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Degeneration of Purkinje cells in KO mice. a Immunofluorescence labeling of cerebellar cryosections of WT and KO littermates at P16, P25, P30, and P42 using Calbindin antibody (green) and DAPI (blue). Higher-magnification images from lobule VI/VII were shown in the low panel. Arrows pointed to lobule X with no obvious Purkinje cell loss. Lobules are indicated by Roman numerals. b Quantification of the Calbindin labeled Purkinje cells in different ages (P16, P20, P25, P30, and P42). The number of PC declined from P20 in Tmem30a KO. n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001. The data represent means ± SEM. c Calbindin immunostaining (left: DAB staining) revealed loss of dendrites in Tmem30a KO Purkinje cells in lobule V with loss of PC dendrites (left) and deformed, degenerating spiny branchlets (right, arrows) compared with WT control. Boxed areas at the right were shown at higher-magnification as middle insets. d Calbindin immunostaining further revealed axonal spheroids (torpedoes) in the initial axonal segment (left, arrows) and white matter (right, arrows) of KO cerebellum, indicating cell degeneration. e Representative transmission electron microscope (TEM) images of axons in the P16 WT (E1) and KO (E2, E3) white matter in the cerebellar cortex. Arrow in E1 indicates normal WT axon. Arrows in E2 and E3 indicate representative degenerative axons
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Phospholipids are asymmetrically distributed across mammalian plasma membrane with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer. This asymmetric distribution is dependent on a group of P4-ATPases named PS flippases. The proper transport and function of PS flippases require a β-...
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... of the ML was owing to PC loss. Moreover, loss of cells from the internal granular layer might also contribute to the macroscopic cerebellar atrophy observed at subsequent times ( Fig. 2d-f). In contrast, the cerebella from the Tmem30a heterozygous animals appeared structurally normal compared with those from their age-matched control littermates (Fig. S3A, ...
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... and KO mice at P7 (Fig. S4A). In contrast, at P9, character- istics of dystrophic neurons, including shrunken soma and developmental retardation of dendrites, were observed, but no obvious PC loss was evident at this stage (Fig. S4B). PC loss was first detected in the KO cerebellum at P16, and PC degeneration became more pronounced over time (Fig. 3a). At P25, ~ 50% of the PCs had been lost, which is consistent with the reduced thickness of the ML and the early onset of ataxia in the KO mice (Fig. 3b). Interestingly, except for the PCs in lobule X, no PCs survived after P42 (Fig. 3a, arrows). Considering that Tmem30a was also deleted in lobule X, metabolic differ- ences might exist ...
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... dendrites, were observed, but no obvious PC loss was evident at this stage (Fig. S4B). PC loss was first detected in the KO cerebellum at P16, and PC degeneration became more pronounced over time (Fig. 3a). At P25, ~ 50% of the PCs had been lost, which is consistent with the reduced thickness of the ML and the early onset of ataxia in the KO mice (Fig. 3b). Interestingly, except for the PCs in lobule X, no PCs survived after P42 (Fig. 3a, arrows). Considering that Tmem30a was also deleted in lobule X, metabolic differ- ences might exist between lobule X and other parts of the cerebellum 47 . Calbindin immunolabeling also revealed the details of the dendritic and axonal degeneration in ...
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... PC loss was first detected in the KO cerebellum at P16, and PC degeneration became more pronounced over time (Fig. 3a). At P25, ~ 50% of the PCs had been lost, which is consistent with the reduced thickness of the ML and the early onset of ataxia in the KO mice (Fig. 3b). Interestingly, except for the PCs in lobule X, no PCs survived after P42 (Fig. 3a, arrows). Considering that Tmem30a was also deleted in lobule X, metabolic differ- ences might exist between lobule X and other parts of the cerebellum 47 . Calbindin immunolabeling also revealed the details of the dendritic and axonal degeneration in the KO mice during the early period of PC degeneration. The Lobules were indicated by Roman ...
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... number of GC in WT and KO mice (n = 5-7; ***, p < 0.001). The data represent means ± SEM DAB staining of paraffin sections of the Tmem30a KO cerebella showed extensive degeneration of the dendrites prior to the cell body loss at P25, including a decrease in soma size and a marked reduction in the region occupied by dendrites in the KO cerebellum (Fig. 3c). However, these features of PC degeneration were absent in the Tmem30a heterozygous mice (Fig. S3A, ...
