Spatial MIST Technology for Rapid, Highly Multiplexed Detection of Protein Distribution on Brain Tissue

Highly multiplexed analysis of biospecimens significantly advances the understanding of biological basics of diseases, but these techniques are limited by the number of multiplexity and the speed of processing. Here, we present a rapid multiplex method for quantitative detection of protein markers on brain sections with the cellular resolution. This spatial multiplex in situ tagging (MIST) technology is built upon a MIST microarray that contains millions of small microbeads carrying barcoded oligonucleotides. Using antibodies tagged with UV cleavable oligonucleotides, the distribution of protein markers on a tissue slice could be printed on the MIST microarray with high fidelity. The performance of this technology in detection sensitivity, resolution and signal-to-noise level has been fully characterized by detecting brain cell markers. We showcase the codetection of 31 proteins simultaneously within 2 h which is about 10 times faster than the other immunofluorescence-based approaches of similar multiplexity. A full set of computational toolkits was developed to segment the small regions and identify the regional differences across the entire mouse brain. This technique enables us to rapidly and conveniently detect dozens of biomarkers on a tissue specimen, and it can find broad applications in clinical pathology and disease mechanistic studies.

Neurotoxic Potential of Deoxynivalenol in Murine Brain Cell Lines and Primary Hippocampal Cultures

Chronic exposure to the mycotoxin deoxynivalenol (DON) from grain-based food and feed affects human and animal health. Known consequences include entereopathogenic and immunotoxic defects; however, the neurotoxic potential of DON has only come into focus more recently due to the observation of behavioural disorders in exposed farm animals. DON can cross the blood-brain barrier and interfere with the homeostasis/functioning of the nervous system, but the underlying mechanisms of action remain elusive. Here, we have investigated the impact of DON on mouse astrocyte and microglia cell lines, as well as on primary hippocampal cultures by analysing different toxicological endpoints. We found that DON has an impact on the viability of both glial cell types, as shown by a significant decrease of metabolic activity, and a notable cytotoxic effect, which was stronger in the microglia. In astrocytes, DON caused a G1 phase arrest in the cell cycle and a decrease of cyclic-adenosine monophosphate (cAMP) levels. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α was secreted in the microglia in response to DON exposure. Furthermore, the intermediate filaments of the astrocytic cytoskeleton were disturbed in primary hippocampal cultures, and the dendrite lengths of neurons were shortened. The combined results indicated DON’s considerable potential to interfere with the brain cell physiology, which helps explain the observed in vivo neurotoxicological effects.

Correlation studies on pathological changes in brain with neurotransmitters and behavioural changes in Balb/c mice

Aim: To determine the plasma neurotransmitters simultaneously and to find any correlation with pathological changes in the hippocampus and Purkinje cells and their relation with behavioral changes in Balb/c mice. Methodology: In the present study, both sexes of Balb /C mice were divided into two groups (4 males and 4 females; n = 8): Both the groups were given a single dose of either saline or sodium valproate (400mg kg-1) respectively through subcutaneous injection on PND 14. Behavioural tests were conducted on mice pups on various postnatal days till 40th day. On PND 41, blood samples were collected from all the animals for quantification of the neurotransmitters (serotonin, dopamine, and noradrenalin) in plasma, animals were sacrificed by cervical dislocation and whole brain was isolated for histological examination of the Purkinje cells and hippocampus. Results: Sodium valproate exposed animals showed loss of motor skill development (delayed negative geotaxic response), increased locomotor activity, increased anxiety, and retardation in water maze performance, and lower social interaction. Histopathological evolutions of cerebellum purkinje cells and hippocampus showed 40-50% atrophic cells in sodium valproate animals compared to control animals. Interpretation: The results of the present study indicate that Sodium valproate changes specific brain cell population in Balb/C mice, which might be the reason for the altered neurotransmitter levels, leading to behavioural changes in these animals.

Divergent Effect of Central Incretin Receptors Inhibition in a Rat Model of Sporadic Alzheimer’s Disease

The incretin system is an emerging new field that might provide valuable contributions to the research of both the pathophysiology and therapeutic strategies in the treatment of diabetes, obesity, and neurodegenerative disorders. This study aimed to explore the roles of central glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) on cell metabolism and energy in the brain, as well as on the levels of these incretins, insulin, and glucose via inhibition of the central incretin receptors following intracerebroventricular administration of the respective antagonists in healthy rats and a streptozotocin-induced rat model of sporadic Alzheimer’s disease (sAD). Chemical ablation of the central GIP receptor (GIPR) or GLP-1 receptor (GLP-1R) in healthy and diseased animals indicated a region-dependent role of incretins in brain cell energy and metabolism and central incretin-dependent modulation of peripheral hormone secretion, markedly after GIPR inhibition, as well as a dysregulation of the GLP-1 system in experimental sAD.

AGC1 Deficiency: Pathology and Molecular and Cellular Mechanisms of the Disease

AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named “early infantile epileptic encephalopathy 39” (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.

