A three-dimensional human neural cell culture model of Alzheimer's disease.

Alzheimer’s disease (AD) transgenic mice have been used as a standard AD model for basic mechanistic studies and drug discovery. These mouse models showed symbolic AD pathologies including β-amyloid (Aβ) plaques, gliosis and memory deficits but failed to fully recapitulate AD pathogenic cascades including robust phospho tau (p-tau) accumulation, clear neurofibrillary tangles (NFTs) and neurodegeneration, solely driven by familial AD (FAD) mutation(s). Recent advances in human stem cell and three-dimensional (3D) culture technologies made it possible to generate novel 3D neural cell culture models that recapitulate AD pathologies including robust Aβ deposition and Aβ-driven NFT-like tau pathology. These new 3D human cell culture models of AD hold a promise for a novel platform that can be used for mechanism studies in human brain-like environment and high-throughput drug screening (HTS). In this review, we will summarize the current progress in recapitulating AD pathogenic cascades in human neural cell culture models using AD patient-derived induced pluripotent stem cells (iPSCs) or genetically modified human stem cell lines. We will also explain how new 3D culture technologies were applied to accelerate Aβ and p-tau pathologies in human neural cell cultures, as compared the standard two-dimensional (2D) culture conditions. Finally, we will discuss a potential impact of the human 3D human neural cell culture models on the AD drug-development process. These revolutionary 3D culture models of AD will contribute to accelerate the discovery of novel AD drugs.

γ-Secretase is composed of at least four proteins, presenilin (PS), nicastrin (NCT), Aph1, and Pen2. PS is the catalytic subunit of the γ-secretase complex, having aspartic protease activity. PS has two homologs, namely, PS1 and PS2. To compare the activity of these complexes containing different PSs, we reconstituted them in yeast, which lacks γ-secretase homologs. Yeast cells were transformed with PS1 or PS2, NCT, Pen2, Aph1, and artificial substrate C55-Gal4p. After substrate cleavage, Gal4p translocates to the nucleus and activates transcription of the reporter genes ADE2, HIS3, and lacZ. γ-Secretase activity was measured based on yeast growth on selective media and β-galactosidase activity. PS1 γ-secretase was ∼24-fold more active than PS2 γ-secretase in the β-galactosidase assay. Using yeast microsomes containing γ-secretase and C55, we compared the concentration of Aβ generated by PS1 or PS2 γ-secretase. PS1 γ-secretase produced ∼24-fold more Aβ than PS2 γ-secretase. We found the optimal pH of Aβ production by PS2 to be 7.0, as for PS1, and that the PS2 complex included immature NCT, unlike the PS1 complex, which included mature NCT. In this study, we compared the activity of PS1 or PS2 per one γ-secretase complex. Co-immunoprecipitation experiments using yeast microsomes showed that PS1 concentrations in the γ-secretase complex were ∼28 times higher than that of PS2. Our data suggest that the PS1 complex is only marginally less active than the PS2 complex in Aβ production.

Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer's disease (AD), because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel 3D culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of amyloid-β (Aβ) and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix and the analysis of AD pathogenesis. The 3D culture generation takes 1-2 d. The aggregation of Aβ is observed after 6 weeks of differentiation, followed by robust tau pathology after 10-14 weeks.

