Controllable synthesis of barium carbonate nano- and microparticles for SPECT and CT imaging.

The design and synthesis of nano- and microcarriers for preclinical and clinical imaging are highly attractive due to their unique features, for example, multimodal properties. However, broad translation of these carriers into clinical practice is postponed due to the unknown biological reactivity of the new components used for their synthesis. Here, we have developed microcarriers (∼2-3 µm) and  nanocarriers (<200 nm) made of barium carbonate (BaCO3) for multiple imaging applications in vivo. In general, barium in the developed carriers can be used for X-ray computed tomography, and the introduction of a diagnostic isotope (99mTc) into the BaCO3 structure enables in vivo visualization using single-photon emission computed tomography. The bioimaging has shown that the radiolabeled BaCO3 nano- and microcarriers had different biodistribution profiles and tumor accumulation efficiencies after intratumoral and intravenous injections. In particular, in the case of intratumoral injection, all the types of used carriers mostly remained in the tumors (>97%). For intravenous injection, BaCO3 microcarriers were mainly localized in the lung tissues. However, BaCO3 NPs were mainly accumulated in the liver. These results were supported by ex vivo fluorescence imaging, direct radiometry, and histological analysis. The BaCO3-based micro- and nanocarriers showed negligible in vivo toxicity towards major organs such as the heart, lungs, liver, kidneys, and spleen. This study provides a simple strategy for the design and fabrication of the BaCO3-based carriers for the development of dual bioimaging.

Activation of Gq-Coupled Receptors in Astrocytes Restores Cognitive Function in Alzheimer's Disease Mice Model.

Alzheimer's disease (AD) is one of the most widespread neurodegenerative diseases. Most of the current AD therapeutic developments are directed towards improving neuronal cell function or facilitating Aß amyloid clearance from the brain. However, some recent evidence suggests that astrocytes may play a significant role in the pathogenesis of AD. In this paper, we evaluated the effects of the optogenetic activation of Gq-coupled exogenous receptors expressed in astrocytes as a possible way of restoring brain function in the AD mouse model. We evaluated the effects of the optogenetic activation of astrocytes on long-term potentiation, spinal morphology and behavioral readouts in 5xFAD mouse model of AD. We determined that in vivo chronic activation of astrocytes resulted in the preservation of spine density, increased mushroom spine survival, and improved performance in cognitive behavioral tests. Furthermore, chronic optogenetic stimulation of astrocytes resulted in the elevation of EAAT-2 glutamate uptake transporter expression, which could be a possible explanation for the observed in vivo neuroprotective effects. The obtained results suggest that the persistent activation of astrocytes may be considered a potential therapeutic approach for the treatment of AD and possibly other neurodegenerative disorders.

Astrocyte Activation Markers.

Astrocytes are the most common type of glial cells that provide homeostasis and protection of the central nervous system. Important specific characteristic of astrocytes is manifestation of morphological heterogeneity, which is directly dependent on localization in a particular area of the brain. Astrocytes can integrate into neural networks and keep neurons active in various areas of the brain. Moreover, astrocytes express a variety of receptors, channels, and membrane transporters, which underlie their peculiar metabolic activity, and, hence, determine plasticity of the central nervous system during development and aging. Such complex structural and functional organization of astrocytes requires the use of modern methods for their identification and analysis. Considering the important fact that determining the most appropriate marker for polymorphic and multiple subgroups of astrocytes is of decisive importance for studying their multifunctionality, this review presents markers, modern imaging techniques, and identification of astrocytes, which comprise a valuable resource for studying structural and functional properties of astrocytes, as well as facilitate better understanding of the extent to which astrocytes contribute to neuronal activity.

Optogenetic Activation of Astrocytes-Effects on Neuronal Network Function.

Optogenetics approach is used widely in neurobiology as it allows control of cellular activity with high spatial and temporal resolution. In most studies, optogenetics is used to control neuronal activity. In the present study optogenetics was used to stimulate astrocytes with the aim to modulate neuronal activity. To achieve this goal, light stimulation was applied to astrocytes expressing a version of ChR2 (ionotropic opsin) or Opto-α1AR (metabotropic opsin). Optimal optogenetic stimulation parameters were determined using patch-clamp recordings of hippocampal pyramidal neurons' spontaneous activity in brain slices as a readout. It was determined that the greatest increase in the number of spontaneous synaptic currents was observed when astrocytes expressing ChR2(H134R) were activated by 5 s of continuous light. For the astrocytes expressing Opto-α1AR, the greatest response was observed in the pulse stimulation mode (T = 1 s, t = 100 ms). It was also observed that activation of the astrocytic Opto-a1AR but not ChR2 results in an increase of the fEPSP slope in hippocampal neurons. Based on these results, we concluded that Opto-a1AR expressed in hippocampal astrocytes provides an opportunity to modulate the long-term synaptic plasticity optogenetically, and may potentially be used to normalize the synaptic transmission and plasticity defects in a variety of neuropathological conditions, including models of Alzheimer's disease and other neurodegenerative disorders.

Genetic Constructs for the Control of Astrocytes' Activity.

