Ca+2 and Nε-lysine acetylation regulate the CALR-ATG9A interaction in the lumen of the endoplasmic reticulum.

The acetylation of autophagy protein 9 A (ATG9A) in the lumen of the endoplasmic reticulum (ER) by ATase1 and ATase2 regulates the induction of reticulophagy. Analysis of the ER-specific ATG9A interactome identified calreticulin (CALR), an ER luminal Ca+2-binding chaperone, as key for ATG9A activity. Specifically, if acetylated, ATG9A is sequestered by CALR and prevented from engaging FAM134B and SEC62. Under this condition, ATG9A is unable to activate the autophagy core machinery. In contrast, when non-acetylated, ATG9A is released by CALR and able to engage FAM134B and SEC62. In this study, we report that Ca+2 dynamics across the ER membrane regulate the ATG9A-CALR interaction as well as the ability of ATG9A to trigger reticulophagy. We show that the Ca+2-binding sites situated on the C-domain of CALR are essential for the ATG9A-CALR interaction. Finally, we show that K359 and K363 on ATG9A can influence the ATG9A-CALR interaction. Collectively, our results disclose a previously unidentified aspect of the complex mechanisms that regulate ATG9A activity. They also offer a possible area of intersection between Ca+2 metabolism, acetyl-CoA metabolism, and ER proteostasis.

Aß∗56 is a stable oligomer that impairs memory function in mice.

Amyloid-ß (Aß) oligomers consist of fibrillar and non-fibrillar soluble assemblies of the Aß peptide. Aß∗56 is a non-fibrillar Aß assembly that is linked to memory deficits. Previous studies did not decipher specific forms of Aß present in Aß∗56. Here, we confirmed the memory-impairing characteristics of Aß∗56 and extended its biochemical characterization. We used anti-Aß(1-x), anti-Aß(x-40), anti-Aß(x-42), and A11 anti-oligomer antibodies in conjunction with western blotting, immunoaffinity purification, and size-exclusion chromatography to probe aqueous brain extracts from Tg2576, 5xFAD, and APP/TTA mice. In Tg2576, Aß∗56 is a ∼56-kDa, SDS-stable, A11-reactive, non-plaque-dependent, water-soluble, brain-derived oligomer containing canonical Aß(1-40). In 5xFAD, Aß∗56 is composed of Aß(1-42), whereas in APP/TTA, it contains both Aß(1-40) and Aß(1-42). When injected into the hippocampus of wild-type mice, Aß∗56 derived from Tg2576 mice impairs memory. The unusual stability of this oligomer renders it an attractive candidate for studying relationships between molecular structure and effects on brain function.

The citrate transporters SLC13A5 and SLC25A1 elicit different metabolic responses and phenotypes in the mouse.

Cytosolic citrate is imported from the mitochondria by SLC25A1, and from the extracellular milieu by SLC13A5. In the cytosol, citrate is used by ACLY to generate acetyl-CoA, which can then be exported to the endoplasmic reticulum (ER) by SLC33A1. Here, we report the generation of mice with systemic overexpression (sTg) of SLC25A1 or SLC13A5. Both animals displayed increased cytosolic levels of citrate and acetyl-CoA; however, SLC13A5 sTg mice developed a progeria-like phenotype with premature death, while SLC25A1 sTg mice did not. Analysis of the metabolic profile revealed widespread differences. Furthermore, SLC13A5 sTg mice displayed increased engagement of the ER acetylation machinery through SLC33A1, while SLC25A1 sTg mice did not. In conclusion, our findings point to different biological responses to SLC13A5- or SLC25A1-mediated import of citrate and suggest that the directionality of the citrate/acetyl-CoA pathway can transduce different signals.

Aß*56 is a stable oligomer that correlates with age-related memory loss in Tg2576 mice.

