Diabetes Mellitus is associated with increased risk of cognitive and behavioural disorders with hitherto undeciphered role of glia. Glia as majority population in brain serve several vital functions, ...thus require pertinent revelation to further explicate the mechanisms affecting the brain function following diabetes. In this study we have evaluated glial changes in terms of phenotypic switching, proliferation and expression of activation cell surface markers and associated cellular degeneration in hippocampus following STZ-induced diabetes and caused cognitive impairments. Experimental diabetes was induced in Wistar rats by a single dose of STZ (45 mg/kg body weight; intraperitoneally) and changes were studied in 2nd, 4th and 6th week post diabetes confirmation using Barnes maze and T-maze test, immunohistochemistry and image analysis. An increase in GFAP expression sequentially from 2nd to 6th weeks of diabetes was analogous with the phenotypic changes and increased astrocyte number. Elevated level of S100β with defined stellate morphology further confirmed the astrocytosis following diabetes. Enhanced level of Iba-1 and MHC-II revealed the corroborated microglial activation and proliferation following diabetes, which was unresolved till date. Increased caspase-3 activity induced profound cell death upto 6th weeks post diabetes confirmation. Such caspase 3 mediated cellular damage with a concomitant activation of the astrocytes and microglia suggests that diabetes linked cell death activates the astrocytes and microglia in hippocampus which further underpin the progression and severity of brain disorders resulting in cognitive and behavioural impairments.
Abstract Background Metastasis, the spread, and growth of malignant cells at secondary sites within a patient’s body, accounts for over 90% of cancer-related mortality. Breast cancer is the most ...common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10–16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses, which represents a critical barrier for the development of efficient therapies for affected breast cancer patients. Methods Previous research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM, we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP, other cell lines of MCF-7, HCC-1806, and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid. Results We found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover, MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids. Conclusions Our co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture.
Local neocortical circuits play critical roles in information processing, including synaptic plasticity, circuit physiology, and learning, and GABAergic inhibitory interneurons have key roles in ...these circuits. Moreover, specific neurological disorders, including schizophrenia and autism, are associated with deficits in GABAergic transmission in these circuits. GABAergic synapses represent a small fraction of neocortical synapses, and are embedded in complex local circuits that contain many neuron and synapse types. Thus, it is challenging to study the physiological roles of GABAergic inhibitory interneurons and their synapses, and to develop treatments for the specific disorders caused by dysfunction at these GABAergic synapses. To these ends, we report a novel technology that can deliver different genes into pre- and post-synaptic neocortical interneurons connected by a GABAergic synapse: First, standard gene transfer into the presynaptic neurons delivers a synthetic peptide neurotransmitter, containing three domains, a dense core vesicle sorting domain, a GABAA receptor-binding domain, a single-chain variable fragment anti-GABAA ß2 or ß3, and the His tag. Second, upon release, this synthetic peptide neurotransmitter binds to GABAA receptors on the postsynaptic neurons. Third, as the synthetic peptide neurotransmitter contains the His tag, antibody-mediated, targeted gene transfer using anti-His tag antibodies is selective for these neurons. We established this technology by expressing the synthetic peptide neurotransmitter in GABAergic neurons in the middle layers of postrhinal cortex, and the delivering the postsynaptic vector into connected GABAergic neurons in the upper neocortical layers. Targeted gene transfer was 61% specific for the connected neurons, but untargeted gene transfer was only 21% specific for these neurons. This technology may support studies on the roles of GABAergic inhibitory interneurons in circuit physiology and learning, and support gene therapy treatments for specific disorders associated with deficits at GABAergic synapses.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Autophagy is a multifaceted cellular process that not only maintains the homeostatic and adaptive responses of the brain but is also dynamically involved in the regulation of neural cell generation, ...maturation, and survival. Autophagy facilities the utilization of energy and the microenvironment for developing neural stem cells. Autophagy arbitrates structural and functional remodeling during the cell differentiation process. Autophagy also plays an indispensable role in the maintenance of stemness and homeostasis in neural stem cells during essential brain physiology and also in the instigation and progression of diseases. Only recently, studies have begun to shed light on autophagy regulation in glia (microglia, astrocyte, and oligodendrocyte) in the brain. Glial cells have attained relatively less consideration despite their unquestioned influence on various aspects of neural development, synaptic function, brain metabolism, cellular debris clearing, and restoration of damaged or injured tissues. Thus, this review composes pertinent information regarding the involvement of autophagy in neural stem cells and glial regulation and the role of this connexion in normal brain functions, neurodevelopmental disorders, and neurodegenerative diseases. This review will provide insight into establishing a concrete strategic approach for investigating pathological mechanisms and developing therapies for brain diseases.