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... of paraffin sections of the Tmem30a KO cerebella showed extensive degeneration of the dendrites prior to the cell body loss at P25, including a decrease in soma size and a marked reduction in the region occupied by dendrites in the KO cerebellum (Fig. 3c). However, these features of PC degeneration were absent in the Tmem30a heterozygous mice (Fig. S3A, ...
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... examination of the PC dendrites at a high magnifi- cation revealed a substantial decrease in dendritic spine density and significant morphological changes in both the apical and basal dendrites of the KO PCs (Fig. 3c, arrows). These changes resulted in the loss of synaptic contacts between the PCs and target neurons. Calbindin immunolabeling also identified axonal swellings in the internal GCL and within the core of the cerebellar white matter in the KO mice (Fig. 3d, arrows). Such swellings are considered a hallmark of axonal dystrophy 48 . In con- trast, ...
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... and significant morphological changes in both the apical and basal dendrites of the KO PCs (Fig. 3c, arrows). These changes resulted in the loss of synaptic contacts between the PCs and target neurons. Calbindin immunolabeling also identified axonal swellings in the internal GCL and within the core of the cerebellar white matter in the KO mice (Fig. 3d, arrows). Such swellings are considered a hallmark of axonal dystrophy 48 . In con- trast, the axons in the cerebella from the WT mice were slender and displayed a uniform caliber (Fig. 3d, left). The PC axons in the KO mice also degenerated as shown by the reduced number of axons present in the white matter in the KO mice compared with that in ...
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... target neurons. Calbindin immunolabeling also identified axonal swellings in the internal GCL and within the core of the cerebellar white matter in the KO mice (Fig. 3d, arrows). Such swellings are considered a hallmark of axonal dystrophy 48 . In con- trast, the axons in the cerebella from the WT mice were slender and displayed a uniform caliber (Fig. 3d, left). The PC axons in the KO mice also degenerated as shown by the reduced number of axons present in the white matter in the KO mice compared with that in the control cere- bella (Fig. 3d, right). To further confirm the degeneration of the PC axons, the cerebellar GCL tracts in the KO and WT mice were examined by TEM. The electron micro- ...
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... are considered a hallmark of axonal dystrophy 48 . In con- trast, the axons in the cerebella from the WT mice were slender and displayed a uniform caliber (Fig. 3d, left). The PC axons in the KO mice also degenerated as shown by the reduced number of axons present in the white matter in the KO mice compared with that in the control cere- bella (Fig. 3d, right). To further confirm the degeneration of the PC axons, the cerebellar GCL tracts in the KO and WT mice were examined by TEM. The electron micro- graphs revealed axonal pathologies, including the , and P42). The number of PC declined from P20 in Tmem30a KO. n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001. The data represent means ± SEM. ...
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... of KO cerebellum, indicating cell degeneration. e Representative transmission electron microscope (TEM) images of axons in the P16 WT (E1) and KO (E2, E3) white matter in the cerebellar cortex. Arrow in E1 indicates normal WT axon. Arrows in E2 and E3 indicate representative degenerative axons accumulation of organelles in swollen nerve fibers (Fig. 3E3), the presence of vacant areas, and autophagosome-like bodies owing to degeneration of nerve fibers (Fig. 3E2). These changes were widespread in the white matter of the KO cerebella but completely absent from the same regions of the WT cerebella (Fig. 3E1), confirming the immunofluorescence results mentioned above. The degeneration of ...
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... probed with an antibody against ER stress markers BiP and CHOP, GAPDH antibody as loading control. Sample size n = 6, ***, p < 0.001. The data represent means ± SEM caspase-3, which is an important marker of cell apoptosis, was elevated from P16 to P20 (Fig. 6a-d). This is con- sistent with the rapid cell loss that occurred after P16 (Fig. 3a, b). In addition, the number of TUNEL-positive cells in the KO cerebella at P20 was higher (Fig. 6e, arrows) than that in the control cerebella. Collectively, loss of Tmem30a in PCs induced the activation of the ER stress pathway and subsequent cell apoptosis, explaining the rapid loss of PCs in the KO ...
Citations
... [5][6][7] Tmem30a deficiency can cause retinal bipolar cell apoptosis, anemia, cerebellar ataxia, and reduced angiogenesis. [8][9][10][11] Moreover, our previous study showed that podocytespecific Tmem30a loxP/loxP ; NPHS2-Cre mice exhibited albuminuria, podocyte damage and loss, and mesangial cell proliferation with substantial extracellular matrix accumulation, which eventually developed into FSGS. 12 However, the specific effects and mechanisms of Tmem30a in podocyte injury remain unclear. ...
