acase@tulane.edu / Objectives Human Immunodeficiency Virus (HIV) infects immune cells and lowers cell-mediated immunity leading to acquired immune deficiency syndrome or AIDS. The virus causes damage/dysfunction of helper T cells, macrophages, and dendritic cells. One of the long-term complications of untreated AIDS is severe cognitive impairment caused by HIV-associated dementia (HAD). Highly Active Antiretroviral therapy, the HAART regimen, which inhibits virus replication has been shown to reduce the incidence of HAD. HAART includes several mechanistically diverse classes of drugs such as protease inhibitors, nucleoside inhibitors of reverse transcriptase, and non-nucleoside reverse transcriptase inhibitors. The protease inhibitor, nelfinavir, is used in HIV therapy in order to mitigate the effects of the HIV virus by breaking down HIV-1 and HIV-2 proteases, which are essential to the replication of the virus within the host cell. HAART has decreased the incidence of HAD yet milder cognitive dysfunction, considered to be a consequence of anti-HIV drug toxicity, often manifests. Previous studies have shown that protease inhibitors may play a role in causing oxidative stress in endothelial cells. The present study involves understanding the effect of nelfinavir on mitochondrial oxidative stress and its role in the injury to human brain microvascular endothelial cells that form the blood-brain barrier. Methods and Results Our studies utilized primary human brain microvascular endothelial cells (hBMECs). We performed measurements of mitochondrial superoxide levels (ESR Spectrscopy), oxygen consumption rates or OCR (Seahorse XFe Extracellular Flux Analyzer and MitoStress Test Assay), cell viability/proliferation (CCK8 based Cellular Viability Assay). Sub-therapeutic doses of nelfinavir (1 µmol/L) increased the cell proliferation whereas therapeutic (3-5 µmol/L) and supra-therapeutic (10 µmol/L) doses of nelfinavir reduced the cell viability. In addition, treatment with sub-therapeutic levels of nelfinavir has no effect on the levels of mitochondrial superoxide in hBMECs but therapeutic and supra-therapeutic levels of nelfinavir increased mitochondrial superoxide levels. Measurements of OCR showed that sub-therapeutic doses of nelfinavir enhanced the basal and maximal respiration in hBMECs. In contrast, therapeutic concentration of nelfinavir reduced ATP production and spare respiratory capacity although basal respiration, proton leak, and non-mitochondrial respiration were unchanged. However, supra-therapeutic dose of nelfinavir significantly reduced basal respiration, ATP production, and spare respiratory capacity accompanied by reduced non-mitochondrial respiration and proton leak. Conclusions We identified that nelfinavir treatment was associated with a decrease in cellular proliferation at therapeutic and supra-therapeutic levels. Furthermore, we identified an increase in mitochondrial superoxide species in cells treated with nelfinavir in concentrations beyond therapeutic levels which was accompanied by a decrease in basal respiration, ATP production, and mitochondrial spare capacity. These results are indicative of nelfinavir causing cellular cytotoxicity in BMECs that are likely mediated by mitochondrial oxidative stress and impaired mitochondrial respiration. / 1 / Gowthamram Rajaprabhakaran

