Retromer Complex Research Articles

Arabidopsis retromer subunit AtVPS29 is involved in SLY1-mediated gibberellin signaling

Key messageRetromer protein AtVPS29 upregulates the SLY1 protein and downregulates the RGA protein, positively stimulating the development of the root meristematic zone, which indicates an important role of AtVPS29 in gibberellin signaling.In plants, the large retromer complex is known to play roles in multiple development processes, including cell polarity, programmed cell death, and root hair growth in Arabidopsis. However, many of its roles in plant development remain unknown. Here, we show that Arabidopsis trimeric retromer protein AtVPS29 (vacuolar protein sorting 29) modulates gibberellin signaling. The SLEEPY1 (SLY1) protein, known as a positive regulator of gibberellic acid (GA) signaling, exhibited lower abundance in vps29-3 mutants compared to wild-type (WT) plants. Conversely, the DELLA repressor protein, targeted by the E3 ubiquitin ligase SCF (Skp, Cullin, F-box) complex and acting as a negative regulator of GA signaling, showed increased abundance in vps29-3 mutants compared to WT. The vps29-3 mutants exhibited decreased sensitivity to exogenous GA supply in contrast to WT, despite an upregulation in the expression of GA receptor genes within the vps29-3 mutants. In addition, the expression of the GA synthesis genes was downregulated in vps29-3 mutants, implying that the loss of AtVPS29 causes the downregulation of GA synthesis and signaling. Furthermore, vps29-3 mutants exhibited a reduced meristematic zone accompanied by a decreased cell number. Together, these data indicate that AtVPS29 positively regulates SLY1-mediated GA signaling and plant growth.

Partial loss of Sorting Nexin 27 resembles age- and Down syndrome-associated T cell dysfunctions

BackgroundSorting Nexin 27 (SNX27)-retromer complex facilitates cargo recycling from endosomes to the plasma membrane. SNX27 downregulation in neurons, as the result of Trisomy 21 (T21), has been linked with cognitive deficits due to impairment of AMPA and NMDA receptor recycling. Studies in human T cell lines likewise demonstrated that SNX27 regulates the correct delivery of cargoes to the immune synapse limiting the activation of pro-inflammatory pathways. Nevertheless, the physiological consequences of partial SNX27 loss in T cell homeostasis are still unclear.ResultsIn this study, we have explored the consequences of T cell specific partial SNX27 downregulation in mice. T cells with partial SNX27 deficiency show a marked deficit in the CD4+ T cell pool, a hallmark of aging in mice and humans, and a well-characterized comorbidity of individuals with Down syndrome (DS). When analyzed ex vivo, CD4+ T cells with partial SNX27 deletion demonstrate enhanced proliferation but diminished IL-2 production. In contrast, the CD8+ population show enhanced expression of pro-inflammatory cytokines and lytic enzymes.ConclusionsThis mouse model supports the relevance of SNX27 in the organization of the immune synapse, previously described in cell lines, as well as in the control of T cell homeostasis. Individuals with DS experiment an acceleration of the aging process, which particularly affects the immune and central nervous systems. Thus, we hypothesize that reduced SNX27 expression in DS could contribute to the dysregulation of these systems and further research in SNX27 will shed light on the molecular factors underlying the phenotypes observed in people with DS and its contribution to aging.

T‐PEP Related Presentation: Alzheimer’s disease pathogenic protein SORLA forms homodimer potentially required for its function

