Alzheimer's Disease Pathogenesis: The Denied Access Model

Department of Biology, Florida Institute of Technology 150 W. University Blvd, Melbourne, Australia

Corresponding Author:

Shaohua Xu
Department of Biology
Florida Institute of Technology 150 W. University Blvd
Melbourne, Australia
Tel: (321) 674-8430
E-mail: xshaohua@fit.edu

Received date: June 16, 2017; Accepted date: July 12, 2017; Published date: July 18, 2017

Citation: Wade ND, Shaohua X (2017) On Alzheimer’s Disease Pathogenesis: The Denied Access Model. J Alzheimers Dis Parkinsonism 7:359. doi:10.4172/2161-0460.1000359

Copyright: © 2017 Wade ND, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Journal of Alzheimers Disease & Parkinsonism

Abstract

Currently, AD has no cure and only treatments for the symptoms exist. Modern research still debates the toxic component of AD and the exact mechanism causing neurodegeneration. A hallmark of the disease is the production of the amyloid-beta (Aß) peptides and eventual self-assembly of these peptides into fibrils and extracellular Aß plaques. Both plaques and oligomers are proposed to be the direct cause of AD, but it remains unclear how the physical presence of these structures affect neuronal function and pathogenesis. Biomolecule aggregation is known to play a role in the pathogenesis of numerous diseases by restricting diffusion and bulk flow, and the same restriction could occur in the brain due to the dense amyloid plaques forming in the extracellular space. These plaques could prevent proper flow and diffusion of essential nutrients and prevent cellular waste removal by acting as extracellular channel blockades; however, limited models exist that address these issues. Alternative models and molecular tools need to be developed which focus on diffusion and bulk flow in relation to neural function and the physical presence of amyloid plaques. This review aims to evaluate the effect of the plaques on diffusion and bulk flow in relation to neural function in the brain.

Keywords

Neurodegeneration; Alzheimer’s disease; Pathogenesis; Plaques; Peptide fragment

Introduction

Obstruction of bulk flow by aggregated biomolecules is thought to play an important role in the pathogenesis of numerous human diseases, such as kidney stones, gallbladder stones, atherosclerosis, and thrombotic cardiovascular diseases [1]. Amyloid plaques deposited in the human brain are derived from amyloid-beta (Aß) peptides that aggregate and form fibrils which bundle to form plaques [2]. These plaques are also made of other proteins, lipids, and their calcification. How the presence of plaques affects neuronal function and pathogenesis, and whether the amyloid plaques obstruct flow of fluid molecules is unclear. Compared to the centimeter large atherosclerotic plaques or kidney stones, amyloid plaques are smaller, often less than 50 μm in diameter, making them a difficult subject to isolate and study.

The AD field has shifted from studying the potential toxicity of the plaques to studying the toxicity of the intermediate oligomers [3]. Some groups believe the plaques represent a non-toxic byproduct and provide a neuroprotective role by sequestering the oligomers [4-7]. After years of oligomer-focused research, numerous inconsistent results and lack of evident in vivo toxicity exists [8]. New methods are needed to study the potential physical toxicity of the plaques in relation to diffusion and bulk flow and to study the physical relationship of extracellular protein aggregation to viscosity and neuronal function. The cause of the massive amount of cell death found in AD remains unexplained so the search for an alternative toxicity is needed.

Aggregation and transformation of misfolded proteins into amyloid fibrils is a commonly observed phenomenon in dozens of human diseases, ranging from diabetes mellitus type 2 to neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease, and Parkinson’s disease [9]. Since the human body has no known mechanism for removing these amyloids, patients tend to already have extensive amyloid deposits once diagnosed, which leads to very poor prognoses [10]. Specifically in AD, Aβ accumulation with time leads to the formation of amyloid plaques [11].

