Neuronal extracellular microRNAs miR-124 and miR-9 mediate cell-cell communication between neurons and microglia.

In contrast to peripheral macrophages, microglia in the central nervous system (CNS) exhibit a specific deactivated phenotype; however, it is not clear how this phenotype is maintained. Two alternative hypotheses were postulated recently: (a) microglia differ from peripheral macrophages being derived from the yolk sac (YS), whereas peripheral macrophages originate from bone marrow (BM); (b) microglia acquire a specific phenotype under the influence of the CNS microenvironment. We have previously shown that microglia express miR-124, which was also induced in BM-derived macrophages co-cultured with a neurons. We here investigated the possibility of horizontal transfer of the neuron-specific microRNAs miR-124 and miR-9 from primary neurons to microglia/macrophages. We found that after incubation with neuronal conditioned media (NCM), macrophages downregulated activation markers MHC class II and CD45. Neither cultured adult microglia nor YS- and BM-derived macrophages demonstrated intrinsic levels of miR-124 expression. However, after incubation with NCM, miR-124 was induced in both YS- and BM-derived macrophages. Biochemical analysis demonstrated that the NCM contained miR-124 and miR-9 in complex with small proteins, large high-density lipoproteins (HDLs), and exosomes. MiR-124 and miR-9 were promptly released from neurons, and this process was inhibited by tetrodotoxin, indicating an important role of neuronal electric activity in secretion of these microRNAs. Incubation of macrophages with exogenous miR-124 resulted in efficient translocation of miR-124 into the cytoplasm. This study demonstrates an important role of neuronal miRNAs in communication of neurons with microglia, which favors the hypothesis that microglia acquire a specific phenotype under the influence of the CNS microenvironment.

Platelets mediate protective neuroinflammation and promote neuronal plasticity at the site of neuronal injury.

It is generally accepted that inflammation within the CNS contributes to neurodegeneration after traumatic brain injury (TBI), but it is not clear how inflammation is initiated in the absence of infection and whether this neuroinflammation is predominantly beneficial or detrimental. We have previously found that brain-enriched glycosphingolipids within neuronal lipid rafts (NLR) induced platelet degranulation and secretion of neurotransmitters and pro-inflammatory factors. In the present study, we compared TBI-induced inflammation and neurodegeneration in wild-type vs. St3gal5 deficient (ST3-/-) mice that lack major CNS-specific glycosphingolipids. After TBI, microglial activation and CNS macrophage infiltration were substantially reduced in ST3-/- animals. However, ST3-/- mice had a larger area of CNS damage with marked neuronal/axonal loss. The interaction of platelets with NLR stimulated neurite growth, increased the number of PSD95-positive dendritic spines, and intensified neuronal activity. Adoptive transfer and blocking experiments provide further that platelet-derived serotonin and platelet activating factor plays a key role in the regulation of sterile neuroinflammation, hemorrhage and neuronal plasticity after TBI.

The Role of Platelets in the Stimulation of Neuronal Synaptic Plasticity, Electric Activity, and Oxidative Phosphorylation: Possibilities for New Therapy of Neurodegenerative Diseases.

The central nervous system (CNS) is highly vascularized where neuronal cells are located in proximity to endothelial cells, astroglial limitans, and neuronal processes constituting integrated neurovascular units. In contrast to many other organs, the CNS has a blood-brain barrier (BBB), which becomes compromised due to infection, neuroinflammation, neurodegeneration, traumatic brain injury, and other reasons. BBB disruption is presumably involved in neuronal injury during epilepsy and psychiatric disorders. Therefore, many types of neuropsychological disorders are accompanied by an increase in BBB permeability leading to direct contact of circulating blood cells in the capillaries with neuronal cells in the CNS. The second most abundant type of blood cells are platelets, which come after erythrocytes and outnumber ~100-fold circulating leukocytes. When BBB becomes compromised, platelets swiftly respond to the vascular injury and become engaged in thrombosis and hemostasis. However, more recent studies demonstrated that platelets could also enter CNS parenchyma and directly interact with neuronal cells. Within CNS, platelets become activated by recognizing major brain gangliosides on the surface of astrocytes and neurons and releasing a milieu of pro-inflammatory mediators, neurotrophic factors, and neurotransmitters. Platelet-derived factors directly stimulate neuronal electric and synaptic activity and promote the formation of new synapses and axonal regrowth near the site of damage. Despite such active involvement in response to CNS damage, the role of platelets in neurological disorders was not extensively studied, which will be the focus of this review.

