Cell growth and nutrient availability control the mitotic exit signaling network in budding yeast.

Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients can influence cell size by modulating the extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that modulate bud growth in response to nutrient availability. This led to the discovery that nutrients regulate numerous components of the mitotic exit network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multisite phosphorylation during bud growth that is dependent upon bud growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to override a growth requirement for mitotic exit. The data suggest a model in which the MEN ensures that mitotic exit occurs only when an appropriate amount of bud growth has occurred.

Dichotomous effect of methylphenidate on microglia and astrocytes: Insights from in vitro and animal studies.

Methylphenidate (MPH) has been used for decades to treat attention-deficit/hyperactivity disorder (ADHD) and narcolepsy. Moreover, several studies have shown that it is subject to misuse, particularly among college students and adolescents, for cognitive enhancement or as a recreational drug. This phenomenon causes concern, and it is critical to clarify better how MPH impacts brain cells. In fact, data has suggested that MPH could result in neuroinflammation and neurodegeneration across several brain regions; however, little is known about the effect of MPH on glial cells. To address this, we used microglia N9 cell line and primary cultures of cortical astrocytes that were exposed to MPH (0.01 - 2 mM), as well as Wistar Kyoto rats (WKY) chronically administered with MPH (1.5 mg/kg/day). Several parameters were analyzed, and we concluded that MPH has no significant direct effect on microglial cells, apart from cell migration impairment. On the contrary, MPH promotes astrogliosis, oxidative/nitrosative stress, and increases proinflammatory cytokine TNF levels by astrocytes, which was concordant with the results obtained in the hippocampus of WKY rats. Overall, the present results suggest that brain cells respond differently to MPH, with a more prominent direct effect on astrocytes when compared to microglia.

Attention-Deficit/Hyperactivity Disorder Animal Model Presents Retinal Alterations and Methylphenidate Has a Differential Effect in ADHD versus Control Conditions.

Attention-Deficit/Hyperactivity Disorder (ADHD) is one of the most prevalent neurodevelopmental disorders. Interestingly, children with ADHD seem to experience more ophthalmologic abnormalities, and the impact of methylphenidate (MPH) use on retinal physiology remains unclear. Thus, we aimed to unravel the retina's structural, functional, and cellular alterations and the impact of MPH in ADHD versus the control conditions. For that, spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were used as animal models of ADHD and the controls, respectively. Animals were divided into four experimental groups as follows: WKY vehicle (Veh; tap water), WKY MPH (1.5 mg/kg/day), SHR Veh, SHR MPH. Individual administration was performed by gavage between P28-P55. Retinal physiology and structure were evaluated at P56 followed by tissue collection and analysis. The ADHD animal model presents the retinal structural, functional, and neuronal deficits, as well as the microglial reactivity, astrogliosis, blood-retinal barrier (BRB) hyperpermeability and a pro-inflammatory status. In this model, MPH had a beneficial effect on reducing microgliosis, BRB dysfunction, and inflammatory response, but did not correct the neuronal and functional alterations in the retina. Curiously, in the control animals, MPH showed an opposite effect since it impaired the retinal function, neuronal cells, and BRB integrity, and also promoted both microglia reactivity and upregulation of pro-inflammatory mediators. This study unveils the retinal alterations in ADHD and the opposite effects induced by MPH in the retina of ADHD and the control animal models.

Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

A multi-staged neuropeptide response to traumatic brain injury.

PURPOSE: As the most abundant neuropeptides in Central Nervous System, Substance P and Neuropeptide Y are arguably involved in the response to brain trauma. This study aims to characterize a new concept of multi-staged neuropeptide response to TBI. METHODS: This study assessed Substance P, Neuropeptide Y, S100B, standard inflammatory parameters and ionic disturbance in TBI victims, with and without intracranial lesions, and healthy controls. In the group with intracranial lesions, blood samples were drawn until 6 h after initial trauma, at 48 h and 7 days post-TBI. RESULTS: An early increase in Substance P (mean 613.463 ± 49.055 SE 6 h post-TBI with brain contusions vs. 441.441 ± 22.572 SE pg/dL control group) is evident. Concerning TBI without intraparenchymatous lesions, an increase in substance P is also present (825.60 ± 23.690 SE pg/dL). Following an initial increase and subsequent fall in NPY levels (45.997 ± 4.96 SE 6 h post-TBI vs. 32.395 ± 4.056 SE 48 h post-TBI vs. 19.700 ± 1.462 SE pg/mL control group), a late increase in NPY is obvious (43.268 ± 6.260 SE pg/mL 7 day post-TBI). Post-traumatic hypomagnesemia (0.754 ± 0.015 SE 6 h post-TBI vs. 0.897 ± 0.021 SE mmol/L control group) and a peak in S100B (95.668 ± 14.102 SE 6 h post-TBI vs. 30.187 ± 3.347 SE pg/mL control group) are also present. CONCLUSION: A multi-staged neuropeptide response to TBI is obvious and represents a potential therapeutic strategy for the treatment of intraparenchymal lesions and cerebral edema following TBI.

