Pilot Study of the Total and Phosphorylated Tau Proteins in Early-Stage Multiple Sclerosis.

Background and Objectives: Recent findings suggest that neurodegeneration starts early in the course of multiple sclerosis (MS) and significantly contributes to the progression of patients' disability. Tau is a microtubule-binding protein that is known to play a role in the pathophysiology of many neurodegenerative disorders. Newly emerging data on tau protein-induced neurodegenerative processes and its possible involvement in MS suggest that it may be involved in the pathology of early-stage MS. Therefore, this study aimed to test this hypothesis in patients with newly diagnosed MS. Materials and Methods: Cerebrospinal fluid (CSF) was collected from 19 patients with newly diagnosed MS and 19 control subjects. All MS patients underwent neurological examination, lumbar punction, and brain magnetic resonance imaging (MRI). CSF concentrations of total and phosphorylated tau (phospho-tau-181) protein were measured using commercial enzyme-linked immunosorbent assay kits. Results: The total tau concentration was significantly higher in the CSF of MS patients compared to controls (141.67 pg/mL, IQR 77.79-189.17 and 68.77 pg/mL, IQR 31.24-109.17, p = 0.025). In MS patients, the total tau protein positively correlated with total CSF protein (r = 0.471, p = 0.048). Significantly higher total tau concentration was measured in MS patients with higher lesion load in brain MRI (≥9 versus <9 lesions; 168.33 pg/mL, IQR 111.67-222.32 and 73.33 pg/mL, IQR -32.13-139.29-, p = 0.021). The CSF concentration of phospho-tau-181 protein was below the detection limit in both MS and control subjects. Conclusions: The concentration of total tau protein level is elevated, whereas phospho-tau-181 is undetectable in the CSF of patients with early-stage MS.

The Role of Intracellular Ca2+ and Mitochondrial ROS in Small Aß1-42 Oligomer-Induced Microglial Death.

Alzheimer's disease (AD) is the most common form of dementia worldwide, and it contributes up to 70% of cases. AD pathology involves abnormal amyloid beta (Aß) accumulation, and the link between the Aß1-42 structure and toxicity is of major interest. NMDA receptors (NMDAR) are thought to be essential in Aß-affected neurons, but the role of this receptor in glial impairment is still unclear. In addition, there is insufficient knowledge about the role of Aß species regarding mitochondrial redox states in neurons and glial cells, which may be critical in developing Aß-caused neurotoxicity. In this study, we investigated whether different Aß1-42 species-small oligomers, large oligomers, insoluble fibrils, and monomers-were capable of producing neurotoxic effects via microglial NMDAR activation and changes in mitochondrial redox states in primary rat brain cell cultures. Small Aß1-42 oligomers induced a concentration- and time-dependent increase in intracellular Ca2+ and necrotic microglial death. These changes were partially prevented by the NMDAR inhibitors MK801, memantine, and D-2-amino-5-phosphopentanoic acid (DAP5). Neither microglial intracellular Ca2+ nor viability was significantly affected by larger Aß1-42 species or monomers. In addition, the small Aß1-42 oligomers caused mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial depolarization, glutamate release, and neuronal cell death. In microglia, the Aß1-42-induced mtROS overproduction was mediated by intracellular calcium ions and Aß-binding alcohol dehydrogenase (ABAD). The data suggest that the pharmacological targeting of microglial NMDAR and mtROS may be a promising strategy for AD therapy.

Extracellular tau stimulates phagocytosis of living neurons by activated microglia via Toll-like 4 receptor-NLRP3 inflammasome-caspase-1 signalling axis.

In tauopathies, abnormal deposition of intracellular tau protein followed by gradual elevation of tau in cerebrospinal fluids and neuronal loss has been documented, however, the mechanism how actually neurons die under tau pathology is largely unknown. We have previously shown that extracellular tau protein (2N4R isoform) can stimulate microglia to phagocytose live neurons, i.e. cause neuronal death by primary phagocytosis, also known as phagoptosis. Here we show that tau protein induced caspase-1 activation in microglial cells via 'Toll-like' 4 (TLR4) receptors and neutral sphingomyelinase. Tau-induced neuronal loss was blocked by caspase-1 inhibitors (Ac-YVAD-CHO and VX-765) as well as by TLR4 antibodies. Inhibition of caspase-1 by Ac-YVAD-CHO prevented tau-induced exposure of phosphatidylserine on the outer leaflet of neuronal membranes and reduced microglial phagocytic activity. We also show that suppression of NLRP3 inflammasome, which is down-stream of TLR4 receptors and mediates caspase-1 activation, by a specific inhibitor (MCC550) also prevented tau-induced neuronal loss. Moreover, NADPH oxidase is also involved in tau-induced neurotoxicity since neuronal loss was abolished by its pharmacological inhibitor. Overall, our data indicate that extracellular tau protein stimulates microglia to phagocytose live neurons via Toll-like 4 receptor-NLRP3 inflammasome-caspase-1 axis and NADPH oxidase, each of which may serve as a potential molecular target for pharmacological treatment of tauopathies.