ASD-like behaviors, a dysregulated inflammatory response and decreased expression of PLP1 characterize mice deficient for sialyltransferase ST3GAL5.

Gangliosides are glycosphingolipids, which are abundant in brain, are known to modulate ion channels and cell-to-cell communication. Deficiencies can result in aberrant myelination and altered immune responses, which can give rise to neurodevelopmental psychiatric disorders. However, to date, little mechanistic data is available on how ganglioside deficiencies contribute to the behavioural disorders. In humans, the loss of lactosylceramide-alpha-2,3-sialyltransferase (ST3Gal5) leads to a severe neuropathology, but in ST3Gal5 knock-out (St3gal5-/-) mice the absence of GM3 and associated a-, b- and c-series gangliosides is partially compensated by 0-series gangliosides and there is no overt behavioural phenotype. Here, we sought to examine the behavioural and molecular consequences of GM3 loss more closely. Mutants of both sexes exhibited impaired conditioned taste aversion in an inhibitory learning task and anxiety-like behaviours in the open field, moderate motor deficits, abnormal social interactions, excessive grooming and rearing behaviours. Taken together, the aberrant behaviours are suggestive of an autism spectrum disorder (ASD)-like syndrome. Molecular analysis showed decreased gene and protein expression of proteolipid protein-1 (Plp1) and over expression of proinflammatory cytokines, which has been associated with ASD-like syndromes. The inflammatory and behavioural responses to lipopolysaccharide (LPS) were also altered in the St3gal5-/- mice compared to wild-type, which is indicative of the importance of GM3 gangliosides in regulating immune responses. Together, the St3gal5-/- mice display ASD-like behavioural features, altered response to systemic inflammation, signs of hypomyelination and neuroinflammation, which suggests that deficiency in a- and b-series gangliosides could contribute to the development of an ASD-like pathology in humans.

Sex-Specific ADHD-like Behaviour, Altered Metabolic Functions, and Altered EEG Activity in Sialyltransferase ST3GAL5-Deficient Mice.

A deficiency in GM3-derived gangliosides, resulting from a lack of lactosylceramide-alpha-2,3-sialyltransferase (ST3GAL5), leads to severe neuropathology, including epilepsy and metabolic abnormalities. Disruption of ganglioside production by this enzyme may also have a role in the development of neuropsychiatric disorders. ST3Gal5 knock-out (St3gal5-/-) mice lack a-, b-, and c-series gangliosides, but exhibit no overt neuropathology, possibly owing to the production of compensatory 0-series glycosphingolipids. Here, we sought to investigate the possibility that St3gal5-/- mice might exhibit attention-deficit/hyperactivity disorder (ADHD)-like behaviours. In addition, we evaluated potential metabolic and electroencephalogram (EEG) abnormalities. St3gal5-/- mice were subjected to behavioural testing, glucose tolerance tests, and the levels of expression of brain and peripheral A and B isoforms of the insulin receptor (IR) were measured. We found that St3gal5-/- mice exhibit locomotor hyperactivity, impulsivity, neophobia, and anxiety-like behavior. The genotype also altered blood glucose levels and glucose tolerance. A sex bias was consistently found in relation to body mass and peripheral IR expression. Analysis of the EEG revealed an increase in amplitude in St3gal5-/- mice. Together, St3gal5-/- mice exhibit ADHD-like behaviours, altered metabolic and EEG measures providing a useful platform for better understanding of the contribution of brain gangliosides to ADHD and associated comorbidities.

Platelets promote epileptic seizures by modulating brain serotonin level, enhancing neuronal electric activity, and contributing to neuroinflammation and oxidative stress.

