In connection with our research program on the development of new isatin-based anticancer candidates, herein we report the synthesis of two novel series of thiazolidinone-isatin conjugates (4a–n) and thiazolo[3,2-a]benzimidazolone-isatin conjugates (7a–d), and in vitro evaluation of their antiproliferative activity towards two breast cancer cell lines; triple negative MDA-MB-231, and MCF-7. Compounds 4m and 7b emerged as the most active congeners against MDA-MB-231 cells (IC₅₀ = 7.6 ± 0.5 and 13.2 ± 1.1 µM, respectively). Compounds 4m and 7b were able to provoke apoptosis in MDA-MB-231 cells, evidenced by the up-regulation of Bax and down-regulation of Bcl-2, besides boosting caspase-3 levels. Hybrid 4m induced a fourfold increase in the percentage of cells at Sub-G₁, with concurrent arrest in G₂-M phase by 2.5-folds. Furthermore, hybrid 4m resulted in a sixfold increase in the percentage of annexin V-FITC positive apoptotic MDA-MB-231 cells as compared with the control. Moreover, the cytotoxic activities of the active conjugates were assessed towards two nontumorigenic cell lines (breast MCF-10A and lung WI-38) where both conjugates 4m and 7b displayed mean tumor selectivity index: 9.6 and 13.9, respectively. Finally, several ADME descriptors were predicted for the active conjugates via a theoretical kinetic study.

The metabolite lactate (L‐lactate) has been hypothesized to represent an important energy source during brain activation. The contribution of lactate in fueling synchronized synaptic transmission during fast neural network oscillations underlying complex cortex function such as visual perception, memory formation, and motor activity is less clear, however. We explored the role of cellular lactate production and lactate transport (uptake and release) via the monocarboxylate transporters 1 and 2 (glial MCT1 and neuronal MCT2) during persistent gamma oscillations (frequency at around 40 Hz) and recurrent rhythmic events called sharp wave‐ripples (with “ripples” at around 250 Hz) in cultured rat and acute mouse hippocampal slices (ex vivo) that received energy substrate supply with glucose (D‐glucose) only. In addition, we assessed neuronal lactate dynamics during spontaneous activity (“resting state”) and during electrical stimulation (10 Hz) in mouse primary neuron‐astrocyte cultures (in vitro) receiving glucose only. We combined electrophysiology (local field potential recordings), tissue lactate analysis [ultra‐performance liquid chromatography‐mass spectrometry (UPLC‐MS)], and live‐cell fluorescence imaging [Förster resonance energy transfer (FRET) sensor Laconic]. We report that (1) lactate is produced during gamma oscillations when glucose is supplied and oxygen availability is unlimited (high oxygenation) for mitochondrial respiration. (2) The properties of gamma oscillations remain regular in the presence of the MCT1/2 blocker AR‐C155858. (3) By contrast, MCT1/2 blockade fully suppresses gamma oscillations when mainly lactate is supplied. (4) The properties of sharp wave‐ripples remain regular during MCT1/2 inhibition. (5) Lactate is produced in primary hippocampal neurons during spontaneous activity and electric stimulus‐induced excitation, and it accumulates in the neuronal cytosol during MCT1/2 inhibition. In conclusion, lactate is produced in cortical tissue, including neurons fueled by glucose only. Moreover, lactate transport and lactate exchange (“shuttling”) via glial MCT1 and neuronal MCT2 are not required to sustain synchronized synaptic transmission during fast neural network oscillations. image

Background: The cytokine tumor necrosis factor-alpha (TNF-α) regulates inflammatory responses in infectious and neurodegenerative diseases and also affects neuronal function. The role of TNF-α in the activation of microglial cells (resident central nervous system macrophages), including the impact on neuronal survival, excitability, and synaptic transmission is incompletely defined, however. We explored the effects of chronic TNF-α exposure (72 h) on microglia and neurons in organotypic hippocampal slice cultures from male and female rats, i.e., postnatal cortex tissue lacking leukocyte invasion and adaptive immunity. Methods: We applied gene expression analysis, biochemical assays, immunohistochemistry, electrophysiology by extracellular (local field potential) and intracellular (intrinsic membrane properties) recordings, and pharmacological ablation of the microglial cell population. We mainly focused on carbachol-induced neural network oscillations (brain waves) in the gamma frequency band (30–70 Hz) that underlie higher cognitive functions such as perception, attention, and memory. Results: TNF-α induced microglial proliferation and upregulation of genes related to inflammation and oxidative stress such as Il6 (interleukin-6), Nos2 [inducible nitric oxide (NO) synthase, iNOS] and Sod2 (superoxide dismutase 2), which was accompanied by a decreased number of slices showing gamma oscillations in extracellular recordings. Notably, a fraction of slices presented neural bursting reflecting hyperexcitability in the tissue. Neuronal dysfunction was absent during acute TNF-α exposure (30 min). When paired with the lymphocyte cytokine interferon-gamma (IFN-γ), TNF-α induced an amplified neuroinflammation response dominated by bursting or loss of electrical activity. In intracellular recordings, neurons showed a brief burst of action potentials followed by slowing of spiking with pronounced afterhyperpolarization (switch from regular to burst firing behavior) during depolarizing current injection. Notably, the impairments could be attenuated by inhibition of iNOS and NADPH oxidase, glucose supplementation, microglial depletion or blockade of TNF receptor 1 (TNFR1) signaling with small molecule drugs, RIPA-56 and ICCB-19. Conclusions: Our data provide mechanistic insight into TNF-α- and IFN-γ-induced neuronal impairments mediated by microglial NO, metabolic and oxidative stress, and demonstrate functional neuroprotection by pharmacology. Our study extends the pathophysiological understanding of diseases such as sepsis, multiple sclerosis, Alzheimer’s disease, depression and schizophrenia featuring activated microglia, infiltrating monocytes and T cells, and/or blood-brain barrier leakage.