Polyamine Homeostasis in Snyder-Robinson Syndrome

Loss-of-function mutations of the spermine synthase gene (SMS) result in Snyder-Robinson Syndrome (SRS), a recessive X-linked syndrome characterized by intellectual disability, osteoporosis, hypotonia, speech abnormalities, kyphoscoliosis, and seizures. As SMS catalyzes the biosynthesis of the polyamine spermine from its precursor spermidine, SMS deficiency causes a lack of spermine with an accumulation of spermidine. As polyamines, spermine, and spermidine play essential cellular roles that require tight homeostatic control to ensure normal cell growth, differentiation, and survival. Using patient-derived lymphoblast cell lines, we sought to comprehensively investigate the effects of SMS deficiency on polyamine homeostatic mechanisms including polyamine biosynthetic and catabolic enzymes, derivatives of the natural polyamines, and polyamine transport activity. In addition to decreased spermine and increased spermidine in SRS cells, ornithine decarboxylase activity and its product putrescine were significantly decreased. Treatment of SRS cells with exogenous spermine revealed that polyamine transport was active, as the cells accumulated spermine, decreased their spermidine level, and established a spermidine-to-spermine ratio within the range of wildtype cells. SRS cells also demonstrated elevated levels of tissue transglutaminase, a change associated with certain neurodegenerative diseases. These studies form a basis for further investigations into the leading biochemical changes and properties of SMS-mutant cells that potentially represent therapeutic targets for the treatment of Snyder-Robinson Syndrome.

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Polyamine Homeostasis in Snyder-Robinson Syndrome

10.20944/preprints201811.0422.v1 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tracy Murray Stewart ◽

Matthew Dunworth ◽

Jackson R. Foley ◽

Charles E. Schwartz ◽

Robert A. Casero, Jr.

Keyword(s):

Tissue Transglutaminase ◽

Decarboxylase Activity ◽

Loss Of Function ◽

Transport Activity ◽

Spermine Synthase ◽

Catabolic Enzymes ◽

Polyamine Transport ◽

Exogenous Spermine ◽

Mutant Cells ◽

Derivatives Of

Loss-of-function mutations of the spermine synthase gene (SMS) result in Snyder-Robinson Syndrome (SRS), a recessive X-linked syndrome characterized by intellectual disability, osteoporosis, hypotonia, speech abnormalities, kyphoscoliosis, and seizures. As SMS catalyzes the biosynthesis of the polyamine spermine from its precursor spermidine, SMS deficiency causes a lack of spermine with an accumulation of spermidine. As polyamines, spermine and spermidine play essential cellular roles that require tight homeostatic control to ensure normal cell growth, differentiation, and survival. Using patient-derived lymphoblast cell lines, we sought to comprehensively investigate the effects of SMS deficiency on polyamine homeostatic mechanisms including polyamine biosynthetic and catabolic enzymes, derivatives of the natural polyamines, and polyamine transport activity. In addition to decreased spermine and increased spermidine in SRS cells, ornithine decarboxylase activity and its product putrescine were significantly decreased. Treatment of SRS cells with exogenous spermine revealed that polyamine transport was active, as the cells accumulated spermine, decreased their spermidine level, and established a spermidine-to-spermine ratio within the range of wild type cells. SRS cells also demonstrated elevated levels of tissue transglutaminase, a change associated with certain neurodegenerative diseases. These studies form a basis for further investigations into the leading biochemical changes and properties of SMS-mutant cells that potentially represent therapeutic targets for the treatment of Snyder-Robinson Syndrome.

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(R,R)-1,12-Dimethylspermine can mitigate abnormal spermidine accumulation in Snyder–Robinson syndrome

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011572 ◽

2020 ◽

Vol 295 (10) ◽

pp. 3247-3256 ◽

Cited By ~ 1

Author(s):

Tracy Murray Stewart ◽

Maxim Khomutov ◽

Jackson R. Foley ◽

Xin Guo ◽

Cassandra E. Holbert ◽

...

