in-vivo-glioblastoma-tumor-modeling-via-stereotaxic-injection-mice

<em>In Vivo</em> Glioblastoma Tumor Modeling via Stereotaxic Injection in Mice for Tumor Progression Studies

Glioblastoma (GB) is a highly aggressive brain cancer with a median survival of under 2 years despite conventional therapy. Research efforts frequently ...

Brain Health and Metabolism (GERO)

University of Chile

The Buck Institute for Research on Aging

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that affects motoneurons. Mutations in superoxide dismutase 1 (SOD1) have been described as a causative genetic factor for ALS. Mice overexpressing ALS-linked mutant SOD1 develop ALS symptoms accompanied by histopathological alterations and protein aggregation. The protein disulfide isomerase family member ERp57 is one of the main up-regulated proteins in tissue of ALS patients and mutant SOD1 mice, whereas point mutations in ERp57 were described as possible risk factors to develop the disease. ERp57 catalyzes disulfide bond formation and isomerization in the endoplasmic reticulum (ER), constituting a central component of protein quality control mechanisms. However, the actual contribution of ERp57 to ALS pathogenesis remained to be defined. Here, we studied the consequences of overexpressing ERp57 in experimental ALS using mutant SOD1 mice. Double transgenic SOD1/ERp57 animals presented delayed deterioration of electrophysiological activity and maintained muscle innervation compared to single transgenic SOD1 littermates at early-symptomatic stage, along with improved motor performance without affecting survival. The overexpression of ERp57 reduced mutant SOD1 aggregation, but only at disease end-stage, dissociating its role as an anti-aggregation factor from the protection of neuromuscular junctions. Instead, proteomic analysis revealed that the neuroprotective effects of ERp57 overexpression correlated with increased levels of synaptic and actin cytoskeleton proteins in the spinal cord. Taken together, our results suggest that ERp57 operates as a disease modifier at early stages by maintaining motoneuron connectivity.

Protein disulfide isomerase ERp57 protects early muscle denervation in experimental ALS.

Alteration to endoplasmic reticulum (ER) proteostasis is observed in a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR target genes. In this study, we designed an ATF6f/XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has a stronger effect in reducing the abnormal aggregation of mutant huntingtin and α-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson's disease and Huntington's disease. These results support the concept in which directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the UPR in models of neurodegeneration.

Sustaining neuronal proteostasis during the course of our life is a central aspect required for brain function. The dynamic nature of synaptic composition and abundance is a requisite to drive cognitive and motor processes involving a tight control of many aspects of protein biosynthesis and degradation. Through the concerted action of specialized stress sensors, the proteostasis network monitors and limits the accumulation of damaged, misfolded, or aggregated proteins. These stress pathways signal to the cytosol and nucleus to reprogram gene expression, enabling adaptive programs to recover cell function. During aging, the activity of the proteostasis network declines, which may increase the risk of accumulating abnormal protein aggregates, a hallmark of most neurodegenerative diseases. Here, I discuss emerging concepts illustrating the functional significance of adaptive signaling pathways to normal brain physiology and their contribution to age-related disorders. Pharmacological and gene therapy strategies to intervene and boost proteostasis are expected to extend brain healthspan and ameliorate disease states.

Adapting the proteostasis capacity to sustain brain healthspan.

Endoplasmic reticulum (ER) stress is a prominent cellular alteration of diseases impacting the nervous system that are associated to the accumulation of misfolded and aggregated protein species during aging. The unfolded protein response (UPR) is the main pathway mediating adaptation to ER stress, but it can also trigger deleterious cascades of inflammation and cell death leading to cell dysfunction and neurodegeneration. Genetic and pharmacological studies in experimental models shed light into molecular pathways possibly contributing to ER stress and the UPR activation in human neuropathies. Most of experimental models are, however, based on the overexpression of mutant proteins causing familial forms of these diseases or the administration of neurotoxins that induce pathology in young animals. Whether the mechanisms uncovered in these models are relevant for the etiology of the vast majority of age-related sporadic forms of neurodegenerative diseases is an open question. Here, we provide a systematic analysis of the current evidence linking ER stress to human pathology and the main mechanisms elucidated in experimental models. Furthermore, we highlight the recent association of metabolic syndrome to increased risk to undergo neurodegeneration, where ER stress arises as a common denominator in the pathogenic crosstalk between peripheral organs and the nervous system.

Disruption of Endoplasmic Reticulum Proteostasis in Age-Related Nervous System Disorders.

The lysosome is the main catabolic organelle in the cell, also serving as a signaling platform. Lysosomes maintain a low intraluminal pH where dozens of hydrolytic enzymes degrade a wide variety of macromolecules. Besides degradation of polymers, the lysosome is involved in various cellular processes, including energy metabolism, plasma membrane repair and antigen presentation. Recent work has shown that the lysosome is an important calcium store, modulating diverse cellular functions such as membrane fusion and fission, autophagy and lysosomal biogenesis. Precise measurement of free lysosomal calcium concentration has been hampered by its low luminal pH, since the affinity of most calcium probes decreases with higher proton concentration. Here we detailed an adapted protocol for the simultaneous measurement of lysosomal pH and calcium using dextran-conjugated ratiometric fluorescent dyes. As compared with indirect measurements of lysosomal calcium release using genetically-encoded calcium indicators (GECIs), the present method offers the possibility of obtaining pH-corrected, intraluminal calcium concentrations at single lysosome resolution. It also enables simultaneous temporal resolution of lysosomal calcium and pH.