The effect of parthenolide on methamphetamine-induced blood-brain barrier and astrocyte alterations.

BACKGROUND: Methamphetamine abuse is a worldwide concern with long-term health complications. Its impact on neurons has been extensively investigated, and it is currently known that glial cells, including astrocytes, are involved in drug-induced outcomes. Importantly, METH also causes blood-brain barrier (BBB) disruption and astrocytes are critical for BBB (dys)function. Therefore, we aimed to clarify the involvement of neuroinflammation mediated by astrocytes in BBB permeability and brain oedema induced by METH. Further, we aimed to identify a new approach to counteract METH effects. METHODS: Mice were administered with a METH binge regimen (4 × 10 mg/kg) alone or in combination with parthenolide (PTL; 4 × 1 mg/kg), and hippocampi were analysed. For in vitro studies, mouse primary cultures of astrocytes were exposed to 250 µM METH, alone or co-treated with 10 µM PTL. RESULTS: We observed a neuroinflammatory response characterized by astrocytic morphological changes and increased TNF-α, iNOS and ICAM-1 protein levels (213.62%, 205.76% and 191.47% of control, respectively). Additionally, brain oedema and BBB disruption were identified by increased water content (81.30% of tissue weight) and albumin (224.40% of control) in the hippocampal tissue, as well as a significant decrease in vessel coverage by astrocytes after METH exposure. Regarding astrocyte cultures, we further identified TNF-α as a key player in METH-induced cell swelling. Importantly, PTL (present in feverfew plant) prevented both animal and in vitro effects induced by METH. CONCLUSIONS: We provided important insights on brain dysfunction induced by METH, and we also suggest a new approach to counteract such negative effects.

Protective effect of neuropeptide Y2 receptor activation against methamphetamine-induced brain endothelial cell alterations.

Methamphetamine (METH) consumption is a health problem that leads to neurological and psychiatric disturbances. The cellular alterations behind these conditions have been extensively investigated and it is now well-established that METH causes cerebrovascular alterations being a key feature in drug-induced neuropathology. Although promising advances in understanding the blood-brain barrier (BBB) alterations induced by METH, there is still no available approach to counteract or diminish such effects. Interestingly, several studies show that neuropeptide Y (NPY) has an important protective role against METH-induced neuronal and glial toxicity, as well as behavioral deficits. Despite these beneficial effects of the NPY system, nothing is known about its role in brain endothelial cells under conditions of METH exposure. Thus, our aim was to unravel the effect of NPY and its receptors against METH-induced endothelial cell dysfunction. For that, we used a human brain microvascular endothelial cell line (hCMEC/D3) and our results demonstrate that endothelial cells express both NPY Y1 (Y1R) and Y2 (Y2R) receptors, but only Y2R is upregulated after METH exposure. Moreover, this drug of abuse induced endothelial cell death and elicited the production of reactive oxygen species (ROS) by these cells, which were prevented by the activation of Y2R. Additional, cell death and oxidative stress triggered by METH were dependent on the concentration of the drug. In sum, with the present study we identified for the first time the NPY system, and particularly the Y2R subtype, as a promising target to protect against METH-induced neurovascular dysfunction.

Neuromelanin Magnetic Resonance Imaging of the Substantia Nigra in Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder inducing motor, psychiatric changes and cognitive decline, characterized pathologically by striatal atrophy. Pathological changes in the extra-striatal structures, such as the substantia nigra (SN), and abnormalities in pre-synaptic striatal dopamine neurotransmission are also known to occur. Neuromelanin (NM)-sensitive magnetic resonance imaging (NM-MRI) is an innovative technique that was recently developed allowing the in vivo study of pathological changes in the dopaminergic neurons of the SN. OBJECTIVE: To investigate the SN MR signal in HD patients. METHODS: We performed a cross-sectional study using a specific T1-weighted MR sequence to visualize NM. The areas and signal intensity contrast ratios of the T1 hyperintense SN regions were obtained using a semi-automatic segmentation method. RESULTS: A total of 8 HD patients and 12 healthy subjects were evaluated. The SN area was markedly reduced in the HD group compared with the control group (p = 0.02), even after normalization of the SN area with the midbrain area and age correction (p = 0.01). There was a significant reduction in the intensity contrast ratio of the hyperintense SN areas to crus cerebri in HD patients comparing with controls (p = 0.04) after correction for age. CONCLUSIONS: NM-sensitive MR techniques were used for the first time to study the SN in HD patients, showing loss of NM in this region, supporting the implication of dopaminergic neuronal changes in disease pathology. Future research needs to be conducted to evaluate the potential of SN area and intensity contrast as biomarkers for HD.