Imeglimin Is Neuroprotective Against Ischemic Brain Injury in Rats-a Study Evaluating Neuroinflammation and Mitochondrial Functions.

Imeglimin is a novel oral antidiabetic drug modulating mitochondrial functions. However, neuroprotective effects of this drug have not been investigated. The aim of this study was to investigate effects of imeglimin against ischemia-induced brain damage and neurological deficits and whether it acted via inhibition of mitochondrial permeability transition pore (mPTP) and suppression of microglial activation. Ischemia in rats was induced by permanent middle cerebral artery occlusion (pMCAO) for 48 h. Imeglimin (135 µg/kg/day) was injected intraperitoneally immediately after pMCAO and repeated after 24 h. Immunohistochemical staining was used to evaluate total numbers of neurons, astrocytes, and microglia as well as interleukin-10 (IL-10) producing cells in brain slices. Respiration of isolated brain mitochondria was assessed using high-resolution respirometry. Assessment of ionomycin-induced mPTP opening in intact cultured primary rat neuronal, astrocytic, and microglial cells was performed using fluorescence microscopy. Treatment with imeglimin significantly decreased infarct size, brain edema, and neurological deficits after pMCAO. Moreover, imeglimin protected against pMCAO-induced neuronal loss as well as microglial proliferation and activation, and increased the number of astrocytes and the number of cells producing anti-inflammatory cytokine IL-10 in the ischemic hemisphere. Imeglimin in vitro acutely prevented mPTP opening in cultured neurons and astrocytes but not in microglial cells; however, treatment with imeglimin did not prevent ischemia-induced mitochondrial respiratory dysfunction after pMCAO. This study demonstrates that post-stroke treatment with imeglimin exerts neuroprotective effects by reducing infarct size and neuronal loss possibly via the resolution of neuroinflammation and partly via inhibition of mPTP opening in neurons and astrocytes.

The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration.

Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) - activated by extracellular UDP released by stressed neurons - is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aß) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aß-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aß- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases.

Evaluation of the Effectiveness of Post-Stroke Metformin Treatment Using Permanent Middle Cerebral Artery Occlusion in Rats.

Stroke is the second leading cause of death worldwide. Treatment options for ischemic stroke are limited, and the development of new therapeutic agents or combined therapies is imperative. Growing evidence suggests that metformin treatment, due to its anti-inflammatory action, exerts a neuroprotective effect against ischemia/reperfusion-induced brain damage. Experimental assessment has typically been performed in models of cerebral transient ischemia followed by long-term reperfusion. The aim of this study was to evaluate the neuroprotective effect of metformin treatment after permanent middle cerebral artery occlusion (pMCAO) without reperfusion in rats. Neurological deficits were assessed using the Longa scale, which offers a graded scale on body movement following pMCAO. Both infarct size and brain oedema area were measured by staining with 2,3,5-triphenyltetrazolium chloride. The number of neurons and total and activated microglia, as well as interleukin 10 (IL-10) production, in brain sections were evaluated by immunohistochemical staining. Our results show that metformin treatment improves the neurological state and reduces infarct size after 120 h of pMCAO. Metformin also prevents neuronal loss in the ischemic cortex but not in the striatum after 48 h of pMCAO. Moreover, post-stroke treatment with metformin significantly decreases the number of total and activated microglia at 48 h. The anti-inflammatory effect of metformin is associated with increased IL-10 production at 48 h after pMCAO. The results of the present study suggest that post-stroke treatment with metformin exerts anti-inflammatory and neuroprotective effects in a pMCAO model.

Distinct Neurotoxic Effects of Extracellular Tau Species in Primary Neuronal-Glial Cultures.

