PMC:6194691 / 144526-157887 JSONTXT

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of Aβ from ISF\nIn the young, Aβ is present in soluble form and is eliminated as rapidly as it is produced with about 7–8% of the total soluble Aβ being replaced each hour [422, 444]. Monomeric and small oligomeric forms of soluble Aβ are cleared from ISF by at least four routes: incorporation into plaques, metabolism [445–451], efflux across the blood–brain barrier [62, 429, 452–454] and efflux via perivascular routes [25, 85, 128, 455]. The relative importance of each of these routes remains controversial [52, 146, 456–458].\n\nEvidence for transport of soluble Aβ across the blood–brain barrier\nThe ways in which soluble Aβ can be transported across the blood–brain barrier have been investigated by several different groups. Shibata et al. [62] were the first to propose that Aβ could cross the blood–brain barrier by transcytosis mediated by low density lipoprotein receptor related protein (LRP1). This they said could account for the loss of 125I-Aβ1-40 from the brain that they observed. In support of their proposal they found that the loss of total 125I from the brain was reduced by antibodies against LRP1, by receptor (LRP1) associated protein (RAP), which interferes with binding of all known substrates to LRP1, and by absence of apoE seen in knockout mice. (ApoE affects the interaction of Aβ with LRP1). In addition the elimination process appeared to be saturable with Km of 15 nM. All of these observations are consistent with the idea that the elimination of soluble 125I-Aβ1-40 is primarily efflux across the blood–brain barrier and is via an LRP1-dependent process. However it should be kept in mind that demonstrating the importance of LRP1 is not the same as demonstrating elimination via the blood–brain barrier because LRP1 is also present on neurons, astrocytes and vascular smooth muscle cells where it can mediate endocytosis of Aβ leading to its metabolism inside the cells [448, 456, 459] (see [146] for further discussion). Further results supporting the involvement of efflux have been reported by Bell et al. [429], who found that the rate constant for elimination of Aβ1-42 was about half that for Aβ1-40 and also in other papers by Deane, Zhao, Nelson, Zlokovic and coworkers [452, 454, 460].\nResults from several other groups also support the idea that efflux of soluble Aβ does occur at the blood–brain barrier and that LRP1 is involved in this elimination.Jaeger et al. [461] showed that antisense oligonucleotides against LRP-1 substantially decreased the loss of Aβ1-42 after intraparenchymal injection.\nPflanzner et al. [462] demonstrated LRP1-dependent Aβ1-40 transport across monolayers of primary mouse brain capillary endothelial cells, a transport not observed in monolayers of cells with genetically modified LRP1.\nRoberts et al. [457] confirmed that efflux of Aβ from brain to blood occurs in vivo by finding that the concentration in venous blood leaving the brain was 7.5% higher than that in arterial blood.\nQosa et al. [424] using the brain efflux index method found that 62% of added 125I-Aβ1-40 appeared in the blood.\nStorck et al. [453] developed a mouse model in which LRP1 could be knocked out selectively in endothelial cells and showed that the knockout reduced the initial rate of loss of 125I-Aβ1-42 by 48%.\nCollectively the studies discussed above leave little doubt that LRP1 dependent transport across the blood–brain barrier plays a substantial role in Aβ elimination. However, the actual mechanisms governing the net inward or outward flux of Aβ across the blood–brain barrier are considerably more complicated and involve complexing Aβ with soluble factors including clusterin (also called apoJ), apoE and a soluble, truncated form of LRP1 (sLRP1). In addition there are at least four endocytotic/transcytotic systems. Figure 20, based mainly on the views of Zlokovic and colleagues [429, 452, 454, 460, 463–466], is a simplified diagram indicating the mechanisms of Aβ transport across the blood–brain barrier. Notable in this scheme is the involvement of apoE, clusterin and the phosphatidylinositol-binding clathrin assembly protein, PICALM (also called CALM). Genetic variations for each of these have been shown to be associated with increased risk of Alzheimer’s disease [467, 468].