Amyloid-ß (Aß) oligomers consist of fibrillar and non-fibrillar soluble assemblies of the Aß peptide. Tg2576 human amyloid precursor protein (APP)-expressing transgenic mice modeling Alzheimer's disease produce Aß*56, a non-fibrillar Aß assembly that has been shown by several groups to relate more closely to memory deficits than plaques. Previous studies did not decipher specific forms of Aß present in Aß*56. Here, we confirm and extend the biochemical characterization of Aß*56. We used anti-Aß(1-x), anti-Aß(x-40), and A11 anti-oligomer antibodies in conjunction with western blotting, immunoaffinity purification, and size-exclusion chromatography to probe aqueous brain extracts from Tg2576 mice of different ages. We found that Aß*56 is a ∼56-kDa, SDS-stable, A11-reactive, non-plaque-related, water-soluble, brain-derived oligomer containing canonical Aß(1-40) that correlates with age-related memory loss. The unusual stability of this high molecular-weight oligomer renders it an attractive candidate for studying relationships between molecular structure and effects on brain function.

SLC13A5/sodium-citrate co-transporter overexpression causes disrupted white matter integrity and an autistic-like phenotype.

Endoplasmic reticulum-based N ɛ-lysine acetylation serves as an important protein quality control system for the secretory pathway. Dysfunctional endoplasmic reticulum-based acetylation, as caused by overexpression of the acetyl coenzyme A transporter AT-1 in the mouse, results in altered glycoprotein flux through the secretory pathway and an autistic-like phenotype. AT-1 works in concert with SLC25A1, the citrate/malate antiporter in the mitochondria, SLC13A5, the plasma membrane sodium/citrate symporter and ATP citrate lyase, the cytosolic enzyme that converts citrate into acetyl coenzyme A. Here, we report that mice with neuron-specific overexpression of SLC13A5 exhibit autistic-like behaviours with a jumping stereotypy. The mice displayed disrupted white matter integrity and altered synaptic structure and function. Analysis of both the proteome and acetyl-proteome revealed unique adaptations in the hippocampus and cortex, highlighting a metabolic response that likely plays an important role in the SLC13A5 neuron transgenic phenotype. Overall, our results support a mechanistic link between aberrant intracellular citrate/acetyl coenzyme A flux and the development of an autistic-like phenotype.

Increased expression of SLC25A1/CIC causes an autistic-like phenotype with altered neuron morphology.

N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.

ATG9A regulates proteostasis through reticulophagy receptors FAM134B and SEC62 and folding chaperones CALR and HSPB1.

The acetylation of ATG9A within the endoplasmic reticulum (ER) lumen regulates the induction of reticulophagy. ER acetylation is ensured by AT-1/SLC33A1, a membrane transporter that maintains the cytosol-to-ER flux of acetyl-CoA. Defective AT-1 activity, as caused by heterozygous/homozygous mutations and gene duplication events, results in severe disease phenotypes. Here, we show that although the acetylation of ATG9A occurs in the ER lumen, the induction of reticulophagy requires ATG9A to engage FAM134B and SEC62 on the cytosolic side of the ER. To address this conundrum, we resolved the ATG9A interactome in two mouse models of AT-1 dysregulation: AT-1 sTg, a model of systemic AT-1 overexpression with hyperacetylation of ATG9A, and AT-1S113R/+, a model of AT-1 haploinsufficiency with hypoacetylation of ATG9A. We identified CALR and HSPB1 as two ATG9A partners that regulate the induction of reticulophagy as a function of ATG9A acetylation and discovered that ATG9A associates with several proteins that maintain ER proteostasis.

A Cautionary Tale: Endogenous Biotinylated Proteins and Exogenously-Introduced Protein A Cause Antibody-Independent Artefacts in Western Blot Studies of Brain-Derived Proteins.

Antibodies are commonly used to detect or isolate proteins from biological samples. Much attention has been paid to the potential for poorly-characterized antibodies to lead to misleading results, but antibody-independent artefacts may also occur. Here, we recount two examples of antibody-independent artefacts that have confounded the interpretation of results in our search for molecular entities associated with memory loss in Alzheimer's disease (AD). First, when using biotin-avidin systems for antibody detection, endogenous biotinylated proteins created spurious bands in Western blots of brain lysates from AD patients and transgenic mouse models of AD. These artefactual bands occurred in a transgene- and strain-dependent manner. A second, unexpected artefact occurred when Protein A-conjugated Sepharose beads were used to deplete lysates of endogenous immunoglobulins prior to immunopurification of target proteins. In these assays, Protein A shed from the beads, then bound to (and was eluted from) an immunoaffinity matrix designed to capture AD-related proteins. The Protein A then bound detection antibodies when the immunoaffinity eluates were analyzed by Western blot. Both of these artefacts-the endogenous biotinylated proteins and the Protein A artefact-can be monitored by including an "irrelevant" antibody as an experimental control (e.g., running a parallel protocol in which the antibody directed against the target of interest is replaced by a non-specific antibody).