... We found that KLF15 expression was higher in the cerebellum, especially the Purkinje cells, and knocking down cerebellar KLF15 resulted in the loss of Purkinje cells. Several transcription factors such as Znf212 [38], Smad2 [39], and Tmem30a [42] have been reported to be involved in the development of Purkinje cells. The absence of these transcription factors can cause abnormal cerebellar development and loss of Purkinje cells, ultimately leading to ataxia in mice. ...
Kruppel-like factor 15 (KLF15), a member of the KLF family, is closely involved in many biological processes. However, the mechanism by which KLF15 regulates neural development is still unclear. Considering the complexity and importance of neural network development, in this study, we investigated the potent regulatory role of KLF15 in neural network development. KLF15 was detected highly expressed in the cerebellum and enriched in Purkinje cells, with a significant increase in KLF15 expression between 15–20 days of neural development. Knockdown of KLF15 led to loss of Purkinje cells and impaired motility in mice. Therefore, our study aims to elucidate the relationship between KLF15 and Purkinje cells in mice, may provide a new research idea for the developmental mechanism of the mouse cerebellum.
... This neurotoxin undergoes enzymatic action in the brain, where its metabolites lead to the functional loss of niacinamide and NADP (nicotinamide adenine dinucleotide phosphate) with the subsequent hindrance of the transfer of H + ions in niacinamide/NADP-dependent enzymatic reactions. It ultimately compromises the synthesis pathway of adenosine triphosphate (ATP) through the mitochondrial respiratory chain [9,10]. ...
Cerebellar ataxia is a heterogeneous group of neural disorders clinically characterized by cerebellar dysfunction. The diagnosis of patients with progressive cerebellar ataxia is complex due to the direct correlation with other neuron diseases. Although there is still no cure for this pathological condition, some metabolic, hereditary, inflammatory, and immunological factors affecting cerebellar ataxia are being studied and may become therapeutic targets. Advances in studying the neuroanatomy, pathophysiology, and molecular biology of the cerebellum (CE) contribute to a better understanding of the mechanisms behind the development of this disorder. In this study, Wistar rats aged 30 to 35 days were injected intraperitoneally with 3-acetylpyridine (3-AP) and/or metformin (for AMP-activated protein kinase (AMPK) enzyme activation) and euthanized in 24 hours and 4 days after injection. We analyzed the neuromodulatory role of the AMPK on cerebellar ataxia induced by the neurotoxin 3-AP in the brain stem (BS) and CE, after pre-treatment for 7 and 15 days with metformin, a pharmacological indirect activator of AMPK. The results shown here suggest that AMPK activation in the BS and CE leads to a significant reduction in neuroinflammation in these regions. AMPK was able to restore the changes in fatty acid composition and pro-inflammatory cytokines caused by 3-AP, suggesting that the action of AMPK seems to result in a possible neuroprotection on the cerebellar ataxia model.
... Purkinje cells (PC) are the only neurons in the cerebellar cortex capable of transmitting impulses, and Tmem30a Purkinje cell-(PC-) specific knockout (KO) mouse model was used to study the role of this gene in the cerebellum [34]. 2 Journal of Lipids ...
Phospholipids are asymmetrically distributed across mammalian plasma membrane. The function of P4-ATPases is to maintain the abundance of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in the inner leaflet as lipid flippases. Transmembrane protein 30A (TMEM30A, also named CDC50A), as an essential β subunit of most P4-ATPases, facilitates their transport and functions. With TMEM30A knockout mice or cell lines, it is found that the loss of TMEM30A has huge influences on the survival of mice and cells because of PS exposure-triggered apoptosis signaling. TMEM30A is a promising target for drug discovery due to its significant roles in various systems and diseases. In this review, we summarize the functions of TMEM30A in different systems, present current understanding of the protein structures and mechanisms of TMEM30A-P4-ATPase complexes, and discuss how these fundamental aspects of TMEM30A may be applied to disease treatment.
... In particular, TMEM30A can form a complex with ATP8A2 and transports PS and PE across the membrane [230,280,281]. Deletion of TMEM30A from mouse cerebellar Purkinje cells was shown to cause early onset cerebellar ataxia [282], while ATP8A2 mutations are associated with CAMRQ syndrome characterized by cerebellar ataxia and quadrupedal locomotion [283][284][285]. Interestingly, although ATP8A2 is most abundant in the cerebellum, it is not expressed in Purkinje cells, but is expressed in deep cerebellar nuclei [282]. ...