\nFig. 20 Simplified outline of Aβ transport across the blood–brain barrier. Possible movements of Aβ are shown by solid or dashed lines with arrowheads indicating the principal direction. Endocytotic and exocytotic vesicles are shown as invaginations of the membranes. There is intracellular processing once the vesicles have been endocytosed. Aβ from ISF can bind directly to LRP1 on the abluminal membrane with the complex then being incorporated into a clathrin coated pit which can be endocytosed. The Aβ-LRP1 complex is stabilized by binding of the phosphatidylinositol-binding clathrin assembly protein (PICALM). Aβ in ISF can also be complexed with any of the forms of apoE, 2, 3 or 4 or with clusterin. Aβ-apoE2 and Aβ-apoE3 are substrates for interaction with LRP1 and endocytosis. By contrast Aβ-apoE4 inhibits LRP1 mediated endocytosis (dotted line), but can be endocytosed slowly after binding with the very low density lipoprotein receptor (VLDLR). Aβ-clusterin is a substrate for LRP2 mediated endocytois with transport across the blood–brain barrier to plasma. As Aβ-clusterin can also be transported in the opposite direction by LRP2-mediated endocytosis this is almost certainly by transcytosis of vesicles with LRP2 in the membrane. Vesicles with LRP1 in the membrane are also thought to discharge their contents on the far side of the barrier—i.e. this is transcytosis [465]. Some of the intracellular processing steps for the LRP1 vesicles are now known [452, 658]. Aβ is also transported from plasma to ISF. Aβ clusterin can be transported by LRP2 vesicles, but on the plasma side almost all of the LRP2 receptors are occupied by clusterin (dotted double headed arrow) rather than Aβ-clusterin which greatly reduces blood-to-brain transport by this route. Aβ is however, endocytosed after binding to the receptor for advanced glycation products, RAGE, and somehow transported to the brain side. Pgp may, in a manner which has not been well defined, assist transfer of Aβ from the endothelial cells to plasma whether it has entered the cells from ISF, via the LRP1 system, or from plasma, via the RAGE system. Figure based on [452, 464, 465]\nMuch of the soluble Aβ in ISF may be in the form of complexes with apoE or clusterin while in plasma most Aβ is complexed with clusterin or sLRP1, a truncated, soluble form of LRP1 [469]. The apoE gene has three alleles called apoE2, apoE3 and apoE4. Expression of the apoE4 allele is the greatest genetic risk factor known for developing the late-onset form of Alzheimer’s disease [467, 468].\nLRP1 mediated transport of Aβ occurs via clathrin pits, with the LRP1, Aβ, clathrin system stabilized by interaction with PICALM. In addition to this transport of Aβ there is LRP1-mediated transport from brain-to-blood of Aβ complexes with apoE2 or apoE3 and LRP2-mediated transport of Aβ complexes with clusterin. Complexes with apoE4 inhibit LRP1-mediated transport but are transported at a much lower rate by very low-density lipoprotein receptor (VLDLR) mediated transport. This inhibition and slow transport with the resulting tendency to accumulate Aβ in the brain may account for the increased risk of Alzheimer’s disease.\nThe receptor for advanced glycation end products (RAGE) mediates transport of Aβ from blood-to-brain. Aβ-clusterin blood-to-brain transport by LRP2 can also be demonstrated under experimental conditions, however, in vivo it is likely that the Aβ-clusterin complexes are out-competed by clusterin for inwards transport [470–472]. The net flux of complexes via LRP2 is thus brain-to-blood [429, 464]. sLRP1 is released from LRP1 at the luminal membrane by removal of the membrane binding domain. Aβ complexes with sLRP1 are apparently not transported across the blood–brain barrier but can be delivered to the liver. Thus these serve as a sink reducing backflux of Aβ that has emerged from the brain [469].\nThe role of p-glycoprotein (Pgp) has been considered in many studies [243, 473–490] that indicate that it does play a role, but there have also been studies suggesting that it does not [491–494]. P-glycoprotein is present in the luminal membranes of the endothelial cells (see Sect. 4.2.1). With LRP1 mediating entry of Aβ into the endothelial cells from ISF, an obvious role to suggest for p-glycoprotein is Aβ efflux to plasma. Another function of p-glycoprotein may be to return to plasma some of the Aβ brought into the cells by RAGE [423, 495, 496]. However the intervening steps between endocytosis mediated by either LRP1 or RAGE and efflux by p-glycoprotein remain to be established.