Currently, the pathogenesis of Alzheimer’s disease remains unknown. Two leading hypotheses have been proposed: 1) amyloid plaques are pathogenic, and more recently, 2) the oligomer intermediates or as we refer to them, colloids, of fibril formation is pathogenic. Amyloid fibrils can further aggregate into a gel and there is a potential link between gelation and cellular death, as gels are known to eliminate bulk flow at the macroscopic level [12]. However, bulk flow at the microscopic level, under hydrostatic pressure, remains an area of study. New methods need to be developed to determine if amyloid plaques obstruct fluid flow and the circulation of nutrients, waste, and bio-signaling molecules through brain tissues and extracellular space.

Materials and Methods

Ad pathogenesis: Oligomer vs. amyloid hypotheses

Aggregation and accumulation of two different protein molecules causes pathogenesis in AD. Tau, a protein associated with cytoskeleton assembly, accumulates intracellularly to form macromolecule networks called neurofibrillary tangles (NFTs). Aβ, a peptide fragment, accumulates in the extracellular space in structures called plaques. Virtually all neurons that degenerate during the development of AD have accumulation of one or both forms of aggregation; however, Aβ formation alone can attribute to the disease [13].

Amyloid beta oligomers and aggregation

Once the Aß40 or Aß42 peptide is produced, the exact pathways and mechanisms of amyloid aggregation at the molecular level are poorly understood, which hinders rational pharmaceutical interventions. Critical intermediates in this pathway, colloids and their linear aggregates, composed of highly ordered β-sheet complexes [14] were discovered using atomic force microscopy [15] and later confirmed using transmission electron microscopy [16].

Protein molecules initially aggregate into colloidal spheres of uniform size, which then join together linearly, forming amyloid fibrils and eventual plaques (Figure 1) [17,18]. These insoluble plaques are commonly associated with cell death [3] and may lead to progressive degeneration of the blood-brain barrier (BBB) [19]. The accumulation of Aβ plaques in the extracellular space of the brain may initiate a cascade of reactions, beginning with synaptic dysfunction and ultimately resulting in apoptosis of the neuron [3,20-22]. The plaques may form around axons [23] and become associated with activated microglia and reactive astrocytes [11], which produce toxic molecules, such as cytokines, causing dysfunction of neuronal processes.

Figure 1: Proposed mechanism of colloidal aggregation leading to amyloid fibril formation [17].

Cytokines increase the inflammation of the neuron [24] and increase the production of toxic reactive oxygen species [11].

Currently, the popular amyloid cascade hypothesis (ACH), first proposed by Hardy and Higgins [25], has come under question due to formation of plaques in individuals who have normal cognitive function [26,27]. Alternatively, the oligomer hypothesis suggests the colloidal intermediates, also known as oligomers, are the toxic component that leads to neurodegeneration [28,29]. Aß Oligomers (AßOs) are either a step in the formation process or a byproduct of Aβ plaques [30]. The validity of the oligomer hypothesis depends on whether oligomers were present earlier than the fibrils and plaques and exercise their toxicity before substantial plaque formation.

As for the ACH, plaque formation may not have followed the proposed nucleation pathway an oligomers may be a byproduct or produced by other mechanisms [30].

Interestingly, a mouse model supporting the oligomer hypothesis displayed cognitive decline before substantial plaque formation [31]; however, supporting in vivo work at normal concentrations is lacking. The possibility also exists where the plaques provide a neuroprotective role [32] or are simply an innocent bystander in cell death.

The main issue with the oligomer hypothesis is the diversity of toxicity (Figure 2). Countless articles are published which present new mechanistic hypotheses of AßO toxicity. To briefly summarize, AßOs have been linked to AD in the following ways: Oxidative stress and lipid peroxidation in neurons [33-36] toxic ion pore formation on the cell membrane [37,38], synaptic plasticity disruption [39- 42], astrocyte and microglia calcium influx [36], selective neuronal degeneration [43], prion-like infectious behavior [34,44,45], binding to numerous membrane proteins [46-49], insulin resistance [50-52] calcium homeostasis disruption [35,53] and tau hyperphosphorylation induction [54]. One of the main issues is that AßOs lack a common description of structural toxicity and are thought of as “an emperor in need of clothes” since they possess numerous conformations ranging from monomers, to trimers and to eventual fibrils [8]. Moreover, not all oligomers promote toxicity [54,55] and “relevant Aß toxicity has barely been demonstrated” [56].