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular compartment of the brain in the form of congophilic amyloid angiopathy (CAA) and amyloid plaques (APs). Intracellular neurofibrillary tangles (NFTs) (88) formed from the abnormally phosphorylated cytoskeletal protein tau are also seen (52). The identification of the amyloid β protein (Aβ) in CAA and APs (28 ; 58) led to the cloning of the amyloid protein precursor (APP) (44). The discovery of familial AD (FAD) mutations in the APP gene (10 ; 29 ; 63 ; 64 ; 86) has supported the view that a defect in APP metabolism or function is directly involved in AD pathogenesis. The demonstration that mutations in the tau gene can lead to non-Alzheimer dementias with neurofibrillary pathology, lacking Aβ plaques (reviewed by 81), has reinforced the view that the NFTs are a secondary phenomenon in the pathogenesis of AD. It has long been argued that the deposition of amyloid is an early step in AD pathogenesis (58 ; Hardy and Higgins, 1992 ; 57). The term amyloid refers to insoluble proteinaceous deposits that are congophilic and exhibit red-green birefringence in the presence of plane polarized light (45). Implicit in much of the research on the role of APP and Aβ has been the assumption that deposits of amyloid are toxic to the brain (42) and that these deposits are the underlying cause of AD. The observation that Aβ peptides when "aged" (incubated to form amyloid fibrils) become toxic to neurons in culture (94 ; 22 ; 46 ; 66 ; 38) has further supported this view. The amyloid cascade hypothesis of AD, as formalized by Hardy and Higgins (35), states that Aβ"precipitates to form amyloid and, in turn, causes neurofibrillary tangles and cell death." However, this hypothesis has been challenged (see, e.g., 16 ; 34). It has been argued that the deposition of amyloid does not correlate with dementia (89 ; 2 ; 71 ; 72 ; 5), although the failure to observe a correlation may be related to the method by which AP load is measured (14). Whether amyloid deposits have a pathogenic role remains a controversial issue. Some of the neuropathological changes occurring in the AD brain were first described by Alzheimer (1). Extracellular deposits of amyloid in the form of APs and CAA, as well as intracellular NFTs, are major features of AD pathology (68). The major protein constituent of CAA and APs is a 4-kDa polypeptide termed the amyloid protein or Aβ (28 ; 58). A partial amino acid sequence of Aβ was used to clone a cDNA encoding a protein now referred to as the APP, which has features of an integral type I transmembrane glycoprotein (44). The APP gene contains 18 exons spanning >170 kb (95). The region encoding the Aβ sequence comprises part of exons 16 and 17 and contains between 40 and 43 amino acid residues that extend from the ectodomain into the transmembrane domain of the protein (Fig. 1). Primary structure of APP. Upper panel : APP is expressed initially as a type I transmembrane glycoprotein containing a large N-terminal ectodomain, a short transmembrane (TM) domain of hydrophobic amino acid residues, and a relatively short C-terminal cytoplasmic domain. The Aβ sequence makes up part of the ectodomain and extends partly into the TM domain. The APP gene encodes a protein containing a signal peptide sequence (SP), a cysteine-rich domain, a region rich in acidic residues, a domain with homology to Kunitz-type protease inhibitors (KPI), and a region sharing homology to the OX-2 protein. mRNA splicing can produce forms that lack the KPI or OX-2 domain. Lower panel : Amino acid sequence of the Aβ region of APP. Large arrows show the major sites of cleavage for α-, β, and γ-secretases. Small arrows show known FAD mutations. FIG. 1. It is now known that Aβ is a normal product of APP processing (32 ; 30 ; 21). The major route of APP processing is by α-secretase, an enzyme that cleaves within the Aβ sequence (20). Cleavage by β- and γ-secretases at the N- and C-terminal ends of the Aβ sequence liberates the Aβ polypeptide, which can subsequently be secreted from cells (21 ; 30 ; 31). The major form of Aβ that is secreted contains 40 amino acids (Aβ1-40). However, minor species containing 42 or 43 amino acid residues (Aβ1-42/43) are also produced. These extended forms of