaxis. CONCLUSIONS AND IMPLICATIONS: These findings suggest that hCCX-CKR prevents hCXCR3-induced chemotaxis by heteromerization thus representing a novel mechanism of regulation of immune cell migration.

Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes.

BACKGROUND AND PURPOSE: The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers. EXPERIMENTAL APPROACH: CXCR3-CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity. KEY RESULTS: We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific ß-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation. CONCLUSIONS AND IMPLICATIONS: This study indicates that heteromeric CXCR3-CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities.

The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases.

AIMS: HSPB8 is a small heat shock protein that forms a complex with the co-chaperone BAG3. Overexpression of the HSPB8-BAG3 complex in cells stimulates autophagy and facilitates the clearance of mutated aggregation-prone proteins, whose accumulation is a hallmark of many neurodegenerative disorders. HSPB8-BAG3 could thus play a protective role in protein aggregation diseases and might be specifically upregulated in response to aggregate-prone protein-mediated toxicity. Here we analysed HSPB8-BAG3 expression levels in post-mortem human brain tissue from patients suffering of the following protein conformation disorders: Alzheimer's disease, Parkinson's disease, Huntington's disease and spinocerebellar ataxia type 3 (SCA3). METHODS: Western blotting and immunohistochemistry techniques were used to analyse HSPB8 and BAG3 expression levels in fibroblasts from SCA3 patients and post-mortem brain tissues, respectively. RESULTS: In all diseases investigated, we observed a strong upregulation of HSPB8 and a moderate upregulation of BAG3 specifically in astrocytes in the cerebral areas affected by neuronal damage and degeneration. Intriguingly, no significant change in the HSPB8-BAG3 expression levels was observed within neurones, irrespective of their localization or of the presence of proteinaceous aggregates. CONCLUSIONS: We propose that the upregulation of HSPB8 and BAG3 may enhance the ability of astrocytes to clear aggregated proteins released from neurones and cellular debris, maintain the local tissue homeostasis and/or participate in the cytoskeletal remodelling that astrocytes undergo during astrogliosis.

Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3).

AIMS: A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions. METHODS: We used a quantitative analysis of immunohistochemistry of the pons of eight patients with the polyglutamine disorder spinocerebellar ataxia type 3. We employed the anti-polyglutamine antibody 1C2, antibodies against p62 that is involved in delivering ubiquitinated protein aggregates to autophagosomes, antibodies against the chaperones HSPA1A and DNAJB1 and the proteasomal stress marker UBB⁺¹. RESULTS: The 1C2 antibody stained neuronal nuclear inclusions (NNIs), diffuse nuclear staining (DNS), granular cytoplasmic staining (GCS) and combinations, with reproducible distribution. P62 always co-localized with 1C2 in NNI. DNS and GCS co-stained with a lower frequency. UBB⁺¹ was present in a subset of neurones with NNI. A subset of UBB⁺¹-containing neurones displayed increased levels of HSPA1A, while DNAJB1 was sequestered into the NNI. CONCLUSION: Based on our results, we propose a model for the aggregation-associated pathology of spinocerebellar ataxia type 3: GCS and DNS aggregation likely represents early stages of pathology, which progresses towards formation of p62-positive NNI. A fraction of NNI exhibits UBB⁺¹ staining, implying proteasomal overload at a later stage. Subsequently, the stress-inducible HSPA1A is elevated while DNAJB1 is recruited into NNIs. This indicates that the stress response is only induced late when all endogenous protein quality control systems have failed.

Microglia phenotype diversity.

Microglia, the tissue macrophages of the brain, have under healthy conditions a resting phenotype that is characterized by a ramified morphology. With their fine processes microglia are continuously scanning their environment. Upon any homeostatic disturbance microglia rapidly change their phenotype and contribute to processes including inflammation, tissue remodeling, and neurogenesis. In this review, we will address functional phenotypes of microglia in diverse brain regions and phenotypes associated with neuroinflammation, neurogenesis, brain tumor homeostasis, and aging.

Neuron-microglia signaling: chemokines as versatile messengers.

