Dynamics of Lateral Habenula-Ventral Tegmental Area Microcircuit on Pain-Related Cognitive Dysfunctions.

Chronic pain is a health problem that affects the ability to work and perform other activities, and it generally worsens over time. Understanding the complex pain interaction with brain circuits could help predict which patients are at risk of developing central dysfunctions. Increasing evidence from preclinical and clinical studies suggests that aberrant activity of the lateral habenula (LHb) is associated with depressive symptoms characterized by excessive negative focus, leading to high-level cognitive dysfunctions. The primary output region of the LHb is the ventral tegmental area (VTA), through a bidirectional connection. Recently, there has been growing interest in the complex interactions between the LHb and VTA, particularly regarding their crucial roles in behavior regulation and their potential involvement in the pathological impact of chronic pain on cognitive functions. In this review, we briefly discuss the structural and functional roles of the LHb-VTA microcircuit and their impact on cognition and mood disorders in order to support future studies addressing brain plasticity during chronic pain conditions.

Role of Glutamatergic Projections from Lateral Habenula to Ventral Tegmental Area in Inflammatory Pain-Related Spatial Working Memory Deficits.

The lateral habenula (LHb) and the ventral tegmental area (VTA), which form interconnected circuits, have important roles in the crucial control of sensory and cognitive motifs. Signaling in the LHb-VTA pathway can be exacerbated during pain conditions by a hyperactivity of LHb glutamatergic neurons to inhibit local VTA DAergic cells. However, it is still unclear whether and how this circuit is endogenously engaged in pain-related cognitive dysfunctions. To answer this question, we modulated this pathway by expressing halorhodopsin in LHb neurons of adult male rats, and then selectively inhibited the axonal projections from these neurons to the VTA during a working memory (WM) task. Behavioral performance was assessed after the onset of an inflammatory pain model. We evaluated the impact of the inflammatory pain in the VTA synapses by performing immunohistochemical characterization of specific markers for GABAergic (GAD65/67) and dopaminergic neurons (dopamine transporter (DAT), dopamine D2 receptor (D2r) and tyrosine hydroxylase (TH)). Our results revealed that inhibition of LHb terminals in the VTA during the WM delay-period elicits a partial recovery of the performance of pain animals (in higher complexity challenges); this performance was not accompanied by a reduction of nociceptive responses. Finally, we found evidence that the pain-affected animals exhibit VTA structural changes, namely with an upregulation of GAD65/67, and a downregulation of DAT and D2r. These results demonstrate a role of LHb neurons and highlight their responsibility in the stability of the local VTA network, which regulates signaling in frontal areas necessary to support WM processes.

Exploring the Physiological Role of Transthyretin in Glucose Metabolism in the Liver.

Transthyretin (TTR), a 55 kDa evolutionarily conserved protein, presents altered levels in several conditions, including malnutrition, inflammation, diabetes, and Alzheimer's Disease. It has been shown that TTR is involved in several functions, such as insulin release from pancreatic ß-cells, recovery of blood glucose and glucagon levels of the islets of Langerhans, food intake, and body weight. Here, the role of TTR in hepatic glucose metabolism was explored by studying the levels of glucose in mice with different TTR genetic backgrounds, namely with two copies of the TTR gene, TTR+/+; with only one copy, TTR+/-; and without TTR, TTR-/-. Results showed that TTR haploinsufficiency (TTR+/-) leads to higher glucose in both plasma and in primary hepatocyte culture media and lower expression of the influx glucose transporters, GLUT1, GLUT3, and GLUT4. Further, we showed that TTR haploinsufficiency decreases pyruvate kinase M type (PKM) levels in mice livers, by qRT-PCR, but it does not affect the hepatic production of the studied metabolites, as determined by 1H NMR. Finally, we demonstrated that TTR increases mitochondrial density in HepG2 cells and that TTR insufficiency triggers a higher degree of oxidative phosphorylation in the liver. Altogether, these results indicate that TTR contributes to the homeostasis of glucose by regulating the levels of glucose transporters and PKM enzyme and by protecting against mitochondrial oxidative stress.

Radiochemical examination of transthyretin (TTR) brain penetration assisted by iododiflunisal, a TTR tetramer stabilizer and a new candidate drug for AD.

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aß-peptides and prevent Aß aggregation. We have previously shown that treatment of Alzheimer's Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of 131I-labeled IDIF and 131I- and 124I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aß-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice.

The neuronal S100B protein is a calcium-tuned suppressor of amyloid-ß aggregation.

Amyloid-ß (Aß) aggregation and neuroinflammation are consistent features in Alzheimer's disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aß42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aß42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aß42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aß42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aß42 aggregation by interacting with Aß42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface-catalyzed reactions through S100B binding to growing Aß42 oligomers and fibrils. S100B protects cells from Aß42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aß42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.

Transthyretin stability is critical in assisting beta amyloid clearance- Relevance of transthyretin stabilization in Alzheimer's disease.