AbstractBackgroundSo far, many genetic and molecular studies have indicated that the single‐pass transmembrane receptor SORLA can cause Alzheimer’s disease (AD) when mutated, but the detailed mechanism of how SORLA is involved in the pathogenesis of AD is still unknown. SORLA has a unique domain architecture including a huge, multidomain extracellular region with a Vps10p domain and 6 tandem repeats of fibronectin‐type‐III (3Fn) domains plus a short cytoplasmic tail. Although this unique composition must be essential for its potential function as a guiding molecule for protein sorting inside cells, there has been limited information about its overall molecular architecture. We are focusing on the function of SORLA in guiding trafficking along with the retromer complex, and recently found SORLA seems to form homodimers on the cell surface, which correlates with its function.MethodWe employed a highly efficient recombinant expression system for extracellular proteins using mammalian cells that enables detailed biochemical analysis. Using this technique, we produced recombinant secreted protein samples of the whole ectodomain and some fragments derived from human SORLA, and investigated its dimerization property by pull‐down assay followed by SDS PAGE and native PAGE. To investigate the three‐dimensional architecture, we ran a structural prediction using the amino acid sequence of SORLA’s ectodomain only without its signal sequence, propeptide, transmembrane region, and cytoplasmic region, using the program AlphaFold2.ResultOur biochemical experiments showed that human SORLA has homodimer forming property through its Vps10p domain and 3Fn domains. For theVps10p domain especially, it is indicated that the major fraction forms a homodimer in solution. Based on current structural information, we successfully created a mutant that forms covalent bound homodimer with an artificially introduced intermolecular disulfide bond. Interestingly, the predicted model of the ectodomain shows a folded compact structure, indicating that SORLA’s ectodomain forms a large globular shape on the surface of cells.ConclusionOur biochemical experiments showed that SORLA has two putative, opposite‐facing dimeric interfaces. Along with the computational molecular prediction, we propose that SORLA forms polymeric fibril‐like high dimensional structure inside the tubular endosomal membrane, supported by the retromer oligomer from the cytoplasmic side.

SORLA‐retromer complex formation rely on receptor dimerization within the endosome

AbstractBackgroundSORLA acts as an endosome recycling receptor together with the retromer complex to escort cargo through the endo‐lysosomal tubular network. SORLA is encoded by the SORL1 gene, which is the gene most frequently mutated in patients with Alzheimer’s disease, although it is still not clear which of the many variants are (the most) pathogenic. Here, we studied the biochemistry and cell biology of a SORL1 variant that is found in several European families.MethodsWe used transfected cells to study SORLA maturation and shedding of the soluble sSORLA by Western blotting. Expression of non‐mutated and mutated receptor at the cell surface was determined by flow cytometry, and the intracellular localization and overlap with markers of Endoplasmic reticulum and endosome compartments investigated by immunocytochemistry and confocal microscopy. The cellular distribution of receptor dimerization was visualized using bimolecular fluorescence complementation and proximity ligation assays, and quantified using the GFP‐trap method.ResultsWe showed that non‐mutated SORLA forms a dimer in retromer‐coated endosome structures, while the pathogenic SORLA protein is unable to form dimers, although it traffics to the endosome. This lack of dimerization in the endosome correlates with strongly decreased receptor maturation, expression at the cell surface, and shedding of its luminal sSORLA domain.ConclusionSORLA dimer formation is required for its association with retromer, which is important for efficient receptor maturation and delivery to the cell surface. Decreased cell surface expression of mutated protein with impaired dimerization leads to less shedding, showing how maturation and sSORLA production can reliably inform on the pathogenicity of SORL1 genetic variants in Alzheimer’s disease.

The role of membrane trafficking and retromer complex in Parkinson's and Alzheimer's disease.

Membrane trafficking is a physiological process encompassing different pathways involved in transporting cellular products across cell membranes to specific cell locations via encapsulated vesicles. This process is required for cells to mature and function properly, allowing them to adapt to their surroundings. The retromer complex is a complex composed of nexin proteins and peptides that play a vital role in the endosomal pathway of membrane trafficking. In humans, any interference in normal membrane trafficking or retromer complex can cause profound changes such as those seen in neurodegenerative disorders such as Alzheimer's and Parkinson's. Several studies have explored the potential causative mechanisms in developing both disease processes; however, the role of retromer trafficking in their pathogenesis is becoming increasingly significant with promising therapeutic applications. This manuscript describes the processes involved in membrane transport and the roles of the retromer in the onset and progression of Alzheimer's and Parkinson's. Moreover, we will also explore how these aberrant mechanisms may serve as possible avenues for treatment development in both diseases and the prospect of its future application.