Recent data from various experimental models support the link between extracellular tau and neurodegeneration; however, the exact mechanisms by which extracellular tau or its modified forms or aggregates cause neuronal death remain unclear. We have previously shown that exogenously applied monomers and oligomers of the longest tau isoform (2N4R) at micromolar concentrations induced microglial phagocytosis of stressed-but-viable neurons in vitro. In this study, we investigated whether extracellular phosphorylated tau2N4R (p-tau2N4R), isoform 1N4R (tau1N4R) and K18 peptide can induce neuronal death or loss in primary neuronal-glial cell cultures. We found that p-tau2N4R at 30 nM concentration induced loss of viable neurons; however, 700 nM p-tau2N4R caused necrosis of both neurons and microglia, and this neuronal death was partially glial cell-dependent. We also found that extracellular tau1N4R oligomers, but not monomers, at 3 µM concentration caused neuronal death in mixed cell cultures: self-assembly tau1N4R dimers-tetramers induced neuronal necrosis and apoptosis, whereas Aß-promoted tau1N4R oligomers caused glial cell-dependent loss of neurons without signs of increased cell death. Monomeric and pre-aggregated tau peptide containing 4R repeats (K18) had no effect in mixed cultures, suggesting that tau neurotoxicity might be dependent on N-terminal part of the protein. Taken together, our results show that extracellular p-tau2N4R is the most toxic form among investigated tau species inducing loss of neurons at low nanomolar concentrations and that neurotoxicity of tau1N4R is dependent on its aggregation state.

Different effects of metformin and phenformin on hypoxia-induced Ca2+ fluxes in cultured primary neurons.

Recent evidence suggests that metformin and phenformin may exert beneficial effects against neuronal injury in the ischemic brain, however, the difference of action between these two drugs and the molecular mechanism of such protection is not clear. In this study, we investigated whether mild hypoxia-affected neurons exhibit changes in cytosolic calcium handling and whether metformin and phenformin exert any effect on calcium homeostasis in hypoxia-affected neurons. Cultured primary rat cortical cells were stained with calcium sensitive dye Oregon Green 488 BAPTA-1 AM and spontaneous calcium dependent changes of fluorescence were recorded. Using obtained fluorescence traces we estimated changes in relative amplitude of recorded spontaneous signals, changes in frequency of spontaneous activity, and changes in decay of fluorescence traces. We found that hypoxia caused reduction of the relative signal amplitude, increased the spontaneous activity, and slowed the decay of calcium concentration. After pre-treatment of cells with 0.1-0.5 mM metformin, the relative signal amplitude increased and the frequency of spontaneous signals decreased in hypoxia-affected neurons. However, pre-treatment with 1-25 µM phenformin neither increased the relative signal amplitude nor reduced the frequency of spontaneous signals. The decay of fluorescence traces became faster after application of metformin or phenformin comparing to neurons under hypoxic conditions. These results suggest different action of metformin and phenformin in improvement of Ca2+ homeostasis in hypoxia-affected neurons, which may have different effects on neuronal survival and functions after hypoxia/ischemia.

Protective effects of anthocyanins against brain ischemic damage.

Anthocyanins are considered as bioactive components of plant-based diets that provide protection against ischemic cardiovascular pathologies by mechanisms dependent on their antioxidant and reductive capacities. However, it is not clear whether similar anthocyanin-mediated mechanisms can provide protection against ischemia-induced brain mitochondrial injury and cell death. In this study, we compared effects of three cyanidin-3-glycosides - glucoside (Cy3G), galactoside (Cy3Gal) and rutinoside (Cy3R), with pelargonxidin-3-glucoside (Pg3G) and found that at 10-20 µM concentrations they have no direct effect on respiratory functions of mitochondria isolated from normal or ischemia-damaged rat brain slices. However, intravenous injection of Cy3Gal and Cy3G (0,025 mg/kg or 0,05 mg/kg what matches 10 µM or 20 µM respectively) but not Cy3R in rats protected against ischemia-induced caspase activation and necrotic cell death, and reduced infarct size in cerebral cortex and cerebellum. These effects correlated with cytochrome c reducing capacity of cyanidin-3-glycosides. In contrast, intravenous injection of 0,025 mg/kg Pg3G which has the lowest cytochrome c reducing capacity among investigated anthocyanins, had no effect on ischemia-induced caspase activation and necrosis but reduced brain infarct size whereas intravenous injection of 0,05 mg/kg of Pg3G slightly promoted necrosis in the brain. Our data suggest that reductive rather than antioxidant capacities of anthocyanins may be important components in providing protection against ischemic brain damage.

Extracellular tau induces microglial phagocytosis of living neurons in cell cultures.

Tau is a microtubule-associated protein, found at high levels in neurons, and its aggregation is associated with neurodegeneration. Recently, it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are unclear. In this study, we investigated whether extracellular tau2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were examined for neuronal loss, death, and phosphatidylserine exposure, as well as for microglial phagocytosis by fluorescence microscopy. Aggregation of tau2N4R was assessed by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2 days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the 'eat-me' signal phosphatidylserine on the surface of living neurons, and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.