Edward G. Stopa

Multiplicity of cerebrospinal fluid functions: New challenges in health and disease

This review integrates eight aspects of cerebrospinal fluid (CSF) circulatory dynamics: formation rate, pressure, flow, volume, turnover rate, composition, recycling and reabsorption. Novel ways to modulate CSF formation emanate from recent analyses of choroid plexus transcription factors (E2F5), ion transporters (NaHCO3 cotransport), transport enzymes (isoforms of carbonic anhydrase), aquaporin 1 regulation, and plasticity of receptors for fluid-regulating neuropeptides. A greater appreciation of CSF pressure (CSFP) is being generated by fresh insights on peptidergic regulatory servomechanisms, the role of dysfunctional ependyma and circumventricular organs in causing congenital hydrocephalus, and the clinical use of algorithms to delineate CSFP waveforms for diagnostic and prognostic utility. Increasing attention focuses on CSF flow: how it impacts cerebral metabolism and hemodynamics, neural stem cell progression in the subventricular zone, and catabolite/peptide clearance from the CNS. The pathophysiological significance of changes in CSF volume is assessed from the respective viewpoints of hemodynamics (choroid plexus blood flow and pulsatility), hydrodynamics (choroidal hypo- and hypersecretion) and neuroendocrine factors (i.e., coordinated regulation by atrial natriuretic peptide, arginine vasopressin and basic fibroblast growth factor). In aging, normal pressure hydrocephalus and Alzheimers disease, the expanding CSF space reduces the CSF turnover rate, thus compromising the CSF sink action to clear harmful metabolites (e.g., amyloid) from the CNS. Dwindling CSF dynamics greatly harms the interstitial environment of neurons. Accordingly the altered CSF composition in neurodegenerative diseases and senescence, because of adverse effects on neural processes and cognition, needs more effective clinical management. CSF recycling between subarachnoid space, brain and ventricles promotes interstitial fluid (ISF) convection with both trophic and excretory benefits. Finally, CSF reabsorption via multiple pathways (olfactory and spinal arachnoidal bulk flow) is likely complemented by fluid clearance across capillary walls (aquaporin 4) and arachnoid villi when CSFP and fluid retention are markedly elevated. A model is presented that links CSF and ISF homeostasis to coordinated fluxes of water and solutes at both the blood-CSF and blood-brain transport interfaces.Outline1 Overview2 CSF formation2.1 Transcription factors2.2 Ion transporters2.3 Enzymes that modulate transport2.4 Aquaporins or water channels2.5 Receptors for neuropeptides3 CSF pressure3.1 Servomechanism regulatory hypothesis3.2 Ontogeny of CSF pressure generation3.3 Congenital hydrocephalus and periventricular regions3.4 Brain response to elevated CSF pressure3.5 Advances in measuring CSF waveforms4 CSF flow4.1 CSF flow and brain metabolism4.2 Flow effects on fetal germinal matrix4.3 Decreasing CSF flow in aging CNS4.4 Refinement of non-invasive flow measurements5 CSF volume5.1 Hemodynamic factors5.2 Hydrodynamic factors5.3 Neuroendocrine factors6 CSF turnover rate6.1 Adverse effect of ventriculomegaly6.2 Attenuated CSF sink action7 CSF composition7.1 Kidney-like action of CP-CSF system7.2 Altered CSF biochemistry in aging and disease7.3 Importance of clearance transport7.4 Therapeutic manipulation of composition8 CSF recycling in relation to ISF dynamics8.1 CSF exchange with brain interstitium8.2 Components of ISF movement in brain8.3 Compromised ISF/CSF dynamics and amyloid retention9 CSF reabsorption9.1 Arachnoidal outflow resistance9.2 Arachnoid villi vs. olfactory drainage routes9.3 Fluid reabsorption along spinal nerves9.4 Reabsorption across capillary aquaporin channels10 Developing translationally effective models for restoring CSF balance11 Conclusion