The selective degeneration of motoneurons in the spinal cord, the brainstem and the brain cortex is the core pathology of amyotrophic lateral sclerosis (ALS), but evidences suggest that the neighbouring non-neuronal cells are also involved in the disease progression. Beside Riluzole, only drug approved to treat this fatal neurodegenerative disease, new pharmaceutical agents or novel strategies including stem cell therapy are currently under development and evaluated preclinically in front line on mutant SOD1 rodents mimicking all hallmarks of the human disease. Current intravenously delivered drugs tested in ALS therapy assume an intact blood-brain barrier and suppose the passage across the endothelium to hit their targets in the CNS parenchyma. If BBB impairment occurs in ALS, it may lead to revision of planned pharmaceutical treatment. In the first part of the work, we have validated the mutant SOD1 rat model of ALS and we characterized properties and integrity of its BBB. We observed a significant BBB disruption at symptomatic phase of ALS, evidenced by blood protein leakage, IgG accumulation and microhemorrhage. To look for the mechanism of BBB opening, we demonstrated that the expression of key genes involved in the BBB integrity was decreased. At the ultrastructure, the morphology of endothelial cells and vascular astrocyte end-feet was altered. Our results suggest that BBB disruption is a late event in ALS disease course and appears like a consequence of the local degenerative process or neuroinflammation rather than a cause. Since a lot of extracellular oedema and swollen astrocyte end-feet were found in mutant SOD1 rats, we also looked at the expression and localization of aquaporin-4, a key protein involved in CNS water movement. We found that its expression was highly increased in the symptomatic phase of ALS course and we hypothesize that this overexpression might be related to the resolution of oedema after BBB opening. In the second part of the work, we considered an original, easy, non-invasive and safe therapeutical approach of stem cell delivery in ALS rats. Since ALS affects the motoneurons throughout the CNS, we decided to use the bloodstream to deliver neural stem cells. We studied cell homing, survival, proliferation, integration and differentiation. Interestingly, the highest efficiency of cell delivery to the CNS was found in symptomatic ALS and the lowest in healthy animals. Neural stem cells injected into ALS animals preferentially colonized the motor cortex, hippocampus and spinal cord. We detected their successful differentiation into neural lineages by the appearance of MAP2-, GFAP-positive cells and the decrease of nestin expression. One of the realistic near-term clinical goals for ALS is the transplantation of stem cells that counteract the loss of motoneurons by secreting neuroprotective factors. Accordingly, we evaluated in vitro the expression of neurotrophic factors released by stem cells after stimulation with tissue extracts from ALS rats. The aim of this paradigm was to determine whether the ALS environment triggers neuroprotective factors release from stem cells. Mesenchymal stem cells and neural stem cells were able to express a wider range of growth factors than fibroblasts. According to the stem cell population stimulated, we obtained differential expression pattern, raising the choice of cell population for appropriate clinical applications in ALS.

Endogenous peptides modulate a wide range of physiological conditions in the central and peripheral nervous systems, but have not been harnessed to perform similar functions in pharmaceutical roles due to their ease of degradation and difficulty in introducing into the neurovascular unit. We report herein advances that evidence the wide applicability that glycosylation provides as a pathway for improving the drug-like properties of peptides. This is demonstrated by utilizing novel sugar-amino acids to modify the potent mu opioid receptor agonist DAMGO to provide antinociception, and serine glycosides to modify secretin family peptides for neuroprotection and angiotensin-(1-7) to both reduce cognitive impedance following myocardial infarction and as a treatment for peripheral neuropathy. Evidence is presented via a series of in vitro and in vivo models and assays, and demonstrates the advantageous effects of glycosylation through increased persistence in serum, greatly improved blood-brain barrier penetration, and the tolerance of receptor interactions to the addition of a carbohydrate.

No description available.

One of the major limitations in drug development and gene therapy for brain diseases is the natural defensive structure called the blood brain barrier (BBB), which prevents therapeutic polypeptide drugs and viral vectors from entering the brain. Intranasal delivery of therapeutic gene products into the brain offers a non-invasive alternative towards a feasible gene and protein therapy for neurological diseases. From recent studies involving axonal transport, it is tempting to speculate that therapeutic macromolecules including neurotrophic factors and viral vectors can be delivered into the brain by peripheral neurons, such as olfactory receptor neurons (ORNs), which span the BBB. It is thought that the nasal pathway into the brain involves two general mechanisms; intracellular (intraneuronal) or extracellular routes of transport. However the pathways involved have not yet been fully characterized. In this study I firstly investigated the temporal and spatial localisation pattern of both biotinylated and I125 labelled ciliary neurotrophic factor (CNTF) following nasal delivery into Sprague-Dawley rats. Results showed that intranasal delivered CNTF was transported to several brain regions by both intracellular axonal pathway through ORNs and the extracellular trigeminal pathway. Excess unlabelled CNTF competed for receptor binding in the olfactory mucosa confirming receptor mediated intracellular transport to the olfactory bulb via ORNs. Denervation of the olfactory mucosa prior to CNTF delivery failed to prevent CNTF transport to trigeminal and hypothalamic brain regions. Intranasal delivered CNTF was biologically active, resulting in activation of the STAT3 signalling pathway in the thalamus and hypothalamus. To examine the functional activity of intranasal delivered CNTF, I conducted a weight loss trial using an obese Zucker rat (OZR) model to test whether CNTF treatment caused body weight loss. Intranasal administration of CNTF resulted in reduced body weight in the CNTF treated OZR group compared to the BSA control group during the 12 day trial and for 3 days after. Intranasal delivery of CNTF may be a valuable method for the treatment of obesity. In the second study, I investigated the temporal and spatial expression of Enhanced Green Fluorescent Protein (EGFP) transferred by a single nasal delivery of either a recombinant adenovirus vector (Ad5CMV-EGFP) or an adeno-associated virus vector (AAV2-EGFP) into Sprague-Dawley rats. Adenovirus mediated EGFP expression was localized in ORNs throughout the olfactory epithelium after 24 hours. EGFP in the ORNs appeared to be anterogradely transported along their axons to the olfactory bulb and transferred in glomeruli to second-order neurons. EGFP was transferred to several brain regions including the cortex, hippocampus, and brainstem after 7 days. EGFP expression co-localized with Olfactory Marker Protein and was confirmed with EGFP immunofluorescence labelling and western blotting. AAV expressed EGFP localized in similar olfactory and brain regions 6 weeks after delivery. mRNA levels suggested that the AAV-EGFP construct was only incorporated into olfactory mucosa cells and the viral vector was not present in olfactory bulb and brain regions. In conclusion, this simple and non-invasive polypeptide and gene delivery method provides ubiquitous macromolecule distribution throughout the rodent brain and may be useful for the treatment of neurological disorders.

