Mefloquine induces ER stress and apoptosis in BRAFi-resistant A375-BRAFV600E /NRASQ61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model.

Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E -driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. The use of clinical antimalarials for the investigational treatment of malignant melanoma has shown only moderate promise, attributed mostly to inhibition of lysosomal-autophagic adaptations of cancer cells, but identification of specific antimalarials displaying single-agent antimelanoma activity has remained elusive. Here, we have screened a focused library of clinically used artemisinin-combination therapeutic (ACT) antimalarials for the apoptotic elimination of cultured malignant melanoma cell lines, also examining feasibility of overcoming BRAFi-resistance comparing isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E /NRASQ61 vs. BRAFi-resistant A375-BRAFV600E /NRASQ61K ). Among ACT antimalarials tested, mefloquine (MQ) was the only apoptogenic agent causing melanoma cell death at low micromolar concentrations. Comparative gene expression-array analysis (A375-BRAFV600E /NRASQ61 vs. A375-BRAFV600E /NRASQ61K ) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ-induced loss of viability. ER-trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho-eIF2α, XBP-1s, ATF4). Fluo-4 AM-fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375-Luc2 (BRAFV600E /NRASQ61K ) cells, an oral MQ regimen efficiently antagonized brain tumor growth. Taken together, these data suggest that the clinical antimalarial MQ may be a valid candidate for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells, even if metastasized to the brain and BRAFi-resistant.

The angiotensin (1-7) glycopeptide PNA5 improves cognition in a chronic progressive mouse model of Parkinson's disease through modulation of neuroinflammation.

Cognitive decline in Parkinson's Disease (PD) is a prevalent and undertreated aspect of disease. Currently, no therapeutics adequately improve this aspect of disease. It has been previously shown that MAS receptor agonism via the glycosylated Angiotensin (1-7) peptide, PNA5, effectively reduces cognitive decline in models of vascular contributions to cognitive impairment and dementia (VCID). PNA5 has a brain/plasma ratio of 0.255 indicating good brain penetration. The goal of the present study was to determine if (1) systemic administration of PNA5 rescued cognitive decline in a mouse model of PD, and (2) if improvements in cognitive status could be correlated with changes to histopathological or blood plasma-based changes. Mice over-expressing human, wild-type α-synuclein (αSyn) under the Thy1 promoter (Thy1-αSyn mice, "line 61") were used as a model of PD with cognitive decline. Thy1-αSyn mice were treated with a systemic dose of PNA5, or saline (1 mg/kg/day) beginning at 4 months of age and underwent behavioral testing at 6 months, compared to WT. Subsequently, mice brains were analyzed for changes to brain pathology, and blood plasma was examined with a Multiplex Immunoassay for peripheral cytokine changes. Treatment with PNA5 reversed cognitive dysfunction measured by Novel Object Recognition and spontaneous alteration in a Y-maze in Thy1-αSyn mice. PNA5 treatment was specific to cognitive deficits, as fine-motor disturbances were unchanged. Enhanced cognition was associated with decreases in hippocampal inflammation and reductions in circulating levels of Macrophage Induced Protein (MIP-1ß). Additionally, neuronal loss was blunted within the CA3 hippocampal region of PNA5-treated αsyn mice. These data reveal that PNA5 treatment reduces cognitive dysfunction in a mouse model of PD. These changes are associated with decreased MIP-1ß levels in plasma identifying a candidate biomarker for target engagement. Thus, PNA5 treatment could potentially fill the therapeutic gap for cognitive decline in PD.

Enhanced NRF2 expression mitigates the decline in neural stem cell function during aging.