... Deletion of TMEM30A from mouse cerebellar Purkinje cells was shown to cause early onset cerebellar ataxia [282], while ATP8A2 mutations are associated with CAMRQ syndrome characterized by cerebellar ataxia and quadrupedal locomotion [283][284][285]. Interestingly, although ATP8A2 is most abundant in the cerebellum, it is not expressed in Purkinje cells, but is expressed in deep cerebellar nuclei [282]. Thus, although both ATP8A2 and TMEM30A deficiencies cause cerebellar ataxia, the underlying cellular mechanisms differ. ...
... However, at the molecular level, both mutants cause phospholipid dyshomeostasis, especially loss of PS asymmetry on the plasma membrane, which can cause cell death [286,287]. Additionally, since TMEM30A is involved in ER exit and proper targeting of several P4-ATPases [230,282,288], its dysfunction can cause accumulation of related P4-ATPases in the ER, thereby leading to ER stress. Indeed, in the absence of TMEM30A, the expression levels of CHOP and BiP are elevated in Purkinje cells prior to visible cell loss [282]. ...
Cerebellar ataxia is a form of ataxia that originates from dysfunction of the cerebellum, but may involve additional neurological tissues. Its clinical symptoms are mainly characterized by the absence of voluntary muscle coordination and loss of control of movement with varying manifestations due to differences in severity, in the site of cerebellar damage and in the involvement of extracerebellar tissues. Cerebellar ataxia may be sporadic, acquired, and hereditary. Hereditary ataxia accounts for the majority of cases. Hereditary ataxia has been tentatively divided into several subtypes by scientists in the field, and nearly all of them remain incurable. This is mainly because the detailed mechanisms of these cerebellar disorders are incompletely understood. To precisely diagnose and treat these diseases, studies on their molecular mechanisms have been conducted extensively in the past. Accumulating evidence has demonstrated that some common pathogenic mechanisms exist within each subtype of inherited ataxia. However, no reports have indicated whether there is a common mechanism among the different subtypes of inherited cerebellar ataxia. In this review, we summarize the available references and databases on neurological disorders characterized by cerebellar ataxia and show that a subset of genes involved in lipid homeostasis form a new group that may cause ataxic disorders through a common mechanism. This common signaling pathway can provide a valuable reference for future diagnosis and treatment of ataxic disorders.
... Cerebellar degeneration is closely associated with cerebellar motor dysfunction or ataxia (Yang et al., 2018;Liu et al., 2020). The progressive and permanent loss of PCs is a hallmark of many ataxias (Yang et al., 2015). ...
Proper functioning of the cerebellum is crucial to motor balance and coordination in adult mammals. Purkinje cells (PCs), the sole output neurons of the cerebellar cortex, play essential roles in cerebellar motor function. Tripartite motif-containing protein 32 (TRIM32) is an E3 ubiquitin ligase that is involved in balance activities of neurogenesis in the subventricular zone of the mammalian brain and in the development of many nervous system diseases, such as Alzheimer's disease, autism spectrum disorder, attention deficit hyperactivity disorder. However, the role of TRIM32 in cerebellar motor function has never been examined. In this study we found that motor balance and coordination of mid-aged TRIM32 deficient mice were poorer than those of wild-type littermates. Immunohistochemical staining was performed to assess cerebella morphology and TRIM32 expression in PCs. Golgi staining showed that the extent of dendritic arborization and dendritic spine density of PCs were decreased in the absence of TRIM32. The loss of TRIM32 was also associated with a decrease in the number of synapses between parallel fibers and PCs, and in synapses between climbing fibers and PCs. In addition, deficiency of TRIM32 decreased Type I inositol 1,4,5-trisphosphate 5-phosphatase (INPP5A) levels in cerebellum. Overall, this study is the first to elucidate a role of TRIM32 in cerebellar motor function and a possible mechanism, thereby highlighting the importance of TRIM32 in the cerebellum.
... The TMEM30 (also called CDC50) family includes TMEM30A, TMEM30B and TMEM30C, of which TMEM30A interacts with 11 of the 14 P4-ATPases [9][10][11][12][13]. Our previous studies have demonstrated that TMEM30A deficiency causes a series of disorders: retarded retinal angiogenesis, Purkinje cell, retinal bipolar cell and photoreceptor cell degeneration, impaired foetal liver erythropoiesis, intrahepatic cholestasis and chronic myeloid leukaemia [5,[14][15][16][17][18][19]. ...