Our understanding of microglia biology has significantly changed in the last couple of years. Instead of being predominantly detrimental cells showing a stereotypic activation pattern, microglia today are considered highly adaptive elements with many distinct phenotypes. Microglia activity is aimed to protect and to restore and only in case of uncontrolled or impaired microglia function these cells may have detrimental effects. The control of microglia activity is thus an important issue to understand. The family of chemokines are versatile signals specialized to control cell-cell interactions. Neurons express chemokines in a temporarily and spatially regulated manner and microglia respond to these messengers via the appropriate receptors. Due to these features are chemokines ideal messengers for the communication between neurons and microglia.

Expression pattern of voltage-dependent calcium channel subunits in hippocampal inhibitory neurons in mice.

Different subtypes of voltage-dependent calcium channels (VDCCs) generate various types of calcium currents that play important role in neurotransmitter release, membrane excitability, calcium transients and gene expression. Well-established differences in the physiological properties and variable sensitivity of hippocampal GABAergic inhibitory neurons to excitotoxic insults suggest that the calcium homeostasis, thus VDCC subunits expression pattern is likely different in subclasses of inhibitory cells. Using double-immunohistochemistry, here we report that in mice: 1) Cav2.1 and Cav3.1 subunits are expressed in almost all inhibitory neurons; 2) subunits responsible for the L-type calcium current (Cav1.2 and Cav1.3) are infrequently co-localized with calretinin inhibitory cell marker while Cav1.3 subunit, at least in part, tends to compensate for the low expression of Cav1.2 subunit in parvalbumin-, metabotropic glutamate receptor 1alpha- and somatostatin-immunopositive inhibitory neurons; 3) Cav2.2 subunit is expressed in the majority of inhibitory neurons except in calbindin-reactive inhibitory cells; 4) Cav2.3 subunit is expressed in the vast majority of the inhibitory cells except in parvalbumin- and calretinin-immunoreactive neurons where the proportion of expression of this subunit is considerably lower. These data indicate that VDCC subunits are differentially expressed in hippocampal GABAergic interneurons, which could explain the diversity in their electrophysiological properties, the existence of synaptic plasticity in certain inhibitory neurons and their vulnerability to stressful stimuli.

Characterization of the subventricular zone of the adult human brain: evidence for the involvement of Bcl-2.

The subventricular zone (SVZ) is an embryonic remnant that persists and remains mitotically active throughout adulthood. The rodent SVZ harbors neuronal precursors, principally in its anterior part, and generates neuroblasts that migrate tangentially into the olfactory bulb, thus forming the so-called rostral migratory stream. This study aimed at characterizing the SVZ in the human brain. Antibodies raised against the widely used SVZ molecular markers nestin, glial fibrillary acidic protein, beta-tubulin-III and polysialylated neural cell adhesion molecule, have allowed us to characterize in detail a zone similar to the rodent SVZ in humans. Virtually all portions of the lateral ventricle, as well as the ventral (hypothalamic) sector of the third ventricle, displayed immunoreactivity for most of the molecular markers. The midline region of the septum (septal recess) and the ventral portion of the SVZ displayed a particularly intense immunostaining for all SVZ markers. These two regions may represent zones of adult neurogenesis that are unique to primates. Furthermore, the anti-apoptotic protein Bcl-2 was found to be actively synthesized and co-expressed with all the other markers throughout the entire SVZ. This study reveals that a well-developed SVZ exists in the adult human brain and suggests that Bcl-2 might play an important role in the functional organization of such a system.

Differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunits by calretinin-immunoreactive neurons in the human striatum.

