Utilizing human cerebral organoids to model breast cancer brain metastasis in culture.

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.

An identified ensemble within a neocortical circuit encodes essential information for genetically-enhanced visual shape learning.

Advanced cognitive tasks are encoded in distributed neocortical circuits that span multiple forebrain areas. Nonetheless, synaptic plasticity and neural network theories hypothesize that essential information for performing these tasks is encoded in specific ensembles within these circuits. Relatively simpler subcortical areas contain specific ensembles that encode learning, suggesting that neocortical circuits contain such ensembles. Previously, using localized gene transfer of a constitutively active protein kinase C (PKC), we established that a genetically-modified circuit in rat postrhinal cortex, part of the hippocampal formation, can encode some essential information for performing specific visual shape discriminations. However, these studies did not identify any specific neurons that encode learning; the entire circuit might be required. Here, we show that both learning and recall require fast neurotransmitter release from an identified ensemble within this circuit, the transduced neurons; we blocked fast release from these neurons by coexpressing a Synaptotagmin I siRNA with the constitutively active PKC. During learning or recall, specific signaling pathways required for learning are activated in this ensemble; during learning, calcium/calmodulin-dependent protein kinase II, MAP kinase, and CREB are activated; and, during recall, dendritic protein synthesis and CREB are activated. Using activity-dependent gene imaging, we showed that during learning, activity in this ensemble is required to recruit and activate the circuit. Further, after learning, during image presentation, blocking activity in this ensemble reduces accuracy, even though most of the rest of the circuit is activated. Thus, an identified ensemble within a neocortical circuit encodes essential information for performing an advanced cognitive task.

Astrocytic and microglial response in experimentally induced diabetic rat brain.

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 S100ß 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.

Microglia activation and inflammation in hippocampus attenuates memory and mood functions during experimentally induced diabetes in rat.

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.

Connected neurons in multiple neocortical areas, comprising parallel circuits, encode essential information for visual shape learning.

Neocortical areas comprised of multiple neuronal circuits which are encoded with innumerable advanced cognitive tasks. Studies focused on neuronal network and synaptic plasticity has hypothesized that every specific neuron and the circuit process the explicit essential information for the specific tasks. However, the structure of these circuits and the involved critical neurons remain to be elucidated. Considering our previous studies, showing the specificity of rat postrhinal cortex comprising specific neuronal circuit for encoding both the learning and recall of shape discrimination through a fast neurotransmitter release from the transduced neurons, here we have demonstrated that postsynaptic neurons in two distinct areas, perirhinal cortex and the ventral temporal association areas are required for the specific visual shape discriminations learning. The constitutively active PKC was delivered into neuronal cells in postrhinal cortex, and the animals were allowed to learn the new shape discriminations, and then the silencing siRNA was delivered into postsynaptic neurons in either perirhinal cortex or ventral temporal association areas, using a novel technology for gene transfer into connected neurons. We observed that expression of the siRNA caused the deficits in visual performance, via blocking the activity in the neurons, as displayed by activity-dependent gene imaging, and also subsequently obstructed the activation of specific signaling pathways required for further learning, and dendritic protein synthesis and CREB. Thus, ratifying the conclusion that the two parallel circuits are both required for the visual shape discrimination learning.

A high fat, sugar, and salt Western diet induces motor-muscular and sensory dysfunctions and neurodegeneration in mice during aging: Ameliorative action of metformin.

AIMS: To explore the novel linkage between a Western diet combining high saturated fat, sugar, and salt (HFSS) and neurological dysfunctions during aging as well as Metformin intervention, we assessed cerebral cortex abnormalities associated with sensory and motor dysfunctions and cellular and molecular insights in brains using HFSS-fed mice during aging. We also explored the effect of Metformin treatment on these mice. METHODS: C57BL/6 mice were fed with HFSS and treated with metformin from 20 to 22 months of age, resembling human aging from 56 to 68 years of age (an entry phase of the aged portion of lifespan). RESULTS: The motor and sensory cortexes in mice during aging after HFSS diet showed: (A) decreased motor-muscular and sensory functions; (B) reduced inflammation-resolving Arg-1+ microglia; (C) increased inflammatory iNOs+ microglia and TNFα levels; (D) enhanced abundance of amyloid-ß peptide and of phosphorylated Tau. Metformin attenuated these changes. CONCLUSION: A HFSS-combined diet caused motor-muscular and sensory dysfunctions, neuroinflammation, and neurodegeneration, whereas metformin counteracted these effects. Our findings show neuroinflammatory consequences of a HFSS diet in aging. Metformin curbs the HFSS-related neuroinflammation eliciting neuroprotection.

