A 3D Bioengineered Neural Tissue Model Generated from Human iPSC-Derived Neural Precursor Cells.

Available models to study neuropathological diseases include cell cultures and animal models. Brain pathologies, however, are often poorly recapitulated in animal models. 2D cell culture systems are well established and have been used since the early 1900s to grow cells on flat dishes. However, conventional 2D neural culture systems, which lack key features of the brain's 3D microenvironment, often inaccurately represent the diversity and maturation of multiple cell types and their interaction under physiological and pathological conditions.To improve CNS modeling, we have designed a 3D bioengineered neural tissue model generated from human iPSC-derived neural precursor cells (NPCs). This NPC-derived biomaterial scaffold, composed of silk fibroin with an intercalated hydrogel, matches the mechanical properties of native brain tissue and supports the long-term differentiation of neural cells in a donut-shaped sponge within an optically clear central window. This chapter describes integrating iPSC-derived NPCs in these silk-collagen scaffolds and differentiating them into neural cells over time.

3D bioengineered neural tissue generated from patient-derived iPSCs mimics time-dependent phenotypes and transcriptional features of Alzheimer's disease.

Several iPSC-derived three-dimensional (3D) cultures have been generated to model Alzheimer's disease (AD). While some AD-related phenotypes have been identified across these cultures, none of them could recapitulate multiple AD-related hallmarks in one model. To date, the transcriptomic features of these 3D models have not been compared with those of human AD brains. However, these data are crucial to understanding the pertinency of these models for studying AD-related pathomechanisms over time. We developed a 3D bioengineered model of iPSC-derived neural tissue that combines a porous scaffold composed of silk fibroin protein with an intercalated collagen hydrogel to support the growth of neurons and glial cells into complex and functional networks for an extended time, a fundamental requisite for aging studies. Cultures were generated from iPSC lines obtained from two subjects carrying the familial AD (FAD) APP London mutation, two well-studied control lines, and an isogenic control. Cultures were analyzed at 2 and 4.5 months. At both time points, an elevated Aß42/40 ratio was detected in conditioned media from FAD cultures. However, extracellular Aß42 deposition and enhanced neuronal excitability were observed in FAD culture only at 4.5 months, suggesting that extracellular Aß deposition may trigger enhanced network activity. Remarkably, neuronal hyperexcitability has been described in AD patients early in the disease. Transcriptomic analysis revealed the deregulation of multiple gene sets in FAD samples. Such alterations were strikingly similar to those observed in human AD brains. These data provide evidence that our patient-derived FAD model develops time-dependent AD-related phenotypes and establishes a temporal relation among them. Furthermore, FAD iPSC-derived cultures recapitulate transcriptomic features of AD patients. Thus, our bioengineered neural tissue represents a unique tool to model AD in vitro over time.

Pathophysiology of neurodegenerative diseases: An interplay among axonal transport failure, oxidative stress, and inflammation?

Neurodegenerative diseases (NDs) are heterogeneous neurological disorders characterized by a progressive loss of selected neuronal populations. A significant risk factor for most NDs is aging. Considering the constant increase in life expectancy, NDs represent a global public health burden. Axonal transport (AT) is a central cellular process underlying the generation and maintenance of neuronal architecture and connectivity. Deficits in AT appear to be a common thread for most, if not all, NDs. Neuroinflammation has been notoriously difficult to define in relation to NDs. Inflammation is a complex multifactorial process in the CNS, which varies depending on the disease stage. Several lines of evidence suggest that AT defect, axonopathy and neuroinflammation are tightly interlaced. However, whether these impairments play a causative role in NDs or are merely a downstream effect of neuronal degeneration remains unsettled. We still lack reliable information on the temporal relationship between these pathogenic mechanisms, although several findings suggest that they may occur early during ND pathophysiology. This article will review the latest evidence emerging on whether the interplay between AT perturbations and some aspects of CNS inflammation can participate in ND etiology, analyze their potential as therapeutic targets, and the urge to identify early surrogate biomarkers.

Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

Neural cell adhesion molecules 1 (NCAM1) and 2 (NCAM2) belong to the cell adhesion molecules of the immunoglobulin superfamily and have been shown to regulate formation, maturation, and maintenance of synapses. NCAM1 and NCAM2 undergo proteolysis, but the identity of all the proteases involved and how proteolysis is used to regulate their functions are not known. We report here that NCAM1 and NCAM2 are BACE1 substrates in vivo. NCAM1 and NCAM2 overexpressed in HEK cells were both cleaved by metalloproteinases or BACE1, and NCAM2 was also processed by γ-secretase. We identified the BACE1 cleavage site of NCAM1 (at Glu 671) and NCAM2 (at Glu 663) using mass spectrometry and site-directed mutagenesis. Next, we assessed BACE1-mediated processing of NCAM1 and NCAM2 in the mouse brain during aging. NCAM1 and NCAM2 were cleaved in the olfactory bulb of BACE1+/+ but not BACE1-/- mice at postnatal day 10 (P10), 4 and 12 months of age. In the hippocampus, a BACE1-specific soluble fragment of NCAM1 (sNCAM1ß) was only detected at P10. However, we observed an accumulation of full-length NCAM1 in hippocampal synaptosomes in 4-month-old BACE1-/- mice. We also found that polysialylated NCAM1 (PSA-NCAM1) levels were increased in BACE1-/- mice at P10 and demonstrated that BACE1 cleaves both NCAM1 and PSA-NCAM1 in vitro. In contrast, we did not find evidence for BACE1-dependent NCAM2 processing in the hippocampus at any age analyzed. In summary, our data demonstrate that BACE1 differentially processes NCAM1 and NCAM2 depending on the region of brain, subcellular localization, and age in vivo.

Gga3 deletion and a GGA3 rare variant associated with late onset Alzheimer's disease trigger BACE1 accumulation in axonal swellings.

Axonal dystrophy, indicative of perturbed axonal transport, occurs early during Alzheimer's disease (AD) pathogenesis. Little is known about the mechanisms underlying this initial sign of the pathology. This study proves that Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) loss of function, due to Gga3 genetic deletion or a GGA3 rare variant that cosegregates with late-onset AD, disrupts the axonal trafficking of the ß-site APP-cleaving enzyme 1 (BACE1) resulting in its accumulation in axonal swellings in cultured neurons and in vivo. We show that BACE pharmacological inhibition ameliorates BACE1 axonal trafficking and diminishes axonal dystrophies in Gga3 null neurons in vitro and in vivo. These data indicate that axonal accumulation of BACE1 engendered by GGA3 loss of function results in local toxicity leading to axonopathy. Gga3 deletion exacerbates axonal dystrophies in a mouse model of AD before ß-amyloid (Aß) deposition. Our study strongly supports a role for GGA3 in AD pathogenesis, where GGA3 loss of function triggers BACE1 axonal accumulation independently of extracellular Aß, and initiates a cascade of events leading to the axonal damage distinctive of the early stage of AD.

ß-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses.

The ß-secretase ß-site APP-cleaving enzyme 1 (BACE1) is deemed a major culprit in Alzheimer's disease, but accumulating evidence indicates that there is more to the enzyme than driving the amyloidogenic processing of the amyloid precursor protein. For example, BACE1 has emerged as an important regulator of neuronal activity through proteolytic and, most unexpectedly, also through nonproteolytic interactions with several ion channels. Here, we identify and characterize the voltage-gated K+ channel 3.4 (Kv3.4) as a new and functionally relevant interaction partner of BACE1. Kv3.4 gives rise to A-type current with fast activating and inactivating kinetics and serves to repolarize the presynaptic action potential. We found that BACE1 and Kv3.4 are highly enriched and remarkably colocalized in hippocampal mossy fibers (MFs). In BACE1-/- mice of either sex, Kv3.4 surface expression was significantly reduced in the hippocampus and, in synaptic fractions thereof, Kv3.4 was specifically diminished, whereas protein levels of other presynaptic K+ channels such as KCa1.1 and KCa2.3 remained unchanged. The apparent loss of presynaptic Kv3.4 affected the strength of excitatory transmission at the MF-CA3 synapse in hippocampal slices of BACE1-/- mice when probed with the Kv3 channel blocker BDS-I. The effect of BACE1 on Kv3.4 expression and function should be bidirectional, as predicted from a heterologous expression system, in which BACE1 cotransfection produced a concomitant upregulation of Kv3.4 surface level and current based on a physical interaction between the two proteins. Our data show that, by targeting Kv3.4 to presynaptic sites, BACE1 endows the terminal with a powerful means to regulate the strength of transmitter release.SIGNIFICANCE STATEMENT The ß-secretase ß-site APP-cleaving enzyme 1 (BACE1) is infamous for its crucial role in the pathogenesis of Alzheimer's disease, but its physiological functions in the intact nervous system are only gradually being unveiled. Here, we extend previous work implicating BACE1 in the expression and function of voltage-gated Na+ and K+ channels. Specifically, we characterize voltage-gated K+ channel 3.4 (Kv3.4), a presynaptic K+ channel required for action potential repolarization, as a novel interaction partner of BACE1 at the mossy fiber (MF)-CA3 synapse of the hippocampus. BACE1 promotes surface expression of Kv3.4 at MF terminals, most likely by physically associating with the channel protein in a nonenzymatic fashion. We advance the BACE1-Kv3.4 interaction as a mechanism to strengthen the temporal control over transmitter release from MF terminals.

