SMCR8 negatively regulates AKT and MTORC1 signaling to modulate lysosome biogenesis and tissue homeostasis

Physiological Function ◽

Chromosome Region ◽

Tissue Homeostasis ◽

Spine Density ◽

Protein Levels ◽

Lysosome Biogenesis ◽

Mtorc1 Signaling ◽

AbstractThe intronic hexanucleotide expansion in the C9orf72 gene is one of the leading causes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), 2 devastating neurodegenerative diseases. C9orf72 forms a heterodimer with SMCR8 (Smith-Magenis syndrome chromosome region, candidate 8) protein. However, the physiological function of SMCR8 remains to be characterized. Here we report that ablation of SMCR8 in mice results in splenomegaly with autoimmune phenotypes similar to that of C9orf72 deficiency. Furthermore, SMCR8 loss leads to a drastic decrease of C9orf72 protein levels. Many proteins involved in the macroautophagy-lysosome pathways are downregulated upon SMCR8 loss due to elevated activation of MTORC1 and AKT, which also leads to increased spine density in the Smcr8 knockout neurons. In summary, our studies demonstrate a key role of SMCR8 in regulating MTORC1 and AKT signaling and tissue homeostasis.

Regulated proteolysis of p62/SQSTM1 enables differential control of autophagy and nutrient sensing

Science Signaling ◽

10.1126/scisignal.aat6903 ◽

2018 ◽

Vol 11 (559) ◽

pp. eaat6903 ◽

Cited By ~ 15

Author(s):

Julia Sanchez-Garrido ◽

Vanessa Sancho-Shimizu ◽

Avinash R. Shenoy

Keyword(s):

Nutrient Sensing ◽

Caspase 8 ◽

Rimmed Vacuoles ◽

Sequestosome 1 ◽

Inflammatory Conditions ◽

Mtorc1 Signaling ◽

Differential Control ◽

Antimicrobial Immunity ◽

The multidomain scaffold protein p62 (also called sequestosome-1) is involved in autophagy, antimicrobial immunity, and oncogenesis. Mutations in SQSTM1, which encodes p62, are linked to hereditary inflammatory conditions such as Paget’s disease of the bone, frontotemporal dementia (FTD), amyotrophic lateral sclerosis, and distal myopathy with rimmed vacuoles. Here, we report that p62 was proteolytically trimmed by the protease caspase-8 into a stable protein, which we called p62TRM. We found that p62TRM, but not full-length p62, was involved in nutrient sensing and homeostasis through the mechanistic target of rapamycin complex 1 (mTORC1). The kinase RIPK1 and caspase-8 controlled p62TRM production and thus promoted mTORC1 signaling. An FTD-linked p62 D329G polymorphism and a rare D329H variant could not be proteolyzed by caspase-8, and these noncleavable variants failed to activate mTORC1, thereby revealing the detrimental effect of these mutations. These findings on the role of p62TRM provide new insights into SQSTM1-linked diseases and mTORC1 signaling.

Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia

Blood ◽

10.1182/blood.2020005693 ◽

2021 ◽

Author(s):

Amanda G Davis ◽

Daniel T. Johnson ◽

Dinghai Zheng ◽

Ruijia Wang ◽

Nathan D. Jayne ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Alternative Polyadenylation ◽

Hematopoietic Stem ◽

Global Trends ◽

Protein Levels ◽

Mtorc1 Signaling ◽

Differentiation Block ◽

Acute Myeloid

Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site (PAS) usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in acute myeloid leukemia (AML) patients by performing 3' Region Extraction And Deep Sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in PAS usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS dataset. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block of AML patients.

Abstract 536: Lysosome Insufficiency in Atherosclerosis Prone DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Drop Clearance

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.536 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Peggy Robinet ◽

Jonathan D Smith

Keyword(s):

Cathepsin L ◽

Foam Cells ◽

Lysosomal Degradation ◽

Over Expression ◽

Protein Levels ◽

Lysosome Biogenesis ◽

Mouse Macrophages ◽

Macrophage Foam Cells ◽

And Function

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05), suggesting that lysosome number and function may be impaired in DBA/2 foam cells. To assess lysosome function and number, cells were labeled with Lysotracker red DND-99 (LyT) and analyzed by flow cytometry. We found that AcLDL loading did not affect LyT intensity. However, in both unloaded and loaded conditions, DBA/2 cells exhibited a 30 to 50% lower LyT intensity suggesting that they have intrinsically decreased lysosome number/function. Lysosomal degradation capacity was assayed by incubation with DQ-ovalbumin and we observed a 27% decrease in lysosome function in DBA/2 vs. AKR foam cells (p<0.01). In addition, upon loading, the mature form of cathepsin L was increased in AKR (43%, p<0.05) but not DBA/2 cells. Together these data suggest an impairment of lysosomal degradation capacity in DBA/2 foam cells. Finally, we investigated the role of TPC2, a lysosomal membrane protein which over expression has been previously linked to a defect in autolysosome formation. We found that upon AcLDL loading TPC2 protein levels were increased by 35% in DBA/2 cells, which are defective in autolysosome formation, while they were unchanged in AKR cells. In conclusion, we found that DBA/2 vs. AKR foam cells express more TPC2 and have fewer and/or dysfunctional lysosomes that may explain the autolysosome formation defect in these cells.