Iba1 expressing microglia in the dorsal root ganglia become activated following peripheral nerve injury in rats.

The presence of microglia in dorsal root ganglia (DRG) has not been reported earlier. The dorsal root ganglia contain satellite glial cells (SGCs) and macrophages, which are considered to have infiltrated from the systemic blood. An attempt was made to investigate whether microglia as found in the central nervous system are also present in the dorsal root ganglia of untreated rats and following experimental peripheral nerve injury. Female adult Wistar rats were subjected to sciatic nerve transection injury on the right hand side. The DRGs of the right side were studied with the contralateral DRGs of the left side serving as controls. The tissues, harvested at different time points after injury, following intracardial perfusion fixation, and frozen sections were immunolabeled with anti-GFAP as a marker for SGCs and anti-Iba1 and OX-6 as markers for microglia and activated macrophagic microglia, respectively. These antibodies were also used in combination to ascertain if Iba1+ cells are the SGCs or otherwise and also if macrophagic OX-6+ cells are Iba1 positive microglia. The results indicate that Iba1 positive microglial cells are different from the SGCs in the DRGs. The Iba1 positive microglial cells respond to the sciatic nerve injury becoming activated and macrophagic and express MHCII molecules. Such activated microglia apparently may serve as neurosupportive cells, providing neuroprotection and scavenging cellular debris in response to the injury.

Separate Gene Transfers into Pre- and Postsynaptic Neocortical Neurons Connected by mGluR5-Containing 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.

Delivery of different genes into presynaptic and postsynaptic neocortical neurons connected by a BDNF-TrkB synapse.

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.

Efficient gene transfers into neocortical neurons connected by NMDA NR1-containing synapses.

BACKGROUND: Within a circuit, specific neurons and synapses are hypothesized to have essential roles in circuit physiology and learning, and dysfunction in these neurons and synapses causes specific disorders. These critical neurons and synapses are embedded in complex circuits containing many neuron and synapse types. NEW METHOD: We established technology that can deliver different genes into pre- and post-synaptic neurons connected by a specific synapse type. The first, presynaptic gene transfer employs standard gene transfer technology to express a synthetic peptide neurotransmitter which has three domains, a dense core vesicle sorting domain for processing the protein as a peptide neurotransmitter, a receptor-binding domain, here a small peptide that binds to NMDA NR1 subunits, and the His tag. Upon release, this peptide neurotransmitter binds to its cognate receptor on postsynaptic neurons. Gene transfer selectively into these postsynaptic neurons employs antibody-mediated, targeted gene transfer and anti-His tag antibodies, which recognize the His tag domain in the synthetic peptide neurotransmitter. RESULTS: For the model system, we studied the connection from projection neurons in postrhinal cortex to specific neurons in perirhinal cortex. In our initial report, gene transfer to connected neurons was 20+1% specific. Here, we optimized the technology; we improved the transfection for packaging by using a modern using a modern lipid, Lipofectamine 3000, and used a modern confocal microscope to collect data. We now report 80+2% specific gene transfer to connected neurons. COMPARISON WITH EXISTING METHODS: There is no existing method with this capability. CONCLUSIONS: This technology may enable studies on the roles of specific neurons and synapses in circuit physiology and learning, and support gene therapy treatments for specific disorders.

Delivery of different genes into pre- and post-synaptic neocortical interneurons connected by GABAergic synapses.

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.

Experimentally induced diabetes causes glial activation, glutamate toxicity and cellular damage leading to changes in motor function.

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.