Incidence of cognitive and emotional alterations are reportedly two times more in diabetic patients than in non-diabetic population with hitherto unexplained causation and mechanism. Purview of the ...hippocampus functional diversity sanctions the accessibility and the necessity to investigate the regional neuro-immunological aspects of neurodegeneration and related functional alterations following diabetes. We examined the possible involvement of microglia activation, macrophage response, oxidative stress and inflammatory stature in both ventral and dorsal hippocampus of rats rendered diabetic by a single injection of streptozotocin (STZ; 45 mg/ kg body weight; intraperitoneal). Cognitive and behavioural alterations were studied using open field test (locomotor activity), elevated plus maze (anxiety), Barnes maze (spatial cognition) and T maze (working memory) at 2nd, 4th, 6th, 8th, 10th and 12th week post diabetic confirmation. Oxidative stress was investigated via measuring the level of lipid peroxidation biochemically. Scenario of microglia activation, macrophage response and inflammation was gauged using qualitative and quantitative analysis. Pronounced macrophage expression and activation directed microglia phenotypic switching was prominent in both ventral and dorsal hippocampus indicating the impact of oxidative stress following diabetes in hippocampus. The resultant inflammatory response was also progressive and persistent in both ventral and dorsal hippocampus parallel to the altered cognitive, locomotor ability and anxiety behaviour in diabetic rats. Conclusively, present data not only comprehends the microglia, macrophage physiology and related immune response in functionally different hippocampal regions associated cognitive and behavioural deficits, but also offers a suggestive region-specific cellular mechanism pathway for developing an imminent therapeutic approach during particular diabetes deficits.
•Alteration in dorsal and ventral hippocampus resulted in memory and mood deficits.•Diabetes linked oxidative stress exaggerate microglia and macrophage response.•Microglia activation instigate inflammatory response in diabetic hippocampus.•Connexion of metabolic stress, gliosis, cytokines cause diabetic cognitive deficits.
•Different genes were delivered into neurons connected by BDNF-TrkB synapses.•A synthetic peptide neurotransmitter containing BDNF and the His tag was developed.•This peptide neurotransmitter is ...designed to bind to TrkB on postsynaptic neurons.•Antibody-mediated, targeted gene transfer to postsynaptic neurons used anti-His tag.•The roles of neurons connected by BDNF-TrkB synapses may now be studied.
Brain-Derived Neurotrophic Factor (BDNF) signaling through TrkB receptors has important roles in synapse formation, synaptic plasticity, learning, and specific diseases. However, it is challenging to relate BDNF-TrkB synapses to circuit physiology or learning, as BDNF-TrkB synapses are embedded in complex circuits that contain numerous neuron and synapse types. Thus, analyzing the physiology of neurons connected by BDNF-TrkB synapses would be advanced by a technology to deliver different genes into presynaptic and postsynaptic neurons, connected by a BDNF-TrkB synapse. Here, we report selective gene transfer across BDNF-TrkB synapses: The model system was the large projection from rat postrhinal to perirhinal cortex. The first gene transfer, into presynaptic neurons in postrhinal cortex, used a virus vector and standard gene transfer procedures. This vector expresses a synthetic peptide neurotransmitter composed of three domains, a dense core vesicle sorting domain, BDNF, and the His tag. Upon release, this peptide neurotransmitter binds to TrkB receptors on postsynaptic neurons. The second gene transfer, into connected postsynaptic neurons in perirhinal cortex, uses antibody-mediated, targeted gene transfer and an anti-His tag antibody, as the synthetic peptide neurotransmitter contains the His tag. Confocal microscope images showed that using untargeted gene transfer, only 10–15% of the transduced presynaptic axons were proximal to a transduced postsynaptic dendrite. But using targeted gene transfer, ∼70% of the transduced presynaptic axons were proximal to a transduced postsynaptic dendrite. This technology may support studies on the roles of neurons connected by BDNF-TrkB synapses in circuit physiology and learning.