\nThe overall net flux of Aβ across the blood–brain barrier is thus seen to be the resultant of a number of transport mechanisms mediating both inward and outward fluxes. The use of complexing agents in plasma to reduce Aβ flux from blood-to-brain is one strategy being tried to reduce Aβ accumulation in the brain.\n\nEvidence for Aβ elimination via perivascular routes\nThe perivascular route has also been considered as a likely pathway for elimination of Aβ peptides from the brain. In initial studies, following exogenous Aβ introduction into the brain, aggregates were first found along the external boundaries of arterial walls [497, 498] (see also [499, 500]) but at later times were seen throughout the smooth muscle layer of the arteries ([497], see also [501]). The results from these initial studies are consistent with the idea that growth of the deposits starts occurring adjacent to an efflux route for Aβ along the outside of the arteries, i.e. an extramural periarterial route.\nSubsequent studies followed the routes of exit from the parenchyma of fluorescent dextran. This was used as a non-metabolizable marker for substances of the size of Aβ. Within minutes of its injection fluorescence could be visualized throughout the smooth muscle layer of the arterial walls [70]. From this observation it was proposed that both the fluorescent dextran and the Aβ enter the smooth muscle layer near its end closest to the capillaries and move along the vessel wall towards the subarachnoid space with little further exchange between the smooth muscle layer and the surrounding parenchyma. However, it remains difficult to see how there could be sufficient driving force for movement through the extracellular matrix along the entire length, perhaps a millimeter, of the vessel (compare the discussion in Sect. 3.2.1) while at the same time movement over a 10- to 20-fold shorter distance perpendicular to the vessel wall is prevented. For a different viewpoint see [88, 95, 502–504]).\nThere may be an alternative explanation. The higher observed density of dextran or Aβ within the extracellular spaces of the smooth muscle layer than in the interstitial spaces of the parenchyma [102] might suggest that it binds, reversibly, to some component of the extracellular matrix in the layer. There is in fact good evidence for interaction of the Aβ peptides with some components [505, 506]. If the high concentrations within the basement membranes of the layer reflect binding rather than some form of impermeant sheath, then it is not clear whether Aβ and the dextrans reach the sites of the binding by moving parallel to the vessel wall or by traversing it (see Fig. 21). If the latter, movements parallel to the vessel would be occurring via an extramural route that might have a much lower resistance to flow. Transverse movement has been observed for both horseradish peroxidase and 3H-leucine with large cerebral arteries [507], and no additional impermeant layer is known to exist around smaller arteries inside the parenchyma [98]. There is at present no compelling evidence to decide between the intramural and extramural routes for movement parallel to the vessels.\nFig. 21 Putative routes for periarterial efflux. In the intramural proposal solutes move parallel to the vessel wall along the basement membranes of the smooth muscle layer, shown as blue trajectories. In the extramural proposal movements of solutes parallel to the vessel occur primarily in a perivascular space with lower resistance to flow. They also move in and out of the wall by a combination of diffusion and convection, shown as the red trajectories. As discussed in Sect. 3.1 the nature of the extramural pathway is still controversial including whether it is a space one side or the other of the pial cells or alternatively the pial and glial basement membranes themselves. endo endothelium, s.m smooth muscle, BM basement membrane. Pial cells and pial basement membrane(s) are shown together because they are very thin\nThe importance of the perivascular route for Aβ elimination may be not so much that it removes Aβ from the parenchyma but rather that it delivers Aβ into the vessel walls of arterioles and arteries. Cerebral amyloid angiopathy is often seen before formation of senile plaques within the parenchyma (see e.g. [508]) and the damage to the arterioles and arteries may have secondary consequences for the well-being of parenchymal cells, either by effects on blood flow or via local inflammation [509–511]."}