RAGE, LRP-1, and amyloid-beta protein in Alzheimer’s disease

The receptor for advanced glycation end products (RAGE) is thought to be a primary transporter of β-amyloid across the blood–brain barrier (BBB) into the brain from the systemic circulation, while the low-density lipoprotein receptor-related protein (LRP)-1 mediates transport of β-amyloid out of the brain. To determine whether there are Alzheimer’s disease (AD)-related changes in these BBB-associated β-amyloid receptors, we studied RAGE, LRP-1, and β-amyloid in human elderly control and AD hippocampi. In control hippocampi, there was robust RAGE immunoreactivity in neurons, whereas microvascular staining was barely detectable. LRP-1 staining, in contrast, was clearly evident within microvessels but only weakly stained neurons. In AD cases, neuronal RAGE immunoreactivity was significantly decreased. An unexpected finding was the strongly positive microvascular RAGE immunoreactivity. No evidence for colocalization of RAGE and β-amyloid was seen within either microvessels or senile plaques. A reversed pattern was evident for LRP-1 in AD. There was very strong staining for LRP-1 in neurons, with minimal microvascular staining. Unlike RAGE, colocalization of LRP-1 and β-amyloid was clearly present within senile plaques but not microvessels. Western blot analysis revealed a much higher concentration of RAGE protein in AD hippocampi as compared with controls. Concentration of LRP-1 was increased in AD hippocampi, likely secondary to its colocalization with senile plaques. These data confirm that AD is associated with changes in the relative distribution of RAGE and LRP-1 receptors in human hippocampus. They also suggest that the proportion of amyloid within the brains of AD patients that is derived from the systemic circulation may be significant.

Microvascular injury and blood–brain barrier leakage in Alzheimer's disease

Thinning and discontinuities within the vascular basement membrane (VBM) are associated with leakage of the plasma protein prothrombin across the blood-brain barrier (BBB) in Alzheimers disease (AD). Prothrombin immunohistochemistry and ELISA assays were performed on prefrontal cortex. In severe AD, prothrombin was localized within the wall and neuropil surrounding microvessels. Factor VIII staining in severe AD patients indicated that prothrombin leakage was associated with shrinkage of endothelial cells. ELISA revealed elevated prothrombin levels in prefrontal cortex AD cases that increased with the Braak stage (Control=1.39, I-II=1.76, III-IV=2.28, and V-VI=3.11 ng prothrombin/mg total protein). Comparing these four groups, there was a significant difference between control and Braak V-VI (p=0.0095) and also between Braak stages I-II and V-VI (p=0.0048). There was no significant difference in mean prothrombin levels when cases with versus without cerebral amyloid angiopathy (CAA) were compared (p-value=0.3627). When comparing AD patients by APOE genotype (ApoE3,3=2.00, ApoE3,4=2.49, and ApoE4,4=2.96 ng prothrombin/mg total protein) an analysis of variance indicated a difference between genotypes at the 10% significance level (p=0.0705). Tukeys test indicated a difference between the 3,3 and 4,4 groups (p=0.0607). These studies provide evidence that in advanced AD (Braak stage V-VI), plasma proteins like prothrombin can be found within the microvessel wall and surrounding neuropil, and that leakage of the blood-brain barrier may be more common in patients with at least one APOE4 allele.

Neural Heme Oxygenase-1 Expression in Idiopathic Parkinson's Disease☆

Heme oxygenase-1 is a cellular stress protein expressed in brain and other tissues in response to oxidative challenge and other noxious stimuli. In the present study, immunohistochemistry was used to assess HO-1 expression in various postmortem human brain specimens derived from PD and control subjects. In the substantia nigra of both PD and control specimens, moderate HO-1 immunoreactivity was consistently observed in neuromelanin-containing (dopaminergic) neurons. Lewy bodies in PD nigra neurons exhibited intense HO-1 immunostaining in their peripheries. In both PD and control specimens, neuronal HO-1 staining was faint or nondetectable in the other brain regions surveyed. The fraction of GFAP-positive astroglia expressing HO-1 in PD substantia nigra (77.1 +/- 12.3) was significantly greater than that observed in the substantia nigra of control subjects (18.7 +/- 7.1; P = 0.0015). In the other regions examined, percentages of GFAP-positive astroglia coexpressing HO-1 were relatively low and did not differ significantly (P > 0.05) between control and PD specimens. Upregulation of HO-1 in the substantia nigra of PD subjects supports the view that the affected tissue is experiencing chronic oxidative stress. In addition, excessive cellular levels of heme-derived free iron and carbon monoxide resulting from HO-1 overactivity may contribute to the pathogenesis of PD.