Aβ aggregate more readily and may seed amyloid fibril polymerization during the early stages of plaque formation (42). The strongest evidence for a pathogenic role for APP or Aβ comes from genetic studies of early-onset FAD (33). Several FAD mutations have been found in the APP gene. All of these mutations have been found to cluster close to the amyloid sequence in APP. Mutations at codon 716 (Florida), 717 (London), and 723 (Australian) cause an increased proportion of γ-secretase cleavage at position 42 or 43 in the amyloid sequence (Fig. 1 and Table 1). A mutation found at codons 670 and 671 in a Swedish kindred results in increased β-secretase cleavage (8 ; 43), whereas a point mutation at codon 612 (Flemish) inhibits α-secretase cleavage (32). The consequence of the Swedish and Flemish mutations is to increase processing of APP via the β-secretase pathway. All of the FAD mutations in the APP gene result in increased production of Aβ1-42/43 (Table 1). More than 40 FAD mutations in the presenilin 1 and presenilin 2 genes have also been reported (17 ; 75). The common feature of all these mutations is that they also cause an increase in the production of Aβ1-42/43 (73). Thus, presenilins are involved in regulating the proteolytic breakdown of APP by γ-secretase (18). However, the mechanism by which this occurs is unknown. TABLE 1. Studies on transgenic mice that overexpress FAD mutant forms of human APP also strongly argue for a central role of APP or Aβ in disease pathogenesis. Games et al. (26) demonstrated Alzheimer-like pathology in a transgenic mouse overexpressing a mutant (V717F) form of APP. Subsequently, Hsiao et al., (40) demonstrated similar pathology in a mouse expressing another mutant form of APP (Swedish mutation). It is interesting that the Hsiao mouse had behavioral changes indicative of a cognitive defect occurring before the appearance of robust plaque pathology. More recently, AD-like pathology has been reported in two other APP transgenic mice carrying FAD mutations (84). Although these mice do not show NFTs, there are behavioral abnormalities, abnormal neuritic processes, neuron and synapse loss, and biochemical abnormalities reminiscent of AD (26 ; 40 ; 56 ; 26 ; 9 ; 23 ; 41 ; 65 ; 79). The validity of these transgenic mice as models of AD pathology is reinforced by the observation that the AD-like phenotype is accelerated in APP transgenic mice that also contain an FAD mutant presenilin 1 transgene (37). Studies on the genetics of AD and on APP transgenic mice provide compelling evidence that a disturbance in APP metabolism or function is the underlying cause of AD. However, these studies do not prove that Aβ is the causative agent. The strongest evidence implicating Aβ in the pathogenesis of AD comes from the observation that Aβ peptides are toxic to neurons in culture (94 ; 22 ; 46 ; 66 ; 38). This toxicity is enhanced if the peptides are "aged" (incubated from hours to days), a procedure that increases amyloid fibril formation (66). Although the process of aging increases the number of amyloid fibrils formed from Aβ, this is not proof per se that fibrils are the major toxic form of Aβ. It is likely that the levels of soluble oligomeric species of Aβ are also increased by the process of aging (see next section). The mechanism of neurotoxicity is unclear. Some studies suggest that Aβ can disrupt calcium homeostasis (Mattson et al., 59, 61,60), perhaps by interfering with L-type voltage-dependent calcium channels (15 ; 90), Aβ may reduce Na+,K+-ATPase activity (55), thereby influencing membrane depolarization. Furukawa and Mattson (25) have reported that cytochalasin D, a compound that inhibits actin polymerization and calcium entry, can reduce Aβ neurotoxicity. Other studies suggest a role for reactive oxygen species in Aβ toxicity (4 ; 6 ; 36). Disturbances in redox potential may lead to disruption of calcium homeostasis, as reactive oxygen species can impair ATPase activities (55). Aβ may cause lipid peroxidation and affect superoxide dismutase, which may contribute to its neurotoxicity in culture (7). The receptor that transduces the effects of Aβ is unknown, although the receptor for advanced glycation end products (RAGE) (91) has been implicated. Studies by Yan et