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of Aβ from ISF\nIn the young, Aβ is present in soluble form and is eliminated as rapidly as it is produced with about 7–8% of the total soluble Aβ being replaced each hour [422, 444]. Monomeric and small oligomeric forms of soluble Aβ are cleared from ISF by at least four routes: incorporation into plaques, metabolism [445–451], efflux across the blood–brain barrier [62, 429, 452–454] and efflux via perivascular routes [25, 85, 128, 455]. The relative importance of each of these routes remains controversial [52, 146, 456–458].\n\nEvidence for transport of soluble Aβ across the blood–brain barrier\nThe ways in which soluble Aβ can be transported across the blood–brain barrier have been investigated by several different groups. Shibata et al. [62] were the first to propose that Aβ could cross the blood–brain barrier by transcytosis mediated by low density lipoprotein receptor related protein (LRP1). This they said could account for the loss of 125I-Aβ1-40 from the brain that they observed. In support of their proposal they found that the loss of total 125I from the brain was reduced by antibodies against LRP1, by receptor (LRP1) associated protein (RAP), which interferes with binding of all known substrates to LRP1, and by absence of apoE seen in knockout mice. (ApoE affects the interaction of Aβ with LRP1). In addition the elimination process appeared to be saturable with Km of 15 nM. All of these observations are consistent with the idea that the elimination of soluble 125I-Aβ1-40 is primarily efflux across the blood–brain barrier and is via an LRP1-dependent process. However it should be kept in mind that demonstrating the importance of LRP1 is not the same as demonstrating elimination via the blood–brain barrier because LRP1 is also present on neurons, astrocytes and vascular smooth muscle cells where it can mediate endocytosis of Aβ leading to its metabolism inside the cells [448, 456, 459] (see [146] for further discussion). Further results supporting the involvement of efflux have been reported by Bell et al. [429], who found that the rate constant for elimination of Aβ1-42 was about half that for Aβ1-40 and also in other papers by Deane, Zhao, Nelson, Zlokovic and coworkers [452, 454, 460].\nResults from several other groups also support the idea that efflux of soluble Aβ does occur at the blood–brain barrier and that LRP1 is involved in this elimination.Jaeger et al. [461] showed that antisense oligonucleotides against LRP-1 substantially decreased the loss of Aβ1-42 after intraparenchymal injection.\nPflanzner et al. [462] demonstrated LRP1-dependent Aβ1-40 transport across monolayers of primary mouse brain capillary endothelial cells, a transport not observed in monolayers of cells with genetically modified LRP1.\nRoberts et al. [457] confirmed that efflux of Aβ from brain to blood occurs in vivo by finding that the concentration in venous blood leaving the brain was 7.5% higher than that in arterial blood.\nQosa et al. [424] using the brain efflux index method found that 62% of added 125I-Aβ1-40 appeared in the blood.\nStorck et al. [453] developed a mouse model in which LRP1 could be knocked out selectively in endothelial cells and showed that the knockout reduced the initial rate of loss of 125I-Aβ1-42 by 48%.\nCollectively the studies discussed above leave little doubt that LRP1 dependent transport across the blood–brain barrier plays a substantial role in Aβ elimination. However, the actual mechanisms governing the net inward or outward flux of Aβ across the blood–brain barrier are considerably more complicated and involve complexing Aβ with soluble factors including clusterin (also called apoJ), apoE and a soluble, truncated form of LRP1 (sLRP1). In addition there are at least four endocytotic/transcytotic systems. Figure 20, based mainly on the views of Zlokovic and colleagues [429, 452, 454, 460, 463–466], is a simplified diagram indicating the mechanisms of Aβ transport across the blood–brain barrier. Notable in this scheme is the involvement of apoE, clusterin and the phosphatidylinositol-binding clathrin assembly protein, PICALM (also called CALM). Genetic variations for each of these have been shown to be associated with increased risk of Alzheimer’s disease [467, 468].