Modulating Golgi Stress Signaling Ameliorates Cell Morphological Phenotypes Induced by CHMP2B with Frontotemporal Dementia-Associated p.Asp148Tyr.

Some charged multivesicular body protein 2B (CHMP2B) mutations are associated with autosomal-dominant neurodegenerative frontotemporal dementia and/or amyotrophic lateral sclerosis type 7 (FTDALS7). The main aim of this study is to clarify the relationship between the expression of mutated CHMP2B protein displaying FTD symptoms and defective neuronal differentiation. First, we illustrate that the expression of CHMP2B with the Asp148Tyr (D148Y) mutation, which preferentially displays FTD phenotypes, blunts neurite process elongation in rat primary cortical neurons. Similar results were observed in the N1E-115 cell line, a model that undergoes neurite elongation. Second, these effects were also accompanied by changes in neuronal differentiation marker protein expression. Third, wild-type CHMP2B protein was indeed localized in the endosomal sorting complexes required to transport (ESCRT)-like structures throughout the cytoplasm. In contrast, CHMP2B with the D148Y mutation exhibited aggregation-like structures and accumulated in the Golgi body. Fourth, among currently known Golgi stress regulators, the expression levels of Hsp47, which has protective effects on the Golgi body, were decreased in cells expressing CHMP2B with the D148Y mutation. Fifth, Arf4, another Golgi stress-signaling molecule, was increased in mutant-expressing cells. Finally, when transfecting Hsp47 or knocking down Arf4 with small interfering (si)RNA, cellular phenotypes in mutant-expressing cells were recovered. These results suggest that CHMP2B with the D148Y mutation, acting through Golgi stress signaling, is negatively involved in the regulation of neuronal cell morphological differentiation, providing evidence that a molecule controlling Golgi stress may be one of the potential FTD therapeutic targets at the molecular and cellular levels.

The identification of high-performing antibodies for Charged multivesicular body protein 2b for use in Western Blot, immunoprecipitation and immunofluorescence.

Charged multivesicular body protein 2B is a subunit of the endosomal sorting complex required for transport III (ESRCT-III), a complex implicated in the lysosomal degradation pathway and formation of multivesicular bodies. Mutations to the CHMP2B gene can result in abnormal protein aggregates in neurons and is therefore predicted to be associated in neurodegenerative diseases, including across the ALS-FTD spectrum. Through our standardized experimental protocol which compares read-outs in knockout cell lines and isogenic parental controls, this study aims to enhance the reproducibility of research on this target by characterizing eight commercial antibodies against charged multivesicular body protein 2b using Western Blot, immunoprecipitation, and immunofluorescence. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

FTD/ALS Type 7-Associated Thr104Asn Mutation of CHMP2B Blunts Neuronal Process Elongation, and Is Recovered by Knockdown of Arf4, the Golgi Stress Regulator.

Frontotemporal dementia and/or amyotrophic lateral sclerosis type 7 (FTD/ALS7) is an autosomal dominant neurodegenerative disorder characterized by the onset of FTD and/or ALS, mainly in adulthood. Patients with some types of mutations, including the Thr104Asn (T104N) mutation of charged multivesicular body protein 2B (CHMP2B), have predominantly ALS phenotypes, whereas patients with other mutations have predominantly FTD phenotypes. A few mutations result in patients having both phenotypes approximately equally; however, the reason why phenotypes differ depending on the position of the mutation is unknown. CHMP2B comprises one part of the endosomal sorting complexes required for transport (ESCRT), specifically ESCRT-III, in the cytoplasm. We describe here, for the first time, that CHMP2B with the T104N mutation inhibits neuronal process elongation in the N1E-115 cell line, a model line undergoing neuronal differentiation. This inhibitory phenotype was accompanied by changes in marker protein expression. Of note, CHMP2B with the T104N mutation, but not the wild-type form, was preferentially accumulated in the Golgi body. Of the four major Golgi stress signaling pathways currently known, the pathway through Arf4, the small GTPase, was specifically upregulated in cells expressing CHMP2B with the T104N mutation. Conversely, knockdown of Arf4 with the cognate small interfering (si)RNA recovered the neuronal process elongation inhibited by the T104N mutation. These results suggest that the T104N mutation of CHMP2B inhibits morphological differentiation by triggering Golgi stress signaling, revealing a possible therapeutic molecular target for recovering potential molecular and cellular phenotypes underlying FTD/ALS7.

