Jasmina B. Mackic

Cerebrovascular Accumulation and Increased Blood‐Brain Barrier Permeability to Circulating Alzheimer's Amyloid β Peptide in Aged Squirrel Monkey with Cerebral Amyloid Angiopathy

Abstract: Senescent squirrel monkey is a valuable model to study pathogenesis of cerebrovascular amyloid angiopathy (CAA). Cerebrovascular sequestration and blood‐brain barrier (BBB) permeability to 125I‐amyloid β(1‐40) synthetic peptide (sAβ1‐40) were studied in adult versus aged squirrel monkey 1 h after a single intravenous injection. In aged monkey, the half‐time of elimination of sAβ1‐40, te1/2, was prolonged by 0.6 h, the systemic clearance, ClSS, was reduced from 1.8 to 1.1 ml/min/kg, and the mean residence time of intact peptide in the circulation was increased by 1 h (45%). In adult monkey, cerebrovascular sequestration of intact sAβ1‐40 was significant, and the BBB permeability was 18.6‐fold higher than for inulin. In aged monkey, the sequestration of intact sAβ1‐40 by cortical and leptomeningeal microvessels and the BBB permeability were increased by 5.9, 1.8‐, and 2.1‐fold, respectively, in the presence of an unchanged barrier to inulin. In brain parenchyma of aged animals, 76.1% of circulating sAβ1‐40 remained intact versus 45.7% in adult. We conclude that multiple age‐related systemic effects, i.e., reduced body elimination and systemic clearance of sAβ1‐40, and reduced peripheral metabolism, may act in concert with BBB mechanisms, i.e., increased transendothelial transport and microvascular accumulation of blood‐borne sAβ1‐40, and reduced brain metabolism to enhance the development of CAA.

Isoform-Specific Effects of Apolipoproteins E2, E3, and E4 on Cerebral Capillary Sequestration and Blood-Brain Barrier Transport of Circulating Alzheimer's Amyloid β

Abstract: Cerebral capillary sequestration and blood‐brain barrier (BBB) permeability to apolipoproteins E2 (apoE2), E3 (apoE3), and E4 (apoE4) and to their complexes with sAβ1–40, a peptide homologous to the major form of soluble Alzheimers amyloid β, were studied in perfused guinea pig brain. Cerebrovascular uptake of three apoE isoforms was low, their blood‐to‐brain transport undetectable, but uptake by the choroid plexus significant. Binding of all three isoforms to sAβ1–40 in vitro was similar with a KD between 11.8 and 12.9 nM. Transport into brain parenchyma and sequestration by BBB and choroid plexus were negligible for sAβ1–40‐apoE2 and sAβ1–40‐apoE3, but significant for sAβ1–40‐apoE4. After 10 min, 85% of sAβ1–40‐apoE4 taken up at the BBB remained as intact complex, whereas free sAβ1–40 was 51% degraded. Circulating apoE isoforms have contrasting effects on cerebral capillary uptake of and BBB permeability of sAβ. ApoE2 and apoE3 completely prevent cerebral capillary sequestration and blood‐to‐brain transport of sAβ1–40. Conversely, apoE4, by entering brain microvessels and parenchyma as a stable complex with sAβ, reduces peptide degradation and may predispose to cerebrovascular and possibly enhance parenchymal amyloid formation under pathological conditions.

Circulating amyloid-β peptide crosses the blood–brain barrier in aged monkeys and contributes to Alzheimer's disease lesions