BACE1 elevation engendered by GGA3 deletion increases ß-amyloid pathology in association with APP elevation and decreased CHL1 processing in 5XFAD mice.

BACKGROUND: ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in the production of amyloid beta (Aß), the toxic peptide that accumulates in the brains of Alzheimer's disease (AD) patients. Our previous studies have shown that the clathrin adaptor Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) plays a key role in the trafficking of BACE1 to lysosomes, where it is normally degraded. GGA3 depletion results in BACE1 stabilization both in vitro and in vivo. Moreover, levels of GGA3 are reduced and inversely related to BACE1 levels in post-mortem brains of AD patients. METHOD: In order to assess the effect of GGA3 deletion on AD-like phenotypes, we crossed GGA3 -/- mice with 5XFAD mice. BACE1-mediated processing of APP and the cell adhesion molecule L1 like protein (CHL1) was measured as well as levels of Aß42 and amyloid burden. RESULTS: In 5XFAD mice, we found that hippocampal and cortical levels of GGA3 decreased while BACE1 levels increased with age, similar to what is observed in human AD brains. GGA3 deletion prevented age-dependent elevation of BACE1 in GGA3KO;5XFAD mice. We also found that GGA3 deletion resulted in increased hippocampal levels of Aß42 and amyloid burden in 5XFAD mice at 12 months of age. While levels of BACE1 did not change with age and gender in GGAKO;5XFAD mice, amyloid precursor protein (APP) levels increased with age and were higher in female mice. Moreover, elevation of APP was associated with a decreased BACE1-mediated processing of CHL1 not only in 12 months old 5XFAD mice but also in human brains from subjects affected by Down syndrome, most likely due to substrate competition. CONCLUSION: This study demonstrates that GGA3 depletion is a leading candidate mechanism underlying elevation of BACE1 in AD. Furthermore, our findings suggest that BACE1 inhibition could exacerbate mechanism-based side effects in conditions associated with APP elevation (e.g. Down syndrome) owing to impairment of BACE1-mediated processing of CHL1. Therefore, therapeutic approaches aimed to restore GGA3 function and to prevent the down stream effects of its depletion (e.g. BACE1 elevation) represent an attractive alternative to BACE inhibition for the prevention/treatment of AD.

Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells.

Three-dimensional in vitro cell culture models, particularly for the central nervous system, allow for the exploration of mechanisms of organ development, cellular interactions, and disease progression within defined environments. Here we describe the development and characterization of three-dimensional tissue models that promote the differentiation and long-term survival of functional neural networks. These tissue cultures show diverse cell populations including neurons and glial cells (astrocytes) interacting in 3D with spontaneous neural activity confirmed through electrophysiological recordings and calcium imaging over at least 8 months. This approach allows for the direct integration of pluripotent stem cells into the 3D construct bypassing early neural differentiation steps (embryoid bodies and neural rosettes), which streamlines the process while also providing a system that can be manipulated to support a variety of experimental applications. This tissue model has been tested in stem cells derived from healthy individuals as well as Alzheimer's and Parkinson's disease patients, with similar growth and gene expression responses indicating potential use in the modeling of disease states related to neurodegenerative diseases.

Genetic Deletion of the Clathrin Adaptor GGA3 Reduces Anxiety and Alters GABAergic Transmission.

Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) is a monomeric clathrin adaptor that has been shown to regulate the trafficking of the Beta-site APP-cleaving enzyme (BACE1), which is required for production of the Alzheimer's disease (AD)-associated amyloid ßpeptide. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that depletion of GGA3 results in increased BACE1 levels and activity owing to impaired lysosomal trafficking and degradation. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We report here that GGA3 deletion results in novelty-induced hyperactivity and decreased anxiety-like behaviors. Given the pivotal role of GABAergic transmission in the regulation of anxiety-like behaviors, we performed electrophysiological recordings in hippocampal slices and found increased phasic and decreased tonic inhibition in the dentate gyrus granule cells (DGGC). Moreover, we found that the number of inhibitory synapses is increased in the dentate gyrus of GGA3 null mice in further support of the electrophysiological data. Thus, the increased GABAergic transmission is a leading candidate mechanism underlying the reduced anxiety-like behaviors observed in GGA3 null mice. All together these findings suggest that GGA3 plays a key role in GABAergic transmission. Since BACE1 levels are elevated in the brain of GGA3 null mice, it is possible that at least some of these phenotypes are a consequence of increased processing of BACE1 substrates.