A novel splice-site mutation in CHMP2B associated with frontotemporal dementia: The first report from China and literature review.

BACKGROUND: Frontotemporal dementia (FTD) has genetic heterogeneity, and the endosomal ESCRTIII-complex subunit CHMP2B variant is a rare cause of FTD. The mutations in CHMP2B were first identified in a large Danish pedigree with autosomal dominant FTD, and have also been found in several individuals from Belgium, France, the United States, and Türkiye. In the Chinese population, cases of CHMP2B variant-associated FTD have never been reported. METHODS: The spectrum of clinical symptoms and the genetic analysis of the presented patient were identified and investigated. Besides this case, we assessed previously reported cases with CHMP2B gene mutations. RESULTS: This study presents a Chinese patient harboring a novel heterozygous A-to-T variant (NM_014043:c.532-2A>T) in CHMP2B with a phenotype compatible with FTD. Although previous reports suggested cases of CHMP2B variant-associated FTD initially presented with personality changes and stereotypical movements at the age of 50, this case was characterized by psychosis involving delusion of persecution, auditory hallucination, and suspiciousness at the earlier onset age of 44. Minigene splicing assay revealed that the splice-site variant could result in the retention of intron 5. CONCLUSION: This is the first case of CHMP2B variant-associated FTD reported in the Chinese population. The novel c.532-2A>T variant in the acceptor splice site of exon 6 retaining intron 5 was predicted to cause truncated protein and protein conformation changes. This discovery may expand the genetic and phenotypic spectrum of CHMP2B variant-associated FTD.

Cytoplasmic YAP1-mediated ESCRT-III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer.

BACKGROUND: Nuclear Yes1-associated transcriptional regulator (YAP1) promotes tumor progression. However, the function of cytoplasmic YAP1 in breast cancer cells and its impact on the survival of breast cancer patients remain unclear. Our research aimed to explore the biological function of cytoplasmic YAP1 in breast cancer cells and the possibility of cytoplasmic YAP1 as a predictive marker of breast cancer survival. METHODS: We constructed cell mutant models, including NLS-YAP15SA (nuclear localized), YAP1S94A (incapable of binding to the TEA domain transcription factor family) and YAP1S127D (cytoplasmic localized), and used Cell Counting Kit-8 (CCK-8) assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, and Western blotting (WB) analysis to detect cell proliferation and apoptosis. The specific mechanism of cytoplasmic YAP1-mediated endosomal sorting complexes required for transport III (ESCRT-III) assembly was studied by co-immunoprecipitation, immunofluorescence staining, and WB analysis. Epigallocatechin gallate (EGCG) was used to simulate YAP1 retention in the cytoplasm in in vitro and in vivo experiments to study the function of cytoplasmic YAP1. YAP1 binding to NEDD4-like E3 ubiquitin protein ligase (NEDD4L) was identified using mass spectrometry and was verified in vitro. Breast tissue microarrays were used to analyze the relationship between cytoplasmic YAP1 expression and the survival of breast cancer patients. RESULTS: YAP1 was mainly expressed in the cytoplasm in breast cancer cells. Cytoplasmic YAP1 promoted autophagic death of breast cancer cells. Cytoplasmic YAP1 bound to the ESCRT-III complex subunits charged multivesicular body protein 2B (CHMP2B) and vacuolar protein sorting 4 homolog B (VPS4B), promoting assembly of CHMP2B-VPS4B and activating autophagosome formation. EGCG retained YAP1 in the cytoplasm, promoting the assembly of CHMP2B-VPS4B to promote autophagic death of breast cancer cells. YAP1 bound to NEDD4L, and NEDD4L mediated ubiquitination and degradation of YAP1. Breast tissue microarrays revealed that high levels of cytoplasmic YAP1 were beneficial to the survival of breast cancer patients. CONCLUSIONS: Cytoplasmic YAP1 mediated autophagic death of breast cancer cells by promoting assembly of the ESCRT-III complex; furthermore, we established a new breast cancer survival prediction model based on cytoplasmic YAP1 expression.