Single-nuclei isoform RNA sequencing reveals combination patterns of transcript elements across human brain cell types

Single-nuclei RNA-Seq is being widely employed to investigate cell types, especially of human brain and other frozen samples. In contrast to single-cell approaches, however, the majority of single-nuclei RNA counts originate from partially processed RNA leading to intronic cDNAs, thus hindering the investigation of complete isoforms. Here, using microfluidics, PCR-based artifact removal, target enrichment, and long-read sequencing, we developed single-nuclei isoform RNA-sequencing ('SnISOr-Seq'), and applied it to the analysis of human adult frontal cortex samples. We found that exons associated with autism exhibit coordinated and more cell-type specific inclusion than exons associated with schizophrenia or ALS. We discovered two distinct modes of combination patterns: first, those distinguishing cell types in the human brain. These are enriched in combinations of TSS-exon, exon-polyA site, and distant (non-adjacent) exon pairs. Second, those with all isoform combinations found within one neural cell type, which are enriched in adjacent exon pairs. Furthermore, adjacent exon pairs are predominantly mutually associated, while distant pairs are frequently mutually exclusive. Finally, we observed that human-specific exons are as tightly coordinated as conserved exons, pointing to an efficient evolutionary mechanism underpinning coordination. SnISOr-Seq opens the door to single-nuclei long-read isoform analysis in the human brain, and in any frozen, archived or hard-to-dissociate sample.

Autophagy and Glycative Stress: A Bittersweet Relationship in Neurodegeneration

Autophagy is a fine-tuned proteolytic pathway that moves dysfunctional/aged cellular components into the lysosomal compartment for degradation. Over the last 3 decades, global research has provided evidence for the protective role of autophagy in different brain cell components. Autophagic capacities decline with age, which contributes to the accumulation of obsolete/damaged organelles and proteins and, ultimately, leads to cellular aging in brain tissues. It is thus well-accepted that autophagy plays an essential role in brain homeostasis, and malfunction of this catabolic system is associated with major neurodegenerative disorders. Autophagy function can be modulated by different types of stress, including glycative stress. Glycative stress is defined as a cellular status with abnormal and accelerated accumulation of advanced glycation end products (AGEs). It occurs in hyperglycemic states, both through the consumption of high-sugar diets or under metabolic conditions such as diabetes. In recent years, glycative stress has gained attention for its adverse impact on brain pathology. This is because glycative stress stimulates insoluble, proteinaceous aggregation that is linked to the malfunction of different neuropathological proteins. Despite the emergence of new literature suggesting that autophagy plays a major role in fighting glycation-derived damage by removing cytosolic AGEs, excessive glycative stress might also negatively impact autophagic function. In this mini-review, we provide insight on the status of present knowledge regarding the role of autophagy in brain physiology and pathophysiology, with an emphasis on the cytoprotective role of autophagic function to ameliorate the adverse effects of glycation-derived damage in neurons, glia, and neuron-glia interactions.

The landscape of regulatory genes in brain-wide neuronal phenotypes of a vertebrate brain

Multidimensional landscapes of regulatory genes in neuronal phenotypes at whole-brain levels in the vertebrate remain elusive. We generated single-cell transcriptomes of ~67,000 region- and glutamatergic/neuromodulator-identifiable cells from larval zebrafish brains. Hierarchical clustering based on effector gene profiles ('terminal features') distinguished major brain cell types. Sister clusters at hierarchical termini displayed similar terminal features. It was further verified by a population-level statistical method. Intriguingly, glutamatergic/GABAergic sister clusters mostly expressed distinct transcriptional factor (TF) profiles ('convergent pattern'), whereas neuromodulator-type sister clusters predominantly expressed the same TF profiles ('matched pattern'). Interestingly, glutamatergic/GABAergic clusters with similar TF profiles could also display different terminal features ('divergent pattern'). It led us to identify a library of RNA-binding proteins that differentially marked divergent pair clusters, suggesting the post-transcriptional regulation of neuron diversification. Thus, our findings reveal multidimensional landscapes of transcriptional and post-transcriptional regulators in whole-brain neuronal phenotypes in the zebrafish brain.

The Effect of the Classical TSPO Ligand PK 11195 On in Vitro Cobalt Chloride Model of Hypoxia in Non-Neuronal and Brain Cell Lines

The mitochondrial translocator protein (TSPO) is a modulator of the apoptotic pathway involving reactive oxygen species (ROS) generation, mitochondrial membrane potential (Δψm) collapse, activation of caspases and eventually initiation of the apoptotic process. In this in vitro study, H1299 lung cells and BV-2 microglial cells were exposed to the hypoxic effect of CoCl2 with or without PK 11195. Exposing the H1299 cells to 0.5 mM CoCl2 for 24 hours resulted in decreases in cell viability (63%, p<0.05), elevation of cardiolipin peroxidation levels (38%, p<0.05), mitochondrial membrane potential depolarization (13%, p<0.001), and apoptotic cell death (117%, p<0.05). Pretreatment with PK 11195 (25 µM) exhibited significant protective capacity on CoCl2-induced alterations in the mentioned processes. Exposure of BV-2 cells to increasing concentrations of CoCl2 (0.3, 0.5, 0.7 mM) for 4 hours resulted in alterations in the same cellular processes. These alterations were obtained in a dose-dependent manner, except the changes in caspases 3 and 9. The novel ligands as well as PK 1195 attenuated the in vitro hypoxic effects of CoCl2.It appears that the TSPO ligand PK 11195 can prevent CoCl2-induced cellular damage in both non-neuronal and brain cell lines, and they may offer new therapeutic options in hypoxia-related lung and brain diseases which fail to respond to conventional therapies.