Javier S. Burgos

Statins as Neuroprotectants: A Comparative In Vitro Study of Lipophilicity, Blood-Brain-Barrier Penetration, Lowering of Brain Cholesterol, and Decrease of Neuron Cell Death

There is growing evidence to support the hypothesis that statins may act as neuroprotectants in several neuropathological conditions, including Alzheimers disease. The mechanisms for neuroprotection are only partially understood, however, and pleiotropic phenomena could be involved. We have made a comparative study of 9 statins (lovastatin, mevastatin, pravastatin, simvastatin, cerivastatin, atorvastatin, fluvastatin, pitavastatin, and rosuvastatin), analyzing several parameters that could be related to neuroprotection, such as chemical structure, lipophilicity, potential blood-brain-barrier penetration (BBB), 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibition, cholesterol modulation in neurons, glia, and human hepatocyte cell lines, and protection against neurodegeneration caused by tau hyperphosphorylation induced by okadaic acid. Our results indicate that monacolin J derivatives (natural and semi-synthetic statins) are the best candidates for the prevention of neurodegenerative conditions due to their higher potential BBB penetration capacity, cholesterol lowering effect on neurons with a satisfactory safety profile, and in vitro protection against cell death caused by okadaic acid in culture. Among the nine statins studied, simvastatin presented the best characteristics for preventing neurodegenerative conditions.

Effect of orally administered guanosine on seizures and death induced by glutamatergic agents.

Intraperitoneal guanosine has been shown to prevent quinolinic acid-induced seizures in mice. In this study, we investigated the effect of orally administered guanosine on seizures induced by the glutamate agonists quinolinic acid and kainate, and the endogenous glutamate releaser alpha-dendrotoxin. Guanosine (7.5 mg/kg, per os), administered 75 min in advance, prevented 70% of seizures induced by i.c.v. quinolinic acid, being as efficient as the NMDA channel blocker MK-801 administered intraperitoneally. Guanosine was ineffective against kainate-induced seizures, but significantly reversed the potentiation of seizures and death caused by the concomitant injection of MK-801. Guanosine also significantly prevented seizures and death induced by i.c.v. alpha-dendrotoxin, whereas MK-801 and phenobarbital only prevented death. Altogether, our findings underscore the therapeutic potential of oral administration of guanosine for treating diseases involving glutamatergic excitotoxicity, including epilepsy.

Effect of Apolipoprotein E on the Cerebral Load of Latent Herpes Simplex Virus Type 1 DNA

ABSTRACT Herpes simplex virus type 1 (HSV-1) is neurotropic and enters a latent state lasting the lifetime of the host. This pathogen has recently been proposed as a risk factor for Alzheimers disease (AD) in conjunction with apolipoprotein E4 (ApoE4). In a murine acute infection model, we showed that viral neuroinvasiveness depends directly on the overall ApoE dosage and especially on the presence of isoform ApoE4. If an interaction between ApoE and HSV-1 is involved in AD, it may occur during latency rather than during acute infection. Certainly, ApoE plays an important role in late-onset AD, i.e., at a time in life when the majority of people harbor HSV-1 in their nervous system. In the present work, wild-type, APOE knockout, APOE3, and APOE4 transgenic mice were used to analyze the influence of the ApoE profile on the levels of latent virus DNA. The knockout mice had significantly lower concentrations of the virus in the nervous system than the wild-type mice, while the APOE4 mice had very high levels in the brain compared to the APOE3 animals. ApoE4 seems to facilitate HSV-1 latency in the brain much more so than ApoE3. The APOE dosage correlated directly with the HSV-1 DNA concentration in the brain, strengthening the hypothesis that HSV-1, together with ApoE, might be involved in AD.

A robust method for quantitative high-throughput analysis of proteomes by 18O labeling

MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by 18O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail.

Involvement of Apolipoprotein E in the Hematogenous Route of Herpes Simplex Virus Type 1 to the Central Nervous System

ABSTRACT Apolipoprotein E (ApoE), a constituent of the lipoproteins, may be relevant in herpes simplex virus type 1 (HSV-1) infection of the central nervous system (CNS), since HSV-1 binds to human serum ApoE lipoproteins. This study demonstrates the involvement of ApoE in the hematogenous route of HSV-1 to the CNS.

Simvastatin is the Statin that Most Efficiently Protects Against Kainate-Induced Excitotoxicity and Memory Impairment

Statins have recently been shown to act as protectants against several neuropathological conditions. They have received special attention in the field of Alzheimers disease (AD), where epidemiological studies indicating a lower prevalence of AD/dementia in statin-prescribed populations. Excitotoxicity, which derives from the overstimulation of glutamate receptors, is a major cause of neuron death in several neurological diseases, including AD and epilepsy. We have carried out a comparative study to investigate the effects of all the commercially available statins (simvastatin, lovastatin, fluvastatin, pravastatin, and atorvastatin) on neuron damage and memory impairment. To this end, we studied neurodegeneration in a mouse model by systemic administration of kainate. Simvastatin was the most effective statin in reducing the deleterious effects caused by kainate, including the severity of seizures, excitotoxicity, oxidative damage, neuritic dystrophy and apoptosis in the hippocampus and other limbic structures of the brain cortex. Lovastatin was the second most efficient statin in preventing seizures and histopathological signs of excitotoxicity, whilst fluvastatin, pravastatin, and atorvastatin showed neither antiepileptic nor neuroprotective effects. Only simvastatin enhanced episodic-like memory. To the best of our knowledge this is the first in vivo study to analyze the neuroprotective effect of all the commercially available statins. Our results suggest that both simvastatin and lovastatin (but especially simvastatin) may well have therapeutic potential in the treatment of neurodegenerative diseases involving excitotoxicity and memory impairment, including AD.