The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias.

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

SIRT6 Protects Against Myocardial Ischemia-Reperfusion Injury by Attenuating Aging-Related CHMP2B Accumulation.

Impaired autophagic flux induces aging-related ischemia vulnerability, which is the hallmark pathology in cardiac aging. Our previous work has confirmed that the accumulation of charged multivesicular body protein 2B (CHMP2B), a subunit of the ESCRT-III complex, in the heart can impair autophagy flux. However, whether CHMP2B accumulation contributes to aging-related intolerance to ischemia/reperfusion (I/R) injury and the regulatory mechanism for CHMP2B in aged heart remain elusive. The cardiac CHMP2B level was significantly higher in aged human myocardium than that in young myocardium. Increased CHMP2B were shown to inhibit autophagic flux leading to the deterioration of MI/R injury in aged mice hearts. Interestingly, a negative correlation was observed between SIRT6 and CHMP2B expression in human heart samples. Specific activation of SIRT6 suppressed CHMP2B accumulation and ameliorated autophagy flux in aged hearts. Using myocardial-specific SIRT6 heterozygous knockout mice and recovery experiments confirmed that SIRT6 regulated myocardial CHMP2B levels. Finally, activation of SIRT6 decreased acetylation of FoxO1 to promote its transcriptional function on Atrogin-1, a muscle-specific ubiquitin ligase, which subsequently enhanced the degradation of CHMP2B by Atrogin-1. This is a novel mechanism for SIRT6 against aging-related myocardial ischemia vulnerability, particularly by preventing excessive accumulation of autophagy key factors.

Six generations of CHMP2B-mediated Frontotemporal Dementia: Clinical features, predictive testing, progression, and survival.

OBJECTIVES: Chromosome 3-linked frontotemporal dementia (FTD-3) is caused by a c.532-1G > C mutation in the CHMP2B gene. It is extensively studied in a Danish family comprising one of the largest families with an autosomal dominantly inherited frontotemporal dementia (FTD). This retrospective cohort study utilizes demographics to identify risk factors for onset, progression, life expectancy, and death in CHMP2B-mediated FTD. The pedigree of 528 individuals in six generations is provided, and clinical descriptions are presented. Choices of genetic testing are evaluated. MATERIALS AND METHODS: Demographic and lifestyle factors were assessed in survival analysis in all identified CHMP2B mutation carriers (44 clinically affected FTD-3 patients and 16 presymptomatic CHMP2B mutation carriers). Predictors of onset and progression included sex, parental disease course, education, and vascular risk factors. Life expectancy was established by matching CHMP2B mutation carriers with average life expectancies in Denmark. RESULTS: Disease course was not correlated to parental disease course and seemed unmodified by lifestyle factors. Diagnosis was recognized at an earlier age in members with higher levels of education, probably reflecting an early dysexecutive syndrome, unmasked earlier in people with higher work-related requirements. Carriers of the CHMP2B mutation had a significant reduction in life expectancy of 13 years. Predictive genetic testing was chosen by 20% of at-risk family members. CONCLUSIONS: CHMP2B-mediated FTD is substantiated as an autosomal dominantly inherited disease of complete penetrance. The clinical phenotype is a behavioral variant FTD. The disease course is unpredictable, and life expectancy is reduced. The findings may be applicable to other genetic FTD subtypes.

A novel synaptopathy-defective synaptic vesicle protein trafficking in the mutant CHMP2B mouse model of frontotemporal dementia.

Mutations in the ESCRT-III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C-terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse.