Figure 2: The diverse oligomer theory [48].

If AßOs are the toxic component, the massive cell loss, role in cognitive alterations and existence in non-AD patients cannot be fully explained [8]. More in vivo work using naturally occurring concentrations of AßOs along with other models of toxicity must be examined. Also, if they are toxic at low concentrations, such as 100 nM, why is toxicity specific to neurons and not elsewhere in the body where AßOs are also found?

The failure of many clinical trials following the ACH has caused researchers to change focus towards the oligomer theory. Most of the failures are due to the pitfalls of the transgenic mouse AD models and their inability to successfully correlate clinical trials to the complex human system [57,58]. Interestingly, the only FDA approved clinical drugs were never tested in the transgenic mouse model [57].

The ACH is also very unappealing for therapeutic treatment [59], since even if clearance of the plaques is successful, the surrounding neurons are already dead or damaged, so cognition cannot be restored. From a clinical view, attacking the oligomers appears much more appealing, and has made them the key drug target [60] and the focus of recent proposals. Clinical trials targeting oligomers have also failed across the board, again likely linked to the imperfect mouse model; however, trials have not focused on all aspects of the disease. Future trials must focus on all aspects including oligomers, fibrils, plaques and even tau [61]. Although some groups believe it, the plaques should not be considered a non-toxic byproduct, and should remain as a research focus based on their potential physical toxicity. Since existing models have not been successful, new models and methods must be proposed to address the issue of plaque’s physical presence. Much debate remains as to which aggregation state is toxic to the cells [62,63] and more work elucidating the physical effects of plaques within the brain must be investigated. It is likely the two disease states are linked, and both play a role in toxicity.

Results

Why denied access model?

In vitro, amyloid fibers have been found to be capable of further aggregating and forming gels [12,64]. Gels are known to be capable of eliminating bulk flow and reduce diffusion. Can amyloid plaques in AD be gels of porous matrix? TEM images of amyloid plaques either in AD tissue or isolated by density gradient centrifugation reveal these plaques are biogels with dense fiber network [64,65]. A transformation of brain fluid to a gel-like state due to amyloid fibril formation could have drastic neurophysiological effects on viscosity-dependent processes and tortuosity, or obstructive geometry, of the extracellular space.

Denied access model

When surrounded by amyloid plaques/protein gel, neurites are denied to have free access to interstitial ions, nutrients, signaling molecules and metabolic waste drainage pathways, which results in the inhibition of the compound action potential propagation. This is an alternative model for the AD community to consider, although continued investigations are required to verify the model and to fulfill the details. We hypothesize that gel formation around axons may inhibit the heart-pulse-driven fluid flow and then the circulation of nutrients and ions around the axon and affect the propagation of the action potential.

Denied access model explains numerous observations associated with ad tissue analysis

In Alzheimer’s disease, extensive neuronal cell death is found in anatomical regions of the brain where there is no presence or accumulation of neuritic senile plaques, and/or paired-helical filaments (PHF). Based on our model, plaques wrapped around axons may affect the vitality of the neuronal cell bodies located in a different anatomical region. Cognitive impairment and the degree of dementia do not correlate with the amyloid-beta plaques burden in many Alzheimer’s disease patients. However, the amyloid-beta plaque burden is quantified not based on the type of the plaque but rather a sum of all types. Based on our model, primitive plaques may not change the bulk flow or viscosity as much as the classical plaque. Thus, there exists a possibility that the classical plaque burden correlates better with the stages of Alzheimer’s disease. Our model also explains the observation that in transgenic mice amyloid plaques, although not classical ones, are found, but neuronal and synapse loss are rather minimal.