1. We studied cerebrovascular sequestration and blood-brain barrier (BBB) permeability to [125I]- or [123I]-labeled amyloid-beta peptides (A beta) in aged rhesus and aged squirrel monkey, the nonhuman primate models of cerebral beta-amyloidosis and cerebrovascular amyloid angiopathy (CAA), respectively. 2. In aged rhesus, the half-time of elimination of [125I]A beta 1-40, t1/2e, was faster by 1.34 h, the systemic clearance, Clss, increased by 4.21 ml/min/kg and the mean residence time of intact peptide in the circulation shortened by 2 h. 3. Cerebrovascular sequestration of [125I]A beta 1-40 was significant in aged squirrel monkey (20.8 ml/g x 10(2)), but undetectable in the rhesus. 4. The permeability surface area product, PS, for [14C]inulin was low in both species (0.11-0.18 ml/g/s x 10(6)) indicating an intact barrier. 5. The BBB permeability to A beta 1-40 was 34.8- and 13.7-fold higher than for [14C]inulin in aged squirrel and rhesus, respectively, suggesting a specialized A beta transport across the BBB. 6. The single photon computed emission tomography studies confirmed a saturable [123I]A beta 1-40 transport at the BBB in primates (Km = 40 nM). 7. Brain autoradiographic analysis of [125I]A beta 1-42 or [125I]A beta 1-40 after intracarotid infusions of radiotracers confirmed co-localization of the signal with A beta-immunoreactive plaques in rhesus monkeys. 8. Metabolism of [125I]A beta 1-40 in brain and plasma was slower in aged squirrel compared to aged rhesus, by 2.9- and 2.6-fold, respectively. 9. Thus, transport of circulating A beta across the BBB contributes to brain parenchymal accumulation of amyloid in aged nonhuman primates. Negligible capillary binding, rapid systemic and brain degradation, and accelerated body elimination of blood-borne A beta, may prevent the development of CAA in rhesus in contrast to squirrel monkeys.

Substitution at codon 22 reduces clearance of Alzheimer’s amyloid-β peptide from the cerebrospinal fluid and prevents its transport from the central nervous system into blood

A point mutation of G to C at codon 693 of the amyloid-beta (Abeta) precursor protein gene results in Glu to Gln substitution at position 22 of the Abeta (AbetaQ22) associated with hereditary cerebrovascular amyloidosis with hemorrhage Dutch type. Factors that regulate AbetaQ22 levels in the central nervous system (CNS) are largely unknown. By using ventriculo-cisternal perfusion technique in guinea pigs, we demonstrated that clearance from the cerebrospinal fluid and transport from the CNS to blood of [(125)I]-AbetaQ22 (1 nM) were reduced by 36% and 52%, respectively, in comparison to the wild type Abeta(1-40) peptide. In contrast to significant uptake and transport of Abeta(1-40) across the brain capillaries and leptomeningeal vessels, AbetaQ22 was not taken up at these CNS vascular transport sites, which was associated with its 53% greater accumulation in the brain. The CNS clearance of Abeta(1-40) was half-saturated at 23.6 nM; AbetaQ22 had about 6.8-fold less affinity for the CNS efflux transporters and its elimination relied mainly on transport across the choroid plexus. Thus, the Dutch mutation impairs elimination of Abeta from brain by reducing its rapid transport across the blood-brain barrier and the vascular drainage pathways, which in turn may result in accumulation of the peptide around the blood vessels and in brain.

Brain Clearance of Alzheimer's Amyloid-β40 in the Squirrel Monkey: A SPECT Study in a Primate Model of Cerebral Amyloid Angiopathy

Squirrel monkey is a valuable model to study pathogenesis of cerebrovascular amyloid angiopathy (CAA). Previous studies suggested that circulating amyloid- β 40 peptide (A β 40) crosses the blood-brain barrier (BBB) and may therefore enhance cerebrovascular amyloidosis in aged squirrel monkeys. In the present study, we used single photon emission computed tomography (SPECT) to determine elimination of 123 I-A β 40 and 99m Tc-DTPA, an extracellular marker, from the brain in squirrel monkeys at different age. Following intracerebral microinfusions, the time-activity brain clearance curves indicated bi-exponential removal of 123 I-A β 40 with an initial rapid washout (1.1 ≤ t 1/2 ≤ 2.7 h). This, plus the observed appearance of 123 I-radioactivity in plasma suggest significant brain-to-blood transport. In contrast, 99m Tc-DTPA was removed slowly by brain interstitial fluid bulk flow (monoexponential decay with 6.8 ≤ t 1/2 ≤ 16.8 h) . A comparison of three middle aged (11-16 years old) vs. two old (22 yrs old) monkeys was consistent with an age-related decline in the BBB capacity to remove 123 I-A β from the brain. This correlated with an age-dependent increase in A β 40/42 cerebrovascular immunoreactivity and amyloid deposition. Thus, vascular clearance plays an important role in reducing A β levels in the squirrel monkey brain and impaired A β 40 elimination across the BBB may contribute to the development of CAA.