Autophagy and ALS: mechanistic insights and therapeutic implications.

Mechanisms of protein homeostasis are crucial for overseeing the clearance of misfolded and toxic proteins over the lifetime of an organism, thereby ensuring the health of neurons and other cells of the central nervous system. The highly conserved pathway of autophagy is particularly necessary for preventing and counteracting pathogenic insults that may lead to neurodegeneration. In line with this, mutations in genes that encode essential autophagy factors result in impaired autophagy and lead to neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). However, the mechanistic details underlying the neuroprotective role of autophagy, neuronal resistance to autophagy induction, and the neuron-specific effects of autophagy-impairing mutations remain incompletely defined. Further, the manner and extent to which non-cell autonomous effects of autophagy dysfunction contribute to ALS pathogenesis are not fully understood. Here, we review the current understanding of the interplay between autophagy and ALS pathogenesis by providing an overview of critical steps in the autophagy pathway, with special focus on pivotal factors impaired by ALS-causing mutations, their physiologic effects on autophagy in disease models, and the cell type-specific mechanisms regulating autophagy in non-neuronal cells which, when impaired, can contribute to neurodegeneration. This review thereby provides a framework not only to guide further investigations of neuronal autophagy but also to refine therapeutic strategies for ALS and related neurodegenerative diseases.Abbreviations: ALS: amyotrophic lateral sclerosis; Atg: autophagy-related; CHMP2B: charged multivesicular body protein 2B; DPR: dipeptide repeat; FTD: frontotemporal dementia; iPSC: induced pluripotent stem cell; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PINK1: PTEN induced kinase 1; RNP: ribonuclear protein; sALS: sporadic ALS; SPHK1: sphingosine kinase 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; ULK: unc-51 like autophagy activating kinase; UPR: unfolded protein response; UPS: ubiquitin-proteasome system; VCP: valosin containing protein.

Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

Cortical Frontoparietal Network Dysfunction in CHMP2B-Frontotemporal Dementia.

A rare cause of inherited frontotemporal dementia (FTD) is a mutation in the CHMP2B gene on chromosome 3 leading to the autosomal dominantly inherited FTD (CHMP2B-FTD). Since CHMP2B-FTD is clinically well-characterized, and patients show a distinct pattern of executive dysfunction, the condition offers possible insight in the early electroencephalographic (EEG) changes in the cortical networks. Specifically, EEG microstate analysis parses the EEG signals into topographies believed to represent discrete network activations. We investigated the EEG dynamics in patients with symptomatic CHMP2B-FTD (n = 5) as well as pre-symptomatic mutation carriers (n = 5) compared to non-carrier family members (n = 6). The data was parsed into four archetypal microstates and global power was calculated. A trend was found for lower occurrence in microstate D in CHMP2B-FTD (p-value = 0.177, F-value = 2.036). Patients with recent symptom onset (<1 year) showed an increased duration of microstate D, whereas patients who had been symptomatic for longer periods (>2 years) showed decreased duration. Patients with CHMP2B-FTD present with executive dysfunction, and microstate D has previously been shown to be associated with the fronto-parietal network. The biphasic pattern may represent the pathophysiological changes in brain dynamics during neurodegeneration, which may apply to other neurodegenerative diseases.

The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei.

Nuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

Lessons learned from CHMP2B, implications for frontotemporal dementia and amyotrophic lateral sclerosis.

Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are two neurodegenerative diseases with clinical, genetic and pathological overlap. As such, they are commonly regarded as a single spectrum disorder, with pure FTD and pure ALS representing distinct ends of a continuum. Dysfunctional endo-lysosomal and autophagic trafficking, leading to impaired proteostasis is common across the FTD-ALS spectrum. These pathways are, in part, mediated by CHMP2B, a protein that coordinates membrane scission events as a core component of the ESCRT machinery. Here we review how ALS and FTD disease causing mutations in CHMP2B have greatly contributed to our understanding of how endosomal-lysosomal and autophagic dysfunction contribute to neurodegeneration, and how in vitro and in vivo models have helped elucidate novel candidates for potential therapeutic intervention with implications across the FTD-ALS spectrum.