A new look at cerebrospinal fluid circulation

According to the traditional understanding of cerebrospinal fluid (CSF) physiology, the majority of CSF is produced by the choroid plexus, circulates through the ventricles, the cisterns, and the subarachnoid space to be absorbed into the blood by the arachnoid villi. This review surveys key developments leading to the traditional concept. Challenging this concept are novel insights utilizing molecular and cellular biology as well as neuroimaging, which indicate that CSF physiology may be much more complex than previously believed. The CSF circulation comprises not only a directed flow of CSF, but in addition a pulsatile to and fro movement throughout the entire brain with local fluid exchange between blood, interstitial fluid, and CSF. Astrocytes, aquaporins, and other membrane transporters are key elements in brain water and CSF homeostasis. A continuous bidirectional fluid exchange at the blood brain barrier produces flow rates, which exceed the choroidal CSF production rate by far. The CSF circulation around blood vessels penetrating from the subarachnoid space into the Virchow Robin spaces provides both a drainage pathway for the clearance of waste molecules from the brain and a site for the interaction of the systemic immune system with that of the brain. Important physiological functions, for example the regeneration of the brain during sleep, may depend on CSF circulation.

Pathologic evaluation of the human suprachiasmatic nucleus in severe dementia.

Sleep disruption and other circadian rhythm disturbances are frequently seen in dementia patients. In this study, we examined the suprachiasmatic nucleus (SCN), the putative site of the hypothalamic circadian pacemaker, to determine the nature and degree of pathologic changes caused by severe dementia. Neuropathologic examination indicated that among 30 patients with a clinical history of severe dementia, 22 had Braak and Braak stage V-VI Alzheimer disease, 3 had combined Alzheimer and Parkinson disease, 3 had Pick disease and 2 had severe hippocampal sclerosis. Comparisons were made with a control group composed of 13 age-matched patients with no clinical or pathological evidence of dementia or other CNS disorders. To determine the pathologic involvement within the SCN, human hypothalami were stained with: Nissl, Bielchowsky silver, thioflavin S and specific antibodies directed against vasopressin (VP), neurotensin (NT), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), beta-amyloid (B/A4) and glial fibrillary acidic protein (GFAP). Pathologic damage was primarily limited to neuronal loss and neurofibrillary tangle formation. Only rare diffuse plaques were noted. The pathologic changes within the SCN were less severe than in the other brain regions. Morphometric analysis was accomplished using a stereological approach to sample the average total number of positively stained neurons and astrocytes in 10 different 0.1mm2 microscopic fields in the dorsal subdivision of the SCN. Patients with Alzheimer disease exhibited a significant decrease in vasopressin (9.75 vs 16.7, p < 0.001) and neurotensin (6.82 vs 9.63, p < 0.002) neurons, as well as a corresponding increase in the GFAP-stained astrocyte/Nissl-stained neuron ratio (0.54 vs 0.10, p < 0.009). These studies provide evidence that both vasopressin and neurotensin neurons are lost in Alzheimer disease, and that the astrocyte/neuron ratio is a reliable indicator of disease-related pathology within the SCN. Taken collectively, our data support the hypothesis that damage to the SCN may be an underlying anatomical substrate for the clinically observed changes in circadian rhythmicity that have been observed in Alzheimer patients.

