Michael L. Niehoff

Effect of LPS on the permeability of the blood–brain barrier to insulin

Insulin has emerged as an important neuropeptide. Central actions of insulin appear to oppose those in the periphery. Insulin is transported across the blood-brain barrier (BBB) by a saturable transport system. The permeability of the BBB to insulin is altered by various events, but no studies exist that have examined the permeability of the BBB to insulin during infection or inflammation, states which can induce peripheral insulin resistance. We looked at the effects of lipopolysaccharide (LPS), a bacterial endotoxin and a powerful cytokine releaser, on the permeability of the BBB to human insulin in CD-1 mice. Intraperitoneal injections of LPS significantly increased the uptake by the brain of 131I-insulin and disrupted the BBB to 125I-albumin. After subtraction of the brain/serum ratio for 125I-albumin, brain/serum ratios for insulin were increased: 10.38 +/- 0.70 microl/g (LPS) vs. 3.62 +/- 0.27 microl/g (no LPS), P<0.0001, showing that LPS increased the uptake of insulin independent of BBB disruption. This increase in insulin uptake was due to enhanced saturable transport. Pretreatment with indomethacin 10 min before LPS injections enhanced BBB disruption, but not insulin transport. Pretreatment with the nitric oxide (NO) synthase inhibitor aminoguanidine had no effect on insulin or albumin uptake, but pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) enhanced insulin transport, but not BBB disruption. We conclude that LPS increases the saturable transport of insulin across the BBB independent of disruption and prostaglandins with potentiation by NO inhibition. Such increased transport could potentiate the central effects of insulin and so contribute to the peripheral insulin resistance seen with infection and inflammation.

Leptin transport across the blood-brain barrier of the Koletsky rat is not mediated by a product of the leptin receptor gene

Obesity in humans is thought to be caused by a resistance to leptin. Currently, the evidence suggests that this resistance is caused by an impaired transport of leptin across the blood-brain barrier (BBB). It has been assumed that the short form of the leptin receptor, which is a splice variant of the gene which produces all known leptin receptors, is the leptin transporter, but evidence for this is mixed. The Koletsky rat model should provide a clear answer as to whether transport is dependent on leptin receptors as it does not express any functional receptors. The transport of intravenous leptin across the BBB of the Koletsky rat has been found to be greatly reduced, but evidence for a residual of transport makes it unclear whether the transporter is essentially absent or simply saturated by the high levels of leptin in the serum. Here we used the brain perfusion method to negate the influence of serum levels. We found that, whereas no transport of intravenous leptin occurred in the obese Koletsky, the rate of transport was no different from controls when brain perfusion was used. Leptin was transported completely across the BBB, was saturable, and had the same distribution among brain regions as previously found in normal weight mice (highest transport into the hippocampus and hypothalamus, lowest in the frontal cortex). We conclude that a leptin transporter and possibly its gene have yet to be identified and that the short form likely plays a role in the modulation of transport activity.

Permeability of the blood-brain barrier to a novel satiety molecule nesfatin-1.

Nesfatin-1 has recently been identified as a hypothalamic and brain stem peptide that regulates feeding behavior. Here, we determined the ability of nesfatin-1 to cross the blood-brain barrier (BBB) of mice. We used multiple-regression analysis to determine that radioactively labeled nesfatin-1 injected intravenously entered the brain. The entry rate (K(i)) of (131)I-nesfatin-1 from blood-to-brain was 0.20+/-0.02 microl/g min. This modest rate of entry was not inhibited by the administration of nonradioactive nesfatin-1, suggesting that BBB transport of nesfatin-1 into the brain is by a nonsaturable mechanism. High performance liquid chromatography (HPLC) and acid precipitation showed that most of the injected radiolabeled nesfatin-1 reached the brain as intact peptide, and capillary depletion with vascular washout revealed that 67% of (131)I-nesfatin-1 crossed the BBB to reach the brain parenchyma. Efflux of labeled nesfatin-1 from brain back into blood was by way of bulk flow. These findings demonstrate that nesfatin-1 crosses the BBB in both the blood-to-brain and brain-to-blood directions by nonsaturable mechanisms.

Testing the Neurovascular Hypothesis of Alzheimer’s Disease: LRP-1 Antisense Reduces Blood-Brain Barrier Clearance, Increases Brain Levels of Amyloid-β Protein, and Impairs Cognition

Decreased clearance is the main reason amyloid-beta protein (Abeta) is increased in the brains of patients with Alzheimers disease (AD). The neurovascular hypothesis states that this decreased clearance is caused by impairment of low density lipoprotein receptor related protein-1 (LRP-1), the major brain-to-blood transporter of Abeta at the blood-brain barrier (BBB). As deletion of the LRP-1 gene is a lethal mutation, we tested the neurovascular hypothesis by developing a cocktail of phosphorothioate antisenses directed against LRP-1 mRNA. We found these antisenses in comparison to random antisense selectively decreased LRP-1 expression, reduced BBB clearance of Abeta42, increased brain levels of Abeta42, and impaired learning ability and recognition memory in mice. These results support dysfunction of LRP-1 at the BBB as a mechanism by which brain levels of Abeta could increase and AD would be promoted.