Glutamate-glutamine homeostasis is perturbed in neurons and astrocytes derived from patient iPSC models of frontotemporal dementia.

Frontotemporal dementia (FTD) is amongst the most prevalent early onset dementias and even though it is clinically, pathologically and genetically heterogeneous, a crucial involvement of metabolic perturbations in FTD pathology is being recognized. However, changes in metabolism at the cellular level, implicated in FTD and in neurodegeneration in general, are still poorly understood. Here we generate induced human pluripotent stem cells (hiPSCs) from patients carrying mutations in CHMP2B (FTD3) and isogenic controls generated via CRISPR/Cas9 gene editing with subsequent neuronal and glial differentiation and characterization. FTD3 neurons show a dysregulation of glutamate-glutamine related metabolic pathways mapped by 13C-labelling coupled to mass spectrometry. FTD3 astrocytes show increased uptake of glutamate whilst glutamate metabolism is largely maintained. Using quantitative proteomics and live-cell metabolic analyses, we elucidate molecular determinants and functional alterations of neuronal and glial energy metabolism in FTD3. Importantly, correction of the mutations rescues such pathological phenotypes. Notably, these findings implicate dysregulation of key enzymes crucial for glutamate-glutamine homeostasis in FTD3 pathogenesis which may underlie vulnerability to neurodegeneration. Neurons derived from human induced pluripotent stem cells (hiPSCs) of patients carrying mutations in CHMP2B (FTD3) display major metabolic alterations compared to CRISPR/Cas9 generated isogenic controls. Using quantitative proteomics, 13C-labelling coupled to mass spectrometry metabolic mapping and seahorse analyses, molecular determinants and functional alterations of neuronal and astrocytic energy metabolism in FTD3 were characterized. Our findings implicate dysregulation of glutamate-glutamine homeostasis in FTD3 pathogenesis. In addition, FTD3 neurons recapitulate glucose hypometabolism observed in FTD patient brains. The impaired mitochondria function found here is concordant with disturbed TCA cycle activity and decreased glycolysis in FTD3 neurons. FTD3 neuronal glutamine hypermetabolism is associated with up-regulation of PAG expression and, possibly, ROS production. Distinct compartments of glutamate metabolism can be suggested for the FTD3 neurons. Endogenous glutamate generated from glutamine via PAG may enter the TCA cycle via AAT (left side of neuron) while exogenous glutamate taken up from the extracellular space may be incorporated into the TCA cycle via GDH (right side of the neuron) FTD3 astrocytic glutamate uptake is upregulated whilst glutamate metabolism is largely maintained. Finally, pharmacological reversal of glutamate hypometabolism manifesting from decreased GDH expression should be explored as a novel therapeutic intervention for treating FTD3.

Neuroprotective activity of ursodeoxycholic acid in CHMP2BIntron5 models of frontotemporal dementia.

Frontotemporal dementia (FTD) is one of the most prevalent forms of early-onset dementia. It represents part of the FTD-Amyotrophic Lateral Sclerosis (ALS) spectrum, a continuum of genetically and pathologically overlapping disorders. FTD-causing mutations in CHMP2B, a gene encoding a core component of the heteromeric ESCRT-III Complex, lead to perturbed endosomal-lysosomal and autophagic trafficking with impaired proteostasis. While CHMP2B mutations are rare, dysfunctional endosomal-lysosomal signalling is common across the FTD-ALS spectrum. Using our established Drosophila and mammalian models of CHMP2BIntron5 induced FTD we demonstrate that the FDA-approved compound Ursodeoxycholic Acid (UDCA) conveys neuroprotection, downstream of endosomal-lysosomal dysfunction in both Drosophila and primary mammalian neurons. UDCA exhibited a dose dependent rescue of neuronal structure and function in Drosophila pan-neuronally expressing CHMP2BIntron5. Rescue of CHMP2BIntron5 dependent dendritic collapse and apoptosis with UDCA in rat primary neurons was also observed. UDCA failed to ameliorate aberrant accumulation of endosomal and autophagic organelles or ubiquitinated neuronal inclusions in both models. We demonstrate the neuroprotective activity of UDCA downstream of endosomal-lysosomal and autophagic dysfunction, delineating the molecular mode of action of UDCA and highlighting its potential as a therapeutic for the treatment of FTD-ALS spectrum disorders.