\nFig. 20 Simplified outline of Aβ transport across the blood–brain barrier. Possible movements of Aβ are shown by solid or dashed lines with arrowheads indicating the principal direction. Endocytotic and exocytotic vesicles are shown as invaginations of the membranes. There is intracellular processing once the vesicles have been endocytosed. Aβ from ISF can bind directly to LRP1 on the abluminal membrane with the complex then being incorporated into a clathrin coated pit which can be endocytosed. The Aβ-LRP1 complex is stabilized by binding of the phosphatidylinositol-binding clathrin assembly protein (PICALM). Aβ in ISF can also be complexed with any of the forms of apoE, 2, 3 or 4 or with clusterin. Aβ-apoE2 and Aβ-apoE3 are substrates for interaction with LRP1 and endocytosis. By contrast Aβ-apoE4 inhibits LRP1 mediated endocytosis (dotted line), but can be endocytosed slowly after binding with the very low density lipoprotein receptor (VLDLR). Aβ-clusterin is a substrate for LRP2 mediated endocytois with transport across the blood–brain barrier to plasma. As Aβ-clusterin can also be transported in the opposite direction by LRP2-mediated endocytosis this is almost certainly by transcytosis of vesicles with LRP2 in the membrane. Vesicles with LRP1 in the membrane are also thought to discharge their contents on the far side of the barrier—i.e. this is transcytosis [465]. Some of the intracellular processing steps for the LRP1 vesicles are now known [452, 658]. Aβ is also transported from plasma to ISF. Aβ clusterin can be transported by LRP2 vesicles, but on the plasma side almost all of the LRP2 receptors are occupied by clusterin (dotted double headed arrow) rather than Aβ-clusterin which greatly reduces blood-to-brain transport by this route. Aβ is however, endocytosed after binding to the receptor for advanced glycation products, RAGE, and somehow transported to the brain side. Pgp may, in a manner which has not been well defined, assist transfer of Aβ from the endothelial cells to plasma whether it has entered the cells from ISF, via the LRP1 system, or from plasma, via the RAGE system. Figure based on [452, 464, 465]\nMuch of the soluble Aβ in ISF may be in the form of complexes with apoE or clusterin while in plasma most Aβ is complexed with clusterin or sLRP1, a truncated, soluble form of LRP1 [469]. The apoE gene has three alleles called apoE2, apoE3 and apoE4. Expression of the apoE4 allele is the greatest genetic risk factor known for developing the late-onset form of Alzheimer’s disease [467, 468].\nLRP1 mediated transport of Aβ occurs via clathrin pits, with the LRP1, Aβ, clathrin system stabilized by interaction with PICALM. In addition to this transport of Aβ there is LRP1-mediated transport from brain-to-blood of Aβ complexes with apoE2 or apoE3 and LRP2-mediated transport of Aβ complexes with clusterin. Complexes with apoE4 inhibit LRP1-mediated transport but are transported at a much lower rate by very low-density lipoprotein receptor (VLDLR) mediated transport. This inhibition and slow transport with the resulting tendency to accumulate Aβ in the brain may account for the increased risk of Alzheimer’s disease.\nThe receptor for advanced glycation end products (RAGE) mediates transport of Aβ from blood-to-brain. Aβ-clusterin blood-to-brain transport by LRP2 can also be demonstrated under experimental conditions, however, in vivo it is likely that the Aβ-clusterin complexes are out-competed by clusterin for inwards transport [470–472]. The net flux of complexes via LRP2 is thus brain-to-blood [429, 464]. sLRP1 is released from LRP1 at the luminal membrane by removal of the membrane binding domain. Aβ complexes with sLRP1 are apparently not transported across the blood–brain barrier but can be delivered to the liver. Thus these serve as a sink reducing backflux of Aβ that has emerged from the brain [469].\nThe role of p-glycoprotein (Pgp) has been considered in many studies [243, 473–490] that indicate that it does play a role, but there have also been studies suggesting that it does not [491–494]. P-glycoprotein is present in the luminal membranes of the endothelial cells (see Sect. 4.2.1). With LRP1 mediating entry of Aβ into the endothelial cells from ISF, an obvious role to suggest for p-glycoprotein is Aβ efflux to plasma. Another function of p-glycoprotein may be to return to plasma some of the Aβ brought into the cells by RAGE [423, 495, 496]. However the intervening steps between endocytosis mediated by either LRP1 or RAGE and efflux by p-glycoprotein remain to be established.