al. (92) suggest that Aβ may also bind an intracellular hydrosteroid dehydrogenase known as ERAB. The neurotoxicity may be mediated by an indirect action of Aβ on a nonneuronal cell. For example, microglial cells are often found in association with neuritic plaques (69), and Aβ has been shown to activate microglia in culture (11). Therefore, the possibility that Aβ stimulates release of an unidentified neurotoxic agent from a nonneuronal compartment must also be considered. Recent work by Geula et al. (27) has shown that when aged Aβ is injected into the brains of old rhesus monkeys, it is neurotoxic. However, injection of the same material into young monkeys has little toxic effect. This suggests that although Aβ may be pathogenic, there must be other age-related susceptibility factors that are also important to generate a toxic reaction in vivo. There is now considerable evidence that the type 4 allele of the apoE gene is a major susceptibility factor for late-onset AD (82). ApoE is a 299-amino acid glycoprotein that is principally involved in lipid transport and related functions (reviewed by 54). The N-terminal domain (residues 1-191) contains a receptor-binding region (residues 136-150) that exists as a four-helix bundle. The C terminus is hydrophobic and contains the lipoprotein-binding determinants. Genetic heterogeneity leads to three common isoforms, designated as apoE2, apoE3, and apoE4, encoded by three alleles called ε2, ε3, and ε4, respectively. The isoforms differ from each other by cysteine-arginine interchanges at positions 112 and 158 (54). The ε4 allele frequency is significantly increased in late-onset AD patients (82). Furthermore, those individuals with one or two copies of the ε4 allele have higher amounts of Aβ immunoreactivity in their brains than those individuals without ε4 (74), suggesting that apoE4 promotes fibrillation of Aβ to form amyloid. There is evidence from several studies to suggest that apoE4 could be involved in the polymerization of Aβ to form amyloid in vivo. For example, apoE4 has been found to promote Aβ fibrillogenesis in vitro more readily than apoE3 (Strittmatter et al., 82,83 ; 53). Also studies in which apoE knockout mice have been crossed with human APP transgenic mice show that the expression of apoE is necessary for amyloid deposition in vivo (3). However, the mechanism by which apoE4 influences the risk of AD is still unknown. Although apoE can bind Aβ (Strittmatter et al., 82,83), most studies showing that apoE4 stimulates Aβ aggregation have used apoprotein, i.e., delipidated, forms of apoE, which do not possess a native conformation. Indeed, studies using native forms of apoE isoforms suggest that apoE4 binds less well to Aβ than the other isoforms (49 ; 96 ; 93). On the basis of this finding it has been proposed that apoE may be involved in clearance of Aβ (92), although this hypothesis would not explain why apoE knockout inhibits Aβ deposition (3). Molecular genetic studies indicate a central role for Aβ in AD pathogenesis. However, these studies do not indicate the form or site of action of Aβ neurotoxicity. Until the mechanism of Aβ neurotoxicity is understood, it will be difficult to explain the topography of neurodegeneration (77). It has been presumed that deposits of amyloid constitute the entire Aβ load, but recent studies indicate that some of the Aβ in the brain exists in a soluble form. Soluble Aβ is unlikely to be detected by routine fixation and immunostaining. Aβ is probably secreted as a monomer and subsequently aggregates into soluble oligomers or fibrils (67). There is good evidence for the existence of low-molecular-weight Aβ oligomers in the brain. Studies by Kuo et al. (47) have isolated watersoluble Aβ oligomers from normal and AD brains. Perhaps of greatest interest in this study was the finding that not only was the level of soluble Aβ greater in the AD brain compared with controls, but as with some familial mutations, the proportion of soluble Aβ1-42/43 was significantly increased over soluble Aβ1-40 species in AD patients. Similar results have been obtained by Funato et al. (24). In the study by Kuo et al. (47), the watersoluble Aβ species ranged in size from monomers of <10 kDa to oligomers of > 100 kDa. Studies by