Metformin-Enhanced Cardiac AMP-Activated Protein Kinase/Atrogin-1 Pathways Inhibit Charged Multivesicular Body Protein 2B Accumulation in Ischemia-Reperfusion Injury.

Background: Cardiac autophagic flux is impaired during myocardial ischemia/reperfusion (MI/R). Impaired autophagic flux may exacerbate MI/R injury. Charged multivesicular body protein 2B (CHMP2B) is a subunit of the endosomal sorting complex required for transport (ESCRT-III) complex that is required for autophagy. However, the reverse role of CHMP2B accumulation in autophagy and MI/R injury has not been established. The objective of this article is to elucidate the roles of AMP-activated protein kinase (AMPK)/atrogin-1 pathways in inhibiting CHMP2B accumulation in ischemia-reperfusion injury. Methods: Male C57BL/6 mice (3-4 months) and H9c2 cardiomyocytes were used to evaluate MI/R and hypoxia/reoxygenation (H/R) injury in vivo and in vitro, respectively. MI/R was built by a left lateral thoracotomy and occluded the left anterior descending artery. H9c2 cells were firstly treated in 95% N2 and 5% CO2 for 15 h and reoxygenation for 1 h. Metformin (100 mg/kg/d) and CHMP2B (Ad-CHMP2B) transfected adenoviruses were administered to the mice. The H9c2 cells were treated with metformin (2.5 mM), MG-132 (10 µM), bafilomycin A1 (10 nM), and compound C (20 µM). Results: Autophagic flux was found to be inhibited in H/R-treated cardiomyocytes and MI/R mice, with elevated cardiac CHMP2B accumulation. Upregulated CHMP2B levels in the in vivo and in vitro experiments were shown to inhibit autophagic flux leading to the deterioration of H/R-cardiomyocytes and MI/R injury. This finding implies that CHMP2B accumulation increases the risk of myocardial ischemia. Metformin suppressed CHMP2B accumulation and ameliorated H/R-induced autophagic dysfunction by activating AMPK. Activated AMPK upregulated the messenger RNA expression and protein levels of atrogin-1, a muscle-specific ubiquitin ligase, in the myocardium. Atrogin-1 significantly enhanced the interaction between atrogin-1 and CHMP2B, therefore, promoting CHMP2B degradation in the MI/R myocardium. Finally, this study revealed that metformin-inhibited CHMP2B accumulation induced autophagic impairment and ischemic susceptibility in vivo through the AMPK-regulated CHMP2B degradation by atrogin-1. Conclusion: Impaired CHMP2B clearance in vitro and in vivo inhibits autophagic flux and weakens the myocardial ischemic tolerance. Metformin treatment degrades CHMP2B through the AMPK-atrogin-1-dependent pathway to maintain the homeostasis of autophagic flux. This is a novel mechanism that enriches the understanding of cardioprotection.

Serum Neurofilament Light in Patients with Frontotemporal Dementia Caused by CHMP2B Mutation.

