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. 2020 Jun 26;29(14):2395–2407. doi: 10.1093/hmg/ddaa121

Ornithine decarboxylase, the rate-limiting enzyme of polyamine synthesis, modifies brain pathology in a mouse model of tuberous sclerosis complex

David Kapfhamer 1, James McKenna III 1, Caroline J Yoon 1, Tracy Murray-Stewart 2, Robert A Casero Jr 2, Michael J Gambello 1,✉
PMCID: PMC7424721  PMID: 32588887

Abstract

Tuberous sclerosis complex (TSC) is a rare autosomal dominant neurodevelopmental disorder characterized by variable expressivity. TSC results from inactivating variants within the TSC1 or TSC2 genes, leading to constitutive activation of mechanistic target of rapamycin complex 1 signaling. Using a mouse model of TSC (Tsc2-RG) in which the Tsc2 gene is deleted in radial glial precursors and their neuronal and glial descendants, we observed increased ornithine decarboxylase (ODC) enzymatic activity and concentration of its product, putrescine. To test if increased ODC activity and dysregulated polyamine metabolism contribute to the neurodevelopmental defects of Tsc2-RG mice, we used pharmacologic and genetic approaches to reduce ODC activity in Tsc2-RG mice, followed by histologic assessment of brain development. We observed that decreasing ODC activity and putrescine levels in Tsc2-RG mice worsened many of the neurodevelopmental phenotypes, including brain growth and neuronal migration defects, astrogliosis and oxidative stress. These data suggest a protective effect of increased ODC activity and elevated putrescine that modify the phenotype in this developmental Tsc2-RG model.

Introduction

The tuberous sclerosis complex (TSC) (OMIM 191100, 613 254) is a rare autosomal dominant disease that often causes substantial central nervous system pathology. Brain phenotypes include cortical tubers, subependymal nodules (SENs), subependymal giant cell astrocytomas (SEGAs) and other morphologic abnormalities. Morbidity and mortality are often due to epilepsy, intellectual disability, autism spectrum disorders and neuropsychiatric disease (1). TSC is caused by inactivating variants in either TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins form a complex with TBC1D7 to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway (2–4). The hyperactivity of mTORC1 signaling due to loss of TSC1 or TSC2 induces an anabolic state with an increase in nucleotide, protein, lipid and other macromolecular synthesis to fuel cell growth and proliferation (5). A hallmark of TSC is the intrafamilial and interfamilial variable expressivity among patients. A patient can remain undiagnosed due to relatively benign symptoms, only to be diagnosed after having a severely affected child suffering from recalcitrant epilepsy and developmental delay. While some of the variable expressivity is due to specific pathogenic variants in TSC1 or TSC2 (6,7), limited success has been made in associating disease variability with specific TSC1 or TSC2 mutations, degree of mosaicism, genetic modifiers and environmental factors. The identification of novel metabolic targets of mTORC1 hyperactivity may improve our general understanding of TSC biology and its inherent variability.

Using a mouse model of TSC in which the Tsc2 gene was conditionally targeted in most developing neurons and glial cells of the CNS (Tsc2-RG, (8)), we performed untargeted metabolomic profiling studies and found elevated levels of the polyamine putrescine, the product of a rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase (ODC). The activity of ODC was also elevated in the Tsc2-RG brains (9), with no change in the downstream polyamine metabolites spermidine or spermine.

Polyamines are small aliphatic polycations with diverse biological functions. Due to their positive charge, polyamines can interact with nucleic acids and proteins and regulate specific ion channels, thereby exerting wide-ranging effects on transcription, translation, RNA and protein stability and cell signaling (10). Polyamine synthesis is a tightly controlled process involving multiple feedback loops, underscoring the biological importance of maintaining proper levels of these metabolites. In eukaryotes, the primary polyamines, putrescine, spermidine and spermine are synthesized mainly from the amino acid ornithine. ODC, a rate-limiting enzyme in polyamine synthesis, converts ornithine to putrescine. Spermidine and spermine are sequentially produced from putrescine by aminopropylation using decarboxylated S-adenosylmethionine (dcSAM) as the aminopropyl donor and catalyzed by spermidine synthase and spermine synthase, respectively (11) (Fig. 1A). dcSAM is the product of the second rate-limiting enzyme in the pathway, S-adenosylmethionine decarboxylase, the processing and stabilization of which is mTORC1-dependent (12). Functionally, polyamines have been shown to play critical roles in cell growth, proliferation and migration; cellular stress; aging; and neurodegenerative diseases (10,13). The observations that (1) ODC1 is a transcriptional target of proto-oncogene cMYC (14), (2) polyamines are involved in cell growth and proliferation and (3) polyamines are upregulated in malignancies have made them a focus of cancer research (15). Currently, multiple clinical studies investigating the therapeutic effects of the irreversible ODC inhibitor 2-difluoromethylornithine (DFMO) on neuroblastoma, astroglioma and other cancers are underway (16–18).

Figure 1.

Figure 1

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ODC expression in human TSC tuber and mouse Tsc2-RG brain. (A) Polyamine synthetic pathway. ODC indicates ornithine decarboxylase; SpdS, spermidine synthase; SpmS, spermine synthase; dcSAM, decarboxylated S-adenosylmethionine; MTA, 5′-methylthioadenosine. (B, C) IHC analysis showing intense ODC1 staining in giant cells of cortical tuber tissue (C) compared with adjacent cortical non-tuber tissue (B) from a TSC patient. (D–K, D′–K′) IHC analysis of ODC1 immunoreactivity in brains of control (D, D′, H, H′), untreated Tsc2-RG (E, E′, I, I′), Tsc2-RG;Odc1+/− (F, F′, J, J′) and DFMO-treated Tsc2-RG (G, G′, K, K′) mice. ODC1 immunoreactivity in cortex (E, E′) and hippocampal CA1 pyramidal cells (I, I′) of untreated Tsc2-RG mice and appears localized to both the nucleus and cytoplasm (black arrows), in contrast to control animals (D, D′, H, H′) where expression is primarily cytoplasmic (white arrows). Odc1 haploinsufficiency and DFMO treatment of Tsc2-RG mice partially reverse ODC expression levels and nuclear localization. D′–K′ show increased magnification of boxed inset fields indicated in D–K, respectively. CL indicates control; RG, Tsc2-RG; RG;Odc+/−, Tsc2-RG;Odc1+/−; RG + DFMO, Tsc2-RG + DFMO.