Roher et al. (70) suggest that the watersoluble dimeric species are neurotoxic, whereas in a recent study Lambert et al. (50) found that small, low-molecular-weight oligomers of Aβ1-42 are several orders of magnitude more potent neurotoxins than high-molecular-weight fibrillar species of Aβ1-40. These studies have implications for our view of amyloid toxicity. If oligomeric soluble forms of Aβ have a pathogenic role, then is it possible that APs are not the major toxic form of Aβ in the brain (Fig. 2) ? Model describing pathways of APP processing. APP can be cleaved by β- and γ-secretases to yield Aβ1-42/43 (γ42/43) or Aβ1-40 (γ40), which can be actively secreted. Alternatively, APP can be cleaved by α-secretase (α) to yield sAPPα, which several studies (reviewed by 78) have shown may have neuroprotective or trophic functions. Aβ1-42/43 can aggregate to form soluble oligomeric species or may seed the polymerization of Aβ1-40 to form insoluble amyloid fibrils, which are deposited in the form of APs. FIG. 2. Recently, Crook et al. (13) described an unusual variant of AD involving a deletion of exon 9 of the presenilin 1 gene from the mRNA. Both NFTs and Aβ-immunopositive plaques were present, but the plaques were of the diffuse nonneuritic (nonfibrillar) type. Like the other FAD mutations in the presenilin 1 gene, the exon 9 deletion also increases Aβ1-42/43 production (62). There are at least two possible explanations for the existence of an AD variant with only diffuse plaques. As previously considered, 42- or 43-residue-long forms of Aβ may be secreted to exert a neurotoxic action (Fig. 2). However, a second possibility is that intraneuronal Aβ plays a role in pathogenesis. Although the molecular genetic studies strongly argue for a direct role of Aβ1-42/43 in AD pathogenesis, they do not provide any indication of whether the Aβ is extracellular or intracellular. Skovronsky et al. (76) have shown that Aβ1-42/43 accumulates preferentially in an insoluble intracellular fraction where it is more abundant than Aβ1-40. Lee et al. (51) have localized intracellular Aβ1-42/43 to the endoplasmic reticulum. Clearly, if Aβ accumulates within intracellular organelles, it could have profound effects on normal cellular protein trafficking and metabolism. If Aβ1-42/43 is the real culprit in AD, then an inverse correlation between the amount of Aβ1-42/43 production and the age of onset of the disease might be predicted. Mutations causing high levels of Aβ1-42/43 in cell culture should cause an early age of onset of clinical symptoms. However, studies with fibroblasts taken from FAD patients (73) show that this is not the case (17). There are several possible explanations for this. It is possible that the level of Aβ1-42/43 production in cell culture does not reflect the level in brain. Another possibility is that FAD mutations influence other cellular events that affect the age of onset. A third possibility is that Aβ1-42/43 is not the only (or even the major) pathogenic form of Aβ. Although Aβ peptides terminating in positions 39-43 are the major forms produced, it is possible that very low levels of previously undetected longer forms of Aβ may also exist (12). If they do exist, they could also be neurotoxic. This would be consistent with observations that C-terminal fragments of APP containing the Aβ sequence are more toxic than Aβ (80). There is very good evidence that Aβ accumulation is the underlying cause of FAD, and there is strong circumstantial evidence to suggest that a similar process underlies the pathogenesis of sporadic (late-onset) AD. Although amyloid deposits (APs and CAA) are markers of the disease, insoluble fibrillar Aβ may not be the main neurotoxic form. Low-molecular-weight diffusible forms of Aβ1-42/43 may also be important. The fact that aged Aβ in vitro contains more amyloid fibrils does not necessarily prove that amyloid is neurotoxic. It is likely that aging also produces increased amounts of soluble oligomeric Aβ species. Therefore, more work is needed to define the precise nature of the toxic form of Aβ and to delineate the mechanism of this toxicity. D.H.S. is supported by grants from the National Health and Medical Research Council of Australia and the Rebecca L. Cooper Foundation.

