Accumulating evidence supports the idea that continually decreasing choroid plexus (CP) function in advanced ageing exacerbates Alzheimer's disease (AD). As part of a new paradigm to explain brain interstitium deterioration in age-related dementias, increasingly more attention is being paid to the role of compromised blood-CSF 1 and blood-brain 2 barriers. Structural alterations and functional failures in CP as well as brain capillary transport systems adversely affect fluid dynamics and composition 34. This review treats mainly CP dysfunction and the compensatory reactions that occur in this epithelium in AD.

Efficient CSF turnover is essential for a healthy brain. It depends upon an exquisite balance between CSF formation and reabsorption 5. Compromised secretory phenomena at the CP 'upstream' predispose the brain to AD-type problems. On the other hand, defective clearance of CSF at the arachnoid membrane 'downstream' leads to normal pressure hydrocephalus (NPH) 6. The pivotal role of the CP in CSF homeostasis and brain viability becomes more evident when the system fails. More information is needed to evaluate how diminishing choroidal functions affect the surrounding brain in the face of AD and other dementias.

In serving the brain's metabolic needs by supplying 'biochemical goods', the CP uses CSF as a conduit for convecting substances 7. Numerous solutes ranging from ions to large proteins are entrained in the CSF that percolates through the ventricular axis (Fig. 1). CSF intimately contacts the periventricular brain tissue with which it exchanges materials bidirectionally by diffusion and bulk flow. Continually undergoing chemical modification as it flows downstream, the CSF completes the volume transmission process 7 by draining into venous blood at distal arachnoidal sites (Fig. 1).

Reduced formation of CSF and stagnated flow in ageing and AD 4 limits the delivery of substances to neurons. This debilitates brain function 3. Numerous proteins are synthesized and secreted by CP into CSF. Other substances are transported from blood. Table 1 overviews molecules normally distributed by the CP-CSF nexus. Arginine vasopressin (AVP) is a neuropeptide synthesized by CP epithelium and secreted into CSF 89; it regulates CSF formation and modulates hippocampal memory mechanisms 1011. Growth factors foster cell growth, blood supply and water balance 1. Trace element distribution across CP is complex and involves many mechanisms. Iron transport into CSF, for example, is regulated by several proteins 1 alterations in which may predispose to amyloidogenesis 12. Vitamins B and C are actively transported at the blood-CSF barrier 13. This stabilizes their concentration in CSF, except in late-onset AD 14. The net entry of nucleoside bases into CSF is also determined by active transporters in CP 1516. Cysteine protease inhibitors like cystatin C are synthesized in CP epithelium for transport into CSF 17. Hormones such as leptin and prolactin are translocated from plasma to CSF by saturable choroidal receptors 1819. Overall it is striking that the CP-CSF interface engages in such prolific transport. Disease-associated interference with CP transporters and volume transmission limits the availability of CSF molecules for the brain 120.

Structure intimately relates to function in various epithelia. Therefore it is useful to analyze histopathological damage to CP at various stages of AD in order to clarify the onset of functional losses at the blood-CSF barrier. Ageing and AD cause similar degeneration in CP. Structural changes in AD though are usually greater than in non-demented control counterparts 21. Serot and colleagues have delineated modifications in the choroidal epithelium, stroma and vessels in AD subjects 222324. There are substantial alterations in all tissue compartments.

Several features characterizing the CP in AD are highlighted in Table 2. Epithelial cells are typically truncated, with an average volume 70% of that at birth. Such epithelial atrophy likely affects cellular functions. Particularly prominent is an increase in the number of Biondi bodies in AD 25. These bodies are fibrillar inclusions in the cytoplasm of very old epithelial cells. Lipofuchsin vacuoles also occur frequently in the cytoplasm. The basement membrane underlying the cell often thickens to 350 nm, compared to a much thinner membrane (ca. 100 nm) in neonates 21. Another liability in AD is immunological deposition of C1q, IgG and IgM along the epithelial basement membrane. As fibrosis intensifies with age and disease, the stroma attains a thickness of a few tenths of a micron 21. At the inner core of the choroidal villus, the blood vessel walls thicken. This coincides with the appearance of amyloid, hyaline bodies, psammomas and calcifications. Altogether, the histopathologic changes in CP compartments point to grossly-declining secretory functions in AD. Such structural abnormalities coincide with diminished CSF production in ageing 26 and AD patients 4.

Due to the functional nexus of the CSF with brain interstitial fluid, the neuronal microenvironment in AD is impacted by markedly altered transport and permeability in CP. When neurodegenerative diseases, ischemia 2728 and elevated pressure 2930 inflict damage on the choroidal epithelium, there are resultant adverse changes in CSF composition and volume. Because neurons are sensitive to instabilities in CSF dynamics and constituents, it is important to assess the nature and progression of disrupted CP function in chronic diseases. In view of the expanding number of AD victims, it is timely to consider patterns of expression of chaperone proteins, receptors and transporters in CP at various Braak stages. Such information may abet future attempts to stabilize choroid plexus functional integrity perturbed in early AD dementia.