The denied access model also applies to other diseases where accumulation of protein aggregates may affect local diffusion, bulk flow and bioenergy-driven transportation. Since the toxic effect is physical rather than chemical, the model applies to not only AD but also alphasynuclein aggregation-induced Parkinson’s disease, aggregation of prion protein induced transmissible spongiform encephalopathies and islet amyloid polypeptide aggregation induced death of the beta cell in type II diabetes. Movement of molecules, ions and subcellular particles will all be affected due to the aggregation of proteins and then gelation of the amyloid fibers.

The denied access model is simple and inclusive of the physics and engineering perspective of gels or plaques, in addition to the biochemical nature. The inclusion of physical properties such as bulk flow and diffusion is seen in the illustration of atherosclerosis, thrombosis, gallbladder stones and kidney stones, where aggregation of biomolecules, large and small, is also an early event of disease pathogenesis. The transportation phenomenon, or bulk flow and diffusion, through gels are an active research area in engineering.

Denied Access by Attenuated Diffusion and Bulk Flow Through the Amyliod Plaque

The extracellular space (ECS) defines the channel-like environment where interstitial fluids (ISF), consisting of nutrients, neurotransmitters, signaling molecules, ions, and wastes, travel through the all tissues to maintain cellular homeostasis and function. The widths of the channels range from 20-40 nm and encompass around 20% of total brain volume [66,67]. Variations of the ECS geometry and volume fraction can arise during sleep, development, aging and neurological disorders, so understanding any alterations in function are vital for neurological research and drug delivery. These variations can affect proper clearance routes, cellular uptake, diffusion, and flow of nutrients and waste across the BBB [68].

Diffusion

Diffusion is the net movement of individual molecules down a concentration gradient due to random motion or thermal movement. Diffusion was first defined by Adolf Fick (Eq. 1) in the 19th century as,

(1)

Where J is the flux through an area, D is the diffusion coefficient, dC is the change in concentration of material, and dx is the change in distance. Thus, flux is proportional to the concentration gradient. Diffusion through the ECS is determined by the volume fraction or porosity, defined as α (Eq. 2) and the tortuosity or obstructive geometric capabilities of the environment, defined as λ (Eq. 3) HYPERLINK\l “page36” [68]. Volume fraction is defined as,

(2)

where V_ECS is the volume of the extracellular space devoid of matrix components and V_Tissue is the volume occupied by the tissue, and tortuosity is defined as,

(3)

where Df is the free diffusion coefficient and D* is the effective diffusion coefficient, typically determined using radiotracers or fluorescent dyes. The volume fraction of the ECS has been well studied using electron microscopy and occupies approximately 20% of total brain volume [69,70]. This value has been recently debated due to the difficulty in determining the ECS volume fraction in living tissues, where there is a high likelihood of ischemia; therefore, new methods, such as in vivo studies using quantum dots [71] and fluorescent dyes [72], have been developed and remain an area of study.

Due to the physiological independence between volume fraction and tortuosity, it is difficult to derive a relationship between the two. There are also many non-geometric factors such as ISF viscosity (discussed in bulk flow), molecular size, ECS channel interaction [73] and the uptake of molecules by cells or across the BBB [74]. The Nicholson group attempted to derive an empirical relationship using Archie’s law (Eq.4) HYPERLINK \l “page27” [75-77],

(4)

Which relates sedimentary porosity and saturation. It was determined that the values remained independent since the tortuosity exponent, n, is a variable according to the original law. However, if the tortuosity exponent is variable due to the variance between regions of white and gray matter, the relationship displays some dependence [78,79]. This relationship remains an important area of investigation when discussing diffusion throughout the brain.

Bulk flow

Bulk flow is the movement of water and solutes in response to a pressure gradient. Analysis of fluid flow through a porous matrix can be defined using a simplified version of

Darcy’s law (Eq. 5) which states,

(5)

Where Q is the flux of fluid per unit area through a porous matrix, Ks is the Darcy permeability coefficient, ∇P is the hydrostatic pressure gradient across the matrix and η is the viscosity of the ISF. According to the equation, bulk flow is strongly dependent on the viscosity of the media and the permeability through a porous matrix. The movement of fluids through the ECS of the brain has numerous implications, including cell to cell communication, drug delivery, distribution, waste clearance, ionic homeostasis, and immune function [80].