Circadian Locomotor Activity and Core-Body Temperature Rhythms in Alzheimer's Disease

Sleep-wake cycle disturbances suggest that circadian rhythms may be disrupted in patients with Alzheimers disease (AD). In this study, we examined the circadian rhythms of core-body temperature and locomotor activity in 28 patients with probable AD and 10 healthy controls. AD patients had higher percent nocturnal activity than controls, corresponding to the clinical picture of fragmented sleep. The amplitude of the activity cycle in the AD patients was lower than that of controls and the acrophase of this cycle in AD patients was 4.5 h later. There was no difference in the amplitude of the core-body temperature circadian rhythm, but AD patients had delayed temperature acrophases. A subgroup of AD patients with large mean time differences between the acrophases of their activity and temperature cycles had lower temperature amplitudes and greater activity during the night. These findings suggest that a subgroup of AD patients with impaired endogenous pacemaker function may have a diminished capacity to synchronize the rhythm of core-body temperature with the circadian cycle of rest-activity. This circadian rhythm dysfunction may partly explain the fragmented nocturnal sleep exhibited by these patients.

Basic fibroblast growth factor in Alzheimer's disease

We have examined the presence of basic fibroblast growth factor (FGF) in normal and in Alzheimer brains, studied the distribution of the mitogen by immunohistochemical techniques, measured the quantities of growth factor in selected areas of the brain (Brodmann areas 10/11 and 20/21), characterized the molecular forms by Western blotting and determined its sites of synthesis by in situ hybridization. Although the same molecular forms of basic FGF are found in control and Alzheimer brains, basic FGF is increased in the brains of Alzheimers patients. Furthermore, basic FGF is not distributed in an identical fashion to normal and Alzheimer brains, but is found in association with the lesions that characterize this disease. In normal controls (n = 5), basic FGF was found to be widely distributed throughout the three brain regions examined (prefrontal cortex, hippocampus, and hypothalamus). Immunoreactivity was observed within astrocytes in both the grey and white matter, as well as within neuronal perikarya. Brain tissues that were obtained from Alzheimer patients (N = 4) showed a substantial increase in the overall specific staining of astrocytes and neurons, particularly in areas of reactive gliosis. Focal concentration of immunoreactive basic FGF was evident within the neuritic plaques, and could be clearly seen in association with the neurofibrillary tangles present within neuronal perikarya. The possibility that basic FGF expression in the CNS is linked to the pathogenesis of the disease is discussed.

Anti-striatal antibodies in Tourette syndrome cause neuronal dysfunction

Serologic studies of children with Tourette syndrome (TS) have detected anti-neuronal antibodies but their role in TS has not been explored. Stereotypies and episodic utterances, analogous to involuntary movements seen in TS, were induced in rats by intrastriatal microinfusion of TS sera or gamma immunoglobulins (IgG) under noninflammatory conditions, as found in TS. Immunohistochemical analysis confirmed the presence of IgG selectively bound to striatal neurons. These data support the hypothesis that binding of an anti-neuronal antibody from some children with TS induced striatal dysfunction and suggest a possible cause for the basal ganglia alterations observed in children with TS.

Agrin and microvascular damage in Alzheimer’s disease

Abstract Heparan sulfate proteoglycans (HSPGs) are ubiquitously present within the perivascular basement membrane, and have been shown to be altered in patients with Alzheimer’s Disease (AD). Although the HSPG agrin clearly orchestrates the differentiation of the neuromuscular junction, its role in the brain remains unclear. Growing evidence suggests that agrin may be an important vascular basement membrane (VBM)-associated HSPG. In previous studies, we demonstrated that agrin is present throughout the brain microvasculature, as well as in neuronal cell bodies. AD brains exhibited fragmentation of VBM-associated agrin. Agrin immunoreactivity was also seen within senile plaques and neurofibrillary tangles. These changes were accompanied by the appearance of an additional pool of insoluble agrin. In the present study, we provide further evidence for microvascular damage in AD, by examining the distribution of agrin and laminin within the VBM, and by measuring the agrin concentration within hippocampus and prefrontal cortex. Furthermore, we assessed blood-brain-barrier (BBB) leakage by examining the perivascular distribution of prothrombin immunoreactivity. Soluble agrin levels were increased approximately 30% in Braak stage III–VI AD patients relative to age-matched controls. Furthermore, agrin and laminin exhibited identical patterns of VBM fragmentation in AD and colocalized with beta-amyloid in senile plaques. Microvascular changes were associated with the appearance of perivascular prothrombin immunoreactivity. Our data suggest that agrin is an important VBM-associated HSPG in the brain and that agrin levels are altered in association with microvascular damage in AD.