Simultaneous determination of intraluminal lysosomal calcium and pH by dextran-conjugated fluorescent dyes.

The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.  In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known as regulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress. Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

Assays to Study IRE1 Activation and Signaling.

A long proportion of the population is resilient to the negative consequences of stress. Glucocorticoids resulting from endocrine responses to stress are essential adaptive mediators, but also drive alterations to brain function, negatively impacting neuronal connectivity, synaptic plasticity, and memory-related processes. Recent evidence has indicated that organelle function and cellular stress responses are relevant determinant of vulnerability and resistance to environmental stress. At the molecular level, a fundamental mechanism of cellular stress adaptation is the maintenance of proteostasis, which also have key roles in sustaining basal neuronal function. Here, we discuss recent evidence suggesting that proteostasis unbalance at the level of the endoplasmic reticulum, the main site for protein folding in the cell, represents a possible mechanistic link between individuals and cellular stress.

Proteostasis and resilience: on the interphase between individual's and intracellular stress.

The development of the central nervous system requires a series of morphogenetic events that shape brain and spinal cord structures. Several brain regions and neural circuits are formed by differential gene expression patterns and cell migration events involving neurons. During neurogenesis and neuritogenesis, increased demand for protein synthesis occurs to express key neuronal proteins to generate axons, dendrites, and synapsis. The endoplasmic reticulum (ER) is a central hub controlling protein homeostasis (proteostasis), impacting a wide range of cellular processes required for brain function. Although most of the field has focused on studying the role of ER stress in neurodegenerative diseases marked by abnormal protein aggregation, accumulating evidence indicates that ER proteostasis contributes to brain development and may impact neurodevelopmental processes such as neuronal migration, differentiation, and function. Here, we review emerging evidence linking neurodevelopment with ER proteostasis and its relevance to human disorders.

Emerging roles of endoplasmic reticulum proteostasis in brain development.

A chosen STING with a PERKy trail.

Protein disulfide isomerases (PDIs) constitute a family of oxidoreductases promoting redox protein folding and quality control in the endoplasmic reticulum. PDIs catalyze disulfide bond formation, isomerization, and reduction, operating in concert with molecular chaperones to fold secretory cargoes in addition to directing misfolded proteins to be refolded or degraded. Importantly, PDIs are emerging as key components of the proteostasis network, integrating protein folding status with central surveillance mechanisms to balance proteome stability according to cellular needs. Recent advances in the field driven by the generation of new mouse models, human genetic studies, and omics methodologies, in addition to interventions using small molecules and gene therapy, have revealed the significance of PDIs to the physiology of the nervous system. PDIs are also implicated in diverse pathologies, ranging from neurodevelopmental conditions to neurodegenerative diseases and traumatic injuries. Here, we review the principles of redox protein folding in the ER with a focus on current evidence linking genetic mutations and biochemical alterations to PDIs in the etiology of neurological conditions.

Critical roles of protein disulfide isomerases in balancing proteostasis in the nervous system.

Alteration in the buffering capacity of the proteostasis network is an emerging feature of Alzheimer's disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is the main adaptive pathway to cope with protein folding stress at the ER. Inositol-requiring enzyme-1 (IRE1) operates as a central ER stress sensor, enabling the establishment of adaptive and repair programs through the control of the expression of the transcription factor X-box binding protein 1 (XBP1). To artificially enforce the adaptive capacity of the UPR in the AD brain, we developed strategies to express the active form of XBP1 in the brain. Overexpression of XBP1 in the nervous system using transgenic mice reduced the load of amyloid deposits and preserved synaptic and cognitive function. Moreover, local delivery of XBP1 into the hippocampus of an 5xFAD mice using adeno-associated vectors improved different AD features. XBP1 expression corrected a large proportion of the proteomic alterations observed in the AD model, restoring the levels of several synaptic proteins and factors involved in actin cytoskeleton regulation and axonal growth. Our results illustrate the therapeutic potential of targeting UPR-dependent gene expression programs as a strategy to ameliorate AD features and sustain synaptic function.

The unfolded protein response transcription factor XBP1s ameliorates Alzheimer's disease by improving synaptic function and proteostasis.

Calcium is a crucial messenger of intracellular and extracellular signals, regulating a great variety of cellular processes such as cell death, proliferation, and metabolism. Inside the cell, calcium signaling is one of the main interorganelle communication mediators, with central functional roles at the endoplasmic reticulum (ER), mitochondria, Golgi complex, and lysosomes. Lysosomal function is highly dependent on lumenal calcium and most of the lysosomal membrane-localised ion channels regulate several lysosomal functions and properties such as lumenal pH. One of these functions configures a specific type of cell death involving lysosomes, named lysosome-dependent cell death (LDCD), which contributes to maintenance of tissue homeostasis, development and pathology when deregulated. Here, we cover the fundamental aspects of LDCD with a special focus on recent advances in calcium signaling in LDCD.