Figure 3: Denied access model. The neurite regions surrounded by amyloid gel plaques are denied from accessing to nutrient and signaling molecules as well as to the drainage pathways for metabolic wastes. This denied access leads to the inhibition of the action potential propagation and eventually the degeneration of the disused neurites.

Bulk flow through the ECS remains a debated issue [81]. Flow typically occurs via osmotically balanced fluid secretion across the BBB but can also be generated as fluid secretion from cells [82] and the choroid plexuses [83]. One research group determined bulk flow only occurs in perivascular spaces surrounding capillaries and little to no flow occurs throughout the remaining ECS [84]. Varying flow rates were also found when comparing white to gray matter regions of the brain. White matter regions expressed a flow rate of 10.5 μm/min towards the ventricles, but regions of gray matter only displayed flow under osmotic stress [85]. The issue of diffusion also returns, since some groups believe that diffusion cannot be the sole clearance mechanism due to a constant rate of clearance for various sized molecules [86-88]. However, another group that used albumin and large dextrans suggests diffusion is responsible for clearance and is independent from bulk flow [89]. As mentioned, quantitative measurements of flow rates have been made by injecting tracers into the brain and observing their movements [85,90], but no work has examined direct effects of Aß plaques.

Limited research has been conducted towards understanding how the formation of amyloid plaques affects diffusion. Researchers utilizing diffusion-weighted magnetic resonance imaging (DW-MRI) have demonstrated a reduced interstitial fluid diffusion in the brain of APP23 transgenic mice [91] and reduced diffusivity in the white matter of patients suffering from sporadic Jakob-Creutzfeldt disease [92], another neuroencephalopathy associated with the formation of insoluble amyloid. Sykova [93] analyzed the ECS and the volume fraction and diffusion of the cerebral cortex in APP23 mice using realtime tetramethylammonium (TMA) and DW-MRI methods. They determined that the volume fraction increased in aged APP23 mice by approximately 3.0% in both female and male mice and apparent diffusion of TMA decreased by 7.0% in females and 1.7% in males. Plaque load was also twice as high in females as it was in males. The control group decreased their ECS volume fraction by approximately 7% in males and approximately 4% in females, and displayed a<1% increased apparent TMA diffusion in aged mice [93]. These results suggest that plaques may decrease diffusion by increasing overall brain tortuosity; however, the direct correlation remains unknown.

Another physical impact of plaque formations may be fluid clearance from the brain by increasing the tortuosity, as previously noted by Sykova et al. [93]. Clearance is important across the BBB to prevent buildup of toxic molecules and to help maintain cellular homeostasis (Figure 4A). Clearance also prevents high concentrations of soluble Aβ and eventual plaque formation [94,95] and an age-related imbalance of Aß release could drive the progression of AD [3]. The accumulation of plaques in the extracellular space (Figure 4B) and drainage pathways (Figures 4C and 4D) could reduce the clearance of high molecular weight substances by impeding the normal clearance routes [68,70,96,]. Plaques can become very large, up to 150 μm in diameter [97], so it is likely that the dense plaques can restrict fluid movement through the less than 0.1 μm extracellular space pathways. Moreover, AD patients display a significant reduction in ISF flow [98], which may be linked to the increased plaque load. Future work must develop new methods to evaluate the effect of the presence of amyloid plaques on neuronal function and fluid flow in brain tissue.

Figure 4: Bulk flow and fluid clearance in the brain. (A) Nutrients and toxins must be able to freely move into and out of the brain (Image from the University of New Hampshire). (B) Plaque formations can become very large, up to 150 μm in diameter [97] and can likely block the (C) less than 0.1 μm extracellular channels and synaptic junctions [70]. (D) Factors within the extracellular space such as charged residues, binding, and dead space can also affect fluid flow [68].