Although it is known that aging affects neural stem progenitor cell (NSPC) biology in fundamental ways, the underlying dynamics of this process are not fully understood. Our previous work identified a specific critical period (CP) of decline in NSPC activity and function during middle age (13-15 months), and revealed the reduced expression of the redox-sensitive transcription factor, NRF2, as a key mediator of this process. Here, we investigated whether augmenting NRF2 expression could potentially mitigate the NSPC decline across the identified CP. NRF2 expression in subventricular zone (SVZ) NSPCs was upregulated via GFP tagged recombinant adeno-associated viral vectors (AAV-NRF2-eGFP), and its cellular and behavioral effects compared to animals that received control vectors (AAV-eGFP). The vectors were administered into the SVZs of aging rats, at time points either before or after the CP. Results indicate that animals that had received AAV-NRF2-eGFP, prior to the CP (11 months of age), exhibited substantially improved behavioral function (fine olfactory discrimination and motor tasks) in comparison to those receiving control viruses. Further analysis revealed that NSPC proliferation, self-renewal, neurogenesis, and migration to the olfactory bulb had significantly increased upon NRF2 upregulation. On the other hand, increasing NRF2 after the CP (at 20 months of age) produced no notable changes in NSPC activity at either cellular or behavioral levels. These results, for the first time, indicate NRF2 pathway modulation as a means to support NSPC function with age and highlight a critical time-dependency for activating NRF2 to enhance NSPC function.

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization.

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy.

The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

Effects of the iron oxide nanoparticle Molday ION Rhodamine B on the viability and regenerative function of neural stem cells: relevance to clinical translation.

An essential component of developing successful neural stem cell (NSC)-based therapies involves the establishment of methodologies to noninvasively monitor grafted NSCs within brain tissues in real time. In this context, ex vivo labeling with ultrasmall superparamagnetic iron oxide (USPIO) particles has been shown to enable efficient tracking of transplanted NSCs via magnetic resonance imaging (MRI). However, whether and how USPIO labeling affects the intrinsic biology of NSCs is not thoroughly understood, and remains an active area of investigation. Here, we perform a comprehensive examination of rat NSC survival and regenerative function upon labeling with the USPIO, Molday ION Rhodamine B (MIRB), which allows for dual magnetic resonance and optical imaging. After optimization of labeling efficiency, two specific doses of MIRB (20 and 50 µg/mL) were chosen and were followed for the rest of the study. We observed that both MIRB doses supported the robust detection of NSCs, over an extended period of time in vitro and in vivo after transplantation into the striata of host rats, using MRI and post hoc fluorescence imaging. Both in culture and after neural transplantation, the higher 50 µg/mL MIRB dose significantly reduced the survival, proliferation, and differentiation rate of the NSCs. Interestingly, although the lower 20 µg/mL MIRB labeling did not produce overtly negative effects, it increased the proliferation and glial differentiation of the NSCs. Additionally, application of this dose also changed the morphological characteristics of neurons and glia produced after NSC differentiation. Importantly, the transplantation of NSCs labeled with either of the two MIRB doses upregulated the immune response in recipient animals. In particular, in animals receiving the 50 µg/mL MIRB-labeled NSCs, this immune response consisted of an increased number of CD68(+)-activated microglia, which appeared to have phagocytosed MIRB particles and cells contributing to an exaggerated MRI signal dropout in the animals. Overall, these results indicate that although USPIO particles, such as MIRB, may have advantageous labeling and magnetic resonance-sensitive features for NSC tracking, a further examination of their effects might be necessary before they can be used in clinical scenarios of cell-based transplantation.

Reduced Nrf2 expression mediates the decline in neural stem cell function during a critical middle-age period.