We recently reported the existence of medium and large intemeurons immunoreactive for the calcium-binding protein calretinin in the human striatum. We also showed a selective sparing of all medium, but not all large, calretinin-immunoreactive striatal neurons in Huntington's disease striatum. Because glutamate receptor-mediated excitotoxicity has been implicated in the massive loss of striatal projection neurons that characterizes Huntington's disease, we have applied a double-antigen localization procedure to post mortem tissue from eight normal human subjects to determine the expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor subunits 1/2/4 by the calretinin-immunoreactive interneurons. The two types of calretinin-immunoreactive neurons were found to display various patterns of glutamate receptor subunit expression and a specific regionalization was also noted in the expression of these glutamate receptor subunits. Approximately half of the large calretinin-immunoreactive neurons displayed immunoreactivity for glutamate receptor subunits 1 and 2, and about the same proportion of medium calretinin-immunoreactive neurons expressed glutamate receptor subunits 1 and 4. These double-labeled neurons were rather uniformly distributed in the caudate nucleus and putamen. In contrast, as much as 70.1% of the large calretinin-immunoreactive neurons displayed glutamate receptor subunit 4 immunoreactivity in the postcommissural portion of the putamen, an area that corresponds to the sensorimotor striatal territory. For their part, the medium calretinin-immunoreactive neurons were markedly enriched with glutamate receptor subunit 2, 76% of them being double labeled in the caudate nucleus, which corresponds to the striatal associative territory, compared with 85.5% in the postcommissural putamen. Receptor subunit composition plays a key role in determining the functional properties of glutamate receptors, including their permeability to calcium and susceptibility to excitotoxic insults. Thus, the differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor subunits reported here may help to explain the selective sparing of certain types of calretinin-immunoreactive striatal interneurons in Huntington's disease, although other factors, such as post-transcriptional editing, are also likely to be involved.

Radiation pressure and stability of interferometric gravitational-wave detectors.

The effect of radiation pressure on the stability of Fabry-Perot cavities with hanging mirrors is investigated. Such cavities will form an integral part of the laser interferometric gravitational-wave detectors that are being constructed around the globe. The mirrors are hung by means of a pendulum suspension and are locked by servo controls. We assume a realistic servo-control transfer function that satisfies the standard stability criteria. We find that for positive offsets from the resonance of the cavity the system is stable. However, we show that for negative offsets instabilities can occur, although the servo system has the effect of increasing the instability threshold, compared with the nonservoed case. Conditions for stability are finally given, involving the finesse of the cavity, the input power, the mass of the mirrors, the servo gain, and the phase detuning from perfect resonance. Gravitational-wave detectors with arm cavities having a finesse as low as approximately 200 could exhibit instabilities. Some implications for the locking of these detectors are finally given.

Stability of giant Fabry-Perot cavities of interferometric gravitational-wave detectors.

We consider the coupling of the thermoelastic mirror deformations to the resonance of giant cavities involved in interferometric detectors of gravitational waves. As this problem is coupled and nonlinear, instabilities could occur a priori. We analytically solve the coupled problem of thermoelastic deformations and their effect on the laser field, perturbatively, and we show that within the realm of our (physically reasonable) assumptions there are no instabilities that can simulate a false event in the observational frequency range of 1 Hz to 1 kHz.

A comparative study of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes in foods.

Nineteen laboratories across Canada took part in a comparative study of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes in foods and environmental samples. The results show that the enrichment period of the FDA method can be shortened from 7 to 2 days without substantially reducing the number of positive samples. With a limited number of samples, the USDA method proved to be slightly more efficient in isolating L. monocytogenes than the FDA method. Fraser broth, in principle, proved to be useful as a screening tool but is not very selective. Oxford agar and lithium chloride-phenylethanol-moxalactam medium were better than modified McBride's agar in isolating this microorganism.

Kerr effect in all-fiber cavities of optical gyros.

The transmission spectrum of an all-fiber cavity is theoretically analyzed, in order to evaluate the perturbation induced in the eigenfrequency measurement by the Kerr effect. Existence of a threshold separating the bistable operation region from the monostable is shown. In the weakly nonlinear operation, the asymmetry of the line is evaluated and the resulting error in the frequency measurement is analytically derived.

Continuous laser amplification in a monomode fiber longitudinally pumped by evanescent field coupling.

Amplification of continuous laser radiation propagating in a monomode optical fiber was obtained by evanescent field coupling within a pumped dye solution deposited in the neighborhood of the fiber core. A factor-of-25 signal gain was recorded for 632.8-nm He-Ne radiation and DCM dye solution.

Elastooptical antenna for detection of gravitational radiation.

Using the photoelastic effect in transparent solids to detect stresses induced by a gravitational wave is proposed. The induced birefringence is measured by a polarimeter. The phase difference is amplified by a Fabry-Perot cavity containing the medium. The sensitivity of this optical strain sensor is discussed, and that of the antenna is estimated.