... A conditional knockout (cKO) allele carrying a floxed Tmem30a allele (Tmem30a loxp/loxp ) has previously been described [15][16][17] ...
... Compared with the normal controls, the significantly reduced TMEM30A expression was observed in the glomeruli of patients with MCD or MN ( Fig. 1 B, C) and rod bipolar cells [16,17]. In the cerebellum, Tmem30a loss results in early-onset ataxia and cerebellar atrophy [15]. In the liver, Tmem30a deficiency impairs mouse foetal liver erythropoiesis and causes intrahepatic cholestasis by affecting the normal expression and localization of bile salt transporters and causes intrahepatic cholestasis [5,18]. ...
Phosphatidylserine (PS) is asymmetrically concentrated in the cytoplasmic leaflet of eukaryotic cell plasma membranes. This asymmetry is regulated by a group of P4 ATPases (named PS flippases) and its β-subunit TMEM30A. The disruption of PS flippase leads to severe human diseases. Tmem30a is essential in the mouse retina, cerebellum and liver. However, the role of Tmem30a in the kidney, where it is highly expressed, remains unclear. Podocytes in the glomerulus form a branched interdigitating filtration barrier that can prevent the traversing of large cellular elements and macromolecules from the blood into the urinary space. Damage to podocytes can disrupt the filtration barrier and lead to proteinuria and podocytopathy, including focal segmental glomerulosclerosis, minimal change disease, membranous nephropathy, and diabetic nephropathy. We observed reduced TMEM30A expression in patients with minimal change disease and membranous nephropathy, indicating potential roles of TMEM30A in podocytopathy. To investigate the role of Tmem30a in the kidney, we generated a podocyte-specific Tmem30a knockout (KO) mouse model using the NPHS2-Cre line. Tmem30a KO mice displayed albuminuria, podocyte degeneration, mesangial cell proliferation with prominent extracellular matrix accumulation and eventual progression to focal segmental glomerulosclerosis (FSGS). Our data demonstrate a critical role of Tmem30a in maintaining podocyte survival and glomerular filtration barrier integrity. Understanding the dynamic regulation of the PS distribution in the glomerulus provides a unique perspective to pinpoint the mechanism of podocyte damage and potential therapeutic targets.
... Although Cul3 haploinsufficient mice have a slightly reduced body weight at birth, their weight is comparable to control animals as adults ( Supplementary Fig. 1c), while the brain to body weight ratio is unaffected in mutant newborn and adult mice ( Supplementary Fig. 1d). Adult Cul3 +/− mice present with hind limb clasping (Fig. 1a) and mild gait abnormalities, such as increased sway and stance length (Fig. 1b, c and Supplementary Fig. 2a), phenotypes which are observed in other ASD mouse models 19,20 and indicative of cerebellar dysfunctions 21 . Further indicating motor defects, Cul3 +/− mice underperform when challenged on the accelerating RotaRod (Fig. 1d, d′), a task requiring formation and consolidation of a repetitive motor routine 22,23 . ...
De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs. De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). Here, the authors show that Cul3 is essential to regulate neuronal migration by tightly regulating Plastin3 (Pls3). Pls3 cell-autonomously regulates cell migration by regulating the actin cytoskeleton organization.
... 11,[13][14][15][16] Moreover, emerging studies using mouse models have indicated that Tmem30a deficiency leads to multiple disease phenotypes in various tissues, including retina, liver, and cerebellum. [17][18][19][20][21][22] These studies highlight the fundamental roles of Tmem30a in maintaining the normal physiology of various tissues, which function via vesicle-mediated protein transport. However, the role of Tmem30a in the endocrine system, another system with high secretory activity, remains elusive. ...
... To corroborate a previous study in which Tmem30a expression was shown in human and rat pancreatic islets, 27 we began by examining the expression of Tmem30a in mouse pancreatic islets. Frozen sections of mouse pancreas were double-immunostained with a specific antibody against TMEM30A [17][18][19]21,22 and an insulin antibody to mark b cells. Confocal imaging confirmed that TMEM30A is expressed in pancreatic islets, including in insulinmarked b cells ( Figure 1A), implying an association between Tmem30a expression and insulin synthesis and/or secretion. ...
