The multifaceted functions of NFE2L1 in metabolism and associated disorders.

Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as Nrf1) is a crucial member of the CNC-bZIP subfamily of transcription factors expressed ubiquitously throughout our body. Recent findings have revealed its association with various metabolic processes, encompassing glucose, lipid, and protein metabolism. In the realm of glucose metabolism, NFE2L1 exerts regulatory control by modulating pancreatic ß cells and insulin production. It also influences glucose metabolism in liver and the insulin sensitivity of adipose tissue. Regarding lipid metabolism, NFE2L1 governs this process by influencing the expression of specific adipogenic and lipolysis genes in both liver and adipose tissue. Additionally, NFE2L1 regulates specific lipids, such as cholesterol. These involvements underlie various manifestations of NFE2L1 deficiency such as adipocyte hypertrophy, inflammation, and steatohepatitis. In the realm of protein metabolism, NFE2L1 serves as a major transcription factor regulating the 26S proteasome genes expression, which dysfunction has been related with multiple diseases including neurodegenerative diseases, cancers, autoimmune conditions, etc. In this comprehensive review, we summarize the diverse roles that NFE2L1 plays in glucose, lipid, and protein metabolism, as well as its impact on diseases related to these metabolic processes.

Protein folding, cellular stress and cancer.

Proteins are acknowledged as the phenotypical manifestation of the genotype, because protein-coding genes carry the information for the strings of amino acids that constitute the proteins. It is widely accepted that protein function depends on the corresponding "native" structure or folding achieved within the cell, and that native protein folding corresponds to the lowest free energy minimum for a given protein. However, protein folding within the cell is a non-deterministic dissipative process that from the same input may produce different outcomes, thus conformational heterogeneity of folded proteins is the rule and not the exception. Local changes in the intracellular environment promote variation in protein folding. Hence protein folding requires "supervision" by a host of chaperones and co-chaperones that help their client proteins to achieve the folding that is most stable according to the local environment. Such environmental influence on protein folding is continuously transduced with the help of the cellular stress responses (CSRs) and this may lead to changes in the rules of engagement between proteins, so that the corresponding protein interactome could be modified by the environment leading to an alternative cellular phenotype. This allows for a phenotypic plasticity useful for adapting to sudden and/or transient environmental changes at the cellular level. Starting from this perspective, hereunder we develop the argument that the presence of sustained cellular stress coupled to efficient CSRs may lead to the selection of an aberrant phenotype as the resulting adaptation of the cellular proteome (and the corresponding interactome) to such stressful conditions, and this can be a common epigenetic pathway to cancer.

The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

The Thioredoxin Fold Protein (TFP2) from Extreme Acidophilic Leptospirillum sp. CF-1 Is a Chaperedoxin-like Protein That Prevents the Aggregation of Proteins under Oxidative Stress.

Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The "tfp2 cluster" (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H2O2). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H2O2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H2O2, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

Dysregulated ribosome quality control in human diseases.

Precise regulation of mRNA translation is of fundamental importance for maintaining homeostasis. Conversely, dysregulated general or transcript-specific translation, as well as abnormal translation events, have been linked to a multitude of diseases. However, driven by the misconception that the transient nature of mRNAs renders their abnormalities inconsequential, the importance of mechanisms that monitor the quality and fidelity of the translation process has been largely overlooked. In recent years, there has been a dramatic shift in this paradigm, evidenced by several seminal discoveries on the role of a key mechanism in monitoring the quality of mRNA translation - namely, Ribosome Quality Control (RQC) - in the maintenance of homeostasis and the prevention of diseases. Here, we will review recent advances in the field and emphasize the biological significance of the RQC mechanism, particularly its implications in human diseases.

An HSF1-JMJD6-HSP feedback circuit promotes cell adaptation to proteotoxic stress.