The CP is multifunctional, performing a wide range of homeostatic functions for the CNS 5. CSF homeostasis is mediated mainly by CP. It involves the activity of many protein transporters and receptors at the basolateral and apical surfaces of the epithelial cells 31. In addition to providing organic solutes for nutritive and trophic support of the brain, the CP secretions into the ventricles adjust the pH, osmolality, [K⁺] and immune molecule content of the CNS extracellular fluid 7. Accordingly, healthy neurons are fundamentally dependent upon transport at the blood-CSF and blood-brain interfaces.

Moreover to provide steady neuroprotection by regulating the extracellular milieu, the CP must protect itself against various stressor agents that build up during ageing and disease. There have been few investigations of the homeostatic systems within CP that stabilize choroidal functions in the face of ageing and AD. We have explored some candidate systems for compensatory responses by CP. Cytoplasmic heat shock proteins chaperone and perform housekeeping to maintain a healthy steady-state intraepithelial milieu 132. Growth factors critically minimize cell morbidity and mortality 27. Fluid-regulating proteins correct ion and water imbalances that occur in neurodegenerative diseases. Consequently our hypothesis is that the upregulation of certain proteins in CP (such as those discussed above) thwarts certain untoward effects of ageing and disease progression. The protein expression aspect of the hypothesis was tested by analyzing immunostaining patterns in human CP specimens from patients with varying severity of AD.

It is difficult to functionally assess the CP in vivo 3033, particularly in man. Alternatively one can evaluate the status of human CP in disease by analyzing autopsied tissues for variable protein expression 34. Such findings are compared to appropriate age-matched controls. Immumocytochemical and biochemical data gleaned from human CP highlight directions to pursue with living animal models: transgenic mice with an AD phenotype or aged rats with CSF pathophysiology. Because protein expression relates to disease progression, it is essential to standardize grading for the severity of AD in subjects. We use a modified Braak & Braak staging system 35: stages I/II (mild; disease involves hippocampus and entorhinal cortex); stages III/IV (moderate; AD spreading to the rest of the limbic lobe, e.g., amygdala); and stages V/VI (severe; further AD spreading to the prefrontal neocortex).

Information about functional proteins in human CP is scarce. However protein expression was recently analyzed in autopsied lateral ventricle CP from normal adult brains and in those with confirmed AD 3637. The ages investigated were generally between 65 and 90 yr for all subjects. Control individuals typically died from cardiac disease or tumors. AD specimens covered all Braak stages. Causes of the AD deaths were usually cardiac complications or pneumonia 37. Fortunately the postmortem intervals (mainly between 2 and 18 hr) did not affect antibody staining 37. For specimen quality it is desirable to procure choroidal tissues quickly after death. Human CP banks are needed to systematically catalog tissues from various stages of AD.

Ageing and AD dementia tax the CP and other CNS transport interfaces. In late life the deteriorating brain presents many potentially-destructive metabolites to the CSF for multi-site excretion into blood. The greater burden of macromolecule disposal in AD occurs when CP and arachnoid membrane, due to ageing debilities, are less able to transfer solutes. Nevertheless the CP seemingly attempts to maintain its 'epithelial soundness' when challenged to perform additional cleansing acts for the brain extracellular fluid (CSF).

In healthly, young adults the CP epithelium sensitively acclimates to chemical and physical distortions in blood, CSF and parenchyma. Following cortical stabbing in rats, the CP upregulates TGFβ presumably to provide this CSF-borne growth factor for repairing the injury 38. A similar phenomenon occurs with IGF-II 39, which is manufactured in the CNS mainly by CP. In diabetes there is enhanced expression of the NKCC1 cotransporter in CP for adjusting CSF dynamics and water distribution 40. Such compensatory responses at the blood-CSF barrier in early adulthood raise the question about CP's ability in later life, when besieged by the deficits of aging and AD, to adequately respond by expressing certain 'homeostatic proteins'.