BACKGROUND: The absence of transthyretin (TTR) in AD mice decreases brain Aß clearance and reduces the low-density lipoprotein receptor-related protein 1 (LRP1). It is possible that neuroprotection by TTR is dependent on its tetramer structural stability, as studies using TTR mutants showed that unstable L55P TTR has low affinity for Aß, and TTR tetrameric stabilizers such as iododiflunisal ameliorate AD features in vivo. METHODS: We firstly investigated TTR folding status in human plasma measuring the resistance to urea denaturation. The importance of TTR stability on Aß internalization was studied in human cerebral microvascular endothelial (hCMEC/D3) and hepatoma cells (HepG2), by flow cytometry. To investigate the fate of Aß at the blood-brain barrier, Aß efflux from hCMEC/D3 cells seeded on transwells was measured using ELISA. Further, to assess Aß colocalization with lysosomes, Lysotracker was used. Moreover, levels of LRP1 were assessed in the liver and plasma of mice with different TTR backgrounds or treated with iododiflunisal. RESULTS: We showed that TTR stability is decreased in AD and that WT TTR and drug-stabilized L55P TTR are able to increase uptake of Aß. Furthermore, measurement of Aß efflux showed that stable or stabilized TTR increased Aß efflux from the basolateral to the apical side. Moreover, HepG2 cells incubated with Aß in the presence of WT TTR, but not L55P TTR, showed an increased number of lysosomes. Further, in the presence of WT TTR, Aß peptide colocalized with lysosomes, indicating that only stable TTR assists Aß internalization, leading to its degradation. Finally, we demonstrated that only stable TTR can increase LRP1 levels. CONCLUSION: TTR stabilization exerts a positive effect on Aß clearance and LRP1 levels, suggesting that TTR protective role in AD is dependent on its stability. These results provide relevant information for the design of TTR-based therapeutic strategies for AD.

Insights on the Interaction between Transthyretin and Aß in Solution. A Saturation Transfer Difference (STD) NMR Analysis of the Role of Iododiflunisal.

Several strategies against Alzheimer disease (AD) are directed to target Aß-peptides. The ability of transthyretin (TTR) to bind Aß-peptides and the positive effect exerted by some TTR stabilizers for modulating the TTR-Aß interaction have been previously studied. Herein, key structural features of the interaction between TTR and the Aß(12-28) peptide (3), the essential recognition element of Aß, have been unravelled by STD-NMR spectroscopy methods in solution. Molecular aspects related to the role of the TTR stabilizer iododiflunisal (IDIF, 5) on the TTR-Aß complex have been also examined. The NMR results, assisted by molecular modeling protocols, have provided a structural model for the TTR-Aß interaction, as well as for the ternary complex formed in the presence of IDIF. This basic structural information could be relevant for providing light on the mechanisms involved in the ameliorating effects of AD symptoms observed in AD/TTR± animal models after IDIF treatment and eventually for designing new molecules toward AD therapeutic drugs.

Transthyretin participates in beta-amyloid transport from the brain to the liver--involvement of the low-density lipoprotein receptor-related protein 1?

Transthyretin (TTR) binds Aß peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer's disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aß levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aß efflux and/or peripheral clearance. Here we showed that TTR promotes Aß internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aß permeability in hCMEC/D3, suggesting that TTR interacts directly with Aß at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aß from, but not into the brain. Furthermore, TTR increased Aß internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR-/- animals. We propose that TTR-mediated Aß clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR-/- mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aß at the blood-brain-barrier and liver, using LRP1.

Anti-inflammatory effect of seeds and callus of Nigella sativa L. extracts on mix glial cells with regard to their thymoquinone content.

Anti-inflammatory effect of the alcoholic extracts of N. sativa seeds and its callus on mix glial cells of rat with regard to their thymoquinone (TQ) content was investigated. Callus induction was achieved for explants of young leaf, stem, petiole, and root of N. sativa on solid Murashige and Skoog (MS) medium containing 2,4-D (1 mg/l) and kinetin (2.15 mg/l). TQ content of the alcoholic extracts was measured by HPLC. Total phenols were determined using Folin-Ciocalteu method and antioxidant power was estimated using FRAP tests. The mix glial cells, inflamed by lipopolysaccharide, were subjected to anti-inflammatory studies in the presence of various amounts of TQ and the alcoholic extracts. Viability of the cells and nitric oxide production were measured by MTT and Griess reagent, respectively. The leaf callus obtained the highest growth rate (115.4 mg/day) on MS medium containing 2,4-D (0.22 mg/l) and kinetin (2.15 mg/l). Analyses confirmed that TQ content of the callus of leaf was 12 times higher than that measured in the seeds extract. However, it decreased as the calli aged. Decrease in the TQ content of the callus was accompanied with an increase in its phenolic content and antioxidant ability. Studies on the inflamed rat mix glial cells revealed significant reduction in the nitric oxide production in the presence of 0.2 to 1.6 mg/ml of callus extract and 1.25 to 20 µl/ml of the seed extracts. However, the extent of the effects is modified assumingly due to the presence of the other existing substances in the extracts.