Zebrafish as a new model for herpes simplex virus type 1 infection.

The zebrafish (Danio rerio) is rapidly gaining ground as a disease model. However, until now, the use of this species with human pathogens has been restricted to just three bacteria; no studies involving viruses that infect humans are recorded. In this study, the zebrafish was used as a model of herpes simplex virus type 1 (HSV-1) infection of the nervous system. Fish infected using viral culture supernatants showed detectable HSV-1 DNA concentrations 1-4 days after inoculation, indicating that this virus can experimentally infect and persist in this host. The kinetics of infection was dose dependent, especially in the head. Histological immunodetection of HSV-1 glycoproteins confirmed the presence of HSV-1 in the organs studied; infection led to histopathological changes. Moreover, the suppression of the immune system by cyclophosphamide and the antiviral effect of acyclovir were demonstrated. The infection of the encephalon was studied in detail, and the time course of viral colonization recorded. Immunofluorescence studies provided immunoreactive evidence of viral antigens in the encephalon and spinal cord. Viruses cleared from infected brains showed the ability to infect human neuroblastoma cells. This study is the first to demonstrate HSV-1 infection in the zebrafish and manifests the potential use of this species in herpesvirus studies.

Kainate administered to adult zebrafish causes seizures similar to those in rodent models.

Glutamate is the major excitatory neurotransmitter of the central nervous system in vertebrates. Excitotoxicity, caused by over‐stimulation of the glutamate receptors, is a major cause of neuron death in several brain diseases, including epilepsy. We describe here how behavioural seizures can be triggered in adult zebrafish by the administration of kainate and are very similar to those observed in rodent models. Kainate induced a dose‐dependent sequence of behavioural changes culminating in clonus‐like convulsions. Behavioural seizures were suppressed by DNQX (6,7‐dinitroquinoxaline‐2,3‐dione) dose‐dependently, whilst MK‐801 (a non‐competitive NMDA receptor antagonist) had a lesser effect. Kainate triggers seizures in adult zebrafish, and thus this species can be considered as a new model for studying seizures and subsequent excitotoxic brain injury.

Herpes Simplex Virus Type 1 Infection via the Bloodstream with Apolipoprotein E Dependence in the Gonads Is Influenced by Gender

ABSTRACT Herpes simplex virus type 1 (HSV-1) causes disease in humans and animals. Infection usually occurs via the neural route and possibly occurs via the hematogenous route. The latter, however, is the main route by which immunosuppressed individuals and neonates are infected. Gender-dependent differences in the incidence and severity of some viral infections have been reported. To detect differences between the sexes with respect to HSV-1 colonization and disease, the characteristics of both acute and latent infections in hematogenously infected male and female mice were compared. In acute infection, the female mice had a poorer outcome: HSV-1 colonization was more effective, especially in the gonads and brain. In the encephalon, the midbrain had the highest viral load. In latent infection, brain viral loads were not significantly different with respect to sex. Significant differences were seen, however, in the blood and trigeminal ganglia: HSV-1 seroprevalence was observed in females, with no virus detected in males. In brain dissections, only the cerebral cortex of the females had viral loads statistically higher than those observed in the males. The spread of the virus to several organs of interest during acute infection was examined immunohistochemically. Female mice showed greater viral immunostaining, especially in the adrenal cortex, gonads, and midbrain. In male mice, HSV-1 was detected predominantly in the adrenal cortex. It was also found that apolipoprotein E promotes virus colonization of the ovaries, the APOE gene dose being directly related to viral invasiveness.

Is Periodontitis a Risk Factor for Cognitive Impairment and Dementia? A Case‐Control Study

BACKGROUND Dementia is a multi-etiologic syndrome characterized by multiple cognitive deficits but not always by the presence of cognitive impairment. Cognitive impairment is associated with multiple non-modifiable risk factors but few modifiable factors. Epidemiologic studies have shown an association between periodontitis, a potentially modifiable risk factor, and cognitive impairment. The objective of this study is to determine whether clinical periodontitis is associated with the diagnosis of cognitive impairment/dementia after controlling for known risk factors, including age, sex, and education level. METHODS A case-control study was conducted in Granada, Spain, in two groups of dentate individuals aged >50 years: 1) cases with a firm diagnosis of mild cognitive impairment or dementia of any type or severity and 2) controls with no subjective memory loss complaints and a score >30 in the Phototest cognitive test (screening test for cognitive impairment). Periodontitis was evaluated by measuring tooth loss, plaque and bleeding indexes, probing depths, and clinical attachment loss (AL). RESULTS The study included 409 dentate adults, 180 with cognitive impairment and 229 without. A moderate and statistically significant association was observed between AL and cognitive impairment after controlling for age, sex, education level, oral hygiene habits, and hyperlipidemia (P = 0.049). No significant association was found between tooth loss and cognitive impairment. CONCLUSION Periodontitis appears to be associated with cognitive impairment after controlling for confounders such as age, sex, and education level.