Accordingly to assess pathophysiologic consequences of AD we investigated human CP's ability to upregulate certain functional proteins (as distinguished from structural ones) in advanced states of AD dementia. With advancing knowledge about intricate cell physiology (coordinated interactions among organelles, cytoplasm and membrane-bound proteins) it is relevant to evaluate components of cellular homeostasis. The term 'homeostatic proteins'refers to chaperones, receptors and transporters that stabilize the internal environment of the cell. CSF stability depends in large part upon CP epithelial homeostasis. Our group has analyzed several 'homeostatic proteins' involved in CP intracellular milieu stabilization:

CP is highly equipped, homeostatically speaking, to help the brain adapt to the metabolic distortions of dementia. Injured neurons undergo compositional changes. These cellular perturbations are transmitted to the extracellular space and distort the interstitial fluid composition. Many catabolites in the interstitium eventually gain access to the ventricles where they contact the CPs. By accepting a host of CSF-borne molecules, for either transport or receptor stimulation, the CP mediates a wide scope of renal- and hepatic-like activities 1398. This epithelial interface also integrates many neurohumoral activities of the endocrine and immune systems 9899. Diseases afflicting the CNS generate injury metabolites and cytokines that are conveyed to the ependyma, pia-glia, arachnoid membrane and CP epithelium. These interfaces handle catabolites and peptide fragments 100 by reabsorbing them into the systemic circulation for clearance or by sequestering toxic substances in lysosomes for metabolic conversion. Harmful substances are thereby effectively removed from the brain-CSF system.

Signaling molecules are also carried by CSF from diseased regions to the CP. There they bind to specific receptors in the apical membrane and consequently elicit a variety of bioreactive responses. Binding sites for a wide array of peptides, proteins and other organic substances abound in the mammalian CP 73. Choroidal epithelial cells can thus be regarded as 'bioreactors' that respond to chemical changes in extracellular fluid. Their response includes the synthesis of peptides, growth factors and sundry molecules for homeostatically repairing injured neurons.

Currently, little is known about the spectrum of responses by CP to the disrupted CNS homeostasis in AD. It would be informative to compare AD stages for expression abilities of CP vs. the ependyma and meninges. Differences as well as similarities are anticipated. The initial studies of gene expression in human CP reported herein reveal that in Braak stages V/VI there is still strong expression of HSP90 and NKCC1 (Figs. 2 &4). Therefore even though the CP in advanced AD shows extensive histopathology (Table 2) and has reduced enzymatic activities and fluid formation 4626, it evidently retains the ability to react to biochemical perturbations by expressing housekeeping proteins. This stabilizing effect on the blood-CSF barrier epithelium enables regulatory phenomena that ultimately support AD-stressed neurons.

Insight can be gained by investigating CSF neurochemical composition as a function of AD severity. Additionally it would be instructive to analyze how cultured CP epithelium reacts to 'pathological' CSF from patients at progressive Braak stages. The Z310 cultured cell line and primary cultures of CP are useful for such analyses 101102. Given the significance of CP in facilitating repair of CNS structures, it should also be fruitful to analyze in vivo gene expressions that reflect pathophysiological interactions between CP-CSF and brain.

Therapeutic strategies to halt CNS deterioration should include ways to defend the CP epithelium against the oxidative ravages of ageing and AD. Prolonging CP viability and work efficiency may be important in maintaining the well being of geriatric patients. Even without a cure for AD, if deleterious changes in the brain interstitium were minimized in the elderly by stabilizing the CP-CSF (as well as the BBB), then it might be feasible to prevent the early manifestations of AD (Braak stages I/II) from intensifying into the debilitating pathology of V/VI.

One class of agents with considerable potential for AD therapeutics is the growth factor group. A useful paradigm for CSF growth factors and neuroprotection has evolved from experiments on transient forebrain ischemia in rats 5960. The TFI experiments characterized the destructive effects of acute ischemia on the lateral ventricle CP and the nearby CA1 region of hippocampus 50; and the time course of cell recovery (or death) in these adjacent regions protected (or not) by supplemental infusion of FGF2 via CSF prior to the ischemic insult 61. Exogenous FGF2 administered before or after the induced ischemia lessened cell death in CA1, probably in part by stabilizing CP functions 285061. Moreover the CP substantially repaired its epithelial cell barrier by 24 hr post-TFI, even without supplemental FGF2 60. Collectively these findings manifest the impressive plasticity of CP to reconstitute itself after disruption; and reveal the potential therapeutic value of pharmacologically-administered growth factors to reduce harm to CP and hippocampus.

Several other growth factors synthesized by CP and secreted into CSF should be explored in translational research dealing with interacting ischemia, hydrocephalus and AD 14628365055103104. Following TFI the CP upregulates TGFβ, another peptide that helps the CNS adjust to injury 58. Consequently choroidally-manufactured growth factors in disease states can benefit the plexus locally (by autocrine and paracrine mechanisms) and the brain more globally (by endocrine-like bulk flow of CSF). Our ischemia findings for the CP-CSF-hippocampus compartments 275860 relate to AD in that reduced blood flow to the ageing CNS exacerbates AD progression. Growth factor supplements (e.g., VEGF, NGF, IGF-II & HGF) could augment viability in AD-vulnerable regions by enhancing vascularization, preventing programmed cell death or promoting stem cell conversion in the subventricular zone (SVZ). Newly-formed neurons in the SVZ might then migrate to atrophic regions to replace destroyed cells. One pharmacologic approach to stall AD onset is to administer a combination of growth factors designed to increase neuroprotection while minimizing fibrosis 55. We theorize that an optimal regimen of growth factors and neuropeptides would restore CP function or prevent further loss of homeostatic capabilities.