Discussion

Techniques to study amyloid plaques on neuronal function

We identified four new methods being developed in our laboratory to study if and how the amyloid plaque affects the neuronal function. Specifically, these methods will be used to analyze the following:

Fibrin as an aß plaque proxy

To study the effect of amyloid plaque on neuronal function, we need to have the plaque prepared in the presence of the functional neurons, under physiological conditions and within seconds to minutes. The constraints of the physiological condition and the short time period force us to look for a proxy of amyloid fibril gel as they take hours to days to produce, often under non-physiological conditions.

Volume fraction and pore size: Ideally, the use of Aβ is preferred, but time, attainability, and practicality must be considered. Fortunately, amyloid fibrils are not the only example of a fibrous network created by proteins. The hindrance of diffusion and convective transport are also elementary properties of wound healing, a process that relies on blood clotting due to the formation of a fibrin gel network. Fibrin gels are formed within a few minutes between the interactions of the protein fibrinogen, from blood plasma and the activating enzyme thrombin [99]. Thrombin cleaves fibrinopeptides located in the middle of fibrinogen, producing a fibrin monomer and subsequent protofibril formation [100]. Compared to amyloid gels, fibrin is easily attainable, found within the body, and forms rapidly under physiological conditions [99]. Amyloid gels, on the other hand, require hours or even days to form in vitro. Importantly, fibrin is physically similar to amyloid gels and provides an ideal proxy. With these factors considered, a convenient analog to study the physical effects of toxicity must be used. This would allow for rapid and feasible studies of protein aggregation in cellular environments.

Fibrin provides an ideal analog for studying the physical effects of amyloid plaques or gels and the potentially abolished bulk flow on neuronal function; however, the gel must have the appropriate porosity to simulate the amyloid plaque. To qualify, fibrin gel must have higher porosity compatible to amyloid gel. Scanning electron microscopy (SEM) (Figure 5A) and atomic force microscopy (AFM) (Figure 5B) images reveal a dense fibrous network with pore sizes less than 1 μm, which are comparable to Aβ (Figure 5C).

Figure 5: Scanning electron micrograph of fibrin (A), atomic force micrograph of fibrin (B), and atomic force micrograph of amyloid-beta (C). Fibrin was generated using a 10:1 ratio of fibrinogen (20 mg/ml in 0.9% NaCl) and thrombin (50 U/ml in 0.1% BSA, pH 7.4). Amyloid fibril formation of 50 uM Aβ42 fibrils incubated at 37°C is seen in 1 week [128].

Chemically, fibrin is different from Aβ, and although fibrin is not a true amyloid, its regulation is similar to the enzymatic control of peptide hormone aggregation [101]. Fibrin has a high β-sheet content of 40% [102] and can stain using Congo red [103], an amyloid-specific stain. Kranenburg [103] believe fibrin can form cross-β structures and amyloid fibrils, and that these β-sheet structures function as the scaffold to activate plasmin, which eventually cleaves aggregated fibrin.

Viscosity of gels using molecular rotors: Increased viscosity is also thought to play a role in the pathogeneses of AD linked to amyloid plaque formation. De la Torre and Mussivand [19] hypothesized that blood viscosity can increase due to amyloid deposits causing degeneration of brain capillaries at the BBB; however, the link between increased local viscosity and degeneration has remained elusive. Protein aggregation into a gel, as seen in vitro, possibly also occurs in vivo.

