Joseph F. Poduslo

Permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors : NGF, CNTF, NT-3, BDNF

A comparison was made of the permeabilities of different neurotrophic factors at the blood-brain barrier (BBB) and blood-nerve barrier (BNB) in normal adult rats by quantifying the permeability coefficient-surface area (PS) product after correction for the residual plasma volume (Vp) occupied by the protein in the capillary bed of the nerve endoneurium or different brain regions. The i.v. bolus injection technique was used in the cannulated brachial vein and artery using the same protein radioiodinated with a second isotope of iodine (125I vs. 131I) to separately determine the PS and Vp values. The plasma washout showed a decreasing plasma half-life in the order of brain-derived neurotrophic factor (BDNF) < neurotrophin-3 (NT-3) < ciliary neurotrophic factor (CNTF) < nerve growth factor (NGF). The PS at the BNB for NGF was 1.40 +/- 0.15 x 10(-6) ml/g/s (mean +/- SEM). The other neurotrophic proteins were all significantly higher than NGF (CNTF: 9.5 x ; NT-3: 20.8 x ; BDNF: 18.9 x ). The Vp for NGF at the BNB was 1.92 +/- 0.12 microliters/g and was not significantly different from the other proteins except for NGF vs. BDNF (P < 0.05). The PS for NGF at the BBB ranged from 1.5 to 2.7 x 10(-6) ml/g/s for six different brain regions. The PS for CNTF ranged from 6.0 to 8.0-fold higher than NGF; NT-3: 10.6 to 15.2-fold higher; and BDNF: 11.3 to 16.4-fold higher. The Vp values were not significantly different except for CNTF in the hippocampus and cortex (P < 0.05). SDS-PAGE analyses of all the radioiodinated neurotrophic proteins after 60 min of uptake revealed intact protein in the endoneurium and in the six different brain regions with exposure times of 2-42 days. The quantification of the permeability of these neurotrophic proteins provides baseline values for comparison of different protein modifications that enhance the PS while still preserving the neurotrophic activity (e.g., protein glycation; Poduslo and Curran, Mol. Brain Res., 23 (1994) 157). Enhanced permeability following modification might allow the use of systematic delivery of these proteins for practical therapeutic treatment of various neurodegenerative disorders.

Nerve Glucose, Fructose, Sorbitol, myo-Inositol, and Fiber Degeneration and Regeneration in Diabetic Neuropathy

We measured the alcohol sugars in sural nerves from 11 controls, 21 conventionally treated patients with diabetes and neuropathy, and 4 diabetics without neuropathy. The results were related to metabolic control and to clinical, neuropathological, and morphometric abnormalities in the nerves. The mean endoneurial glucose, fructose, and sorbitol values were higher in diabetic patients than in controls. Linear regression analysis revealed that nerve sorbitol content in the diabetics was inversely related to the number of myelinated fibers (P = 0.003). Mean nerve levels of myo-inositol were not decreased in the diabetic patients, with or without neuropathy, and were not associated with any of the neuropathological end points of diabetes. Our results indicate that myo-inositol deficiency is not part of the pathogenesis of human diabetic neuropathy, as had been hypothesized. Other accumulated alcohol sugars, however, were increased in diabetes and were associated with the severity of neuropathy. On repeat biopsy, six diabetics, treated for a year with the aldose reductase inhibitor sorbinil, had decreased endoneurial levels of sorbitol (P less than 0.01) and fructose (0.05 less than P less than 0.1), but unchanged levels of myo-inositol.

Molecular targeting of Alzheimer's amyloid plaques for contrast-enhanced magnetic resonance imaging.