In searching for agents to modify CP epithelial protein expression, the route of delivery of the active drug is of primary importance. Unlike the brain with its impermeable microvessels, the CP readily takes up water-soluble drugs from the plasma due to the highly-permeable choroidal capillaries. Consequently water-soluble agents freely diffuse to receptors or binding sites at the basolateral surface of the epithelium. Access of blood-borne hydrophilic compounds to the CSF-side of CP however is problematic because the tight junctions and basolateral membrane impede diffusing molecules as small as mannitol 105. Molecular sieving at the basolateral membrane restricts the permeation of hydrophilic molecules as small as urea (m.w. = 60) into the CP-CSF compartments 106.

To circumvent the blood-CSF barrier, therapeutic agents are delivered into CSF by lateral ventricle catheters in experimental animals 55 or hydrocephalic patients. Gene therapy offers the additional challenge of finding a viral vector that selectively targets the CP epithelium for transduction 107. Timely, innovative strategies are in order to find specific ways to target and improve CP function in neurodegenerative states.

A compelling aspect of CSF translational research is to identify agents that effectively regulate fluid formation by CP. Whereas the difficulty in congenital hydrocephalus is to downregulate CSF formation, the challenge in AD is to enhance CSF turnover perhaps by accelerating fluid production as well as outflow. Augmented flow of CSF enhances 'sink action' 108109. This would expedite clearance of toxic molecules like Aβ out of the brain interstitial fluid into the ventricles. New agents that stimulate CP to secrete CSF more rapidly should be tested in aged animals and those with AD-phenotypes. A consistent finding of decreased CNS burdens of Aβ in animals with increased CSF turnover could spur the development of drugs for brain 'cleansing' in AD.

Another CP pathology meriting pharmacological attention is the massive fibrosis that progressively envelops the interstitium in old age. This interstitial fibrosis is even more extensive in AD 21. Fibrosis undoubtedly impedes efficient movement of molecules between blood and CSF. It is pertinent to ascertain if therapeutic minimization of CP fibrosis in later life would permit a brisk turnover of CSF to be sustained. Attenuating the formation of Biondi bodies and other choroidal cellular inclusions (Table 2) would be a unique approach to prevent age- and disease-related curtailment of CSF production. Optimal vascular-interstitial-epithelial interactions in the CP are foundational for vigorous CSF dynamics. The longer the blood-CSF interface retains its epithelial secretory capabilities, the more successfully it can conduct homeostatic activities to ward off AD.

The CP has the main responsibility for CSF homeostasis. Therefore the functional status of the blood-CSF barrier is of great consequence to the CNS. Maintaining the CSF at a stable, specialized composition is of the utmost importance to neurons. CSF is prominent in regulating brain interstitial fluid with which it exchanges nutrients and waste products. Diseases markedly affect these molecular exchanges. Maintaining healthy bidirectional transport across the CP epithelium (CSF-blood) and ependyma (CSF-brain) is thus integral to a sound brain fluid environment.

CSF macrocirculation through the ventriculo-subarachnoid system together with CSF microcirculation in the perivascular Virchow-Robin spaces 110111 perform distributive as well as collective functions. By gathering waste products, the CSF is a quasi-lymphatic system with critical functions in excreting harmful peptides and proteins. In early life the upregulated secretions of CP play a central role in brain ontogeny by furnishing growth factors to the germinal matrix. At the end of life with disease onset or aging consequences, the CP transporters are upregulated again to rescue failing neurons by providing neurotrophic materials to CSF. Equally important, peptides in excess such as amyloid beta (Aβ) fragments in AD must be eliminated from CNS by perivascular pathways 112. To facilitate clearance of Aβ, the continual production of CSF by CP sustains 'sink action' on the brain interstitium 109.

As the main generator of CSF, the CP has a pivotal role in helping the brain cope with the twin stressors of ageing and disease. More attention should be focused on the blood-CSF interface for pharmacologic opportunities to stave off CP dysfunction. Our findings on human CP expression of HSP90, FGFr and NKCC1 demonstrate that this epithelium in AD reacts to metabolic insults by upregulating certain proteins. This suggests that even diseased CP could respond to therapeutic agents, thus opening new vistas for treating CSF dysfunction in age-related dementias.