Bulk viscosity is known to be different from molecular viscosity. For fibrin to be qualified as an amyloid fibril gel proxy for diffusivity study, the viscosity of fibrin gel, at the molecular level, needs to be comparable or lower than that of amyloid fibril gel. To study ion or molecule diffusion across the amyloid plaque, the molecular viscosity is of interest. A successful technique used to study viscosity applies fluorescent molecules known as molecular rotors. These rotors are able to alter their fluorescence by changing the rate of an aromatic conformational change [104,105]. This rate of change is dependent on the microviscosity of the environment decreasing bond rotation and increasing fluorescent yield [106]. Previous work in the lab used 9-(2-carboxy-2-cyanovinyl) julolidine (CCVJ) as the molecular rotor to determine how the formation of fibrin altered environmental viscosity [107]. Results were compared to varying concentrations of glycerol, a known viscous solution, where increasing concentrations amplified the fluorescence intensity. In 20 mg/ml fibrin, fluorescence intensity increased during the incubation period with the greatest increase occurring within the first 60 s.

Only a small increase occurred in 10 mg/ml fibrin (Figure 6), suggesting 20 mg/ml would be an optimal concentration to study gelation in a time dependent manner. This work established that gelation results in an increased environmental microviscosity.

Figure 6: Relative fluorescence intensity of 5 μM CCVJ in two different fibrin gels. One gel was composed of 20 mg/mL fibrinogen (�?�), and the other was composed of 10 mg/mL fibrinogen (�?�). Thrombin concentration was kept constant (10-75 U/mL).

Sciatic nerve as a CNS proxy

A goal is to determine whether the physical presence of plaques causes toxicity. Since in vivo experiments using mice or human models are difficult to perform, an ideal analog is the sciatic nerve from frogs, such as the Xenopus laevis, as it has long been successfully employed in studying numerous physiological properties of axons and is well characterized. The sciatic nerve is a large bundle of many axons surrounded by an epineurium membrane (Figure 7A). The individual axons are structurally supported with collagen, known as the endoneurium, and encompassed with myelin containing nodes of Ranvier (Figures 7B and 7C), which allows for rapid conduction of action potentials.

Figure 7: Scanning electron micrograph images of Xenopus laevis sciatic nerve. (A) The sciatic nerve image was generated by fixing the nerve bundle in 3% glutaraldehyde, washing in 0.1% PBS, staining in 1% osmium tetroxide/0.1% PBS, dehydrating in increasing concentrations of ethanol and ethyl-acetate, and critical point drying. Cryostat sections were also prepared in the cross sectional (C) and longitudinal (D) directions at 20 μm thickness.

In AD, white matter atrophy due to plaque formation may be as important as gray matter atrophy. Plaques are more numerous in associative regions of the brain higher in gray matter content, such as the extracellular matrix of the hippocampus where learning and memory take place [108] and where the most severe neuropathological changes occur [22]; however, plaques are also found in myelinated white matter regions such as the cortex. The question arises as to whether the myelinated sciatic nerve would serve as an appropriate proxy for neurodegenerative studies. Although the gray matter regions are more intensely studied, white matter atrophy occurs in AD [109] and is found in roughly 60% of AD cases [110]. White matter atrophy is often considered secondary to gray matter damage [111], but recent developments determined white matter damage occurs in pre-AD stages and cannot be linked to gray matter atrophy [112,113]. Furthermore, studies analyzing white matter attributed the damage to cognitive decline [114] and discovered higher levels of soluble Aβ in AD patients [115].

Many physiological experiments using the frog sciatic nerve have been conducted examining the compound action potential (CAP) and the biochemical effects that may cause degeneration. One group examined the time-dependent effects acrylamide had on the antioxidative stress in rat nerve tissues. With increased concentrations injected into rats over a few weeks, decreases were found in both CAP duration and CAP amplitude [116].

Furthermore, [116] determined that acrylamide may be associated with an increase in lipid peroxidation and reduction of the antioxidative capacity. Another experiment examined the direct effects of polyethylene glycol (PEG) on isolated rabbit sciatic nerves [117]. They found 40% PEG abolished the compound action potential within an hour due to increased osmotic pressures creating a hypertonic environment and shrinkage of the axon. These structural changes may lead to reduced ion permeability, neuronal stress, and eventually death [117]. These results suggest the frog sciatic nerve may be an ideal tissue for neurophysiology experimentation to test the effect of viscosity on neuronal function. Completing these experiments on gray matter would be extremely difficult; whereas, the sciatic nerve is easy to dissect and shares common physiological features as central nervous system neurons [118]. If plaques are the culprit for neuronal loss, similar reasons for toxicity may be seen in both white and gray matter.