Smart molecular probes for both diagnostic and therapeutic purposes are expected to provide significant advances in clinical medicine and biomedical research. We describe such a probe that targets beta-amyloid plaques of Alzheimers disease and is detectable by magnetic resonance imaging (MRI) because of contrast imparted by gadolinium labeling. Three properties essential for contrast enhancement of beta-amyloid plaques on MRI exist in this smart molecular probe, putrescine-gadolinium-amyloid-beta peptide: (1) transport across the blood-brain barrier following intravenous injection conferred by the polyamine moiety, (2) binding to plaques with molecular specificity by putrescine-amyloid-beta, and (3) magnetic resonance imaging contrast by gadolinium. MRI was performed on ex vivo tissue specimens at 7 T at a spatial resolution approximating plaque size (62.5 microm(3)), in order to prove the concept that the probe, when administered intravenously, can selectively enhance plaques. The plaque-to-background tissue contrast-to-noise ratio, which was precisely correlated with histologically stained plaques, was enhanced more than nine-fold in regions of cortex and hippocampus following intravenous administration of this probe in AD transgenic mice. Continuing engineering efforts to improve spatial resolution are underway in MRI, which may enable in vivo imaging at the resolution of individual plaques with this or similar contrast probes. This could enable early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed.

In vivo visualization of Alzheimer's amyloid plaques by magnetic resonance imaging in transgenic mice without a contrast agent.

One of the cardinal pathologic features of Alzheimers disease (AD) is the formation of senile, or amyloid, plaques. Transgenic mice have been developed that express one or more of the genes responsible for familial AD in humans. Doubly transgenic mice develop “human‐like” plaques, providing a mechanism to study amyloid plaque biology in a controlled manner. Imaging of labeled plaques has been accomplished with other modalities, but only MRI has sufficient spatial and contrast resolution to visualize individual plaques noninvasively. Methods to optimize visualization of plaques in vivo in transgenic mice at 9.4 T using a spin echo sequence based on adiabatic pulses are described. Preliminary results indicate that a spin echo acquisition more accurately reflects plaque size, while a T2* weighted gradient echo sequence reflects plaque iron content, not plaque size. In vivo MRI–ex vivo MRI–in vitro histologic correlations are provided. Histologically verified plaques as small as 50 μm in diameter were visualized in living animals. To our knowledge this work represents the first demonstration of noninvasive in vivo visualization of individual AD plaques without the use of a contrast agent. Magn Reson Med 52:1263–1271, 2004.

In Vivo Magnetic Resonance Microimaging of Individual Amyloid Plaques in Alzheimer's Transgenic Mice

The ability to detect individual Alzheimers amyloid plaques in vivo by magnetic resonance microimaging (MRI) should improve diagnosis and also accelerate discovery of effective therapeutic agents for Alzheimers disease (AD). Here, we perform in vivo and ex vivo MRI on double transgenic AD mice as well as wild-type mice at varying ages and correlate these with thioflavin-S and iron staining histology. Quantitative counts of individual plaques on MRI increase with age and correlate with histologically determined plaque burden. Plaques 20 μm in diameter can be detected in AD mice as young as 3 months of age with ex vivo MRI. Plaques 35 μm in diameter can be detected by 9 months of age with in vivo MRI. In vivo MRI of individual Alzheimers amyloid plaques provides a noninvasive estimate of plaque burden in transgenic AD mice that might be useful in assessing the efficacy of amyloid reduction therapies.

Defects in Mitochondrial Dynamics and Metabolomic Signatures of Evolving Energetic Stress in Mouse Models of Familial Alzheimer's Disease