Kinetic Analysis of Leucine‐Enkephalin Cellular Uptake at the Luminal Side of the Blood‐Brain Barrier of an In Situ Perfused Guinea‐Pig Brain

Abstract: The uptake of enkephalin‐(5‐L‐leucine) (Leu‐en‐kephalin) at the luminal side of the blood‐brain barrier was measured by means of an in situ vascular brain perfusion technique in the anaesthetized guinea pig. This method allows measurements of cerebrovascular peptide uptake over periods of up to 20 min, and excludes the solute under study from the general circulation and systemic metabolic influences. A capillary unidirectional transfer constant, Kin, for [tyrosyl‐3,5‐3H]Leu‐enkephalin was estimated graphically from the multiple‐time brain uptake data in the presence of different concentrations of unlabelled peptide, and dose‐dependent self‐inhibition was demonstrated. Analysis of unidirectional influx of blood‐borne Leu‐en kephalin into the brain revealed Michaelis‐Menten saturation kinetics in the parietal cortex, caudate nucleus, and hippocampus, with Vmax between 0.14 and 0.16 nmol min−1 g−1 and Km ranging from 34 to 41 μM, for the saturable component, whereas the estimated diffusion constant, Kd, was not significantly different from zero. Entry of [3H]Leu‐enkephalin was not inhibited in the presence of either a 5 mM concentration of unlabelled L‐tyrosine, tyro‐sylglycine, and tyrosylglycylglycine, or aminopeptidase inhibitor, bestatin (0.5 mM), suggesting that the saturable mechanism of the tracer at the luminal side of the blood‐brain barrier does not involve uptake of the peptides N‐terminal amino acid and/or its tyrosine‐containing fragments. The specific δ‐opioid antagonist, allyl2‐Tyr‐AIB‐Phe‐OH, and μ‐opioid receptor agonist, Tyr‐D‐Ala‐Gly‐Me‐Phe‐NH(CH2)20H, at concentrations in the perfusate above the Km value for the saturable transport of Leu‐enkephalin, did not affect significantly uptake of [3H]Leu‐enkephalin. The present study provides, for the first time, a characterization of the kinetic parameters of the unidirectional uptake of a peptide from the luminal side of the blood‐brain barrier

Blood-brain barrier uptake of the 40 and 42 amino acid sequences of circulating Alzheimer's amyloid β in guinea pigs

An intracarotid brain infusion/capillary depletion technique was used in guinea pigs to examine cerebral capillary sequestration and transport into brain parenchyma of sA beta 1-40 and sA beta 1-42, synthetic peptides identical to two forms of the amyloid beta peptide found in Alzheimers disease lesions: the 40 residue form, found primarily in vascular deposits, and the 42 residue form, found primarily in senile plaques. The peptides crossed well into the brain parenchyma via a specific transport mechanism for which sA beta 1-40 had an approximately two-fold greater affinity than sA beta 1-42. There was significant capillary sequestration of sA beta 1-40, but retention by the microvasculature of sA beta 1-42 was negligible. These data suggest that the level of the 40 residue peptide in cerebral vasculature and of the 42 residue peptide in parenchyma could be regulated by blood-brain barrier sequestration and transport of their respective circulating precursors.