Increased transport of acetyl-CoA into the endoplasmic reticulum causes a progeria-like phenotype.

The membrane transporter AT-1/SLC33A1 translocates cytosolic acetyl-CoA into the lumen of the endoplasmic reticulum (ER), participating in quality control mechanisms within the secretory pathway. Mutations and duplication events in AT-1/SLC33A1 are highly pleiotropic and have been linked to diseases such as spastic paraplegia, developmental delay, autism spectrum disorder, intellectual disability, propensity to seizures, and dysmorphism. Despite these known associations, the biology of this key transporter is only beginning to be uncovered. Here, we show that systemic overexpression of AT-1 in the mouse leads to a segmental form of progeria with dysmorphism and metabolic alterations. The phenotype includes delayed growth, short lifespan, alopecia, skin lesions, rectal prolapse, osteoporosis, cardiomegaly, muscle atrophy, reduced fertility, and anemia. In terms of homeostasis, the AT-1 overexpressing mouse displays hypocholesterolemia, altered glycemia, and increased indices of systemic inflammation. Mechanistically, the phenotype is caused by a block in Atg9a-Fam134b-LC3ß and Atg9a-Sec62-LC3ß interactions, and defective reticulophagy, the autophagic recycling of the ER. Inhibition of ATase1/ATase2 acetyltransferase enzymes downstream of AT-1 restores reticulophagy and rescues the phenotype of the animals. These data suggest that inappropriately elevated acetyl-CoA flux into the ER directly induces defects in autophagy and recycling of subcellular structures and that this diversion of acetyl-CoA from cytosol to ER is causal in the progeria phenotype. Collectively, these data establish the cytosol-to-ER flux of acetyl-CoA as a novel event that dictates the pace of aging phenotypes and identify intracellular acetyl-CoA-dependent homeostatic mechanisms linked to metabolism and inflammation.

Quaternary Structure Defines a Large Class of Amyloid-ß Oligomers Neutralized by Sequestration.

The accumulation of amyloid-ß (Aß) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimer's disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aß oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aß oligomers in vivo.

Characterization of a Novel Mouse Model of Alzheimer's Disease--Amyloid Pathology and Unique ß-Amyloid Oligomer Profile.

Amyloid plaques composed of ß-amyloid (Aß) protein are a pathological hallmark of Alzheimer's disease. We here report the generation and characterization of a novel transgenic mouse model of Aß toxicity. The rTg9191 mice harbor a transgene encoding the 695 amino-acid isoform of human amyloid precursor protein (APP) with the Swedish and London mutations (APPNLI) linked to familial Alzheimer's disease, under the control of a tetracycline-response element, as well as a transgene encoding the tetracycline transactivator, under the control of the promoter for calcium-calmodulin kinase IIα. In these mice, APPNLI is expressed at a level four-fold that of endogenous mouse APP and its expression is restricted to forebrain regions. Transgene expression was suppressed by 87% after two months of doxycycline administration. Histologically, we showed that (1) Aß plaques emerged in cerebral cortex and hippocampus as early as 8 and 10.5-12.5 months of age, respectively; (2) plaque deposition progressed in an age-dependent manner, occupying up to 19% of cortex at ~25 months of age; and (3) neuropathology--such as abnormal neuronal architecture, tau hyperphosphorylation and misfolding, and neuroinflammation--was observed in the vicinity of neuritic plaques. Biochemically, we determined total Aß production at varied ages of mice, and we showed that mice produced primarily fibrillar Aß assemblies recognized by conformation-selective OC antibodies, but few non-fibrillar oligomers (e.g., Aß*56) detectable by A11 antibodies. Finally, we showed that expression of the tetracycline transactivator resulted in reduced brain weight and smaller dentate-gyrus size. Collectively, these data indicate that rTg9191 mice may serve as a model for studying the neurological effects of the fibrillar Aß assemblies in situ.