INTRODUCTION: The potential of neurofilament light (NfL) as a blood-based biomarker is currently being investigated in autosomal dominant neurodegenerative disease. This study explores the clinical utility of serum-NfL in frontotemporal dementia due to CHMP2B mutation (FTD-3). METHODS: This cross-sectional study included serum and CSF data from 38 members of the Danish FTD-3 family: 12 affected CHMP2B mutation carriers, 10 presymptomatic carriers, and 16 noncarriers. Serum-NfL levels measured by single-molecule array (Simoa) technology were tested for associations with the clinical groups and clinical parameters. Serum and CSF data were compared, and CSF/serum-albumin ratio was included as a measure of blood-brain barrier (BBB) function. RESULTS: Serum-NfL concentrations were significantly increased in symptomatic CHMP2B mutation carriers compared to presymptomatic carriers and in both groups compared to healthy family controls. Serum-NfL levels appear to increase progressively with age in presymptomatic carriers, and this is perhaps followed by a change in trajectory when patients become symptomatic. Measurements of NfL in serum and CSF were highly correlated and fold-changes in serum and CSF between clinical groups were similar. Increase in serum-NFL levels was correlated with reduced ACE-score. Higher CSF/serum-albumin ratios were demonstrated in FTD-3 patients, but this did not affect the significant associations between serum-NfL and clinical groups. CONCLUSION: Serum-NfL could be utilized as an accurate surrogate marker of CSF levels to segregate symptomatic CHMP2B carriers, presymptomatic carriers, and non-carriers. The observed indication of BBB dysfunction in FTD-3 patients did not confound this use of serum-NfL. The results support the occurrence of mutation-related differences in NfL dynamics in familial FTD.

Expression of a human variant of CHMP2B linked to neurodegeneration in Drosophila external sensory organs leads to cell fate transformations associated with increased Notch activity.

Proper function of cell signaling pathways is dependent upon regulated membrane trafficking events that lead to the endocytosis, recycling, and degradation of cell surface receptors. The endosomal complexes required for transport (ESCRT) genes play a critical role in the sorting of ubiquitinated cell surface proteins. CHMP2BIntron5 , a truncated form of a human ESCRT-III protein, was discovered in a Danish family afflicted by a hereditary form of frontotemporal dementia (FTD). Although the mechanism by which the CHMP2B mutation in this family causes FTD is unknown, the resulting protein has been shown to disrupt normal endosomal-lysosomal pathway function and leads to aberrant regulation of signaling pathways. Here we have misexpressed CHMP2BIntron5 in the developing Drosophila external sensory (ES) organ lineage and demonstrate that it is capable of altering cell fates. Each of the cell fate transformations seen is compatible with an increase in Notch signaling. Furthermore, this interpretation is supported by evidence that expression of CHMP2BIntron5 in the notum environment is capable of raising the levels of Notch signaling. As such, these results add to a growing body of evidence that CHMP2BIntron5 can act rapidly to disrupt normal cellular function via the misregulation of critical cell surface receptor function.

Longitudinal transcriptomic analysis of altered pathways in a CHMP2Bintron5-based model of ALS-FTD.

Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with currently no cure. These two diseases share a clinical continuum with overlapping genetic causes. Mutations in the CHMP2B gene are found in patients with ALS, FTD and ALS-FTD. To highlight deregulated mechanisms occurring in ALS-FTD linked to the CHMP2B gene, we performed a whole transcriptomic study on lumbar spinal cord from CHMP2Bintron5 mice, a model that develops progressive motor alterations associated with dementia symptoms reminiscent of both ALS and FTD. To gain insight into the transcriptomic changes taking place during disease progression this study was performed at three stages: asymptomatic, symptomatic and end stage. We showed that before appearance of motor symptoms, the major disrupted mechanisms were linked with the immune system/inflammatory response and lipid metabolism. These processes were progressively replaced by alterations of neuronal electric activity as motor symptoms appeared, alterations that could lead to motor neuron dysfunction. To investigate overlapping alterations in gene expression between two ALS-causing genes, we then compared the transcriptome of symptomatic CHMP2Bintron5 mice with the one of symptomatic SOD1G86R mice and found the same families deregulated providing further insights into common underlying dysfunction of biological pathways, disrupted or disturbed in ALS. Altogether, this study provides a database to explore potential new candidate genes involved in the CHMP2Bintron5-based pathogenesis of ALS, and provides molecular clues to further understand the functional consequences that diseased neurons expressing CHMP2B mutant may have on their neighbor cells.