The drugs currently available for treating epilepsy are only partially effective in managing this condition. Therefore, it is crucial to investigate new pathways that induce and promote epilepsy development. Previously, we found that platelets interact with neuronal glycolipids and actively secrete pro-inflammatory mediators during central nervous system (CNS) pathological conditions such as neuroinflammation and traumatic brain injury (TBI). These factors increase the permeability of the blood-brain barrier (BBB), which may create a predisposition to epileptic seizures. In this study, we demonstrated that platelets substantially enhanced epileptic seizures in a mouse model of pentylenetetrazole (PTZ) -induced seizures. We found that platelets actively secreted serotonin, contributed to increased BBB permeability, and were present in the CNS parenchyma during epileptic seizures. Furthermore, platelets directly stimulated neuronal electric activity and induced the expression of specific genes related to early neuronal response, neuroinflammation, and oxidative phosphorylation, leading to oxidative stress in neurons. The intracranial injection of physiological numbers of platelets that mimicked TBI-associated bleeding was sufficient to induce severe seizures, which resembled conventional PTZ-induced epileptic activity. These findings highlight a conceptually new role of platelets in the development of epileptic seizures, and indicate a potential new therapeutic approach targeting platelets to prevent and treat epilepsy.

Fresh evidence for major brain gangliosides as a target for the treatment of Alzheimer's disease.

Although it was suggested that gangliosides play an important role in the binding of amyloid fragments to neuronal cells, the exact role of gangliosides in Alzheimer's disease (AD) pathology remains unclear. To understand the role of gangliosides in AD pathology in vivo, we crossed st3gal5-deficient (ST3-/-) mice that lack major brain gangliosides GM1, GD1a, GD3, GT1b, and GQ1b with 5XFAD transgenic mice that overexpress 3 mutant human amyloid proteins AP695 and 2 presenilin PS1 genes. We found that ST3-/- 5XFAD mice have a significantly reduced burden of amyloid depositions, low level of neuroinflammation, and did not exhibit neuronal loss or synaptic dysfunction. ST3-/- 5XFAD mice performed significantly better in a cognitive test than wild-type (WT) 5XFAD mice, which was comparable with WT nontransgenic mice. Treatment of WT 5XFAD mice with the sialic acid-specific Limax flavus agglutinin resulted in substantial improvement of AD pathology to a level of ST3-/- 5XFAD mice. Thus, our findings highlight an important role for gangliosides as a target for the treatment of AD.

Methods of Study of Neuron Structural Heterogeneity: Flow Cytometry vs. Laser Interferometry.

Neuronal cells are probably the less studied cells regarding their heterogeneity on a single cell or population levels. One of the main problems of studying of individual neurons is the presence of long processes (axons) on differentiated adult neurons that hamper their isolation without significant damage to the cells. Therefore, the most common method to study neuronal cells is immunofluorescent microscopy of sections of the brain, which remains poorly quantitative and allows analyzing a small number of fixed cells. Also, immunofluorescent microscopy has a number of staining artifacts since histology section has high level of autofluorescence and non-specific binding of fluorescent probes. Alternative methods that could overcome disadvantages of immunofluorescent histology include flow cytometry, scanning cytometry, and laser interferometry. Flow cytometry and, to some extent of degree, scanning cytometry allow performing analysis of multiple markers with a low level of non-specific background and very robust statistics. Laser interferometry allows studies intact, alive neurons without staining. Limitations and advantages of these methods are discussed in this chapter.

Usage of Multiparameter Flow Cytometry to Study Microglia and Macrophage Heterogeneity in the Central Nervous System During Neuroinflammation and Neurodegeneration.

The resident macrophages of the central nervous system (CNS), also known as microglia, and blood-derived macrophages play an important role in the functional activity of the normal CNS, as well as in the development of neuroinflammation during various neurodegenerative disorders. Microglia and macrophages represent heterogeneous populations, which can modulate CNS environment and have different effects on neuronal regeneration. In this chapter, the main features of microglial and macrophage subsets and current methods for investigation of their heterogeneity will be discussed.