The Chromatin Regulator Mll1 Supports T Follicular Helper Cell Differentiation by Controlling Expression of Bcl6, LEF-1, and TCF-1.

T follicular helper (TFH) cells are essential for developing protective Ab responses following vaccination. Greater understanding of the genetic program leading to TFH differentiation is needed. Chromatin modifications are central in the control of gene expression. However, detailed knowledge of how chromatin regulators (CRs) regulate differentiation of TFH cells is limited. We screened a large short hairpin RNA library targeting all known CRs in mice and identified the histone methyltransferase mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation. Loss of Mll1 expression reduced formation of TFH cells following acute viral infection or protein immunization. In addition, expression of the TFH lineage-defining transcription factor Bcl6 was reduced in the absence of Mll1. Transcriptomics analysis identified Lef1 and Tcf7 as genes dependent on Mll1 for their expression, which provides one mechanism for the regulation of TFH differentiation by Mll1. Taken together, CRs such as Mll1 substantially influence TFH differentiation.

Dissecting Regulators of Aging and Age-Related Macular Degeneration in the Retinal Pigment Epithelium.

Age-related macular degeneration (AMD), the leading cause of blindness in elderly populations, involves the loss of central vision due to progressive dysfunction of the retinal pigment epithelium (RPE) and subsequent loss of light-sensing photoreceptors. While age is a key risk factor, not every aged individual develops AMD. Thus, the critical question is what specific cellular changes tip the balance from healthy aging to disease. To distinguish between changes associated with aging and AMD, we compared the RPE proteome in human eye bank tissue from nondiseased donors during aging (n = 50, 29-91 years) and in donors with AMD (n = 36) compared to age-matched donors without disease (n = 28). Proteins from RPE cells were separated on two-dimensional gels, analyzed for content, and identified using mass spectrometry. A total of 58 proteins displayed significantly altered content with either aging or AMD. Proteins involved in metabolism, protein turnover, stress response, and cell death were altered with both aging and AMD. However, the direction of change was predominantly opposite. With aging, we detected an overall decrease in metabolism and reductions in stress-associated proteins, proteases, and chaperones. With AMD, we observed upregulation of metabolic proteins involved in glycolysis, TCA, and fatty acid metabolism, with a concurrent decline in oxidative phosphorylation, suggesting a reprogramming of energy utilization. Additionally, we detected upregulation of proteins involved in the stress response and protein turnover. Predicted upstream regulators also showed divergent results, with inhibition of inflammation and immune response with aging and activation of these processes with AMD. Our results support the idea that AMD is not simply advanced aging but rather the culmination of perturbed protein homeostasis, defective bioenergetics, and increased oxidative stress within the aging RPE, exacerbated by environmental factors and the genetic background of an individual.

No association between cataract surgery and mitochondrial DNA damage with age-related macular degeneration in human donor eyes.

PURPOSE: To determine whether age-related macular degeneration (AMD) severity or the frequency of retinal pigment epithelium mitochondrial DNA lesions differ in human donor eyes that have undergone cataract surgery compared to phakic eyes. METHODS: Eyes from human donors aged ≥ 55 years were obtained from the Minnesota Lions Eye Bank. Cataract surgery status was obtained from history provided to Eye Bank personnel by family members at the time of tissue procurement. Donor eyes were graded for AMD severity using the Minnesota Grading System. Quantitative PCR was performed on DNA isolated from macular punches of retinal pigment epithelium to quantitate the frequency of mitochondrial DNA lesions in the donor tissue. Univariable and multivariable analyses were performed to evaluate for associations between (1) cataract surgery and AMD severity and (2) cataract surgery and mitochondrial DNA lesion frequency. RESULTS: A total of 157 subjects qualified for study inclusion. Multivariable analysis with age, sex, smoking status, and cataract surgery status showed that only age was associated with AMD grade. Multivariable analysis with age, sex, smoking status, and cataract surgery status showed that none of these factors were associated with retinal pigment epithelium mitochondrial DNA lesion frequency. CONCLUSIONS: In this study of human donor eyes, neither retinal pigment epithelium mitochondrial DNA damage nor the stage of AMD severity are independently associated with cataract surgery after adjusting for other AMD risk factors. These new pathologic and molecular findings provide evidence against a relationship between cataract surgery and AMD progression and support the idea that cataract surgery is safe in the setting of AMD.

Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease.

The polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) and neurodegeneration, but the mechanisms remain unclear. Here, we found that mHtt promotes ribosome stalling and suppresses protein synthesis in mouse HD striatal neuronal cells. Depletion of mHtt enhances protein synthesis and increases the speed of ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro. Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but its depletion has no discernible effect on protein synthesis or ribosome stalling in HD cells. We found interactions of ribosomal proteins and translating ribosomes with mHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seq indicates a widespread shift in ribosome occupancy toward the 5' and 3' end and unique single-codon pauses on selected mRNA targets in HD cells, compared to controls. Thus, mHtt impedes ribosomal translocation during translation elongation, a mechanistic defect that can be exploited for HD therapeutics.

Stable Isotope Labeling Reveals Novel Insights Into Ubiquitin-Mediated Protein Aggregation With Age, Calorie Restriction, and Rapamycin Treatment.

Accumulation of protein aggregates with age was first described in aged human tissue over 150 years ago and has since been described in virtually every human tissue. Ubiquitin modifications are a canonical marker of insoluble protein aggregates; however, the composition of most age-related inclusions remains relatively unknown. To examine the landscape of age-related protein aggregation in vivo, we performed an antibody-based pulldown of ubiquitinated proteins coupled with metabolic labeling and mass spectrometry on young and old mice on calorie restriction (CR), rapamycin (RP)-supplemented, and control diets. We show increased abundance of many ubiquitinated proteins in old mice and greater retention of preexisting (unlabeled) ubiquitinated proteins relative to their unmodified counterparts-fitting the expected profile of age-increased accumulation of long-lived aggregating proteins. Both CR and RP profoundly affected ubiquitinome composition, half-live, and the insolubility of proteins, consistent with their ability to mobilize these age-associated accumulations. Finally, confocal microscopy confirmed the aggregation of two of the top predicted aggregating proteins, keratins 8/18 and catalase, as well as their attenuation by CR and RP. Stable-isotope labeling is a powerful tool to gain novel insights into proteostasis mechanisms, including protein aggregation, and could be used to identify novel therapeutic targets in aging and protein aggregation diseases.

Age modifies respiratory complex I and protein homeostasis in a muscle type-specific manner.

Changes in mitochondrial function with age vary between different muscle types, and mechanisms underlying this variation remain poorly defined. We examined whether the rate of mitochondrial protein turnover contributes to this variation. Using heavy label proteomics, we measured mitochondrial protein turnover and abundance in slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) from young and aged mice. We found that mitochondrial proteins were longer lived in EDL than SOL at both ages. Proteomic analyses revealed that age-induced changes in protein abundance differed between EDL and SOL with the largest change being increased mitochondrial respiratory protein content in EDL. To determine how altered mitochondrial proteomics affect function, we measured respiratory capacity in permeabilized SOL and EDL. The increased mitochondrial protein content in aged EDL resulted in reduced complex I respiratory efficiency in addition to increased complex I-derived H2 O2 production. In contrast, SOL maintained mitochondrial quality, but demonstrated reduced respiratory capacity with age. Thus, the decline in mitochondrial quality with age in EDL was associated with slower protein turnover throughout life that may contribute to the greater decline in mitochondrial dysfunction in this muscle. Furthermore, mitochondrial-targeted catalase protected respiratory function with age suggesting a causal role of oxidative stress. Our data clearly indicate divergent effects of age between different skeletal muscles on mitochondrial protein homeostasis and function with the greatest differences related to complex I. These results show the importance of tissue-specific changes in the interaction between dysregulation of respiratory protein expression, oxidative stress, and mitochondrial function with age.

Respiratory chain protein turnover rates in mice are highly heterogeneous but strikingly conserved across tissues, ages, and treatments.

The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [(2)H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman's ρ = 0.57; P < 10(-4)), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions.

Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.

Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10-week RP or 40% CR. Protein abundance and turnover were measured in vivo using (2) H3 -leucine heavy isotope labeling followed by LC-MS/MS, and translation was assessed by polysome profiling. We observed 35-60% increased protein half-lives after CR and 15% increased half-lives after RP compared to age-matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions.

Altered proteome turnover and remodeling by short-term caloric restriction or rapamycin rejuvenate the aging heart.