Calcium signaling in lysosome-dependent cell death.

The tumor microenvironment (TME) represents a dynamic network of cancer cells, stromal cells, immune mediators, and extracellular matrix components, crucial for cancer progression. Stress conditions such as oncogene activation, nutrient deprivation, and hypoxia disrupt the endoplasmic reticulum (ER), activating the unfolded protein response (UPR), the main adaptive mechanism to restore ER function. The UPR regulates cancer progression by engaging cell-autonomous and cell-non-autonomous mechanisms, reprogramming the stroma and promoting immune evasion, angiogenesis, and invasion. This review explores the role of UPR beyond cancer cells, focusing on how ER stress signaling reshapes the TME, supporting tumor growth. The therapeutic potential of targeting the UPR is also discussed.

The UPRising connection between endoplasmic reticulum stress and the tumor microenvironment.

Aging trajectories vary among individuals, characterized by progressive functional decline, often leading to disease states. One of the central hallmarks of aging is the deterioration of proteostasis, where the function of the endoplasmic reticulum (ER) is dramatically affected. ER stress is monitored and adjusted by the unfolded protein response (UPR); a signaling pathway that mediates adaptive processes to restore proteostasis. Studies in multiple model organisms (yeast, worms, flies, and mice) in addition to human tissue indicates that adaptive UPR signaling contributes to healthy aging. Strategies to improve ER proteostasis using small molecules and gene therapy reduce the decline of organ function during normal aging in mammals. This article reviews recent advances in understanding the significance of the ER proteostasis network to normal aging and its relationship with other hallmarks of aging such as senescence.

Central role of the ER proteostasis network in healthy aging.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motoneurons and compromised proteostasis. Dysfunction of the endoplasmic reticulum (ER) has been identified as a transversal pathogenic mechanism associated with motoneurons vulnerability in ALS. Protein disulfide isomerases (PDIs) are key enzymes catalyzing protein folding at the ER that are altered in the disease, involving biochemical and genetic perturbations. In ALS cases, we previously identified variants in the gene encoding PDIA3 (also known as Grp58 or ERp57), which were associated with altered neurite outgrowth in cell culture and abnormal motoneuron connectivity in zebrafish. Here, we report the generation of transgenic mice expressing the ALS-associated PDIA3 variant. Moderate PDIA3 overexpression resulted in altered motor capacity accompanied by decreased motoneuron number. The adverse effects of PDIA3 expression were associated with induction of ER stress in the spinal cord and subtle morphological changes in neuromuscular junctions. Our results suggest that the PDIA3 variant is likely pathogenic and its overexpression in mice recapitulate some ALS features, further supporting the concept that altered proteostasis due to PDI dysfunction may predispose an individual to develop the disease.

Article	Year
Protein disulfide isomerase ERp57 protects early muscle denervation in experimental ALS. Acta neuropathologica communications\| PubMed ID: 33541434	2021
Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the UPR in models of neurodegeneration. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 33545358	2021
Adapting the proteostasis capacity to sustain brain healthspan. Cell\| PubMed ID: 33691137	2021
Disruption of Endoplasmic Reticulum Proteostasis in Age-Related Nervous System Disorders. Progress in molecular and subcellular biology\| PubMed ID: 34050870	2021
Simultaneous determination of intraluminal lysosomal calcium and pH by dextran-conjugated fluorescent dyes. Methods in cell biology\| PubMed ID: 34311868	2021
Assays to Study IRE1 Activation and Signaling. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34985699	2022
Proteostasis and resilience: on the interphase between individual's and intracellular stress. Trends in endocrinology and metabolism: TEM\| PubMed ID: 35337729	2022
Emerging roles of endoplasmic reticulum proteostasis in brain development. Cells & development\| PubMed ID: 35483574	2022
A chosen STING with a PERKy trail. Nature cell biology\| PubMed ID: 35501369	2022
Critical roles of protein disulfide isomerases in balancing proteostasis in the nervous system. The Journal of biological chemistry\| PubMed ID: 35654139	2022
The unfolded protein response transcription factor XBP1s ameliorates Alzheimer's disease by improving synaptic function and proteostasis. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 37016577	2023
Calcium signaling in lysosome-dependent cell death. Cell calcium\| PubMed ID: 37178674	2023
The UPRising connection between endoplasmic reticulum stress and the tumor microenvironment. Trends in cancer\| PubMed ID: 39472237	2024
Central role of the ER proteostasis network in healthy aging. Trends in cell biology\| PubMed ID: 39547881	2024
Expression of protein disulfide isomerase A3Q481K variant associated with amyotrophic lateral sclerosis triggers disease features in mice. Neurobiology of disease\| PubMed ID: 40345258	2025

Claudio Hetz

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