Glycosylation may be a general strategy for the transport of biologically active neuro(glyco)peptides into the brain. With that in mind, a series of modified DAMGO analogues were synthesized and subjected to conformational analysis, and in vitro and in vivo studies related to opioid analgesia. Those studies will help to determine the balance of carbohydrate and peptide, to reach maximum BBB transport; in other words this is a study to test the biousian hypothesis. 1) The μ-agonist DAMGO was altered by incorporating moieties of increasing water solubility into the C-terminus, including carboxamide and simple glycosides. The hydrophilic C-terminal moieties were varied from glycinol in DAMGO to L-serine amide (LYM100), L-serine amide β-D-xyloside (LYM50), L-serine amide β-Dglucoside (LYM110), L-serine amide β-lactoside (LYM147). Two phosphopeptides LYM1311 and LYM1312 were synthesized with the phosphate group attached to Lserine amide at the C-terminus. Conformational analysis experiments included: 1HNMR, diffusion, variable temperature experiments to find the temperature coefficient, circular dichroism, 2DNMR noesy and tocsy, and molecular modeling. The peptides associate with SDS micelles with a strong electrostatic component. The SDS micelles stabilized the β-turn that is nascent in water. CSI (chemical shift indexes), temperature coefficients and circular dichroism do not give much insight into the structural conformation. 2D NMR analysis followed by molecular modeling confirmed a β-turn preferred conformation. No specific type of β-turn could be assigned to the DAMGO analogs. 2) Antinociceptive mouse tail-flick studies were performed, and opioid binding was determined. Analgesic potency (i.v.) increased, passing through a maximum (A₅₀ ≈ 0.2 μmol/Kg) for LYM100 & LYM50 as membrane affinity vs. water solubility became optimal, and then dropped off (A₅₀ ≈ 1.0 μmol/Kg) for LYM110 & LYM147 as water-solubility dominated the molecular behavior. Correlation of i.v. A₅₀ values with estimated hydrodynamic values (glucose units) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A₅₀ vs e^(-Awater/Alipid)), provided U-shaped or V-shaped curves, as predicted by the “biousian hypothesis.” The μ-selective opioid agonism was maintained upon modifications at the C-terminus. The optimal “degree of glycosylation” that achieves the maximum degree of transport for the DAMGO peptide message seems to be between the peptide with the carboxamide C-terminal group and the xyloside.