Background The identification of early mechanisms underlying Alzheimers Disease (AD) and associated biomarkers could advance development of new therapies and improve monitoring and predicting of AD progression. Mitochondrial dysfunction has been suggested to underlie AD pathophysiology, however, no comprehensive study exists that evaluates the effect of different familial AD (FAD) mutations on mitochondrial function, dynamics, and brain energetics. Methods and Findings We characterized early mitochondrial dysfunction and metabolomic signatures of energetic stress in three commonly used transgenic mouse models of FAD. Assessment of mitochondrial motility, distribution, dynamics, morphology, and metabolomic profiling revealed the specific effect of each FAD mutation on the development of mitochondrial stress and dysfunction. Inhibition of mitochondrial trafficking was characteristic for embryonic neurons from mice expressing mutant human presenilin 1, PS1(M146L) and the double mutation of human amyloid precursor protein APP(Tg2576) and PS1(M146L) contributing to the increased susceptibility of neurons to excitotoxic cell death. Significant changes in mitochondrial morphology were detected in APP and APP/PS1 mice. All three FAD models demonstrated a loss of the integrity of synaptic mitochondria and energy production. Metabolomic profiling revealed mutation-specific changes in the levels of metabolites reflecting altered energy metabolism and mitochondrial dysfunction in brains of FAD mice. Metabolic biomarkers adequately reflected gender differences similar to that reported for AD patients and correlated well with the biomarkers currently used for diagnosis in humans. Conclusions Mutation-specific alterations in mitochondrial dynamics, morphology and function in FAD mice occurred prior to the onset of memory and neurological phenotype and before the formation of amyloid deposits. Metabolomic signatures of mitochondrial stress and altered energy metabolism indicated alterations in nucleotide, Krebs cycle, energy transfer, carbohydrate, neurotransmitter, and amino acid metabolic pathways. Mitochondrial dysfunction, therefore, is an underlying event in AD progression, and FAD mouse models provide valuable tools to study early molecular mechanisms implicated in AD.

Targeting Alzheimer amyloid plaques in vivo

The only definitive diagnosis for Alzheimer disease (AD) at present is postmortem observation of neuritic plaques and neurofibrillary tangles in brain sections. Radiolabeled amyloid-β peptide (Aβ), which has been shown to label neuritic plaques in vitro, therefore could provide a diagnostic tool if it also labels neuritic plaques in vivo following intravenous injection. In this study, we show that the permeability of Aβ at the blood–brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine. We also show that, following intravenous injection, radiolabeled, putrescine-modified Aβ labels amyloid deposits in vivo in a transgenic mouse model of AD, as well as in vitro in human AD brain sections. This technology, when applied to humans, may be used to detect plaques in vivo, allowing early diagnosis of the disease and therapeutic intervention before cognitive decline occurs.

Permeability of proteins at the blood-brain barrier in the normal adult mouse and double transgenic mouse model of Alzheimer's disease

The permeability of albumin, insulin, and human A beta 1--40 at the blood-brain barrier (BBB) was determined in the normal adult mouse (B6/SJL) and in the double transgenic Alzheimer mouse (APP, PS1) by using an I.V. bolus injection technique to quantify the permeability coefficient-surface area (PS) product for each protein after correction for the residual plasma volume (V(p)) occupied by the protein in the blood vessels of different brain regions using a second aliquot of the same protein radiolabeled with a different isotope of iodine ((125)I vs (131)I) as a vascular space marker. This technology for quantifying BBB permeability of proteins was adapted from the rat to the mouse and involved catheterizing the femoral artery and vein of the mouse instead of the brachial artery and vein as for the rat. Because of the smaller blood volume in the mouse, serial sampling (20 microl) of blood from the femoral artery of the mouse was performed and directly TCA precipitated to generate a whole blood washout curve for the intact protein. When similar blood sampling techniques were used in the rat, the PS values for albumin and insulin at the BBB were similar in these two species. In the double transgenic mouse, the V(p) values for albumin were significantly increased 1.4- to 1.6-fold in five of six brain regions compared to the normal adult mouse, which indicated increased adherence of albumin to vessel walls. As a result, the PS values were significantly decreased, from 1.4- to 3.2-fold, which likely reflected decreased transport of albumin by passive diffusion. In contrast, insulin, which is taken up into the brain by a receptor-mediated transport mechanism at the BBB, showed no significant difference in the V(p) values but a significant increase in the PS values in four of six brain regions. This suggests a compensatory mechanism in the Alzheimers transgenic brain whereby there is an increased permeability to insulin at the BBB. Surprisingly, there was no significant difference in the V(p) or PS values for human A beta 1--40 at the BBB in the double transgenic Alzheimer mouse at 24, 32, or 52 weeks of age, when there is both significant A beta levels in the plasma and amyloid burden in the brains of these animals. These data suggest that there is not an alteration in permeability to human A beta 1--40 at the BBB with increasing amyloid burden in the double transgenic Alzheimer mouse. Although these observations suggest structural alterations at the BBB, they do not support the concept of extensive BBB damage with substantial increases in BBB permeability in Alzheimers disease.