Modeling Clinically Heterogeneous Presenilin Mutations with Transgenic Drosophila

Alzheimer's disease (AD) arises from complex multilevel interactions between genetic, epigenetic, and environmental factors. Recent studies suggest that exposure to the environmental and occupational toxicant formaldehyde (FA) may play a significant role in AD development. However, the effects of FA exposure on Aβ and tau pathologies in human neural cell 3D culture systems remain unexplored. To investigate FA's role in AD initiation, we differentiated 3D-cultured immortalized human neural progenitor ReN cells (ReNcell VM) into neurons and glial cells, followed by FA treatment. FA exposure for 12 weeks resulted in a dose-dependent increase in Aβ40, Aβ42, and phosphorylated tau levels. To further examine FA's role in AD progression, we established a 3D human neural cell culture AD model by transfecting ReN cells with AD-related mutant genes, including mutant APP and PSEN1, which recapitulate key AD pathological events. Our findings demonstrate that FA exposure significantly elevated Aβ40, Aβ42, and phosphorylated tau levels in this 3D-cultured AD model. These results suggest that FA exposure contributes to the initiation and progression of AD pathology in 3D-cultured human neural cells.

Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Abeta(1-42) production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Abeta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Abeta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Abeta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.

Sequential processing of the β-amyloid precursor protein by β- and γ-secretase generates the amyloid β-peptide (Aβ), which is widely believed to play a causative role in Alzheimer disease. Selective lowering of the pathogenic 42-amino acid variant of Aβ by γ-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of γ-secretase, display differential responses to non-steroidal anti-inflammatory drug (NSAID)-type GSMs and more potent second-generation compounds. Although many pathogenic PS mutations resisted lowering of Aβ(42) generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Aβ(42) for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Aβ(42) responses to GSM-1, suggesting that a positive GSM-1 response depends on the spatial environment in γ-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and Leu-166 was identified as a critical residue with respect to the Aβ(42)-lowering response of GSM-1. Finally, we found that GSM-1-responsive and -resistant PS mutants behave very similarly toward other potent second-generation compounds of different structural classes than GSM-1. Taken together, our data show that a positive Aβ(42) response for PS mutants depends both on the particular mutation and the GSM used and that attenuated Aβ(42) responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.

The relationship between amyloid-β (Aβ) species and tau pathology in Alzheimer’s disease (AD) is not fully understood. Here, we provide direct evidence that Aβ42/40 ratio, not total Aβ level, plays a critical role in inducing neurofibrillary tangles (NTFs) in human neurons. Using 3D-differentiated clonal human neural progenitor cells (hNPCs) expressing varying levels of amyloid β precursor protein (APP) and presenilin 1 (PS1) with AD mutations, we show that pathogenic tau accumulation and aggregation are tightly correlated with Aβ42/40 ratio. Roles of Aβ42/40 ratio on tau pathology are also confirmed with APP transmembrane domain (TMD) mutant hNPCs, which display differential Aβ42/40 ratios without mutant PS1. Moreover, naïve hNPCs co-cultured with APP TMD I45F (high Aβ42/40) cells, not with I47F cells (low Aβ42/40), develop robust tau pathology in a 3D non-cell autonomous cell culture system. These results emphasize the importance of reducing the Aβ42/40 ratio in AD therapy.

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the beta-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21-89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.