A Conserved PP2A Regulatory Subunit Enforces Proportional Relationships Between Cell Size and Growth Rate.

Cell size is proportional to growth rate. Thus, cells growing rapidly in rich nutrients can be nearly twice the size of cells growing slowly in poor nutrients. This proportional relationship appears to hold across all orders of life, yet the underlying mechanisms are unknown. In budding yeast, most growth occurs during mitosis, and the proportional relationship between cell size and growth rate is therefore enforced primarily by modulating growth in mitosis. When growth is slow, the duration of mitosis is increased to allow more time for growth, yet the amount of growth required to complete mitosis is reduced, which leads to the birth of small daughter cells. Previous studies have found that Rts1, a member of the conserved B56 family of protein phosphatase 2A regulatory subunits, works in a TORC2 signaling network that influences cell size and growth rate. However, it was unclear whether Rts1 influences cell growth and size in mitosis. Here, we show that Rts1 is required for the proportional relationship between cell size and growth rate during mitosis. Moreover, nutrients and Rts1 influence the duration and extent of growth in mitosis via Wee1 and Pds1/securin, two conserved regulators of mitotic progression. Together, the data are consistent with a model in which global signals that set growth rate also set the critical amount of growth required for cell cycle progression, which would provide a simple mechanistic explanation for the proportional relationship between cell size and growth rate.

Effect of chronic methylphenidate treatment on hippocampal neurovascular unit and memory performance in late adolescent rats.

Methylphenidate (MPH) is the classic treatment for attention deficit hyperactivity disorder (ADHD) among children and adults. Despite its beneficial effects, non-medical use of MPH is nowadays a problem with high impact on society. Thus, our goal was to uncover the neurovascular and cognitive effects of MPH chronic use during a critical period of development in control conditions. For that, male Wistar Kyoto rats were treated with MPH (1.5 or 5 mg/kg/day at weekdays, per os) from P28 to P55. We concluded that the higher dose of MPH caused hippocampal blood-brain barrier (BBB) hyperpermeability by vesicular transport (transcytosis) concomitantly with the presence of peripheral immune cells in the brain parenchyma. These observations were confirmed by in vitro studies, in which the knockdown of caveolin-1 in human brain endothelial cells prevented the increased permeability and leukocytes transmigration triggered by MPH (100 µM, 24 h). Furthermore, MPH led to astrocytic atrophy and to a decrease in the levels of several synaptic proteins and impairment of AKT/CREB signaling, together with working memory deficit assessed in the Y-maze test. On the contrary, we verified that the lower dose of MPH (1.5 mg/kg/day) increased astrocytic processes and upregulated several neuronal proteins as well as signaling pathways involved in synaptic plasticity culminating in working memory improvement. In conclusion, the present study reveals that a lower dose of MPH in normal rats improves memory performance being associated with the modulation of astrocytic morphology and synaptic machinery. However, a higher dose of MPH leads to BBB dysfunction and memory impairment.

Impact of developmental exposure to methylphenidate on rat brain's immune privilege and behavior: Control versus ADHD model.

Attention deficit hyperactivity disorder (ADHD) is the most prevalent childhood mental disorders that often persists into adulthood. Moreover, methylphenidate (MPH) is the mainstay of medical treatment for this disorder. Yet, not much is known about the neurobiological impact of MPH on control versus ADHD conditions, which is crucial to simultaneously clarify the misuse/abuse versus therapeutic use of this psychostimulant. In the present study, we applied biochemical and behavioral approaches to broadly explore the early-life chronic exposure of two different doses of MPH (1.5 and 5 mg/kg/day) on control and ADHD rats (Wistar Kyoto and Spontaneously Hypertensive rats, respectively). We concluded that the higher dose of MPH promoted blood-brain barrier (BBB) permeability and elicited anxiety-like behavior in both control and ADHD animals. BBB dysfunction triggered by MPH was particularly prominent in control rats, which was characterized by a marked disruption of intercellular junctions, an increase of endothelial vesicles, and an upregulation of adhesion molecules concomitantly with the infiltration of peripheral immune cells into the prefrontal cortex. Moreover, both doses of MPH induced a robust neuroinflammatory and oxidative response in control rats. Curiously, in the ADHD model, the lower dose of MPH (1.5 mg/kg/day) had a beneficial effect since it balanced both immunity and behavior relative to vehicle animals. Overall, the contrasting effects of MPH observed between control and ADHD models support the importance of an appropriate MPH dose regimen for ADHD, and also suggest that MPH misuse negatively affects brain and behavior.