Heat Shock Factor 1 (HSF1) is best known as the master transcriptional regulator of the heat-shock response (HSR), a conserved adaptive mechanism critical for protein homeostasis (proteostasis). Combining a genome-wide RNAi library with an HSR reporter, we identified Jumonji domain-containing protein 6 (JMJD6) as an essential mediator of HSF1 activity. In follow-up studies, we found that JMJD6 is itself a noncanonical transcriptional target of HSF1 which acts as a critical regulator of proteostasis. In a positive feedback circuit, HSF1 binds and promotes JMJD6 expression, which in turn reduces heat shock protein 70 (HSP70) R469 monomethylation to disrupt HSP70-HSF1 repressive complexes resulting in enhanced HSF1 activation. Thus, JMJD6 is intricately wired into the proteostasis network where it plays a critical role in cellular adaptation to proteotoxic stress.

Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21, but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Compared to euploids, aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results provide implications for other aneuploidy disorders including Down syndrome.

Proteostasis in health and disease: a conversation with Professor Rick Morimoto.

Professor Richard (Rick) Morimoto is the Bill and Gayle Cook Professor of Biology and Director of the Rice Institute for Biomedical Research at Northwestern University. He has made foundational contributions to our understanding of how cells respond to various stresses, and the role played in those responses by chaperones. Working across a variety of experimental models, from C. elegans to human neuronal cells, he has identified a number of important molecular components that sense and respond to stress, and he has dissected how stress alters cellular and organismal physiology. Together with colleagues, Professor Morimoto has coined the term "proteostasis" to signify the homeostatic control of protein expression and function, and in recent years he has been one of the leaders of a consortium trying to understand proteostasis in healthy and disease states. I took the opportunity to talk with Professor Morimoto about proteostasis in general, the aims of the consortium, and how autophagy is playing an important role in their research effort.

The antagonistic role of an E3 ligase pair in regulating plant NLR-mediated autoimmunity and fungal pathogen resistance.

Plant immune homeostasis is achieved through a balanced immune activation and suppression, enabling effective defense while averting autoimmunity. In Arabidopsis, disrupting a mitogen-activated protein (MAP) kinase cascade triggers nucleotide-binding leucine-rich-repeat (NLR) SUPPRESSOR OF mkk1/2 2 (SUMM2)-mediated autoimmunity. Through an RNAi screen, we identify PUB5, a putative plant U-box E3 ligase, as a critical regulator of SUMM2-mediated autoimmunity. In contrast to typical E3 ligases, PUB5 stabilizes CRCK3, a calmodulin-binding receptor-like cytoplasmic kinase involved in SUMM2 activation. A closely related E3 ligase, PUB44, functions oppositely with PUB5 to degrade CRCK3 through monoubiquitylation and internalization. Furthermore, CRCK3, highly expressed in roots and conserved across plant species, confers resistance to Fusarium oxysporum, a devastating soil-borne fungal pathogen, in both Arabidopsis and cotton. These findings demonstrate the antagonistic role of an E3 ligase pair in fine-tuning kinase proteostasis for the regulation of NLR-mediated autoimmunity and highlight the function of autoimmune activators in governing plant root immunity against fungal pathogens.

Multiple Roles of Apolipoprotein E4 in Oxidative Lipid Metabolism and Ferroptosis During the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease worldwide and has a great socio-economic impact. Modified oxidative lipid metabolism and dysregulated iron homeostasis have been implicated in the pathogenesis of this disorder, but the detailed pathophysiological mechanisms still remain unclear. Apolipoprotein E (APOE) is a lipid-binding protein that occurs in large quantities in human blood plasma, and a polymorphism of the APOE gene locus has been identified as risk factors for AD. The human genome involves three major APOE alleles (APOE2, APOE3, APOE4), which encode for three subtly distinct apolipoprotein E isoforms (APOE2, APOE3, APOE4). The canonic function of these apolipoproteins is lipid transport in blood and brain, but APOE4 allele carriers have a much higher risk for AD. In fact, about 60% of clinically diagnosed AD patients carry at least one APOE4 allele in their genomes. Although the APOE4 protein has been implicated in pathophysiological key processes of AD, such as extracellular beta-amyloid (Aß) aggregation, mitochondrial dysfunction, neuroinflammation, formation of neurofibrillary tangles, modified oxidative lipid metabolism, and ferroptotic cell death, the underlying molecular mechanisms are still not well understood. As for all mammalian cells, iron plays a crucial role in neuronal functions and dysregulation of iron homeostasis has also been implicated in the pathogenesis of AD. Imbalances in iron homeostasis and impairment of the hydroperoxy lipid-reducing capacity induce cellular dysfunction leading to neuronal ferroptosis. In this review, we summarize the current knowledge on APOE4-related oxidative lipid metabolism and the potential role of ferroptosis in the pathogenesis of AD. Pharmacological interference with these processes might offer innovative strategies for therapeutic interventions.

