Gali, Chaitanya Chakravarthi

ABSTRACT Studies in humans and murine models have pointed towards a possible link between metabolic syndrome, which shows insulin resistance and metabolic dysregulation, and Alzheimer’s disease (AD) pathology marked by amyloid-beta (Aβ) accumulation at the BBB and hypometabolism in the brain. Yet, the underlying biological mechanisms by which metabolic syndrome affects these pathological changes in AD brain remain unknown. We hypothesized that insulin resistance is responsible for alterations in blood-brain barrier (BBB) transport of Aβ peptides and glucose. This hypothesis was tested by employing radiolabeled tracers ( 125 I-Aβ40, 125 I-Aβ42, and 18 F-FDG) in high-fat diet (HFD)-fed mouse models that manifest metabolic syndrome. Further, we assessed alterations in the expression of various molecular mediators within the brain microcapillaries harvested from both low-fat diet (LFD)-fed and HFD-fed mice. As expected, our studies show that HFD- fed mice developed peripheral insulin resistance and obesity. In addition, HFD-fed mice demonstrated an increase in the influx rate of Aβ peptides and a reduction in 18 F-FDG (a glucose surrogate) influx rate at the BBB compared to LFD-fed mice. These transport changes are associated with increase in the BBB endothelial expression of RAGE (receptor to traffic Aβ from plasma-to-brain) and a reduction of GLUT1 (glucose transporter) expression in HFD-fed mice compared to LFD-fed mice. Moreover, disruption in insulin signaling, as indicated by reduced pAKT and pERK expression, was observed in HFD-fed mice. Inhibiting AKT or ERK phosphorylation with specific inhibitors resulted in similar changes in Aβ and glucose uptake in polarized BBB endothelial cell monolayers in vitro. These results indicate that HFD induced metabolic syndrome may lead to BBB dysfunction, characterized by increased plasma-to-brain Aβ trafficking and diminished glucose transport at the BBB, thereby aggravating the expression of AD pathological hallmarks.

Background: Histone deacetylases (HDACs) are the enzymes that catalyze the removal of the acetyl group from lysine residues and regulate several biological processes. Suberoylanilide hydroxamic acid (SAHA) is a notable HDAC inhibitor that exhibited remarkable anti-proliferative efficiency by alleviating gene regulation against solid and hematologic cancers. Aim: The aim of this study was to develop new chemotherapeutic agents for breast cancer treatment, therefore, a novel series of Suberoylanilide hydroxamic acid (SAHA) analogs were investigated as anticancer agents. Methods: We designed and synthesized a novel series of analogs derived from SAHA by substituting alkyl, alkoxy, halo, and benzyl groups at different positions of the phenyl ring. The newly synthesized analogs were assessed for their cytotoxic potential against four human cancer cell lines in comparison with healthy cell lines, using several biological assays Results: SAHA analogs displayed significant cytotoxic potential with IC50 values ranging from 1.6 to 19.2 μM in various tumor cell lines. Among these analogs, 2d (containing 3-chloro, 4-floro substitutions on phenyl moiety), 2h (containing 3,4-di chloro substitutions on phenyl moiety), and 2j (containing 4-chloro, 3-methyl substitutions on phenyl moiety) showed significant cytotoxic potential with IC50 values ranging from 1.6 to 1.8 μM in MCF-7 (breast carcinoma) cell line. More importantly, these analogs were found to be non-toxic towards healthy primary human hepatocytes (PHH) and mouse fibroblast cells (NIH3T3), which represent their tumor selectivity. These analogs were further analyzed for their effect on cell migration, BrdU incorporation, Annexin V-FITC and cell cycle arrest (Sub-G1 phase). Remarkably, analogs 2d, 2h, and 2j displayed significant HDAC inhibition than the parent SAHA molecule. Further studies also confirmed that these SAHA analogs are efficient in inducing apoptosis, as they regulated the expression of several proteins involved in mitochondrial or intrinsic apoptosis pathways. Findings in the Chick Chorioallantoic Membrane (CAM) assay studies revealed anti-angiogenic properties of the currently described SAHA analogs Conclusion: From anti-proliferative study results, it is clearly evident that 3,4-substitution at the SAHA phenyl ring improves the anti-proliferative activity of SAHA. Based on these findings, we presume that the synthesized novel SAHA analogs could be potential therapeutic agents in treating breast cancer.

AbstractBackgroundDecreased brain insulin levels exacerbate cognitive decline in AD. Insulin in the brain is derived from systemic circulation via the blood‐brain barrier (BBB). We hypothesize that type II diabetes (T2D) sequelae and Aβ peptide exposure disrupt insulin signaling at the BBB and inhibit insulin delivery to brain. Further, we propose insulin signaling defects at the BBB contribute to Aβ accumulation in AD brain.MethodsThe following studies were performed in wild‐type (WT) mice on regular chow (RC) diet, WT mice on high fat (HF) diet (manifest insulin resistance), APP/PS1 transgenic mice on RC diet (overexpress Aβ), and APP/PS1 mice on HF diet (overexpress Aβ + insulin resistance). After femoral injection of 125I‐insulin or 125I‐Aβ42, the brain accumulation was monitored between 0‐40 min by dynamic SPECT/CT imaging. The brain influx clearance was estimated by the slope obtained from Gjedde‐Patlak graphical analysis. Cerebral microvessels were harvested, and reverse phase protein array (RPPA) was performed to examine insulin signaling changes. Differentially expressed targets were subsequently confirmed by western blot. The brain influx of 125I‐insulin and 125I‐Aβ42 were further assessed in WT‐RC mice after internal carotid infusion with AG1024, a kinase inhibitor of the insulin receptor (IR) and insulin‐like growth factor receptor (IGF‐1R).ResultsCompared to WT‐RC, 125I‐insulin influx was decreased in WT‐HF and APP/PS1‐RC, and was further decreased in APP/PS1‐HF (4.1, 3.4, 3.5 and 2.6*10‐4 mL/min, respectively). Compared to WT‐RC, 125I‐Aβ42 influx was increased in WT‐HF and APP/PS1‐RC (8.6, 28 and 23*10‐4 mL/min, respectively). RPPA analysis revealed global disruptions in BBB insulin signaling pathways. Western blots confirmed reduced expression of IR‐β, p‐AKT and p‐GSK3β in WT‐HF and APP/PS1‐RC compared to WT‐RC. Moreover, the largest decreases were observed in APP/PS1‐HF. Infusion with AG1024 was shown to decrease 125I‐insulin influx, but increase 125I‐Aβ42 influx.ConclusionsBoth T2D and AD mice exhibited decreased brain influx of insulin and increased influx of Aβ42. This was associated with altered expression/activity of insulin signaling kinases at the BBB. Further, IR and/or IGF‐1R kinase activity were shown to differentially regulate BBB trafficking of insulin and Aβ42. Thus, BBB insulin signaling is important for delivering insulin to brain and restricting pathological uptake of Aβ.