A role of the frontotemporal lobar degeneration risk factor TMEM106B in myelination

Brain ◽

10.1093/brain/awaa154 ◽

2020 ◽

Vol 143 (7) ◽

pp. 2255-2271 ◽

Cited By ~ 6

Author(s):

Tuancheng Feng ◽

Rory R Sheng ◽

Santiago Solé-Domènech ◽

Mohammed Ullah ◽

Xiaolai Zhou ◽

...

Keyword(s):

Risk Factor ◽

Cathepsin D ◽

Proteolipid Protein ◽

Wild Type ◽

Protein Levels ◽

Lysosomal Protease ◽

Precursor Cell Line ◽

Lysosomal Membrane Protein

Abstract TMEM106B encodes a lysosomal membrane protein and was initially identified as a risk factor for frontotemporal lobar degeneration. Recently, a dominant D252N mutation in TMEM106B was shown to cause hypomyelinating leukodystrophy. However, how TMEM106B regulates myelination is still unclear. Here we show that TMEM106B is expressed and localized to the lysosome compartment in oligodendrocytes. TMEM106B deficiency in mice results in myelination defects with a significant reduction of protein levels of proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), the membrane proteins found in the myelin sheath. The levels of many lysosome proteins are significantly decreased in the TMEM106B-deficient Oli-neu oligodendroglial precursor cell line. TMEM106B physically interacts with the lysosomal protease cathepsin D and is required to maintain proper cathepsin D levels in oligodendrocytes. Furthermore, we found that TMEM106B deficiency results in lysosome clustering in the perinuclear region and a decrease in lysosome exocytosis and cell surface PLP levels. Moreover, we found that the D252N mutation abolished lysosome enlargement and lysosome acidification induced by wild-type TMEM106B overexpression. Instead, it stimulates lysosome clustering near the nucleus as seen in TMEM106B-deficient cells. Our results support that TMEM106B regulates myelination through modulation of lysosome function in oligodendrocytes.

Transcription Factor EB Is Selectively Reduced in the Nuclear Fractions of Alzheimer’s and Amyotrophic Lateral Sclerosis Brains

Neuroscience Journal ◽

10.1155/2016/4732837 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 17

Author(s):

Hongjie Wang ◽

Ruizhi Wang ◽

Shaohua Xu ◽

Madepalli K. Lakshmana

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Transcription Factor ◽

Normal Control ◽

Stage Iv ◽

Braak Stage ◽

Master Regulator ◽

Protein Levels ◽

Lysosome Biogenesis ◽

Transcription Factor Eb ◽

Multiple studies suggest that autophagy is strongly dysregulated in Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS), as evidenced by accumulation of numerous autophagosomes, lysosomes with discontinuous membranes, and aggregated proteins in the patients’ brains. Transcription factor EB (TFEB) was recently discovered to be a master regulator of lysosome biogenesis and autophagy. To examine whether aberrant autophagy in AD and ALS is due to alterations in TFEB expression, we systematically quantified the levels of TFEB in these brains by immunoblotting. Interestingly, cytoplasmic fractions of AD brains showed increased levels of normalized (to tubulin) TFEB only at Braak stage IV (61%, p<0.01). Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p<0.01), stage V (67%, p<0.01), and stage VI (85%, p<0.01) when compared to normal control (NC) brains. In the ALS brains also, nuclear TFEB levels were reduced by 62% (p<0.001). These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent. Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders.

The role of mutant TAR DNA-binding protein 43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

Biochemical Society Transactions ◽

10.1042/bst0390954 ◽

2011 ◽

Vol 39 (4) ◽

pp. 954-959 ◽

Cited By ~ 13

Author(s):

Jonathan Janssens ◽

Gernot Kleinberger ◽

Hans Wils ◽

Christine Van Broeckhoven

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Current Status ◽

Disease Spectrum ◽

Pathological Disease ◽

TDP-43 (TAR DNA-binding protein 43) has been identified as a key protein of ubiquitinated inclusions in brains of patients with ALS (amyotrophic lateral sclerosis) or FTLD (frontotemporal lobar degeneration), defining a new pathological disease spectrum. Recently, coding mutations have been identified in the TDP-43 gene (TARDBP), which further confirmed the pathogenic nature of the protein. Today, several animal models have been generated to gain more insight into the disease-causing pathways of the FTLD/ALS spectrum. This mini-review summarizes the current status of TDP-43 models, with a focus on mutant TDP-43.