Wheat TaNACα18 functions as a positive regulator of high-temperature adaptive responses and improves cell defense machinery.

Global wheat production amounted to >780 MMT during 2022-2023 whose market size are valued at >$128 billion. Wheat is highly susceptible to high-temperature stress (HTS) throughout the life cycle and its yield declines 5-7% with the rise in each degree of temperature. Previously, we reported an array of HTS-response markers from a resilient wheat cv. Unnat Halna and described their putative role in heat acclimation. To complement our previous results and identify the key determinants of thermotolerance, here we examined the cytoplasmic proteome of a sensitive cv. PBW343. The HTS-triggered metabolite reprograming highlighted how proteostasis defects influence the formation of an integrated stress-adaptive response. The proteomic analysis identified several promising HTS-responsive proteins, including a NACα18 protein, designated TaNACα18, whose role in thermotolerance remains unknown. Dual localization of TaNACα18 suggests its crucial functions in the cytoplasm and nucleus. The homodimerization of TaNACα18 anticipated its function as a transcriptional coactivator. The complementation of TaNACα18 in yeast and overexpression in wheat demonstrated its role in thermotolerance across the kingdom. Altogether, our results suggest that TaNACα18 imparts tolerance through tight regulation of gene expression, cell wall remodeling and activation of cell defense responses.

Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

ProteoCure: A European network to fine-tune the proteome.

Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tuning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).

A Plant Model of α-Synucleinopathy: Expression of α-Synuclein A53T Variant in Hairy Root Cultures Leads to Proteostatic Stress and Dysregulation of Iron Metabolism.

Synucleinopathies, typified by Parkinson's disease (PD), entail the accumulation of α-synuclein (αSyn) aggregates in nerve cells. Various αSyn mutants, including the αSyn A53T variant linked to early-onset PD, increase the propensity for αSyn aggregate formation. In addition to disrupting protein homeostasis and inducing proteostatic stress, the aggregation of αSyn in PD is associated with an imbalance in iron metabolism, which increases the generation of reactive oxygen species and causes oxidative stress. This study explored the impact of αSyn A53T expression in transgenic hairy roots of four medicinal plants (Lobelia cardinalis, Artemisia annua, Salvia miltiorrhiza, and Polygonum multiflorum). In all tested plants, αSyn A53T expression triggered proteotoxic stress and perturbed iron homeostasis, mirroring the molecular profile observed in human and animal nerve cells. In addition to the common eukaryotic defense mechanisms against proteostatic and oxidative stresses, a plant stress response generally includes the biosynthesis of a diverse set of protective secondary metabolites. Therefore, the hairy root cultures expressing αSyn A53T offer a platform for identifying secondary metabolites that can ameliorate the effects of αSyn, thereby aiding in the development of possible PD treatments and/or treatments of synucleinopathies.

RIT2 regulates autophagy lysosomal pathway induction and protects against α-synuclein pathology in a cellular model of Parkinson's disease.