Extra virgin olive oil improves learning and memory in SAMP8 mice.

Polyphenols are potent antioxidants found in extra virgin olive oil (EVOO); antioxidants have been shown to reverse age- and disease-related learning and memory deficits. We examined the effects of EVOO on learning and memory in SAMP8 mice, an age-related learning/memory impairment model associated with increased amyloid-β protein and brain oxidative damage. We administered EVOO, coconut oil, or butter to 11 month old SAMP8 mice for 6 weeks. Mice were tested in T-maze foot shock avoidance and one-trial novel object recognition with a 24 h delay. Mice which received EVOO had improved acquisition in the T-maze and spent more time with the novel object in one-trial novel object recognition versus mice which received coconut oil or butter. Mice that received EVOO had improve T-maze retention compared to the mice that received butter. EVOO increased brain glutathione levels suggesting reduced oxidative stress as a possible mechanism. These effects plus increased glutathione reductase activity, superoxide dismutase activity, and decreased tissue levels of 4-hydroxynoneal and 3-nitrotyrosine were enhanced with enriched EVOO (3 × and 5 × polyphenols concentration). Our findings suggest that EVOO has beneficial effects on learning and memory deficits found in aging and diseases, such as those related to the overproduction of amyloid-β protein, by reversing oxidative damage in the brain, effects that are augmented with increasing concentrations of polyphenols in EVOO.

Aluminum complexing enhances amyloid β protein penetration of blood-brain barrier

A significant co-morbidity of Alzheimers disease and cerebrovascular impairment suggests that cerebrovascular dysregulation is an important feature of dementia. Amyloid beta protein (Abeta), a relevant risk factor in Alzheimers disease, has neurotoxic properties and is thought to play a critical role in the cognitive impairments. Previously, we demonstrated that the 42mer of Abeta (Abeta42) complexed with aluminum (Al-Abeta42) is much more cytotoxic than non-complexed Abeta42. The level of Abeta in the brain is a balance between synthesis, degradation, and fluxes across the blood-brain barrier (BBB). In the present paper, we determined whether complexing with aluminum affected the ability of radioactively iodinated Abeta to cross the in vivo BBB. We found that the rates of uptake of Al-Abeta42 and Abeta42 were similar, but that Al-Abeta42 was sequestered by brain endothelial cells much less than Abeta42 and so more readily entered the parenchymal space of the brain. Al-Abeta42 also had a longer half-life in blood and had increased permeation at the striatum and thalamus. Brain-to-blood transport was similar for Al-Abeta42 and Abeta42. In conclusion, complexing with aluminum affects some aspects of blood-to-brain permeability so that Al-Abeta42 would have more ready access to brain cells than Abeta42.

Isolation of Peptide Transport System-6 from Brain Endothelial Cells: Therapeutic Effects with Antisense Inhibition in Alzheimer and Stroke Models:

By isolating for the first time ever a peptide transporter from the blood—brain barrier (BBB) and developing an antisense that selectively targets the brain-to-blood efflux component, we were able to deliver a therapeutic concentration of the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) 27 to brain in animal models of Alzheimers and stroke. Efflux pumps at the BBB are major causes of BBB impermeability to peptides. PACAP is neuroprotective in vitro in femtomole amounts, but brain uptake of PACAP27 is limited by an efflux component of peptide transport system-6 (PTS-6). Here, we characterized, isolated, and sequenced this component of PTS-6, identifying it as β-F1 ATPase, and colocalized it with PACAP27 on BBB endothelial cells. Antisenses targeting the BBB inhibited PACAP27 efflux, thus increasing brain uptake of PACAP27. Treatment with antisense +PACAP27 improved cognition in a mouse model of Alzheimers disease and reduced infarct size after cerebral ischemia. This represents the first isolation from BBB tissue of a peptide transporter and shows that inhibition of peptide efflux pumps is a potential strategy for drug delivery to brain.