Keyword(s):

Molecular Mechanisms ◽

Dietary Supplementation ◽

Supporting Cell ◽

Control Mechanisms ◽

Loss Of Function ◽

Spermine Synthase ◽

Treatment Plans ◽

Underlying Causes ◽

Exogenous Spermine ◽

Potential Use

Snyder–Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (R,R)-1,12-dimethylspermine (Me2SPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. Me2SPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate Me2SPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. Me2SPM administration to mice revealed that Me2SPM significantly decreases spermidine levels in multiple tissues. Importantly, Me2SPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (R,R)-diastereomer of 1,12-Me2SPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.

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ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922342117 ◽

2020 ◽

Vol 117 (49) ◽

pp. 31198-31207

Author(s):

Stephanie Vrijsen ◽

Laura Besora-Casals ◽

Sarah van Veen ◽

Jeffrey Zielich ◽

Chris Van den Haute ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Response ◽

Antioxidant Properties ◽

Loss Of Function ◽

Mitochondrial Oxidative Stress ◽

C Elegans ◽

Polyamine Transport ◽

Exogenous Spermine ◽

The Impact

Recessive loss-of-function mutations inATP13A2(PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in aCaenorhabditis elegansstrain deficient in the ATP13A2 orthologcatp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated byatfs-1, theC. elegansortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

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Role of unsaturated derivatives of spermidine as substrates for spermine synthase and in supporting growth of SV-3T3 cells

Biochemical Journal ◽

10.1042/bj2740167 ◽

1991 ◽

Vol 274 (1) ◽

pp. 167-171 ◽

Cited By ~ 8

Author(s):

A E Pegg ◽

S Nagarajan ◽

S Naficy ◽

B Ganem

Keyword(s):

Feedback Regulation ◽

3T3 Cells ◽

Spermine Synthase ◽

Polyamine Transport ◽

Spermidine Analogues ◽

Derivatives Of ◽

Cis Isomer ◽

Endogenous Synthesis ◽

Stimulation Of

Synthetic unsaturated analogues of the natural polyamine were examined as possible substrates for spermine synthase and as replacements for spermidine in supporting the growth of SV-3T3 cells. It was found that N-(3-aminopropyl)-1,4-diamino-cis-but-2-ene [the cis isomer of the alkene analogue of spermidine] was a good substrate for spermine synthase, but that the trans isomer [N-(3-aminopropyl)-1,4-diamino-trans-but-2-ene] and the alkene analogue [N-(3-aminopropyl)-1,4-diaminobut-2-yne] were not substrates. These results provide the first demonstration of stereospecificity in the spermine synthase reaction. All three of the unsaturated spermidine analogues described above and the cis-alkene analogue of spermine [N1N4-bis-(3-aminopropyl)-1,4-diamino-cis-but-2-ene] were able to support the growth of SV-3T3 cells that were prevented from the endogenous synthesis of spermidine by treatment with alpha-difluoromethylornithine. Since N-(3-aminopropyl)-1,4-diamino-trans-but-2-ene] and N-(3-aminopropyl)-1,4-diaminobut-2-yne were not converted into a spermine derivative, it is apparent that this conversion is not needed for the stimulation of growth. However, since N1N4-bis-(3-aminopropyl)-1,4-diamino-cis-but-2-ene was also able to support growth and was not degraded to the spermidine derivative, it appears that either polyamine can be effective in this respect. All of the unsaturated analogues tested accumulated in the SV-3T3 cells to a much greater extent than spermidine itself. This indicates that these compounds are substrates for the polyamine transport system, but that they are less effective than the natural polyamines in the feedback regulation of this system.

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ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization

International Journal of Molecular Sciences ◽

10.3390/ijms22052689 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2689

Author(s):

Jianmin Si ◽

Chris Van den Haute ◽

Evy Lobbestael ◽

Shaun Martin ◽

Sarah van Veen ◽

...

Keyword(s):

Membrane Integrity ◽

Ubiquitin Proteasome System ◽

Regulatory Function ◽

Membrane Association ◽

Loss Of Function ◽

Atypical Form ◽

Polyamine Transport ◽

Independent Functions ◽

Ubiquitin Proteasome ◽

The Impact

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

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PIK3CAmutant tumors depend on oxoglutarate dehydrogenase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617922114 ◽

2017 ◽

Vol 114 (17) ◽

pp. E3434-E3443 ◽

Cited By ~ 17

Author(s):

Nina Ilic ◽

Kıvanç Birsoy ◽

Andrew J. Aguirre ◽

Nora Kory ◽

Michael E. Pacold ◽

...