Cerebellar amyloid-ß plaques: disturbed cortical circuitry in AßPP/PS1 transgenic mice as a model of familial Alzheimer's disease.

Cerebellar amyloid-ß (Aß) deposition in the form of neuritic plaques and Purkinje cell loss are common in certain pedigrees of familial Alzheimer's disease (FAD) mainly linked to PS1 mutations. AßPP/PS1 transgenic mice, here used as a model of FAD, show a few Aß plaques in the molecular layer of the cerebellum at 6 months, and which increase in number with age. Motor impairment is apparent in transgenic mice aged 12 months. Combined methods have shown degenerated parallel fibers as the main component of dystrophic neurites of Aß plaques, loss of synaptic contacts between parallel fibers and dendritic spines of Purkinje cells, and degeneration of granule cells starting at 12 months and increasing in mice 18/20 months old. In addition, abnormal mitochondria and focal loss of Purkinje and basket cells, together with occasional axonal torpedoes and increased collaterals of Purkinje cells in mice aged 18/20 months, is suggested to be a concomitant defect presumably related to soluble extracellular or intracellular Aß. These observations demonstrate serious deterioration of the neuronal circuitry in the cerebellum of AßPP/PS1 transgenic mice, and they provide support for the interpretation of similar alterations occurring in certain pedigrees with FAD.

Amyloid generation and dysfunctional immunoproteasome activation with disease progression in animal model of familial Alzheimer's disease.

Double-transgenic amyloid precursor protein/presenilin 1 (APP/PS1) mice express a chimeric mouse/human APP bearing the Swedish mutation (Mo/HuAPP695swe) and a mutant human PS1-dE9 both causative of familial Alzheimer's disease (FAD). Transgenic mice show impaired memory and learning performance from the age of 6 months onwards. Double-transgenic APP/PS1 mice express altered APP and PS1 mRNAs and proteins, reduced ß-secretase 1 (BACE1) mRNA and normal BACE1 protein, all of which suggest a particular mechanism of amyloidogenesis when compared with sporadic AD. The first ß-amyloid plaques in APP/PS1 mice appear at 3 months, and they increase in number and distribution with disease progression in parallel with increased levels of brain soluble ß-amyloid 1-42 and 1-40, but also with reduced 1-42/1-40 ratio with age. Amyloid deposition in plaques is accompanied by altered mitochondria and increased oxidative damage, post-translational modifications and accumulation of altered proteins at the dystrophic neurites surrounding plaques. Degradation pathways are also modified with disease progression including activation of the immunoproteasome together with variable alterations of the different protease activities of the ubiquitin-proteasome system. Present observations show modifications in the production of ß-amyloid and activation and malfunction of the subcellular degradation pathways that have general implications in the pathogenesis of AD and more particularly in specificities of FAD amyloidogenesis.

Dysfunction of the ubiquitin-proteasome system in the cerebellum of aging Ts65Dn mice.

In the cerebellum of adult-aging Ts65Dn mice, a murine model of Down syndrome, Purkinje cells undergo degeneration. Searching for the cause of Purkinje cell degeneration, we have studied the ubiquitin-proteasome system (UPS) in the cerebellum of aging Ts65Dn mice. Inhibition of UPS is sufficient to induce neuron degeneration and death. Proteasome chymotrypsin-like proteolytic activity was reduced by 35% in the cerebellum of Ts65Dn mice in comparison with euploid animals. Accordingly, Western blot analysis of ubiquitin showed an increase in ubiquitinated proteins. Immunocytochemistry for ubiquitin revealed strongly positive intranuclear inclusions in Purkinje cells and large neurons of cerebellar nuclei. The Western blot analysis of ubiquitin in nuclear protein extracts confirmed the increase of ubiquitinated proteins in the cell nuclei. After FUS immunocytochemistry, large intranuclear inclusions were visible in Purkinje cells and large neurons of cerebellar nuclei in Ts65Dn mice. Together, data indicate a possible role for proteasome inhibition in the cerebellar neurodegeneration in Ts65Dn mice.

A single episode of neonatal seizures alters the cerebellum of immature rats.