VPS35 promotes gastric cancer progression through integrin/FAK/SRC signalling-mediated IL-6/STAT3 pathway activation in a YAP-dependent manner

VPS35 is a key subunit of the retromer complex responsible for recognising cytosolic retrieval signals in cargo and is involved in neurodegenerative disease and tumour progression. However, the function and molecular mechanism of VPS35 in gastric cancer (GC) remains largely unknown. Here, we demonstrated that VPS35 was significantly upregulated in GC, which was associated with poor survival. VPS35 promoted GC cell proliferation and metastasis both in vitro and in vivo. Mechanistically, VPS35 activated FAK-SRC kinases through integrin-mediated outside-in signalling, leading to the activation of YAP and subsequent IL-6 expression induction in tumour cells. What’s more, combined mass spectrometry analysis of MGC-803 cell and bioinformatic analysis, we found that phosphorylation of VPS35 was enhanced in GC cells, and phosphorylated VPS35 has enhanced interaction with ITGB3. VPS35 interacted with ITGB3 and affected the recycling of ITGB3 in GC cells. Gain- and loss-of-function experiments revealed that VPS35 promoted tumour proliferation and metastasis via the IL-6/STAT3 pathway. Interestingly, we also found that STAT3 directly bound to the VPS35 promoter and increased VPS35 transcription, thereby establishing a positive regulatory feedback loop. In addition, we demonstrated that VPS35 knockdown sensitised GC cells to 5-FU and cisplatin. These findings provide evidence that VPS35 promotes tumour proliferation and metastasis, and highlight the potential of targeting VPS35- and IL-6/STAT3-mediated tumour interactions as promising therapeutic strategies for GC.

Identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence.

Vacuolar protein sorting-associated protein 35 is a subunit of the retromer complex, a vital constituent of the endosomal protein sorting pathway. The D620N mutation in the VPS35 gene has been reported to be linked to type 17 Parkinson's Disease progression, the exact molecular mechanism remains to be solved. The scientific community would benefit from the accessibility of validated and high-quality anti-hVPS35 antibodies. In this study, we characterized thirteen hVPS35 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. 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.

A missense mutation in Ehd1 associated with defective spermatogenesis and male infertility.

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7-2.1g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans.

Porcine deltacoronavirus accessory protein NS6 harnesses VPS35-mediated retrograde trafficking to facilitate efficient viral infection.

Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has attracted considerable attention. However, whether retrograde transport is associated with PDCoV infection remains unclear. Our present study demonstrates that retromer protein VPS35 acts as a critical host factor that is required for PDCoV infection. Mechanically, VPS35 interacts with PDCoV NS6, mediating the retrograde transport of NS6 from endosomes to the Golgi and preventing it from lysosomal degradation. Recombinant PDCoVs with an NS6 deletion display resistance to VPS35 deficiency. Our work reveals a novel evasion mechanism of PDCoV that involves the manipulation of the retrograde transport pathway by VPS35, providing new insight into the mechanism of PDCoV infection.

Cell-specific secretory granule sorting mechanisms: the role of MAGEL2 and retromer in hypothalamic regulated secretion.

Intracellular protein trafficking and sorting are extremely arduous in endocrine and neuroendocrine cells, which synthesize and secrete on-demand substantial quantities of proteins. To ensure that neuroendocrine secretion operates correctly, each step in the secretion pathways is tightly regulated and coordinated both spatially and temporally. At the trans-Golgi network (TGN), intrinsic structural features of proteins and several sorting mechanisms and distinct signals direct newly synthesized proteins into proper membrane vesicles that enter either constitutive or regulated secretion pathways. Furthermore, this anterograde transport is counterbalanced by retrograde transport, which not only maintains membrane homeostasis but also recycles various proteins that function in the sorting of secretory cargo, formation of transport intermediates, or retrieval of resident proteins of secretory organelles. The retromer complex recycles proteins from the endocytic pathway back to the plasma membrane or TGN and was recently identified as a critical player in regulated secretion in the hypothalamus. Furthermore, melanoma antigen protein L2 (MAGEL2) was discovered to act as a tissue-specific regulator of the retromer-dependent endosomal protein recycling pathway and, by doing so, ensures proper secretory granule formation and maturation. MAGEL2 is a mammalian-specific and maternally imprinted gene implicated in Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. In this review, we will briefly discuss the current understanding of the regulated secretion pathway, encompassing anterograde and retrograde traffic. Although our understanding of the retrograde trafficking and sorting in regulated secretion is not yet complete, we will review recent insights into the molecular role of MAGEL2 in hypothalamic neuroendocrine secretion and how its dysregulation contributes to the symptoms of Prader-Willi and Schaaf-Yang patients. Given that the activation of many secreted proteins occurs after they enter secretory granules, modulation of the sorting efficiency in a tissue-specific manner may represent an evolutionary adaptation to environmental cues.

CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

VPS35 and α-Synuclein fail to interact to modulate neurodegeneration in rodent models of Parkinson’s disease

BackgroundMutations in the vacuolar protein sorting 35 ortholog (VPS35) gene cause late-onset, autosomal dominant Parkinson’s disease (PD), with a single missense mutation (Asp620Asn, D620N) known to segregate with disease in families with PD. The VPS35 gene encodes a core component of the retromer complex, involved in the endosomal sorting and recycling of transmembrane cargo proteins. VPS35-linked PD is clinically indistinguishable from sporadic PD, although it is not yet known whether VPS35-PD brains exhibit α-synuclein-positive brainstem Lewy pathology that is characteristic of sporadic cases. Prior studies have suggested a functional interaction between VPS35 and the PD-linked gene product α-synuclein in lower organisms, where VPS35 deletion enhances α-synuclein-induced toxicity. In mice, VPS35 overexpression is reported to rescue hippocampal neuronal loss in human α-synuclein transgenic mice, potentially suggesting a retromer deficiency in these mice.MethodsHere, we employ multiple well-established genetic rodent models to explore a functional or pathological interaction between VPS35 and α-synuclein in vivo.ResultsWe find that endogenous α-synuclein is dispensable for nigrostriatal pathway dopaminergic neurodegeneration induced by the viral-mediated delivery of human D620N VPS35 in mice, suggesting that α-synuclein does not operate downstream of VPS35. We next evaluated retromer levels in affected brain regions from human A53T-α-synuclein transgenic mice, but find normal levels of the core subunits VPS35, VPS26 or VPS29. We further find that heterozygous VPS35 deletion fails to alter the lethal neurodegenerative phenotype of these A53T-α-synuclein transgenic mice, suggesting the absence of retromer deficiency in this PD model. Finally, we explored the neuroprotective capacity of increasing VPS35 expression in a viral-based human wild-type α-synuclein rat model of PD. However, we find that the overexpression of wild-type VPS35 is not sufficient for protection against α-synuclein-induced nigral dopaminergic neurodegeneration, α-synuclein pathology and reactive gliosis.ConclusionCollectively, our data suggest a limited interaction of VPS35 and α-synuclein in neurodegenerative models of PD, and do not provide support for their interaction within a common pathophysiological pathway.

4-Oxo-2-nonenal-Induced α-Synuclein Oligomers Interact with Membranes in the Cell, Leading to Mitochondrial Fragmentation.

Oxidative stress and formation of cytotoxic oligomers by the natively unfolded protein α-synuclein (α-syn) are both connected to the development of Parkinson's disease. This effect has been linked to lipid peroxidation and membrane disruption, but the specific mechanisms behind these phenomena remain unclear. To address this, we have prepared α-syn oligomers (αSOs) in vitro in the presence of the lipid peroxidation product 4-oxo-2-nonenal and investigated their interaction with live cells using in-cell NMR as well as stimulated emission depletion (STED) super-resolution and confocal microscopy. We find that the αSOs interact strongly with organellar components, leading to strong immobilization of the protein's otherwise flexible C-terminus. STED microscopy reveals that the oligomers localize to small circular structures inside the cell, while confocal microscopy shows mitochondrial fragmentation and association with both late endosome and retromer complex before the SOs interact with mitochondria. Our study provides direct evidence for close contact between cytotoxic α-syn aggregates and membraneous compartments in the cell.