Substantial work has been devoted to better understand the contribution of the myriad of genes that may underly the development of Parkinson's disease (PD) and their role in disease etiology. The small GTPase Ras-like without CAAX2 (RIT2) is one such genetic risk factor, with one single nucleotide polymorphism in the RIT2 locus, rs12456492, having been associated with PD risk in multiple populations. While RIT2 has previously been shown to influence signaling pathways, dopamine transporter trafficking, and LRRK2 activity, its cellular function remains unclear. In the current study, we have situated RIT2 to be upstream of various diverse processes associated with PD. In cellular models, we have shown that RIT2 is necessary for activity-dependent changes in the expression of genes related to the autophagy-lysosomal pathway (ALP) by regulating the nuclear translocation of MiT/TFE3-family transcription factors. RIT2 is also associated with lysosomes and can regulate autophagic flux and clearance by regulating lysosomal hydrolase expression and activity. Interestingly, upregulation of RIT2 can augment ALP flux and protect against α-synuclein aggregation in cortical neurons. Taken together, the present study suggests that RIT2 can regulates gene expression upstream of ALP function and that enhancing RIT2 activity may provide therapeutic benefit in PD.

HLH-30/TFEB rewires the chaperone network to promote proteostasis under conditions of Coenzyme A and Iron-Sulfur Cluster Deficiency.

The maintenance of a properly folded proteome is critical for cellular function and organismal health, and its age-dependent collapse is associated with a wide range of diseases. Here, we find that despite the central role of Coenzyme A as a molecular cofactor in hundreds of cellular reactions, limiting Coenzyme A levels in C. elegans and in human cells, by inhibiting the conserved pantothenate kinase, promotes proteostasis. Impairment of the cytosolic iron-sulfur clusters formation pathway, which depends on Coenzyme A, similarly promotes proteostasis and acts in the same pathway. Proteostasis improvement by Coenzyme A/iron-sulfur cluster deficiencies are dependent on the conserved HLH-30/TFEB transcription factor. Strikingly, under these conditions, HLH-30 promotes proteostasis by potentiating the expression of select chaperone genes providing a chaperone-mediated proteostasis shield, rather than by its established role as an autophagy and lysosome biogenesis promoting factor. This reflects the versatile nature of this conserved transcription factor, that can transcriptionally activate a wide range of protein quality control mechanisms, including chaperones and stress response genes alongside autophagy and lysosome biogenesis genes. These results highlight TFEB as a key proteostasis-promoting transcription factor and underscore it and its upstream regulators as potential therapeutic targets in proteostasis-related diseases.

Metabolic regulation of misfolded protein import into mitochondria.

Mitochondria are the cellular energy hub and central target of metabolic regulation. Mitochondria also facilitate proteostasis through pathways such as the 'mitochondria as guardian in cytosol' (MAGIC) whereby cytosolic misfolded proteins (MPs) are imported into and degraded inside mitochondria. In this study, a genome-wide screen in Saccharomyces cerevisiae uncovered that Snf1, the yeast AMP-activated protein kinase (AMPK), inhibits the import of MPs into mitochondria while promoting mitochondrial biogenesis under glucose starvation. We show that this inhibition requires a downstream transcription factor regulating mitochondrial gene expression and is likely to be conferred through substrate competition and mitochondrial import channel selectivity. We further show that Snf1/AMPK activation protects mitochondrial fitness in yeast and human cells under stress induced by MPs such as those associated with neurodegenerative diseases.

UBQLN1 links proteostasis and mitochondria function to telomere maintenance in human embryonic stem cells.

BACKGROUND: Telomeres consist of repetitive DNA sequences at the chromosome ends to protect chromosomal stability, and primarily maintained by telomerase or occasionally by alternative telomere lengthening of telomeres (ALT) through recombination-based mechanisms. Additional mechanisms that may regulate telomere maintenance remain to be explored. Simultaneous measurement of telomere length and transcriptome in the same human embryonic stem cell (hESC) revealed that mRNA expression levels of UBQLN1 exhibit linear relationship with telomere length. METHODS: In this study, we first generated UBQLN1-deficient hESCs and compared with the wild-type (WT) hESCs the telomere length and molecular change at RNA and protein level by RNA-seq and proteomics. Then we identified the potential interacting proteins with UBQLN1 using immunoprecipitation-mass spectrometry (IP-MS). Furthermore, the potential mechanisms underlying the shortened telomeres in UBQLN1-deficient hESCs were analyzed. RESULTS: We show that Ubiquilin1 (UBQLN1) is critical for telomere maintenance in human embryonic stem cells (hESCs) via promoting mitochondrial function. UBQLN1 deficiency leads to oxidative stress, loss of proteostasis, mitochondria dysfunction, DNA damage, and telomere attrition. Reducing oxidative damage and promoting mitochondria function by culture under hypoxia condition or supplementation with N-acetylcysteine partly attenuate the telomere attrition induced by UBQLN1 deficiency. Moreover, UBQLN1 deficiency/telomere shortening downregulates genes for neuro-ectoderm lineage differentiation. CONCLUSIONS: Altogether, UBQLN1 functions to scavenge ubiquitinated proteins, preventing their overloading mitochondria and elevated mitophagy. UBQLN1 maintains mitochondria and telomeres by regulating proteostasis and plays critical role in neuro-ectoderm differentiation.