PURPOSE: to test whether a single episode of early-life seizures may interfere with the development of the cerebellum. The cerebellum is particularly vulnerable in infants, since it is characterized by an important postnatal histogenesis that leads to the settling of adult circuitry. METHODS: seizures were induced in 10-day-old Wistar rats with a single convulsive dose (80µg/g b.w., s.c.) of pentylentetrazole (PTZ). Immediately after rats were treated with (3)H-thymidine ((3)HTdR, 2.5µCi/g b.w, s.c.). Rats were killed 4h later and paraffin sections of the cerebellar vermis were processed for (3)HTdR autoradiography and immunocytochemistry for 2/3 subunits of AMPA glutamate receptor (GluR2/3), glutamate transporter 1 (GLT1) and calbindin. RESULTS: seizures reduced the proliferation rate of cells in the external germinal layer. Purkinje cells showed increased GluR2/3 immunoreactivity. However, some Purkinje cells were unstained or lost. Increased GLT1 immunoreactivity was present in glial cells surrounding Purkinje cells. Calbindin immunoreaction confirmed that some Purkinje cells were missed. The remaining Purkinje cells showed large spheroids along the course of their axon. CONCLUSIONS: data indicate that seizures lead to a loss and alteration of Purkinje cells in the cerebellum of immature rats. Since at 10 days of life Purkinje cells are no more proliferating, the loss of Purkinje cells should be permanent.

Beta-amyloid overload does not directly correlate with SAPK/JNK activation and tau protein phosphorylation in the cerebellar cortex of Ts65Dn mice.

It is known that in the nervous tissue beta-amyloid overproduction and its extracellular or intracellular deposition can activate mitogen-activated protein kinases involved in tau protein phosphorylation. Hyperphosphorylated tau is not more able to bind neuron microtubules, leading to their disassembly and axon degeneration. We have previously described that at 10 months of age in the cerebellum of Ts65Dn mice, which are partially trisomic for the chromosome 16 and are considered a valuable model for Down syndrome, Purkinje cells undergo axon degeneration. Taking into consideration that Ts65Dn mice carry three copies of the gene encoding for the amyloid precursor protein, to characterize potential signaling events triggering the degenerative phenomenon, specific antibodies were used to examine the role of beta-amyloid overload in the activation of the stress activated kinase/c-jun N-terminal kinase (SAPK/JNK) and tau protein phosphorylation in the cerebellar cortex of 12-month-old Ts65Dn mice. We found small extracellular deposits of beta-amyloid at the borderline between the granule cell layer and the white matter, i.e., in the vicinity of the area where calbindin immunostaining of Purkinje cell axons revealed clusters of newly formed terminals of injured axons. Moreover, intracellular deposits were present in the somata of Purkinje cells. The level of activation of SAPK/JNK was greatly increased. The activation occurred in the "pinceaux" made by basket interneuron axons at the axon hillock of Purkinje cells. Antibody directed against tau protein phosphorylated at Ser-396/Ser-404 revealed positive NG2 cells and Bergman fibers in the molecular layer and oligodendrocytes in the white matter. Data indicate that beta-amyloid extracellular deposits could have exerted a local cytotoxic effect, leading to Purkinje cell axon degeneration. The activation of SAPK/JNK in basket cell "pinceaux" may be a consequence of altered functionality of Purkinje cells and may represent an attempt of basket cells of synaptic remodeling. Moreover, the findings for tau protein phosphorylation suggest that Ts65Dn mice are affected by a cerebellar glial tauopathy.

Axonal abnormalities in cerebellar Purkinje cells of the Ts65Dn mouse.

Ts65Dn mice are a genetic model for Down syndrome. Among others, these mice have cerebellar pathology features which parallel those seen in Down syndrome patients. Both individuals with Down syndrome and Ts65Dn mice have reduced cerebellar volume and numbers of granule and Purkinje cells. In this report, we describe morphological abnormalities of axons of Purkinje cells in the cerebellum of Ts65Dn mice, by using anti-calbindin immunocytochemistry. A consistent number of Purkinje cells shows axons bearing giant varicosities along their transit through the granular layer. The cerebellar arbor vitae made by fasciculated Purkinje cell axons has a patchy appearance, some tracks being devoid of calbindin staining. The infraganglionic plexus, formed by recurrent collaterals of Purkinje cell axons, has enormously increased density, which is evidence for a compensatory reaction to degeneration of distal segments of axons. These alterations are accompanied by strong glial reaction as evidenced by GFAP immunocytochemistry. Moreover, the alterations are more consistent in the anterior lobules of the vermis and intermediate cortex. The axonal pathology of Purkinje cells may explain the impairment in cerebellar functions observed in Ts65Dn mice at the adulthood.