Retromer Stabilization Improves Cognitive Function and Synaptic Plasticity in a Mouse Model of Down Syndrome.

Retromer complex proteins are decreased in postmortem brain tissues from Down syndrome subjects and inversely correlate with the Alzheimer's disease-like neuropathology. However, whether targeting in vivo the retromer system affects cognitive deficits and synaptic function in Down syndrome remains unknown. The aim of the current study was to examine the effects of pharmacological retromer stabilization on cognitive and synaptic functions in a mouse model of Down syndrome. Ts65dn mice were administered the pharmacological chaperone, TPT-172, or vehicle from 4 to 9 months of age and then assessed for changes in cognitive function. To assess the effects of TPT-172 on synaptic plasticity, hippocampal slices from Ts65dn mice were incubated in TPT-172 and used for field potential recordings. Chronic TPT-172 treatment improved performance in cognitive function tests, its incubation with hippocampal slices ameliorated synaptic function response. Pharmacological stabilization of the retromer complex improves synaptic plasticity and memory in a mouse model of Down syndrome. These results support the therapeutic potential of pharmacological retromer stabilization for individual with Down syndrome.

Diversity of retromer-mediated vesicular trafficking pathways in plants.

The plant endomembrane system is organized and regulated by large gene families that encode proteins responsible for the spatiotemporal delivery and retrieval of cargo throughout the cell and to and from the plasma membrane. Many of these regulatory molecules form functional complexes like the SNAREs, exocyst, and retromer, which are required for the delivery, recycling, and degradation pathways of cellular components. The functions of these complexes are well conserved in eukaryotes, but the extreme expansion of the protein subunit families in plants suggests that plant cells require more regulatory specialization when compared with other eukaryotes. The retromer is associated with retrograde sorting and trafficking of protein cargo back towards the TGN and vacuole in plants, while in animals, there is new evidence that the VPS26C ortholog is associated with recycling or 'retrieving' proteins back to the PM from the endosomes. The human VPS26C was shown to rescue vps26c mutant phenotypes in Arabidopsis thaliana, suggesting that the retriever function could be conserved in plants. This switch from retromer to retriever function may be associated with core complexes that include the VPS26C subunit in plants, similar to what has been suggested in other eukaryotic systems. We review what is known about retromer function in light of recent findings on functional diversity and specialization of the retromer complex in plants.

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Rab7/Retromer-based endolysosomal trafficking is essential for proper host invasion in rice blast.

Secretion is a fundamental process that plant pathogens utilize to deliver effectors into the host to downregulate immunity and promote infection. Here, we uncover a fascinating membrane trafficking and delivery route that originates from vacuolar membranes in Magnaporthe oryzae and conduits to the host interface and plasma membrane. To perform such secretory/trafficking function, MoRab7 first recruits the retromer complex to the vacuolar membrane, enabling recognition of a family of SNARE proteins, including MoSnc1. Live-cell imaging confirmed a highly dynamic vesicular trafficking of the retromer complex component(s) and MoSnc1 toward and across the host interface or plasma membrane, and subsequent fusion with target membranes. Interestingly, disruption of the MoRab7/Retromer/MoSnc1-based endolysosomal cascade affects effector secretion and fungal pathogenicity. Taken together, we discovered an unconventional protein and membrane trafficking route starting from the fungal endolysosomes to the M. oryzae-rice interaction interface and dissect the role of MoRab7/Retromer/MoSnc1 sorting machinery in effector secretion during biotrophy and invasive growth in rice blast fungus.

Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two sub-assemblies: Retriever composed of VPS35L, VPS26C, and VPS29; and the CCC complex which contains twelve subunits: COMMD1-COMMD10 and the coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy, and in silico predictions, we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilized by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a 16th subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

Retromer involvement in trafficking of the calcium-activated K channel, KCa3.1

The intermediate-conductance Ca 2+ -activated K + channel KCa3.1 ( KCNN4) regulates many physiological processes, including, water and electrolyte transport in polarised epithelia and vascular tone. Crucial for KCa3.1 function is the regulation of the number of channels at the plasma membrane. Even though, the mechanism of trafficking of KCa3.1 to and from the basolateral membrane (BLM) of epithelial cells has been advanced recently, there is opposing evidence suggesting that KCa3.1 is recycled back to the leading edge of migrating cells; while others have reported that KCa3.1 does not enter recycling endosomes, but is targeted for degradation. The highly conserved multi-protein complex Retromer has been demonstrated to regulate the membrane retrieval and recycling of many membrane-bound proteins, including ion channels. Using mass-spectrometry, it was reported that when VPS35, a core protein of the Retromer complex, was knocked-down, the membrane expression of KCa3.1 was reduced 1.4 fold in HeLa cells; suggesting a link between Retromer and trafficking of KCa3.1. In contrast, our data indicate KCa3.1 was not affected by knockdown of VPS35 in our FRT (Fischer rat thyroid)-KCa3.1 stable cell line. Here, we have examined other key components (sorting nexins SNX1, SNX4, SNX27; cargo binding proteins) of the Retromer complex to determine if those proteins aid in trafficking of KCa3.1 directly to the BLM. We conducted immunoblot and Ussing chamber experiments to examine the effects of knockdown of SNX 1, SNX2 or SNX27 on the BLM population of KCa3.1 and functional expression of the channel (K + current), respectively. Using our FRT-KCa3.1 cell line, we demonstrate knockdown of SNX1, SNX4 or SNX27 decreases KCa3.1 current. Further, we demonstrate knockdown of SNX4 or SNX27 decreases BLM expression of KCa3.1, consistent with the decrease in current observed.These data, along with our previous data, strongly indicate that the role of Retromer in the trafficking of KCa3.1 to the BLM of epithelial cells is quite dynamic. Reducing SNX4 or SNX27 have notable effects on trafficking of KCa3.1. While knockdown of SNX1 might affect the trafficking of KCa3.1 by altering other proteins of the Retromer complex. This work was supported by the National Institutes of Health grants HL083060 and HL092157 to DCD, Lottery Health New Zealand (R-LHR-2019-101706) to FJM, Aim Fund grant to KLH, and the Department of Physiology, University of Otago. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Retromer Proteins Reduced in Down Syndrome and the Dp16 Model: Impact of APP Dose and Preclinical Studies of a γ-Secretase Modulator.

The retromer complex plays an essential role in intracellular endosomal sorting. Deficits in the retromer complex are linked to enhanced Aβ production. The levels of the components of the retromer complex are reported to be downregulated in Alzheimer disease (AD). Down syndrome (DS) shares neuropathological features with AD. Recent evidence points to dysregulation of the retromer complex in DS. The mechanisms underlying retromer deficits in DS and AD are poorly understood. We measured the levels of retromer components in the frontal cortex of cases of DS-AD (AD in DS) as well as DS; the frontal cortex of a person partially trisomic (PT-DS) for human chromosome 21 (HSA21), whose genome had only the normal 2 copies of the APP gene, was also examined. We also analyzed these proteins in the Dp16 mouse model of DS. To further explore the molecular mechanism for changes in the retromer complex, we treated Dp16 mice with a γ-secretase modulator (GSM; 776890), a treatment that reduces the levels of Aβ42 and Aβ40. We found VPS26A, VPS26B, and VPS29, but not VPS35, were significantly reduced in both DS and DS-AD, but not in PT-DS. Downregulation of VPS26A, VPS26B, and VPS29 was recapitulated in the brains of old Dp16 mice (at 16 months of age) and required increased App gene dose. Significantly, GSM treatment completely prevented reductions of the retromer complex. Our studies point to increased APP gene dose as a compromising retromer function in DS and suggest a causal role for Aβ42 and Aβ40. ANN NEUROL 2023;94:245-258.