Proteomics Analysis of Proteotoxic Stress Response in In-Vitro Human Neuronal Models.

Heat stroke, a hazardous hyperthermia-related illness, is characterized by CNS injury, particularly long-lasting brain damage. A root cause for hyperthermic neurological damage is heat-induced proteotoxic stress through protein aggregation, a known causative agent of neurological disorders. Stress magnitude and enduring persistence are highly correlated with hyperthermia-associated neurological damage. We used an untargeted proteomic approach using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify and characterize time-series proteome-wide changes in dose-responsive proteotoxic stress models in medulloblastoma [Daoy], neuroblastoma [SH-SY5Y], and differentiated SH-SY5Y neuron-like cells [SH(D)]. An integrated analysis of condition-time datasets identified global proteome-wide differentially expressed proteins (DEPs) as part of the heat-induced proteotoxic stress response. The condition-specific analysis detected higher DEPs and upregulated proteins in extreme heat stress with a relatively conservative and tight regulation in differentiated SH-SY5Y neuron-like cells. Functional network analysis using ingenuity pathway analysis (IPA) identified common intercellular pathways associated with the biological processes of protein, RNA, and amino acid metabolism and cellular response to stress and membrane trafficking. The condition-wise temporal pathway analysis in the differentiated neuron-like cells detects a significant pathway, functional, and disease association of DEPs with processes like protein folding and protein synthesis, Nervous System Development and Function, and Neurological Disease. An elaborate dose-dependent stress-specific and neuroprotective cellular signaling cascade is also significantly activated. Thus, our study provides a comprehensive map of the heat-induced proteotoxic stress response associating proteome-wide changes with altered biological processes. This helps to expand our understanding of the molecular basis of the heat-induced proteotoxic stress response with potential translational connotations.

Dysregulated energy and protein homeostasis and the loss of GABAergic amacrine cells in aging retina.

Aging is a major risk factor for the development or the worsening of retinal degenerative conditions. The intricate network of the neural retina determined that the retinal aging is a complicated process. The aim of this study is to delineate the transcriptomic changes of major retinal neurons during aging in C57BL/6 mice at single-cell level. We analyzed the transcriptional profiles of the photoreceptor, bipolar, amacrine, and Müller glial cells of 1.5-2 and 24-30 months old mice using single-cell RNA sequencing technique. We selectively confirmed the differences in gene expression using immunofluorescence staining and RNA in situ hybridization analysis. We found that each retinal cell type had unique changes upon aging. However, they all showed signs of dysregulated glucose and energy metabolism, and perturbed proteostasis. In particular, old Müller glia exhibited the most profound changes, including the upregulation of cell metabolism, stress-responses, antigen-presentation and immune responses and metal ion homeostasis. The dysregulated gliogenesis and differentiation was confirmed by the presence of Müller glia expressing rod-specific genes in the inner nuclear layer and the outer plexiform layer of the old retina. We further pinpointed the specific loss of GABAergic amacrine cells in old retina. Our study emphasized changes of amacrine and Müller glia during retinal aging, provided resources for further research on the molecular and cellular regulatory mechanisms underlying aging-associated retinal deterioration.