Polyamine modification increases the permeability of proteins at the blood-nerve and blood-brain barriers

Abstract: The permeability of the blood‐nerve barrier (BNB) and the blood‐brain barrier (BBB) to superoxide dismutase (SOD), insulin, albumin, and IgG in normal adult rats was quantified by measuring the permeability coefficient‐surface area product (PS) with the intravenous bolus injection technique before and after covalent protein modification with the naturally occurring polyamines—putrescine (PUT), spermidine (SPD), and spermine (SPM). The PS value of the BNB for PUT‐SOD was 21.1‐fold greater than the native SOD, and the PS values of the BBB for PUT‐SOD ranged from 17.6‐fold greater for the thalamus to 23.6‐fold greater for the caudate‐putamen compared with native SOD. In a similar manner, polyamine‐modified insulin showed a 1.7–2.0‐fold increase in PS of the BNB and BBB compared with the high values of native insulin. Polyamine‐modified albumin showed a remarkable 54–165‐fold increase in PS of the BNB and BBB compared with native albumin, whereas PUT‐IgG resulted in an even higher increase in the PS that ranged from 111‐ to 349‐fold for nerve and different brain regions compared with native IgG. Polyamine modification of proteins, therefore, can dramatically increase the permeability at the BNB and BBB of a variety of proteins with widely differing Mr and function. It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT > SPD > SPM). Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density. It is suggested that the polyamine transporter may be responsible for the transport of these polyamine‐modified proteins. Systemic administration of polyamine‐modified peptides and proteins might prove to be an efficient approach to deliver therapeutic agents into the CNS and PNS for the treatment of a variety of neurological diseases.

β‐sheet breaker peptide inhibitor of Alzheimer's amyloidogenesis with increased blood–brain barrier permeability and resistance to proteolytic degradation in plasma

Short synthetic peptides homologous to the central region of Abeta but bearing proline residues as beta-sheet blockers have been shown in vitro to bind to Abeta with high affinity, partially inhibit Abeta fibrillogenesis, and redissolve preformed fibrils. While short peptides have been used extensively as therapeutic drugs in medicine, two important problems associated with their use in central nervous system diseases have to be addressed: (a) rapid proteolytic degradation in plasma, and (b) poor blood-brain barrier (BBB) permeability. Recently, we have demonstrated that the covalent modification of proteins with the naturally occurring polyamines significantly increases their permeability at the BBB. We have extended this technology to iAbeta11, an 11-residue beta-sheet breaker peptide that inhibits Abeta fibrillogenesis, by covalently modifying this peptide with the polyamine, putrescine (PUT), and evaluating its plasma pharmacokinetics and BBB permeability. After a single intravenous bolus injection in rats, both 125I-YiAbeta11 and 125I-PUT-YiAbeta11 showed rapid degradation in plasma as determined by trichloroacetic acid (TCA) precipitation and paper chromatography. By switching to the all D-enantiomers of YiAbeta11 and PUT-YiAbeta11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively. The permeability coefficient x surface area product at the BBB was five- sevenfold higher in the cortex and hippocampus for the 125I-PUT-D-YiAbeta11 compared to the 125I-D-YiAbeta11, with no significant difference in the residual plasma volume. In vitro assays showed that PUT-D-YiAbeta11 retains its ability to partially inhibit Abeta fibrillogenesis and dissolve preformed amyloid fibrils. Because of its five- to sevenfold increase in permeability at the BBB and its resistance to proteolysis in the plasma, this polyamine-modified beta-sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vivo and, hence, an important therapy for Alzheimers disease.