In the current review, we aim to discuss the principles and the perspectives of using the genetic constructs based on AAV vectors to regulate astrocytes' activity. Practical applications of optogenetic approaches utilizing different genetically encoded opsins to control astroglia activity were evaluated. The diversity of astrocytic cell-types complicates the rational design of an ideal viral vector for particular experimental goals. Therefore, efficient and sufficient targeting of astrocytes is a multiparametric process that requires a combination of specific AAV serotypes naturally predisposed to transduce astroglia with astrocyte-specific promoters in the AAV cassette. Inadequate combinations may result in off-target neuronal transduction to different degrees. Potentially, these constraints may be bypassed with the latest strategies of generating novel synthetic AAV serotypes with specified properties by rational engineering of AAV capsids or using directed evolution approach by searching within a more specific promoter or its replacement with the unique enhancer sequences characterized using modern molecular techniques (ChIP-seq, scATAC-seq, snATAC-seq) to drive the selective transgene expression in the target population of cells or desired brain regions. Realizing these strategies to restrict expression and to efficiently target astrocytic populations in specific brain regions or across the brain has great potential to enable future studies.

Optogenetic and chemogenetic modulation of astroglial secretory phenotype.

Astrocytes play a major role in brain function and alterations in astrocyte function that contribute to the pathogenesis of many brain disorders. The astrocytes are attractive cellular targets for neuroprotection and brain tissue regeneration. Development of novel approaches to monitor and to control astroglial function is of great importance for further progress in basic neurobiology and in clinical neurology, as well as psychiatry. Recently developed advanced optogenetic and chemogenetic techniques enable precise stimulation of astrocytes in vitro and in vivo, which can be achieved by the expression of light-sensitive channels and receptors, or by expression of receptors exclusively activated by designer drugs. Optogenetic stimulation of astrocytes leads to dramatic changes in intracellular calcium concentrations and causes the release of gliotransmitters. Optogenetic and chemogenetic protocols for astrocyte activation aid in extracting novel information regarding the function of brain's neurovascular unit. This review summarizes current data obtained by this approach and discusses a potential mechanistic connection between astrocyte stimulation and changes in brain physiology.

Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1.

Sulfatases are a family of enzymes (sulfuric ester hydrolases, EC 3.1.6.-) that catalyze the hydrolysis of a wide array of sulfate esters. To date, despite the discovery of many sulfatase genes and the accumulation of data on numerous sulfated molecules, the number of characterized enzymes that are key players in sulfur metabolism remains extremely limited. While mammalian sulfatases are well studied due to their involvement in a wide range of normal and pathological biological processes, lower eukaryotic sulfatases, especially fungal sulfatases, have not been thoroughly investigated at the biochemical and structural level. In this paper, we describe the molecular cloning of Fusarium proliferatum sulfatase (F.p.Sulf-6His), its recombinant expression in Pichia pastoris as a soluble and active cytosolic enzyme and its detailed characterization. Gel filtration and native electrophoretic experiments showed that this recombinant enzyme exists as a tetramer in solution. The enzyme is thermo-sensitive, with an optimal temperature of 25°C. The optimal pH value for the hydrolysis of sulfate esters and stability of the enzyme was 6.0. Despite the absence of the post-translational modification of cysteine into Cα-formylglycine, the recombinant F.p.Sulf-6His has remarkably stable catalytic activity against p-nitrophenol sulfate, with kcat = 0.28 s-1 and Km = 2.45 mM, which indicates potential use in the desulfating processes. The currently proposed enzymatic mechanisms of sulfate ester hydrolysis do not explain the appearance of catalytic activity for the unmodified enzyme. According to the available models, the unmodified enzyme is not able to perform multiple catalytic acts; therefore, the enzymatic mechanism of sulfate esters hydrolysis remains to be fully elucidated.

In vivo analysis of cerebellar Purkinje cell activity in SCA2 transgenic mouse model.

Cerebellar Purkinje cells (PCs) are primarily affected in many spinocerebellar ataxias (SCA). In this study we investigated functional activity of PCs in transgenic mouse model of SCA2, a polyglutamine neurodegenerative hereditary disorder. In our studies we used extracellular single-unit recording method to compare spontaneous activity of PCs in age-matched wild-type mice and SCA2-58Q transgenic mice. We discovered that the fraction of PCs with bursting and an irregular pattern of spontaneous activity dramatically increases in aged SCA2-58Q mice compared with wild-type littermates. Small-conductance calcium-activated potassium (SK) channels play an important role in determining firing rate of PCs. Indeed, we demonstrated that intraperitoneal (IP) injection of SK channel inhibitor NS8593 induces an irregular pattern of PC activity in wild-type mice. Furthermore, we demonstrated that IP injection of SK channel-positive modulator chlorzoxazone (CHZ) decreases spontaneous firing rate of cerebellar PCs. Finally, we have shown that IP injections with CHZ normalize firing activity of cerebellar PCs from aging SCA2-58Q mice. We propose that alterations in PC firing patterns is one of potential causes of ataxic symptoms in SCA2 and in other SCAs and that positive modulators of SK channels can be used to normalize activity of PCs and alleviate ataxic phenotype in patients with SCA.

Restoring calcium homeostasis to treat Alzheimer's disease: a future perspective.

Alzheimer's disease (AD) is a neurodegenerative disorder that primarily compromises memory formation and storage. Several hypotheses regarding the pathogenesis of AD have been proposed; however, no cure is available to date. Here we describe the calcium hypothesis of AD, which is gaining popularity. We present data supporting this hypothesis and focus on a recently discovered calcium-signaling pathway that is dysregulated in AD and propose targets for the development of disease-modifying therapies.