Aquaporin-4 as a New Target against Methamphetamine-Induced Brain Alterations: Focus on the Neurogliovascular Unit and Motivational Behavior.

Methamphetamine (METH) abuse/misuse is a worldwide problem, and despite extensive characterization of its neurotoxicity over the last years, many questions remain unanswered. Recently, it was shown that METH compromises the blood-brain barrier (BBB) and causes a disturbance in the water homeostasis leading to brain edema. Importantly, water transport at BBB is regulated by water channels, aquaporins (AQPs), with AQP4 being expressed in astrocytic end-feet surrounding brain endothelium. Thus, the main goal of this work was to unravel the role of AQP4 under conditions of METH consumption. Our results show that METH (4× 10 mg/kg, 2 h apart, i.p.) interferes with AQP4 protein levels causing brain edema and BBB breakdown in both mice striatum and hippocampus, which culminated in locomotor and motivational impairment. Furthermore, these effects were prevented by pharmacological blockade of AQP4 with a specific inhibitor (TGN-020). Moreover, siRNA knockdown of this water channel protected astrocytes from METH-induced swelling and morphologic alterations. Herein, we unraveled AQP4 as a new therapeutic target to prevent the negative impact of METH.

Effect of Hypoproteic and High-Fat Diets on Hippocampal Blood-Brain Barrier Permeability and Oxidative Stress.

Worldwide, millions of people are exposed to dietary imbalance that impacts in health and quality of life. In developing countries, like in Brazil, in poor settings, dietary habits, traditionally hypoproteic, are changing rapidly to western-type high-fat foods. These rapidly changing dietary habits are imposing new challenges to human health and there are many questions in the field that remain to be answered. Accordingly, we currently do not know if chronic consumption of hypoproteic (regional basic diet, RBD) or high-fat diets (HFD) may impact the brain physiology, contributing to blood-brain barrier (BBB) dysfunction and neuroinflammatory events. To address this issue, mice were challenged by breastfeeding from dams receiving standard, RBD or HFD from suckling day 10 until weaning. Immediately after weaning, mice continued under the same diets until post-natal day 52. Herein, we show that both RBD and HFD cause not only a peripheral but also a consistent central neuroinflammatory response, characterized by an increased production of Reactive Oxygen Species (ROS) and pro-inflammatory cytokines. Additionally, BBB hyperpermeability, accounted by an increase in hippocampal albumin content, a decrease in claudin-5 protein levels and collagen IV immunostaining, was also observed together with an upregulation of vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we also identified a significant astrogliosis, manifested by upregulation of GFAP and S100ß levels and an intensification of arbor complexity of these glial cells. In sum, our data show that dietary imbalance, related with hypoproteic or high-fat content, impairs BBB properties potentially favoring the transmigration of peripheral immune cells and induces both a peripheral and central neuroinflammatory status. Noteworthy, neuroinflammatory events in the hippocampus may cause neuronal malfunction leading to cognitive deficits and long-term persistence of this phenomenon may contribute to age-related neurodegenerative diseases.

Testing the excitation/inhibition imbalance hypothesis in a mouse model of the autism spectrum disorder: in vivo neurospectroscopy and molecular evidence for regional phenotypes.

BACKGROUND: Excitation/inhibition (E/I) imbalance remains a widely discussed hypothesis in autism spectrum disorders (ASD). The presence of such an imbalance may potentially define a therapeutic target for the treatment of cognitive disabilities related to this pathology. Consequently, the study of monogenic disorders related to autism, such as neurofibromatosis type 1 (NF1), represents a promising approach to isolate mechanisms underlying ASD-related cognitive disabilities. However, the NF1 mouse model showed increased γ-aminobutyric acid (GABA) neurotransmission, whereas the human disease showed reduced cortical GABA levels. It is therefore important to clarify whether the E/I imbalance hypothesis holds true. We hypothesize that E/I may depend on distinct pre- and postsynaptic push-pull mechanisms that might be are region-dependent. METHODS: In current study, we assessed two critical components of E/I regulation: the concentration of neurotransmitters and levels of GABA(A) receptors. Measurements were performed across the hippocampi, striatum, and prefrontal cortices by combined in vivo magnetic resonance spectroscopy (MRS) and molecular approaches in this ASD-related animal model, the Nf1+/- mouse. RESULTS: Cortical and striatal GABA/glutamate ratios were increased. At the postsynaptic level, very high receptor GABA(A) receptor expression was found in hippocampus, disproportionately to the small reduction in GABA levels. Gabaergic tone (either by receptor levels change or GABA/glutamate ratios) seemed therefore to be enhanced in all regions, although by a different mechanism. CONCLUSIONS: Our data provides support for the hypothesis of E/I imbalance in NF1 while showing that pre- and postsynaptic changes are region-specific. All these findings are consistent with our previous physiological evidence of increased inhibitory tone. Such heterogeneity suggests that therapeutic approaches to address neurochemical imbalance in ASD may need to focus on targets where convergent physiological mechanisms can be found.