mGluR5-containing synapses have essential roles in synaptic plasticity, circuit physiology, and learning, and dysfunction at these synapses is implicated in specific neurological disorders. As ...mGluR5-containing synapses are embedded in large and complex distributed circuits containing many neuron and synapse types, it is challenging to elucidate the roles of these synapses, and to develop treatments for the associated disorders. Thus, it would be advantageous to deliver different genes into pre- and post-synaptic neurons connected by a mGluR5-containing synapse. Here, we develop this capability: The first gene transfer, into the presynaptic neurons, uses standard techniques to deliver a vector that expresses a synthetic peptide neurotransmitter. This peptide neurotransmitter has three domains, a dense core vesicle sorting domain, a mGluR5-binding domain composed of a single-chain variable fragment anti-mGluR5, and the His tag. Upon release, this peptide neurotransmitter binds to mGluR5, predominately located on the postsynaptic neurons. Selective gene transfer into these neurons uses antibody-mediated, targeted gene transfer and anti-His tag antibodies, as the synthetic peptide neurotransmitter contains the His tag. For the model system, we studied the connection between neurons in two neocortical areas, postrhinal and perirhinal cortices. Targeted gene transfer was over 80 % specific for mGluR5-containing synapses, but untargeted gene transfer was only ~15 % specific for these synapses. This technology may enable studies on the roles of mGluR5-containing neurons and synapses in circuit physiology and learning, and support gene therapy treatments for specific disorders that involve dysfunction at these synapses.
mGluR5-containing synapses have essential roles in synaptic plasticity, circuit physiology, and learning, and dysfunction at these synapses is implicated in specific neurological disorders. As ...mGluR5-containing synapses are embedded in large and complex distributed circuits containing many neuron and synapse types, it is challenging to elucidate the roles of these synapses and to develop treatments for the associated disorders. Thus, it would be advantageous to deliver different genes into pre- and postsynaptic neurons connected by a mGluR5-containing synapse. Here, we develop this capability: The first gene transfer, into the presynaptic neurons, uses standard techniques to deliver a vector that expresses a synthetic peptide neurotransmitter. This peptide neurotransmitter has three domains: a dense core vesicle sorting domain, a mGluR5-binding domain composed of a single-chain variable fragment anti-mGluR5, and the His tag. Upon release, this peptide neurotransmitter binds to mGluR5, predominately located on the postsynaptic neurons. Selective gene transfer into these neurons uses antibody-mediated, targeted gene transfer and anti-His tag antibodies, as the synthetic peptide neurotransmitter contains the His tag. For the model system, we studied the connection between neurons in two neocortical areas: postrhinal and perirhinal cortices. Targeted gene transfer was over 80% specific for mGluR5-containing synapses, but untargeted gene transfer was only ~ 15% specific for these synapses. This technology may enable studies on the roles of mGluR5-containing neurons and synapses in circuit physiology and learning and support gene therapy treatments for specific disorders that involve dysfunction at these synapses.
Behavioral impairments are the most empirical consequence of diabetes mellitus documented in both humans and animal models, but the underlying causes are still poorly understood. As the cerebellum ...plays a major role in coordination and execution of the motor functions, we investigated the possible involvement of glial activation, cellular degeneration and glutamate transportation in the cerebellum of rats, rendered diabetic by a single injection of streptozotocin (STZ; 45 mg/kg body weight; intraperitoneally). Motor function alterations were studied using Rotarod test (motor coordination) and grip strength (muscle activity) at 2nd, 4th, 6th, 8th, 10th, and 12th week post-diabetic confirmation. Scenario of glial (astroglia and microglia) activation, cell death and glutamate transportation was gaged using immunohistochemistry, histological study and image analysis. Cellular degeneration was clearly demarcated in the diabetic cerebellum. Glial cells were showing sequential and marked activation following diabetes in terms of both morphology and cell number. Bergmann glial cells were hypertrophied and distorted. Active caspase-3 positive apoptotic cells were profoundly present in all three cerebellar layers. Reduced co-labeling of GLT-1 and GFAP revealed the altered glutamate transportation in cerebellum following diabetes. These results, exclusively derived from histology, immunohistochemistry and cellular quantification, provide first insight over the associative reciprocity between the glial activation, cellular degeneration and reduced glutamate transportation, which presumably lead to the behavioral alterations following STZ-induced diabetes.
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