The C9ORF72 Gene, Implicated in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia, Encodes a Protein That Functions in Control of Endothelin and Glutamate Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00155-18 ◽

2018 ◽

Vol 38 (22) ◽

Cited By ~ 5

Author(s):

Vitalay Fomin ◽

Patricia Richard ◽

Mainul Hoque ◽

Cynthia Li ◽

Zhuoying Gu ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Frontotemporal Dementia ◽

Morphological Changes ◽

Cell Models ◽

Glutamate Signaling ◽

Protein Levels ◽

Rna Analysis ◽

C9orf72 Gene ◽

ABSTRACT A GGGGCC repeat expansion in the C9ORF72 (C9) gene is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Several mechanisms have been proposed to account for its toxicity, including the possibility that reduced C9 protein levels contribute to disease. To investigate this possibility, we examined the effects of reduced C9 levels in several cell systems. We first showed that C9 knockdown (KD) in U87 glioblastoma cells results in striking morphological changes, including vacuolization and alterations in cell size. Unexpectedly, RNA analysis revealed changes in expression of many genes, including genes involved in endothelin (EDN) signaling and immune system pathways and multiple glutamate cycling genes (e.g., EAAT2), which were verified in several cell models, including astrocytes and brain samples from C9-positive patients. Consistent with deregulation of the glutamate cycling genes, elevated intracellular glutamate was detected in both KD cells and patient astrocytes. Importantly, levels of mRNAs encoding EDN1 and its receptors, known to be elevated in ALS, were sharply increased by C9 KD, likely resulting from an observed activation of NF-κB signaling and/or a possible role of a C9 isoform in gene control.

A multifaceted role of progranulin in regulating amyloid-beta dynamics and responses

10.1101/2021.04.12.439509 ◽

2021 ◽

Author(s):

Huan Du ◽

Man Ying Wong ◽

Tingting Zhang ◽

Mariela Nunez Santos ◽

Charlene Hsu ◽

...

Keyword(s):

Nuclear Localization ◽

Microglial Activation ◽

Nuclear Translocation ◽

Young Male ◽

Disease Mechanism ◽

Lysosome Biogenesis ◽

Age Dependent ◽

Galectin 3

AbstractHaploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN polymorphisms are associated with Alzheimer’s disease (AD). PGRN is highly expressed in the microglia near Aβ plaques and influences plaque dynamics and microglial activation. However, the detailed mechanisms remain elusive. Here we report that PGRN deficiency reduces human APP and Aβ levels in the young male but not female mice. PGRN deficient microglia exhibit increased expression of markers associated with microglial activation, including CD68, Galectin-3, TREM2 and GPNMB, specifically near Aβ plaques. In addition, PGRN loss leads to up-regulation of lysosome proteins and an increase in the nuclear localization of TFE3, a transcription factor involved in lysosome biogenesis. Cultured PGRN deficient microglia show enhanced nuclear translocation of TFE3 and inflammation in response to Aβ fibril treatment. Taken together, our data revealed a sex- and age-dependent effect of PGRN on amyloid metabolism and a role of PGRN in regulating lysosomal activities and inflammation in plaque-associated microglia, which may contribute to the disease mechanism associated with PGRN polymorphisms in AD.

A multifaceted role of progranulin in regulating amyloid-beta dynamics and responses

Life Science Alliance ◽

10.26508/lsa.202000874 ◽

2021 ◽

Vol 4 (7) ◽

pp. e202000874

Author(s):

Huan Du ◽

Man Ying Wong ◽

Tingting Zhang ◽

Mariela Nunez Santos ◽

Charlene Hsu ◽

...

Keyword(s):

Nuclear Localization ◽

Microglial Activation ◽

Nuclear Translocation ◽

Young Male ◽

Lysosome Biogenesis ◽

Age Dependent ◽

App Metabolism ◽

Galectin 3

Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN polymorphisms are associated with Alzheimer’s disease. PGRN is highly expressed in the microglia near Aβ plaques and influences plaque dynamics and microglial activation. However, the detailed mechanisms remain elusive. Here we report that PGRN deficiency reduces human APP and Aβ levels in the young male but not female mice. PGRN-deficient microglia exhibit increased expression of markers associated with microglial activation, including CD68, galectin-3, TREM2, and GPNMB, specifically near Aβ plaques. In addition, PGRN loss leads to up-regulation of lysosome proteins and an increase in the nuclear localization of TFE3, a transcription factor involved in lysosome biogenesis. Cultured PGRN-deficient microglia show enhanced nuclear translocation of TFE3 and inflammation in response to Aβ fibril treatment. Taken together, our data revealed a sex- and age-dependent effect of PGRN on APP metabolism and a role of PGRN in regulating lysosomal activities and inflammation in plaque-associated microglia.