A saturable mechanism for transport of immunoglobulin G across the blood-brain barrier of the guinea pig

The existence of an immunological blood-brain barrier to homologous blood-borne immunoglobulin G (IgG) was investigated in the guinea pig using a vascular brain perfusion technique in situ. Cerebrovascular unidirectional transfer constants (Kin) for 125I-labeled IgG (2.5 micrograms/ml) estimated from the multiple-time brain uptake data, ranged from 0.53 to 0.58 ml min-1 g-1 X 10(3) in the parietal cortex, hippocampus, and caudate nucleus, the transfer rate being some 10 times higher than that for [3H]dextran (MW 70,000). In the presence of 4 mg/ml unlabeled IgG, unidirectional blood to brain transfer of 125I-IgG was markedly inhibited. Immunohistochemical analysis of the brain tissue after vascular perfusion with unlabeled IgG revealed a distribution of the blood-borne immunoglobulin in the endothelial cells of microvessels and in the surrounding perivascular tissue. It is concluded that there is a specific transfer mechanism for IgG at the blood-brain barrier in the guinea pig, which is saturated at physiological plasma levels of IgG.

Transport, uptake, and metabolism of blood-borne vasopressin by the blood-brain barrier

Transport, binding, and metabolism of [phenylalanyl-3,4,5,-3H(N)]arginine vasopressin (AVP) by the blood-brain barrier (BBB) was studied in adult guinea-pigs by means of a novel vascular brain perfusion (VBP)/capillary depletion technique and HPLC. A time-dependent, progressive brain uptake of 3H-radioactivity was measured over the 10 min period of VBP both in brain homogenates and in brain tissue depleted of cerebral microvessels. The unidirectional blood-to-brain transport constant, K(IN), estimated by multiple-time tissue uptake analysis of the homogenate and postcapillary supernatant, indicated that the BBB transfer rate for [3H]AVP (K(IN) = 2.37 +/- 0.25 microliters min-1 per gram brain homogenate) was almost 10 times higher than for simultaneously perfused [14C]sucrose, a cerebrovascular space marker. In contrast to homogenate and postcapillary supernatant, the [3H]radioactivity determined in the vascular pellet after dextran density centrifugation of the brain homogenate was very low and only somewhat higher than for [14C]sucrose. HPLC analysis of the perfused brain tissue revealed time-dependent degradation of the blood-borne neuropeptide. The percentage of intact [3H]AVP as determined in the postcapillary supernatant progressively declined during brain perfusion, from 49% at 1 min to 11.9% at 10 min. The major detectable labeled metabolite was [3H]phenylalanine, the labeled amino acid residue of [3H]AVP. The aminopeptidase inhibitor bestatin (0.5 mM), perfused simultaneously with [3H]AVP by the VBP technique, did not alter tissue uptake of [3H]AVP, indicating that there was no significant hydrolysis of peptide by the luminal BBB surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Cereport™ (RMP-7) Increases the Permeability of Human Brain Microvascular Endothelial Cell Monolayers1

AbstractPurpose. To study Cereport (RMP-7, bradykinin B2 agonist) effects on human brain microvascular endothelial cell (HBMEC) monolayer permeability.nMethods. HBMEC grown on transwell membranes were exposed to Cereport. The monolayer permeability was determined with [I4C]-inulin (MW. 5,200) and [3H]-dextran (MW. 70,000).nResults. Cereport increased the HBMEC permeability to [l4C]-inulin, but not to [3H]-dextran. The effect was transient, maximal at 15 min (i.e., 79.3% increase), and polarized to the basolateral membrane. An inverted U, dose-response curve was observed with active concentrations of Cereport from 0.01 to 0.5 nmol/L, the plateau maximal effect between 0.5 and 10 nmol/L, and loss of activity at the highest concentration, i.e., 20 nmol/L. Cyclic AMP-specific phosphodiesterase 3 (PDE3) inhibitor rolipram (10 μmol/L) abolished Cereport effects, while cGMP-specific PDE5 inhibitor, zaniprast (50 μmol/L) enhanced by 31 % (p < 0.05) the effect of 0.1 nmol/L Cereport. Unlabeled Cereport displaced [125I]-bradykinin and/or [125I]-Cereport from the basolateral side. There was no specific Cereport binding to the apical side.nConclusions. Cereport exerts specific time, dose and size dependent actions on HMBEC monolayer that are restricted to the basolateral membrane. Its effects can be further modulated through changes in cAMP and cGMP second messenger systems.