Although it is known that the regenerative function of neural stem/progenitor cells (NSPCs) declines with age, causal mechanisms underlying this phenomenon are not understood. Here, we systematically analyze subventricular zone (SVZ) NSPCs, in various groups of rats across the aging spectrum, using in vitro and in vivo histological and behavioral techniques. These studies indicate that although NSPC function continuously declines with advancing age, there is a critical time period during middle age (13-15 months) when a striking reduction in NSPC survival and regeneration (proliferation and neuronal differentiation) occurs. The studies also indicate that this specific temporal pattern of NSPC deterioration is functionally relevant at a behavioral level and correlates with the decreasing expression of the redox-sensitive transcription factor, Nrf2, in the NSPCs. When Nrf2 expression was suppressed in 'young' NSPCs, using short interfering RNAs, the survival and regeneration of the NSPCs was significantly compromised and mirrored 'old' NSPCs. Conversely, Nrf2 overexpression in 'old' NSPCs rendered them similar to 'young' NSPCs, and they showed increased survival and regeneration. Furthermore, examination of newborn Nrf2 knockout (Nrf2 -/-) mice revealed a lower number of SVZ NSPCs in these animals, when compared to wild-type controls. In addition, the proliferative and neurogenic potential of the NSPCs was also compromised in the Nrf2-/- mice. These results identify a novel regulatory role for Nrf2 in NSPC function during aging and have important implications for developing NSPC-based strategies to support healthy aging and to treat age-related neurodegenerative disorders.

Systemic human CD34(+) cells populate the brain and activate host mechanisms to counteract nigrostriatal degeneration.

AIM: Here we investigated the neuroprotective potential of systemic CD34(+) human cord blood cells (hCBCs) in a 6-hydroxydopamine rat model of Parkinson's disease. METHODS: Purified CD34(+) hCBCs were intravenously administered to rats subjected to 6-hydroxydopamine 24 h earlier, and behavioral and immunohistological analysis performed. RESULTS: CD34(+) hCBC administration significantly prevented host nigrostriatal degeneration inducing behavioral recovery in treated rats. Although donor hCBCs did not differentiate into neural phenotypes, they stimulated the production of new neuroblasts and angiogenesis, and reduced gliosis in recipient animals. Importantly, surviving donor hCBCs were identified, and their tissue distribution pattern correlated with the observed therapeutic effects. CONCLUSION: Peripherally applied CD34(+) hCBCs can migrate into brain tissues and elicit host-based protective mechanisms to support the survival of midbrain dopamine neurons.

Altered soluble epoxide hydrolase gene expression and function and vascular disease risk in the stroke-prone spontaneously hypertensive rat.

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids and represents a novel therapeutic target in cardiovascular disease treatment. We investigated the relationship among sequence variation in the sEH gene (Ephx2), sEH function, and risk of end-organ injury in strains of spontaneously hypertensive rat (SHRs) differing in their susceptibility to develop brain vascular disease. Brain Ephx2 expression was significantly lower in stroke-prone (SHR/A3) than in stroke-resistant (SHR/N) SHRs (5-fold; P<0.0001). Resequencing of the Ephx2 promoter in the 2 strains identified 3 polymorphisms that significantly influenced promoter transcriptional activity in vitro. Measurements of brain sEH enzyme activity and plasma levels of arachidonate and linoleate metabolites of sEH further suggested significant differences between the 2 strains. Ratios of epoxyoctadecenoic acids to dihydroxyoctadecenoic acids were significantly higher, indicating a lower sEH activity in SHR/A3 than in SHR/N (P<0.0001). Plasma dihydroxyeicosatrienoic acid levels were lower in SHR/A3 than in SHR/N (P<0.0001), but plasma epoxyeicosatrienoic acids levels were similar in the 2 strains. Association analysis of Ephx2 polymorphism in the F2 progeny of an SHR/A3xSHR/N cross showed that animals carrying the SHR/A3 allele of Ephx2 had a greater risk of stroke and associated urinary proteinuria than animals that do not. Investigation of patterns of allelic similarities and differences among multiple stroke-prone and stroke-resistant SHR substrains showed that Ephx2 belongs to a haplotype block shared among all of the stroke-prone but no stroke-resistant substrains. These data support a role for Ephx2 polymorphism on sEH gene expression and function and risk of end-organ injury in the stroke-prone SHR.