Keyword(s):

Cell Proliferation ◽

Glucose Metabolism ◽

Cancer Cell ◽

Mutant Cell ◽

Tca Cycle ◽

Loss Of Function ◽

Cycle Enzyme ◽

Oxoglutarate Dehydrogenase ◽

Mutant Cells ◽

Malate Aspartate Shuttle

OncogenicPIK3CAmutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found thatPIK3CAmutant cancer cells requirePIK3CAbut also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustainPIK3CAmutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy withinPIK3CAmutant cells. Reduced levels of aspartate deregulated the malate–aspartate shuttle, which is important for cytoplasmic NAD+regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, becausePIK3CAmutant cells exhibit a profound reliance on glucose metabolism, malate–aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.

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Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function

Development ◽

10.1242/dev.124.15.2923 ◽

1997 ◽

Vol 124 (15) ◽

pp. 2923-2934 ◽

Cited By ~ 32

Author(s):

K.M. Wassarman ◽

M. Lewandoski ◽

K. Campbell ◽

A.L. Joyner ◽

J.L. Rubenstein ◽

...

Keyword(s):

Normal Development ◽

Motor Nerve ◽

Homeobox Gene ◽

Neural Plate ◽

Mouse Embryos ◽

Loss Of Function ◽

Isthmic Nuclei ◽

Signaling Center ◽

Derivatives Of ◽

Anterior Posterior

Analysis of mouse embryos homozygous for a loss-of-function allele of Gbx2 demonstrates that this homeobox gene is required for normal development of the mid/hindbrain region. Gbx2 function appears to be necessary at the neural plate stage for the correct specification and normal proliferation or survival of anterior hindbrain precursors. It is also required to maintain normal patterns of expression at the mid/hindbrain boundary of Fgf8 and Wnt1, genes that encode signaling molecules thought to be key components of the mid/hindbrain (isthmic) organizer. In the absence of Gbx2 function, isthmic nuclei, the cerebellum, motor nerve V, and other derivatives of rhombomeres 1–3 fail to form. Additionally, the posterior midbrain in the mutant embryos appears to be extended caudally and displays abnormalities in anterior/posterior patterning. The failure of anterior hindbrain development is presumably due to the loss of Gbx2 function in the precursors of the anterior hindbrain. However, since Gbx2 expression is not detected in the midbrain it seems likely that the defects in midbrain anterior/posterior patterning result from an abnormal isthmic signaling center. These data provide genetic evidence for a link between patterning of the anterior hindbrain and the establishment of the mid/hindbrain organizer, and identify Gbx2 as a gene required for these processes to occur normally.

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Mutations inDrosophila myblead to centrosome amplification and genomic instability

Development ◽

10.1242/dev.129.2.347 ◽

2002 ◽

Vol 129 (2) ◽

pp. 347-359 ◽

Cited By ~ 2

Author(s):

Siau-Min Fung ◽

Gary Ramsay ◽

Alisa L. Katzen

Keyword(s):

Cell Cycle ◽

Epidermal Cells ◽

Loss Of Function ◽

Brca1 And Brca2 ◽

Cell Cycles ◽

Myb Gene ◽

Pupal Wing ◽

Complete Cell ◽

Abnormal Mitoses ◽

Mutant Cells

We have previously established that the single myb gene in Drosophila melanogaster, Dm myb, which is related to the proto-oncogene Myb, is required for the G2/M transition of the cell cycle and for suppression of endoreduplication in pupal wing cells. We now report that studies of the abdominal phenotype in loss-of-function Dm myb mutants reveal additional roles for Dm myb in the cell cycle, specifically in mitosis. Abdominal epidermal cells that are mutant for Dm myb proliferate more slowly than wild-type controls throughout pupation, with particularly sluggish progression through the early stages of mitosis. Abnormal mitoses associated with multiple functional centrosomes, unequal chromosome segregation, formation of micronuclei, and/or failure to complete cell division are common in the later cell cycles of mutant cells. Resulting nuclei are often aneuploid and/or polyploid. Similar defects have also been observed in loss-of-function mutations of the tumor suppressor genes p53, Brca1 and Brca2. These data demonstrate that in abdominal epidermal cells, Dm myb is required to sustain the appropriate rate of proliferation, to suppress formation of supernumerary centrosomes, and to maintain genomic integrity.