Objetivo: Investigar a função auditiva, periférica e central, em pacientes com o diagnóstico da síndrome G/BBB, quanto à ocorrência ou não de perda auditiva e, a condução nervosa auditiva periférica e central, em nível do tronco encefálico. Modelo: Análise prospectiva descrevendo os achados audiológicos em indivíduos com a síndrome G/BBB. Local de Execução: Setor de Genética, HRAC-USP. Participantes: 14 pacientes na faixa etária de 7 a 34 anos, do gênero masculino. Variáveis: Limiares audiométricos em decibels nas frequências de 0,25 a 8 kHz nas duas orelhas, tipo de curva timpanométrica nas duas orelhas, latências absolutas das ondas I, III e V; latências interpicos I-V, III-V e I-III e diferença interaural da onda V do PEATE, em milissegundos, para cada orelha. Resultados: Limiares audiométricos normais em 12 (66,7%) pacientes da amostra, e 2 (33,3%) com perda auditiva, sendo 1 do tipo condutiva e 1 neurossensorial. Quanto aos resultados do PEATE, foram encontrados: latências absolutas da onda I dentro dos padrões de normalidade em todos os pacientes, aumento das latências absolutas da onda III e V em 2 e 6 pacientes respectivamente; e as latências interpicos I-III, III-V e I-V se apresentaram aumentadas em 4, 3 e 8 pacientes respectivamente. Conclusões: Frente aos resultados obtidos podemos concluir que pacientes com a síndrome G/BBB podem apresentar perdas auditivas periféricas, condutivas e neurossensoriais, entretanto, não há subsídios para afirmar que as mesmas são em decorrência da síndrome ou da associação com a fissura de palato. Há evidências de comprometimento das vias auditivas centrais em nível do tronco encefálico, embora as alterações estruturais do SNC relatadas nesta síndrome não estejam relacionadas diretamente com as vias auditivas. Estudos com enfoque no perfil audiológico desta população com exames de imagem são necessários para maior clareza dos achados clínicos. / Objective: To investigate the peripheral and central auditory function in patients with G/BBB syndrome and the occurrence of hearing loss in these patients. Model: Prospective study describing the audiological findings in subjects with G/BBB syndrome. Setting: Genetics Department, HRAC-USP. Participants: 14 male patients aged from 7 to 34 years. Variables: Audiometric thresholds in decibels at frequencies of 0.25 to 8 KHz in both ears, tympanometric curve in both ears, absolute latencies of waves I, III and V, interpeak latencies I-V, III-V and I-III and wave V interaural difference of ABR, in milliseconds, for each ear. Results: Normal audiometric thresholds were found in 12 (66.7%) patients, 2 (33.3%) had hearing loss, one type conductive and one sensorioneural. ABR results were: absolute latencies of wave I within normal limits in all patients, an increase of absolute latencies of wave III and V in 2 and 6 patients respectively, and interpeak latencies I-III, IV and V were increased in 4, 3 and 8 patients respectively. Conclusions: Patients with G/BBB syndrome may have peripheral conductive or sensorineural hearing loss; however, there are no subsidies to attribute the etiology to the syndrome itself or to the presence of cleft palate, which was found in all patients. There is evidence of central auditory pathways involvement in the brainstem level, although the structural CNS abnormalities reported in this syndrome are not directly related to the auditory pathways evaluated. Studies focusing on the audiological profile of this population with imaging studies are recommended.

Objetivo: Investigar a função auditiva, periférica e central, em pacientes com o diagnóstico da síndrome G/BBB, quanto à ocorrência ou não de perda auditiva e, a condução nervosa auditiva periférica e central, em nível do tronco encefálico. Modelo: Análise prospectiva descrevendo os achados audiológicos em indivíduos com a síndrome G/BBB. Local de Execução: Setor de Genética, HRAC-USP. Participantes: 14 pacientes na faixa etária de 7 a 34 anos, do gênero masculino. Variáveis: Limiares audiométricos em decibels nas frequências de 0,25 a 8 kHz nas duas orelhas, tipo de curva timpanométrica nas duas orelhas, latências absolutas das ondas I, III e V; latências interpicos I-V, III-V e I-III e diferença interaural da onda V do PEATE, em milissegundos, para cada orelha. Resultados: Limiares audiométricos normais em 12 (66,7%) pacientes da amostra, e 2 (33,3%) com perda auditiva, sendo 1 do tipo condutiva e 1 neurossensorial. Quanto aos resultados do PEATE, foram encontrados: latências absolutas da onda I dentro dos padrões de normalidade em todos os pacientes, aumento das latências absolutas da onda III e V em 2 e 6 pacientes respectivamente; e as latências interpicos I-III, III-V e I-V se apresentaram aumentadas em 4, 3 e 8 pacientes respectivamente. Conclusões: Frente aos resultados obtidos podemos concluir que pacientes com a síndrome G/BBB podem apresentar perdas auditivas periféricas, condutivas e neurossensoriais, entretanto, não há subsídios para afirmar que as mesmas são em decorrência da síndrome ou da associação com a fissura de palato. Há evidências de comprometimento das vias auditivas centrais em nível do tronco encefálico, embora as alterações estruturais do SNC relatadas nesta síndrome não estejam relacionadas diretamente com as vias auditivas. Estudos com enfoque no perfil audiológico desta população com exames de imagem são necessários para maior clareza dos achados clínicos. / Objective: To investigate the peripheral and central auditory function in patients with G/BBB syndrome and the occurrence of hearing loss in these patients. Model: Prospective study describing the audiological findings in subjects with G/BBB syndrome. Setting: Genetics Department, HRAC-USP. Participants: 14 male patients aged from 7 to 34 years. Variables: Audiometric thresholds in decibels at frequencies of 0.25 to 8 KHz in both ears, tympanometric curve in both ears, absolute latencies of waves I, III and V, interpeak latencies I-V, III-V and I-III and wave V interaural difference of ABR, in milliseconds, for each ear. Results: Normal audiometric thresholds were found in 12 (66.7%) patients, 2 (33.3%) had hearing loss, one type conductive and one sensorioneural. ABR results were: absolute latencies of wave I within normal limits in all patients, an increase of absolute latencies of wave III and V in 2 and 6 patients respectively, and interpeak latencies I-III, IV and V were increased in 4, 3 and 8 patients respectively. Conclusions: Patients with G/BBB syndrome may have peripheral conductive or sensorineural hearing loss; however, there are no subsidies to attribute the etiology to the syndrome itself or to the presence of cleft palate, which was found in all patients. There is evidence of central auditory pathways involvement in the brainstem level, although the structural CNS abnormalities reported in this syndrome are not directly related to the auditory pathways evaluated. Studies focusing on the audiological profile of this population with imaging studies are recommended.