The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast.

The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast.

Extended-access methamphetamine self-administration elicits neuroinflammatory response along with blood-brain barrier breakdown.

Methamphetamine (METH) is a highly addictive psychostimulant drug that can lead to neurological and psychiatric abnormalities. Several studies have explored the central impact of METH use, but the mechanism(s) underlying blood-brain barrier (BBB) dysfunction and associated neuroinflammatory processes after chronic METH consumption are still unclear. Important findings in the field are mainly based on in vitro approaches and animal studies using an acute METH paradigm, and not much is known about the neurovascular alterations under a chronic drug use. Thus, the present study aimed to fill this crucial gap by exploring the effect of METH-self administration on BBB function and neuroinflammatory responses. Herein, we observed an increase of BBB permeability characterized by Evans blue and albumin extravasation in the rat hippocampus and striatum triggered by extended-access METH self-administration followed by forced abstinence. Also, there was a clear structural alteration of blood vessels showed by the down-regulation of collagen IV staining, which is an important protein of the endothelial basement membrane, together with a decrease of intercellular junction protein levels, namely claudin-5, occludin and vascular endothelial-cadherin. Additionally, we observed an up-regulation of vascular cell and intercellular adhesion molecule, concomitant with the presence of T cell antigen CD4 and tissue macrophage marker CD169 in the brain parenchyma. Rats trained to self-administer METH also presented a neuroinflammatory profile characterized by microglial activation, astrogliosis and increased pro-inflammatory mediators, namely tumor necrosis factor-alpha, interleukine-1 beta, and matrix metalloproteinase-9. Overall, our data provide new insights into METH abuse consequences, with a special focus on neurovascular dysfunction and neuroinflammatory response, which may help to find novel approaches to prevent or diminish brain dysfunction triggered by this overwhelming illicit drug.

Protein Kinase C Controls Binding of Igo/ENSA Proteins to Protein Phosphatase 2A in Budding Yeast.

Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2ACdc55 We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2ACdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2ACdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2ACdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2ACdc55 by multiple overlapping mechanisms.

Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells.

Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14F mutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.

The TNF-α/NF-κB signaling pathway has a key role in methamphetamine-induced blood-brain barrier dysfunction.

Methamphetamine (METH) is a psychostimulant that causes neurologic and psychiatric abnormalities. Recent studies have suggested that its neurotoxicity may also result from its ability to compromise the blood-brain barrier (BBB). Herein, we show that METH rapidly increased the vesicular transport across endothelial cells (ECs), followed by an increase of paracellular transport. Moreover, METH triggered the release of tumor necrosis factor-alpha (TNF-α), and the blockade of this cytokine or the inhibition of nuclear factor-kappa B (NF-κB) pathway prevented endothelial dysfunction. Since astrocytes have a crucial role in modulating BBB function, we further showed that conditioned medium obtained from astrocytes previously exposed to METH had a negative impact on barrier properties also via TNF-α/NF-κB pathway. Animal studies corroborated the in vitro results. Overall, we show that METH directly interferes with EC properties or indirectly via astrocytes through the release of TNF-α and subsequent activation of NF-κB pathway culminating in barrier dysfunction.

Color capable sub-pixel resolving optofluidic microscope and its application to blood cell imaging for malaria diagnosis.

Miniaturization of imaging systems can significantly benefit clinical diagnosis in challenging environments, where access to physicians and good equipment can be limited. Sub-pixel resolving optofluidic microscope (SROFM) offers high-resolution imaging in the form of an on-chip device, with the combination of microfluidics and inexpensive CMOS image sensors. In this work, we report on the implementation of color SROFM prototypes with a demonstrated optical resolution of 0.66 µm at their highest acuity. We applied the prototypes to perform color imaging of red blood cells (RBCs) infected with Plasmodium falciparum, a particularly harmful type of malaria parasites and one of the major causes of death in the developing world.