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Polyamine metabolism during cardiac hypertrophy

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1980.239.5.e372 ◽

1980 ◽

Vol 239 (5) ◽

pp. E372-E378 ◽

Cited By ~ 1

Author(s):

A. E. Pegg ◽

H. Hibasami

Keyword(s):

Cardiac Hypertrophy ◽

Polyamine Metabolism ◽

Decarboxylase Activity ◽

Elevated Concentration ◽

Enzyme Protein ◽

Carboxylase Activity ◽

Rapid Reduction ◽

Spermine Synthase

Treatment with thyroxine for 7 days to produce myocardial hypertrophy led to an increase in the content of putrescine, spermidine, and spermine in the rat heart. The content of decarboxylated S-adenosylmethionine, the source of the aminopropyl groups needed for polyamine synthesis, was increased by the thyroxine treatment as were the activities of ornithine and S-adenosylmethionine decarboxylases. The enhanced S-adenosylmethionine decarboxylase activity measured in vitro was due to an increase in the amount of enzyme protein as measured by immunotitration with a specific antiserum. In vivo, decarboxylation of S-adenosylmethionine was, therefore, increased both by the increased amount of enzyme protein and by the elevated concentration of putrescine (which activates the enzyme) brought about by the enhanced ornithine carboxylase activity. Spermine synthase did not change significantly during the treatment and spermidine synthase increased only slightly. Therefore, the accumulation of polyamines was mediated predominantly via the increased availability of both putrescine and decarboxylated S-adenosylmethionine. Administration of 1,3-diamino-2-propanol led to a rapid reduction in the activity of ornithine decarboxylase in the heart, and continued exposure to this substance by its inclusion in the drinking water completely prevented the increase in concentration of putrescine and polyamines in response to thyroxine. However, cardiac hypertrophy as measured by the increase in cardiac mass was not prevented by such treatment with 1,3-diaminopropanol, showing that the increased content of polyamines was not essential for the hypertrophic response.

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Response of enzymes involved in the metabolism of polyamines to phytohaemagglutinin-induced activation of human lymphocytes

Biochemical Journal ◽

10.1042/bj1960733 ◽

1981 ◽

Vol 196 (3) ◽

pp. 733-738 ◽

Cited By ~ 14

Author(s):

H Korpela ◽

E Hölttä ◽

T Hovi ◽

J Jänne

Keyword(s):

Ornithine Decarboxylase ◽

Human Lymphocytes ◽

Lymphocyte Activation ◽

Decarboxylase Activity ◽

Spermidine Synthase ◽

Irreversible Inhibitor ◽

Adenosylmethionine Decarboxylase ◽

Spermine Synthase ◽

Diamine Oxidase Activity ◽

Stimulation Of

The stimulation of lymphocyte ornithine decarboxylase and adenosylmethionine decarboxylase produced by phytohaemagglutinin was accompanied by an equally marked, but delayed, stimulation of spermidine synthase, which is not commonly considered as an inducible enzyme. In contrast with the marked stimulation of these biosynthetic enzymes, less marked changes were observed in the biodegradative enzymes of polyamines in response to phytohaemagglutinin. Diamine oxidase activity was undetectable during all stages of the transformation. The activity of polyamine oxidase remained either constant or was slightly decreased several days after addition of the mitogen. The activity of polyamine acetylase (employing all the natural polyamines as substrates) distinctly increased both in the cytosolic and crude nuclear preparations of the cells during later stages of mitogen activation. Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, although powerfully inhibiting ornithine decarboxylase, produced a gradual enhancement of adenosylmethionine decarboxylase activity during lymphocyte activation, without influencing the activities of the two propylamine transferases (spermidine synthase and spermine synthase).

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