Quantifying diffusion through plaques and brain tissue

How amyloid plaques affect the circulation of nutrients, wastes and biosignaling molecules in brain tissue remains unknown. Molecules move through a matrix either by diffusion driven by Brownian motion, or carried by fluid movement or bulk flow. Thus, methods are needed to analyze diffusion and bulk flow. FRAP is a straightforward technique developed to study molecular diffusion in media [119]. Using a confocal laser scanning microscope (CLSM), a user-defined region of interest is selected and bleached, leaving the surrounding area unbleached [120].

With time, the unbleached molecules diffuse into the bleach area and exchange with the bleached molecules [121,122]. Using one of many possible fluorescent dyes, such as rhodamine B (RhoB) or fluorescently tagged proteins, a fluorescent recovery curve can be generated and a diffusion coefficient of the dye through the sample can be calculated [123,124].

Protocols and data analysis of FRAP experiments differ between laboratories causing the existing body of data to be incomparable [125]. Many finite factors affect diffusion, suggesting that each lab must perfect their own calibrations and controls. Numerous studies using this technique and a variety of fluorophores have been completed; however, one vital element of experimental approach arises, fixation of the tissue. Most FRAP experiments analyze real-time protein movement and interactions requiring live tissue samples and cell cultures. If the methods advance for fixed tissue use, countless studies could be completed and experimental procedures would be much simpler.

Effect of plaques on bulk flow

The physical presence of amyloid plaques may hinder brain clearance and deter the movement of nutrients and wastes in the brain. At the tissue level, experiments injecting molecular tracers [85,90] and studies using positron emission tomography [98] have evaluated bulk flow, but they are difficult to perform for standard laboratories. These experiments were focused on movement into and out of the brain rather than through specific regions. Other experiments also focused on either CSF or ISF production rates in relation to bulk flow [126] or used computer based modeling to analyze flow [127]. Feasible, reliable, and straightforward techniques are needed to study fluid movement at the cellular level and to study the relationship of flow due to individual plaques.

It is possible to estimate the bulk flow if the diffusion coefficient of a particular molecule is known through a plaque and free medium. Knowing these will allow the tortuosity and void volume fraction of the plaque to be estimated. That information can then be used to estimate Darcy’s permeability through the plaque and the effect on bulk flow compared to normal flow through the ECS.

Conclusion

Gelation’s relationship to increased viscosity and reduction in bulk flow is a common phenomenon in physical chemistry and engineering, but the increased viscosity has never been confirmed in Aβ plaques found in Alzheimer’s disease (AD). In vitro, proteins, such as Aβ and lysozyme, can undergo conformational changes and self-assemble into amyloid fibrils.

Eventually these fibrils can aggregate and form highly viscous, rigid gels [12,63]. In vivo, it is likely that amyloid plaques act as viscous and structured gels. The elimination of bulk flow and reduced diffusivity due plaque formation in the ECS near axons may be responsible for the inhibition of action potential propagation, which, in time, may cause neuronal death. Here we introduce the denied access model: when surrounded by amyloid gel plaques, neurites are denied access to nutrients, signaling molecules, and metabolic waste drainage pathways, eventually leading to neurotic dystrophy. This is an alternative model for the AD community to consider, although continued investigations are required to verify the model and to fill in the details.

Acknowledgement

The study was supported by NASA, The Florida Space Grant Consortium and The Community Foundation for Brevard. We thank Dr. David Tipton and Dr. Daniel Woodard of Kennedy Space Center for their support. We also thank Dr. Michael Grace for the use of his cryotome, Dr. David Carroll for frog husbandry assistance and the use of a micromanipulator, and the Florida Institute of Technology microscopy center.

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