\nThe overall net flux of Aβ across the blood–brain barrier is thus seen to be the resultant of a number of transport mechanisms mediating both inward and outward fluxes. The use of complexing agents in plasma to reduce Aβ flux from blood-to-brain is one strategy being tried to reduce Aβ accumulation in the brain.\n\nEvidence for Aβ elimination via perivascular routes\nThe perivascular route has also been considered as a likely pathway for elimination of Aβ peptides from the brain. In initial studies, following exogenous Aβ introduction into the brain, aggregates were first found along the external boundaries of arterial walls [497, 498] (see also [499, 500]) but at later times were seen throughout the smooth muscle layer of the arteries ([497], see also [501]). The results from these initial studies are consistent with the idea that growth of the deposits starts occurring adjacent to an efflux route for Aβ along the outside of the arteries, i.e. an extramural periarterial route.\nSubsequent studies followed the routes of exit from the parenchyma of fluorescent dextran. This was used as a non-metabolizable marker for substances of the size of Aβ. Within minutes of its injection fluorescence could be visualized throughout the smooth muscle layer of the arterial walls [70]. From this observation it was proposed that both the fluorescent dextran and the Aβ enter the smooth muscle layer near its end closest to the capillaries and move along the vessel wall towards the subarachnoid space with little further exchange between the smooth muscle layer and the surrounding parenchyma. However, it remains difficult to see how there could be sufficient driving force for movement through the extracellular matrix along the entire length, perhaps a millimeter, of the vessel (compare the discussion in Sect. 3.2.1) while at the same time movement over a 10- to 20-fold shorter distance perpendicular to the vessel wall is prevented. For a different viewpoint see [88, 95, 502–504]).\nThere may be an alternative explanation. The higher observed density of dextran or Aβ within the extracellular spaces of the smooth muscle layer than in the interstitial spaces of the parenchyma [102] might suggest that it binds, reversibly, to some component of the extracellular matrix in the layer. There is in fact good evidence for interaction of the Aβ peptides with some components [505, 506]. If the high concentrations within the basement membranes of the layer reflect binding rather than some form of impermeant sheath, then it is not clear whether Aβ and the dextrans reach the sites of the binding by moving parallel to the vessel wall or by traversing it (see Fig. 21). If the latter, movements parallel to the vessel would be occurring via an extramural route that might have a much lower resistance to flow. Transverse movement has been observed for both horseradish peroxidase and 3H-leucine with large cerebral arteries [507], and no additional impermeant layer is known to exist around smaller arteries inside the parenchyma [98]. There is at present no compelling evidence to decide between the intramural and extramural routes for movement parallel to the vessels.\nFig. 21 Putative routes for periarterial efflux. In the intramural proposal solutes move parallel to the vessel wall along the basement membranes of the smooth muscle layer, shown as blue trajectories. In the extramural proposal movements of solutes parallel to the vessel occur primarily in a perivascular space with lower resistance to flow. They also move in and out of the wall by a combination of diffusion and convection, shown as the red trajectories. As discussed in Sect. 3.1 the nature of the extramural pathway is still controversial including whether it is a space one side or the other of the pial cells or alternatively the pial and glial basement membranes themselves. endo endothelium, s.m smooth muscle, BM basement membrane. Pial cells and pial basement membrane(s) are shown together because they are very thin\nThe importance of the perivascular route for Aβ elimination may be not so much that it removes Aβ from the parenchyma but rather that it delivers Aβ into the vessel walls of arterioles and arteries. Cerebral amyloid angiopathy is often seen before formation of senile plaques within the parenchyma (see e.g. [508]) and the damage to the arterioles and arteries may have secondary consequences for the well-being of parenchymal cells, either by effects on blood flow or via local inflammation [509–511]."}