Chronic caloric restriction (CR) and rapamycin inhibit the mechanistic target of rapamycin (mTOR) signaling, thereby regulating metabolism and suppressing protein synthesis. Caloric restriction or rapamycin extends murine lifespan and ameliorates many aging-associated disorders; however, the beneficial effects of shorter treatment on cardiac aging are not as well understood. Using a recently developed deuterated-leucine labeling method, we investigated the effect of short-term (10 weeks) CR or rapamycin on the proteomics turnover and remodeling of the aging mouse heart. Functionally, we observed that short-term CR and rapamycin both reversed the pre-existing age-dependent cardiac hypertrophy and diastolic dysfunction. There was no significant change in the cardiac global proteome (823 proteins) turnover with age, with a median half-life 9.1 days in the 5-month-old hearts and 8.8 days in the 27-month-old hearts. However, proteome half-lives of old hearts significantly increased after short-term CR (30%) or rapamycin (12%). This was accompanied by attenuation of age-dependent protein oxidative damage and ubiquitination. Quantitative proteomics and pathway analysis revealed an age-dependent decreased abundance of proteins involved in mitochondrial function, electron transport chain, citric acid cycle, and fatty acid metabolism as well as increased abundance of proteins involved in glycolysis and oxidative stress response. This age-dependent cardiac proteome remodeling was significantly reversed by short-term CR or rapamycin, demonstrating a concordance with the beneficial effect on cardiac physiology. The metabolic shift induced by rapamycin was confirmed by metabolomic analysis.

Topograph, a software platform for precursor enrichment corrected global protein turnover measurements.

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.

Mitochondrial DNA damage as a potential mechanism for age-related macular degeneration.

PURPOSE: Increasing evidence suggests a central role for mitochondrial (mt) dysfunction in age-related macular degeneration (AMD). Previous proteomic data from the retinal pigment epithelium (RPE) revealed significant changes to mt proteins, suggesting potential functional defects and damage to mitochondrial DNA (mtDNA) with AMD progression. The present study tests the hypothesis that mtDNA damage increases with aging and AMD. METHODS: Genomic DNA was isolated from the macular region of human donor RPE graded for stages of AMD (Minnesota Grading System [MGS] 1-4). Region-specific mtDNA damage with normal aging was evaluated in 45 control subjects (ages 34-88 years, MGS 1) and AMD-associated damage in diseased subjects (n = 46), compared with that in age-matched control subjects (n = 26). Lesions per 10 kb per genome in the mtDNA and nuclear DNA were measured with long-extension polymerase chain reaction (LX PCR). The level of deleted mtDNA in each donor was measured with quantitative real-time PCR (qPCR). RESULTS: With aging, an increase in mtDNA damage was observed only in the common deletion region of the mt genome. In contrast, with AMD, mtDNA lesions increased significantly in all regions of the mt genome beyond levels found in age-matched control subjects. mtDNA accumulated more lesions than did two nuclear genes, with total damage of the mt genome estimated to be eight times higher. CONCLUSIONS: Collectively, the data indicate that mtDNA is preferentially damaged with AMD progression. These results suggest a potential link between mt dysfunction due to increased mtDNA lesions and AMD.

Mitochondrial proteomics of the retinal pigment epithelium at progressive stages of age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of vision loss in individuals over the age of 65. Histopathological changes become evident in the retinal pigment epithelium (RPE), a monolayer that provides metabolic support for the overlying photoreceptors, even at the earliest stages of AMD that precede vision loss. In a previous global RPE proteome analysis, changes were identified in the content of several mitochondrial proteins associated with AMD. In this study, the subproteome of mitochondria isolated from human donor RPE graded with the Minnesota Grading System (MGS) was analyzed. METHODS: Human donor eye bank eyes were categorized into one of four progressive stages (MGS 1-4) based on the clinical features of AMD. After dissection of the RPE, mitochondrial proteins were isolated and separated by two-dimensional gel electrophoresis based on their charge and mass. Protein spot densities were compared between the four MGS stages. Peptides from spots that changed significantly with MGS stage were extracted and analyzed by using mass spectrometry to identify the protein. RESULTS: Western blot analyses verified that mitochondria were consistently enriched between MGS stages. The densities of eight spots increased or decreased significantly as a function of MGS stage. These spots were identified as the alpha-, beta-, and delta-ATP synthase subunits, subunit VIb of the cytochrome c oxidase complex, mitofilin, mtHsp70, and the mitochondrial translation factor Tu. CONCLUSIONS: The results are consistent with the hypothesis that mitochondrial dysfunction is associated with AMD and further suggest specific pathophysiological mechanisms involving altered mitochondrial translation, import of nuclear-encoded proteins, and ATP synthase activity.