A major problem in traditional cell culture methods, such as Petri dishes and culture flasks, is the very simplified artificial environment around the cells. Traditional cell culture methods lack features of the native cell niche, such as gradients and cell organization. This lack probably explains why pharmaceutics against the neurodegenerative Alzheimer's disease successfully stop the propagation of the disease in the Petri dish, but fail so far in clinical trials. This thesis intends to improve cell culture methods for neuroscience research related to neural developmental questions and neurodegenerative diseases. As the cortex is the main part in our brain, related to memory, emotions and perception, this thesis does focus on cell culture tools and protocols for primary cortical neurons. Currently, dissociated neurons are cultured in pure or co-culture of neural and non-neural cells, but structuring elements and controlled gradient formation is missing. The first part of this thesis discusses different studies that implicate environmental components for neural cells in their native neural cell niche. We will establish a generic neural cell niche, which consists of different neural and non-neural cells, a structured 3D environment, molecular gradients and oriented neurite networks, in a nutshell. Additionally, a simplified model of the generic neural cell niche is introduced that is implemented in a microfluidic base cell culture tool. This novel artificial neural cell niche will provide cell layer structure in 3D and local control of molecular gradients at the microscale. We will use microfluidic technology to integrate missing features in cell culture techniques for primary cortical neurons. The microfluidic device will consist of three parts: (1) a main cell culture channel that is used to organize neural cells in 3D hydrogel layers, side-by-side; (2) parallel perfusion channels to mimic nutrient supply and to control stable gradient formation, (3) interconnecting microchannels, called junction channels, that separate perfusion driven molecular transport from diffusive molecular transport. The perfusion channels are connected to device-incorporated reservoirs that allow maintenance of stable molecular gradients based on perfusion and diffusion without the use of peristaltic or syringe pumps. By injecting cortical neurons entrapped in an agarose-alginate solution in the microfluidic device, we generated 3D micropatterned neural cell layers with stable gradients perpendicular to the layer orientation. We demonstrated neurite outgrowth behavior until three weeks in culture. The application of different cell organization patterns revealed an influence of the pattern on the cell culture response. Using neurotrophic gradients of nerve growth factor (NGF) and nutrient supplements (B27), we showed that neurite guidance and synapse formation followed synergistic NGF/B27 gradients. We found that the gradient induced effects are very sensitive to changes in the environmental structure, such as the cell layer organization. Using a gradient of a phosphatase inhibitor, okadaic acid, we generated locally diseased states of the protein Tau in both 2D and 3D micropatterned neural cell cultures. The diseased form of Tau, hyper-phosphorylated Tau, is a major hallmark of Alzheimer's disease. For the first time, local formation of hyper-phosphorylated Tau was demonstrated to affect a defined cell population in a compartmentalized 2D or 3D neural cell culture. Our results revealed that the propagation mechanism of this Alzheimer's diseased lesion is determined through the formation of the 2D or 3D environment. We think that this new neural cell culture tool has the potential to answer biological questions related to environmental and structural parameters involved in the formation of the cerebral cortex and in the propagation of neurodegenerative diseases. Future studies in modern neuroscience research can now better investigate the effect of gradient parameters such as the slope, average concentration or gradient profile on cell culture and disease propagation in a controlled manner. Furthermore, the influence of cell patterns in 2D and 3D is addressed with the ability to modify cell position, density and type at the microscale. The local formation of neurodegenerative disease lesions in a micropatterned neural cell culture is generic, which makes the integration of other neurodegenerative disease models, such as Parkinson's disease, Amyotrophic lateral sclerosis or Huntington's disease, possible.

Familial Alzheimer's disease (FAD) mutations of the amyloid β-peptide (Aβ) are known to lead to early onset and more aggressive Alzheimer's disease. FAD mutations such as "Iowa" (D23N), "Arctic" (E22G), "Italian" (E22K), and "Dutch" (E22Q) have been shown to accelerate Aβ aggregation relative to the wild-type (WT). The mechanism by which these mutations facilitate increased aggregation is unknown, but each mutation results in a change in the net charge of the peptide. Previous studies have used nonpolarizable force fields to study Aβ, providing some insight into how this protein unfolds. However, nonpolarizable force fields have fixed charges that lack the ability to redistribute in response to changes in local electric fields. Here, we performed polarizable molecular dynamics simulations on the full-length Aβ42 of WT and FAD mutations and calculated folding free energies of the Aβ15-27 fragment via umbrella sampling. By studying both the full-length Aβ42 and a fragment containing mutations and the central hydrophobic cluster (residues 17-21), we were able to systematically study how these FAD mutations impact secondary and tertiary structure and the thermodynamics of folding. Electrostatic interactions, including those between permanent and induced dipoles, affected side-chain properties, salt bridges, and solvent interactions. The FAD mutations resulted in shifts in the electronic structure and solvent accessibility at the central hydrophobic cluster and the hydrophobic C-terminal region. Using umbrella sampling, we found that the folding of the WT and E22 mutants is enthalpically driven, whereas the D23N mutant is entropically driven, arising from a different unfolding pathway and peptide-bond dipole response. Together, the unbiased, full-length, and umbrella sampling simulations of fragments reveal that the FAD mutations perturb nearby residues and others in hydrophobic regions to potentially alter solubility. These results highlight the role electronic polarizability plays in amyloid misfolding and the role of heterogeneous microenvironments that arise as conformational change takes place.

Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell–cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

The three-dimensional (3D) culture of neural cells in extracellular matrix (ECM) gels holds promise for modeling neurodegenerative diseases and pre-clinical evaluation of novel therapeutics. However, most current strategies for fabricating 3D neural cell cultures are not well suited to automated production and analysis. Here, we present a facile, replicable, 3D cell culture system that is compatible with standard laboratory equipment and high-throughput workflows. This system uses aqueous two-phase systems (ATPSs) to confine small volumes (5 and 10 μl) of a commonly used ECM hydrogel (Matrigel) into thin, discrete layers, enabling highly-uniform production of 3D neural cell cultures in a 96-well plate format. These 3D neural cell cultures can be readily analyzed by epifluorescence microscopy and microplate reader. Our preliminary results show that many common polymers used in ATPSs interfere with Matrigel gelation and instead form fibrous precipitates. However, 0.5% hydroxypropyl methylcellulose (HPMC) and 2.5% dextran 10 kDa (D10) were observed to retain Matrigel integrity and had minimal impact on cell viability. This novel system offers a promising yet accessible platform for high-throughput fabrication of 3D neural tissues using readily available and cost-effective materials.

The amyloid hypothesis has dominated the thinking in our attempts to understand, diagnose and develop drugs for Alzheimer's disease (AD). This article presents a new hypothesis that takes into account the numerous familial AD (FAD) mutations in the amyloid precursor protein (APP) and its processing pathways, but suggests a new perspective beyond toxicity of forms of the amyloid beta-peptide (Abeta). Clearly, amyloid deposits are an invariable feature of AD. Moreover, although APP is normally processed to secreted and membrane-bound fragments, sAPPbeta and CTFbeta, by BACE, and the latter is subsequently processed by gamma-secretase to Abeta and CTFgamma, this pathway mostly yields Abeta of 40 residues, and increases in the levels of the amyloidogenic 42-residue Abeta (Abeta42) are seen in the majority of the mutations linked to the disease. The resulting theory is that the disease is caused by amyloid toxicity, which impairs memory and triggers deposition of the microtubule associated protein, Tau, as neurofibrillary tangles. Nevertheless, a few exceptional FAD mutations and the presence of large amounts of amyloid deposits in a group of cognitively normal elderly patients suggest that the disease process is more complex. Indeed, it has been hard to demonstrate the toxicity of Abeta42 and the actual target has been shifted to small oligomers of the peptide, named Abeta derived diffusible ligands (ADDLs). Our hypothesis is that the disease is more complex and caused by a failure of APP metabolism or clearance, which simultaneously affects several other membrane proteins. Thus, a traffic jam is created by failure of important pathways such as gamma-secretase processing of residual intramembrane domains released from the metabolism of multiple membrane proteins, which ultimately leads to a multiple system failure. In this theory, toxicity of Abeta42 will only contribute partially, if at all, to neurodegeneration in AD. More significantly, this theory would predict that focussing on specific reagents such as gamma-secretase inhibitors that hamper metabolism of APP, may initially show some beneficial effects on cognitive performance by elimination of acutely toxic ADDLs, but over the longer term may exacerbate the disease process by reducing membrane protein turnover.