Antisense oligonucleotide against GSK-3β in brain of SAMP8 mice improves learning and memory and decreases oxidative stress: Involvement of transcription factor Nrf2 and implications for Alzheimer disease

Glycogen synthase kinase (GSK)-3β is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimers disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aβ), and neurodegeneration. In this study we used 12-month-old SAMP8 mice, an AD model, to examine the effects GSK-3β may cause regarding the cognitive impairment and oxidative stress associated with AD. To suppress the level of GSK-3β, SAMP8 mice were treated with an antisense oligonucleotide (GAO) directed at this kinase. We measured a decreased level of GSK-3β in the cortex of the mice, indicating the success of the antisense treatment. Learning and memory assessments of the SAMP8 mice were tested post-antisense treatment using an aversive T-maze and object recognition test, both of which observably improved. In cortex samples of the SAMP8 mice, decreased levels of protein carbonyl and protein-bound HNE were measured, indicating decreased oxidative stress. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription factor known to increase the level of many antioxidants, including glutathione-S transferase (GST), and is negatively regulated by the activity of GSK-3β. Our results indicated the increased nuclear localization of Nrf2 and level of GST, suggesting the increased activity of the transcription factor as a result of GSK-3β suppression, consistent with the decreased oxidative stress observed. Consistent with the improved learning and memory, and consistent with GSK-3b being a tau kinase, we observed decreased tau phosphorylation in brain of GAO-treated SAMP8 mice compared to that of RAO-treated SAMP8 mice. Lastly, we examined the ability of GAO to cross the blood-brain barrier and determined it to be possible. The results presented in this study demonstrate that reducing GSK-3 with a phosphorothionated antisense against GSK-3 improves learning and memory, reduces oxidative stress, possibly coincident with increased levels of the antioxidant transcriptional activity of Nrf2, and decreases tau phosphorylation. Our study supports the notion of GAO as a possible treatment for AD.

Delivery of testosterone to the brain by intranasal administration: Comparison to intravenous testosterone

Intranasal (i.n.) administration has emerged as a strategy to deliver therapeutics to the brain. Here, we compared i.n. and intravenous (i.v.) administration for testosterone. About 75% of the i.n. administered testosterone entered the blood. However, whole brain levels of testosterone were about twice as high after i.n. administration as after i.v. administration. About two-thirds of the testosterone entering the brain after i.n. administration did so by direct entry by nasal routes and the remainder indirectly by first entering the blood and then crossing the blood–brain barrier. All brain regions except the frontal cortex had higher levels of testosterone after i.n. administration than after i.v. administration, although the differences among brain regions varied much more for the i.n. route. The olfactory bulb, hypothalamus, striatum, and hippocampus had the highest levels after i.n. administration. The brain uptake pattern suggested a variety of distribution routes likely involving the cerebrospinal fluid, diffusion through brain tissue, and transport through nerve projections. Regional distribution patterns were similar after either i.n. or i.v. administration, suggesting that the dominant factor determining distribution/retention was the same for either route of administration. We conclude that the i.n. administration route delivers testosterone systemically and can target the brain, especially the olfactory bulb, hypothalamus, striatum, and hippocampus.

The GLP-1 Receptor Agonist Liraglutide Improves Memory Function and Increases Hippocampal CA1 Neuronal Numbers in a Senescence-Accelerated Mouse Model of Alzheimer’s Disease

Abstract Recent studies indicate that glucagon-like peptide 1 (GLP-1) receptor agonists, currently used in the management of type 2 diabetes, exhibit neurotrophic and neuroprotective effects in amyloid-β (Aβ) toxicity models of Alzheimer’s disease (AD). We investigated the potential pro-cognitive and neuroprotective effects of the once-daily GLP-1 receptor agonist liraglutide in senescence-accelerated mouse prone 8 (SAMP8) mice, a model of age-related sporadic AD not dominated by amyloid plaques. Six-month-old SAMP8 mice received liraglutide (100 or 500 μg/kg/day, s.c.) or vehicle once daily for 4 months. Vehicle-dosed age-matched 50% back-crossed as well as untreated young (4-month-old) SAMP8 mice were used as control groups for normal memory function. Vehicle-dosed 10-month-old SAMP8 mice showed significant learning and memory retention deficits in an active-avoidance T-maze, as compared to both control groups. Also, 10-month-old SAMP8 mice displayed no immunohistological signatures of amyloid-β plaques or hyperphosphorylated tau, indicating the onset of cognitive deficits prior to deposition of amyloid plaques and neurofibrillary tangles in this AD model. Liraglutide significantly increased memory retention and total hippocampal CA1 pyramidal neuron numbers in SAMP8 mice, as compared to age-matched vehicle-dosed SAMP8 mice. In conclusion, liraglutide delayed or partially halted the progressive decline in memory function associated with hippocampal neuronal loss in a mouse model of pathological aging with characteristics of neurobehavioral and neuropathological impairments observed in early-stage sporadic AD.