Protein mishandling and impaired lysosomal proteolysis generated through calcium dysregulation in Alzheimer's disease.

Impairments in neural lysosomal- and autophagic-mediated degradation of cellular debris contribute to neuritic dystrophy and synaptic loss. While these are well-characterized features of neurodegenerative disorders such as Alzheimer's disease (AD), the upstream cellular processes driving deficits in pathogenic protein mishandling are less understood. Using a series of fluorescent biosensors and optical imaging in model cells, AD mouse models and human neurons derived from AD patients, we reveal a previously undescribed cellular signaling cascade underlying protein mishandling mediated by intracellular calcium dysregulation, an early component of AD pathogenesis. Increased Ca2+ release via the endoplasmic reticulum (ER)-resident ryanodine receptor (RyR) is associated with reduced expression of the lysosome proton pump vacuolar-ATPase (vATPase) subunits (V1B2 and V0a1), resulting in lysosome deacidification and disrupted proteolytic activity in AD mouse models and human-induced neurons (HiN). As a result of impaired lysosome digestive capacity, mature autophagosomes with hyperphosphorylated tau accumulated in AD murine neurons and AD HiN, exacerbating proteinopathy. Normalizing AD-associated aberrant RyR-Ca2+ signaling with the negative allosteric modulator, dantrolene (Ryanodex), restored vATPase levels, lysosomal acidification and proteolytic activity, and autophagic clearance of intracellular protein aggregates in AD neurons. These results highlight that prior to overt AD histopathology or cognitive deficits, aberrant upstream Ca2+ signaling disrupts lysosomal acidification and contributes to pathological accumulation of intracellular protein aggregates. Importantly, this is demonstrated in animal models of AD, and in human iPSC-derived neurons from AD patients. Furthermore, pharmacological suppression of RyR-Ca2+ release rescued proteolytic function, revealing a target for therapeutic intervention that has demonstrated effects in clinically-relevant assays.

Ca2+ Dyshomeostasis Disrupts Neuronal and Synaptic Function in Alzheimer's Disease.

Ca2+ homeostasis is essential for multiple neuronal functions and thus, Ca2+ dyshomeostasis can lead to widespread impairment of cellular and synaptic signaling, subsequently contributing to dementia and Alzheimer's disease (AD). While numerous studies implicate Ca2+ mishandling in AD, the cellular basis for loss of cognitive function remains under investigation. The process of synaptic degradation and degeneration in AD is slow, and constitutes a series of maladaptive processes each contributing to a further destabilization of the Ca2+ homeostatic machinery. Ca2+ homeostasis involves precise maintenance of cytosolic Ca2+ levels, despite extracellular influx via multiple synaptic Ca2+ channels, and intracellular release via organelles such as the endoplasmic reticulum (ER) via ryanodine receptor (RyRs) and IP3R, lysosomes via transient receptor potential mucolipin channel (TRPML) and two pore channel (TPC), and mitochondria via the permeability transition pore (PTP). Furthermore, functioning of these organelles relies upon regulated inter-organelle Ca2+ handling, with aberrant signaling resulting in synaptic dysfunction, protein mishandling, oxidative stress and defective bioenergetics, among other consequences consistent with AD. With few effective treatments currently available to mitigate AD, the past few years have seen a significant increase in the study of synaptic and cellular mechanisms as drivers of AD, including Ca2+ dyshomeostasis. Here, we detail some key findings and discuss implications for future AD treatments.

Human-Induced Neurons from Presenilin 1 Mutant Patients Model Aspects of Alzheimer's Disease Pathology.

Traditional approaches to studying Alzheimer's disease (AD) using mouse models and cell lines have advanced our understanding of AD pathogenesis. However, with the growing divide between model systems and clinical therapeutic outcomes, the limitations of these approaches are increasingly apparent. Thus, to generate more clinically relevant systems that capture pathological cascades within human neurons, we generated human-induced neurons (HiNs) from AD and non-AD individuals to model cell autonomous disease properties. We selected an AD patient population expressing mutations in presenilin 1 (mPS1), which is linked to increased amyloid production, tau pathology, and calcium signaling abnormalities, among other features. While these AD components are detailed in model systems, they have yet to be collectively identified in human neurons. Thus, we conducted molecular, immune-based, electrophysiological, and calcium imaging studies to establish patterns of cellular pathology in this patient population. We found that mPS1 HiNs generate increased Aß42 and hyperphosphorylated tau species relative to non-AD controls, and exaggerated ER calcium responses that are normalized with ryanodine receptor (RyR) negative allosteric modulators. The inflammasome product, interleukin-18 (IL-18), also increased PS1 expression. This work highlights the potential for HiNs to model AD pathology and validates their role in defining cellular pathogenesis and their utility for therapeutic screening.