The rates of opioid prescription and use have continued to increase over the last few decades. In turn, a greater number of patients suffer from opioid tolerance. Treatment of acute pain is a clinical challenge for these patients. Acute pain can arise from common occurrences like surgical pain and pain resulting from the injury. P-glycoprotein (p-gp) is a transporter at the blood-brain barrier (BBB) associated with a decrease in the analgesic efficacy of morphine. Peripheral inflammatory pain (PIP) is a pain state known to cause a change in p-gp trafficking at the BBB. P-gp traffics from the nucleus to the luminal surface of endothelial cells making up the BBB. This surface where circulating blood interfaces with the endothelial cell is where p-gp will efflux morphine back into circulation. Osmotic minipumps were used as a long-term delivery method in this model of opioid tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase when animals were exposed to opioids for 6 days. This observation presents a possible relationship between p-gp trafficking and the challenges of treating post-surgical pain in opioid tolerant patients. This could reveal potential strategies for improving pain management in these patients.

Environmental air pollution is one risk factor associated with the onset and progression of multiple sclerosis (MS). In this project, we investigated the effects of ubiquitous traffic-generated pollutants, namely a mixture of gasoline and diesel vehicle exhaust (MVE), on signaling pathways associated with the pathophysiology of MS in the central nervous system (CNS) of either ovary intact (ov+) or ovariectomized (ov-) female Apolipoprotein (Apo) E-/-. Specifically, we investigated whether a subchronic inhalation exposure to MVE (200 PM μg/m3; 6 hr/d, 7d/wk, 30d) vs. filtered air (FA) controls altered myelination, T cell infiltration, blood-brain barrier (BBB) integrity, or production of reactive oxygen species (ROS) and expression of neuroinflammation markers in the CNS ov+ and ov- Apo E-/- mice. Our results revealed that inhalation exposure to MVE resulted in increased demyelination and CD4+ and CD8+ T cell infiltration, associated with alterations in BBB integrity. Disruption of the BBB was evidenced by decreased tight junction (TJ) protein expression, increased matrix metalloproteinase (MMPs) activity, and increased permeability of immunoglobin (Ig) G, which were more pronounced in the MVE ov- group. Moreover, MVE-exposure also promoted ROS and neuroinflammatory signaling in the CNS of ov+ and ov- mice, compared to FA groups. To analyze mechanisms that may contribute to MVE-exposure mediated inflammatory signaling in the CNS, we examined the NF-κB signaling pathway components, namely IKK subunits, IKKα, and IKKβ, as well as RelA. MVE -exposure did not alter the expression of either IKKα and IKKβ or RelA. However, increased expression of IKKα and IKKβ mRNA was observed in both FA ov- and MVE ov- groups, indicating female sex steroid hormone signaling involvement. Investigation of hormone receptors expression revealed a reduction in cerebral ERα mRNA expression, compared to ov+ mice; however, MVE-exposure resulted in an even further decrease in expression of ERα mRNA, while ERβ and PRO A/B transcript expressions were unchanged across groups. Collectively, these study findings revealed that subchronic inhalation exposure to MVE mediates alterations in ER expression in the CNS of ApoE-/- female mice, associated with altered cerebrovascular integrity and increased ROS production and inflammatory signaling. These detrimental outcomes in the CNS, resulting from MVE-exposure, are further associated with increased CD4+/CD8+ infiltration and local demyelination in the CNS of female ApoE-/-mice, which are hallmarks of MS. Such findings suggest that exposure to ubiquitous traffic-generated air pollutants may contribute to pathologies that exacerbate demyelinating diseases in the CNS of females.