... Mice with a Tmem30a deletion specifically in pancreatic b cells were generated as previously described. [17][18][19][20][21][22] Briefly, Tmem30a loxP/loxP mice were crossed with Tg(Ins2-Cre) 25Mgn (also known as Rip-Cre, Jackson Laboratory, stock no. 003573) transgenic mice, to yield progeny with the genotype Tmem30a loxP/+ ; Ins2-Cre. ...
The processing, maturation and secretion of insulin are under precise regulation, and dysregulation causes profound defects in glucose handling, leading to diabetes. Tmem30a is the β subunit of the phosphatidylserine (PS) flippase, which maintains the membrane asymmetric distribution of PS. Tmem30a regulates cell survival and the localization of subcellular structures, and is thus critical to the normal function of multiple physiological systems. Here, we show that conditional knockout of Tmem30a specifically in pancreatic islet β cells leads to obesity, hyperglycemia, glucose intolerance, hyperinsulinemia and insulin resistance in mice, due to insufficient insulin release. Moreover, we reveal that Tmem30a plays an essential role in clathrin-mediated vesicle transport between the trans Golgi network (TGN) and the PM, which comprises immature secretory granule (ISG) budding at the TGN. We also find that Tmem30a deficiency impairs clathrin-mediated vesicle budding, and thus blocks both insulin maturation in ISGs and the transport of glucose-sensing Glut2 to the PM. Collectively, these disruptions compromise both insulin secretion and glucose sensitivity, thus contributing to impairments in glucose-stimulated insulin secretion. Taken together, our data demonstrate an important role of Tmem30a in insulin maturation and glucose metabolic homeostasis, and suggest the importance of membrane phospholipid distribution in metabolic disorders.
... 11,[13][14][15][16] Moreover, emerging studies using mouse models have indicated that Tmem30a deficiency leads to multiple disease phenotypes in various tissues, including retina, liver, and cerebellum. [17][18][19][20][21][22] These studies highlight the fundamental roles of Tmem30a in maintaining the normal physiology of various tissues, which function via vesicle-mediated protein transport. However, the role of Tmem30a in the endocrine system, another system with high secretory activity, remains elusive. ...
... To corroborate a previous study in which Tmem30a expression was shown in human and rat pancreatic islets, 27 we began by examining the expression of Tmem30a in mouse pancreatic islets. Frozen sections of mouse pancreas were double-immunostained with a specific antibody against TMEM30A [17][18][19]21,22 and an insulin antibody to mark b cells. Confocal imaging confirmed that TMEM30A is expressed in pancreatic islets, including in insulinmarked b cells ( Figure 1A), implying an association between Tmem30a expression and insulin synthesis and/or secretion. ...
... Mice with a Tmem30a deletion specifically in pancreatic b cells were generated as previously described. [17][18][19][20][21][22] Briefly, Tmem30a loxP/loxP mice www.moleculartherapy.org were crossed with Tg(Ins2-Cre) 25Mgn (also known as Rip-Cre, Jackson Laboratory, stock no. 003573) transgenic mice, to yield progeny with the genotype Tmem30a loxP/+ ; Ins2-Cre. ...
The processing, maturation and secretion of insulin are under precise regulation, and dysregulation causes profound defects in glucose handling, leading to diabetes. Tmem30a is the β subunit of the phosphatidylserine (PS) flippase, which maintains the membrane asymmetric distribution of PS. Tmem30a regulates cell survival and the localization of subcellular structures, and is thus critical to the normal function of multiple physiological systems. Here, we show that conditional knockout of Tmem30a specifically in pancreatic islet β cells leads to obesity, hyperglycemia, glucose intolerance, hyperinsulinemia and insulin resistance in mice, due to insufficient insulin release. Moreover, we reveal that Tmem30a plays an essential role in clathrin-mediated vesicle transport between the trans Golgi network (TGN) and the PM, which comprises immature secretory granule (ISG) budding at the TGN. We also find that Tmem30a deficiency impairs clathrin-mediated vesicle budding, and thus blocks both insulin maturation in ISGs and the transport of glucose-sensing Glut2 to the PM. Collectively, these disruptions compromise both insulin secretion and glucose sensitivity, thus contributing to impairments in glucose-stimulated insulin secretion. Taken together, our data demonstrate an important role of Tmem30a in insulin maturation and glucose metabolic homeostasis, and suggest the importance of membrane phospholipid distribution in metabolic disorders.