Reduced presynaptic vesicle stores mediate cellular and network plasticity defects in an early-stage mouse model of Alzheimer's disease.

BACKGROUND: Identifying effective strategies to prevent memory loss in AD has eluded researchers to date, and likely reflects insufficient understanding of early pathogenic mechanisms directly affecting memory encoding. As synaptic loss best correlates with memory loss in AD, refocusing efforts to identify factors driving synaptic impairments may provide the critical insight needed to advance the field. In this study, we reveal a previously undescribed cascade of events underlying pre and postsynaptic hippocampal signaling deficits linked to cognitive decline in AD. These profound alterations in synaptic plasticity, intracellular Ca2+ signaling, and network propagation are observed in 3-4 month old 3xTg-AD mice, an age which does not yet show overt histopathology or major behavioral deficits. METHODS: In this study, we examined hippocampal synaptic structure and function from the ultrastructural level to the network level using a range of techniques including electron microscopy (EM), patch clamp and field potential electrophysiology, synaptic immunolabeling, spine morphology analyses, 2-photon Ca2+ imaging, and voltage-sensitive dye-based imaging of hippocampal network function in 3-4 month old 3xTg-AD and age/background strain control mice. RESULTS: In 3xTg-AD mice, short-term plasticity at the CA1-CA3 Schaffer collateral synapse is profoundly impaired; this has broader implications for setting long-term plasticity thresholds. Alterations in spontaneous vesicle release and paired-pulse facilitation implicated presynaptic signaling abnormalities, and EM analysis revealed a reduction in the ready-releasable and reserve pools of presynaptic vesicles in CA3 terminals; this is an entirely new finding in the field. Concurrently, increased synaptically-evoked Ca2+ in CA1 spines triggered by LTP-inducing tetani is further enhanced during PTP and E-LTP epochs, and is accompanied by impaired synaptic structure and spine morphology. Notably, vesicle stores, synaptic structure and short-term plasticity are restored by normalizing intracellular Ca2+ signaling in the AD mice. CONCLUSIONS: These findings suggest the Ca2+ dyshomeostasis within synaptic compartments has an early and fundamental role in driving synaptic pathophysiology in early stages of AD, and may thus reflect a foundational disease feature driving later cognitive impairment. The overall significance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic vesicle stores, synaptic plasticity, and network propagation, which directly impact memory encoding.

Calcium Signaling Deficits in Glia and Autophagic Pathways Contributing to Neurodegenerative Disease.

SIGNIFICANCE: Numerous cellular processes and signaling mechanisms have been identified that contribute to Alzheimer's disease (AD) pathology; however, a comprehensive or unifying pathway that binds together the major disease features remains elusive. As an upstream mechanism, altered calcium (Ca2+) signaling is a common driving force for many pathophysiological events that emerge during normal aging and development of neurodegenerative disease. Recent Advances: Over the previous three decades, accumulated evidence has validated the concept that intracellular Ca2+ dysregulation is centrally involved in AD pathogenesis, including the aggregation of pathogenic ß-amyloid (Aß) and phospho-τ species, synapse loss and dysfunction, cognitive impairment, and neurotoxicity. CRITICAL ISSUES: Although neuronal Ca2+ signaling within the cytosol and endoplasmic reticulum (ER) has been well studied, other critical central nervous system-resident cell types affected by aberrant Ca2+ signaling, such as astrocytes and microglia, have not been considered as thoroughly. In addition, certain intracellular Ca2+-harboring organelles have been well studied, such as the ER and mitochondria; however other critical Ca2+-regulated organelles, such as lysosomes and autophagosomes, have only more recently been investigated. In this review, we examine Ca2+ dysregulation in microglia and astrocytes, as well as key intracellular organelles important for cellular maintenance and protein handling. Ca2+ dysregulation within these non-neuronal cells and organelles is hypothesized to disrupt the effective clearance of misaggregated proteins and cellular signaling pathways needed for memory networks. FUTURE DIRECTIONS: Overall, we aim to explore how these disrupted mechanisms could be involved in AD pathology and consider their role as potential therapeutic targets. Antioxid. Redox Signal. 29, 1158-1175.