Based on the finding of elevated ODC activity and putrescine levels in Tsc2-RG mice, we empirically treated Tsc2+/− heterozygous mice with the ODC inhibitor DFMO. Tsc2+/− mice have a hippocampal astrogliosis. Amazingly, DFMO treatment dose-dependently reduced hippocampal astrogliosis (9), suggesting a functional consequence of increased ODC/putrescine in TSC pathology. Based on these data, we hypothesized that ODC inhibition and putrescine reduction would similarly improve neurodevelopmental phenotypes of the more severely affected Tsc2-RG mice. Using a combination of genetic and pharmacologic approaches, we observed that ODC inhibition and putrescine reduction worsened neuronal growth and migration defects, astrogliosis and oxidative stress in Tsc2-RG brains, suggesting a protective effect of elevated putrescine and potentially downstream metabolites in our Tsc2-RG model. Moreover, the enzyme ODC is a potent modifier of the TSC brain phenotype. These data establish a role for polyamines in TSC neuropathology and indicate that modulating the polyamine pathway may prove therapeutic for TSC.

Results

Modulation of polyamine synthesis in a mouse model of TSC

Our observation that Tsc2-RG mice have increased ODC enzymatic activity in brain prompted us to investigate ODC protein expression in the brains of Tsc2-RG mice and TSC patient samples. Although the immunohistochemical (IHC) analysis of ODC expression may not accurately reflect enzymatic activity, we observed qualitatively more ODC immunoreactivity in human TSC tuber giant cells compared with non-tuber control tissue (Fig. 1B and C). These data suggest that elevated ODC may be important in human TSC pathogenesis.

To test our hypothesis that increased ODC activity and putrescine levels in the brains of Tsc2-RG mice contribute to TSC neuropathology, we used genetic and pharmacologic approaches to reduce ODC activity in Tsc2-RG brains followed by phenotypic analysis (Fig. 2A). The Tsc2 gene is deleted in radial glial progenitor cells of Tsc2-RG mice by Cre-mediated recombination beginning at embryonic day 12.5 (Fig. 2B). In contrast, the Odc1 gene is expressed much earlier in development with Odc1−/− mice dying soon after implantation (19) (Fig. 2B). To genetically reduce ODC activity, we crossed Tsc2-RG mice onto an Odc1+/− background. Odc1+/− mice are viable and fertile despite a 50% reduction in ODC protein and enzyme activity (19,20). Importantly, Odc1+/− mice are developmentally normal with none of the neurological phenotypes of Tsc2-RG mice. For pharmacologic studies, we reduced ODC activity by treating mice daily from P10 to P21 with intraperitoneal 250 mg/kg DFMO (Fig. 2B). As shown in Figure 2C and D, both treatments reduced ODC activity and putrescine levels, whereas neither treatment altered tuberin levels or mTORC1 activity (Supplementary Material, Fig. S1).

Figure 2.

Figure 2

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Modulation of polyamine synthesis in a mouse model of TSC. (A) Tsc2 loss in radial glia results in mTORC1 hyperactivation, increased ODC activity and putrescine levels. We investigated the effect on TSC neuropathology following genetic and pharmacologic reduction of ODC. (B) Timelines of pharmacologic and genetic reduction of ODC in Tsc2-RG mice. Top: DFMO treatment (daily I.P. injection of 250 mg/kg DFMO from P10 to P21) of Tsc2-RG mice. Bottom: genetic reduction of Odc1 beginning prior to implantation. Gray shading of timeline bars denotes period of ODC reduction. (C, D) Elevated ODC activity (C) and putrescine levels (D) in Tsc2-RG cortex are reduced by DFMO treatment and Odc1 haploinsufficiency. *P < 0.05.

We analyzed experimental mouse brain tissue with ODC immunohistochemistry. While we were unable to quantify ODC antigen, we observed an interesting relocalization of ODC signal. In control cortical and hippocampal pyramidal cells, ODC signal is primarily cytoplasmic (white arrows; Fig. 1D′ and H′). In Tsc2-RG mice, the majority of the ODC signal has relocalized to the nucleus (black arrows; Fig. 1E′ and I′). Both pharmacologic and genetic reductions of ODC generated a mixed pattern of nuclear and cytoplasmic staining cells (Fig. 1F′, G′, J′ and K′). Loss of Tsc2 seems to induce a nuclear rather than cytoplasmic subcellular localization. This localization is reversed with pharmacologic or genetic reduction of Odc1.

To try to quantitate how the loss of Tsc2 and reduction of ODC activity affects Odc1 regulation, we performed immunoblotting analysis of cortical lysates from control and experimental mice to assess ODC antizyme 1 (OAZ1) expression. OAZ1 participates in a highly complex autoregulatory loop to control polyamine levels, in part by binding to and inactivating ODC and targeting the enzyme for degradation (21). OAZ1 synthesis is stimulated by polyamines via a ribosomal frameshift mechanism and is negatively regulated by antizyme inhibitor protein (22,23). As expected, Odc1 haploinsufficiency reduced OAZ1 expression compared with control mice, and DFMO treatment almost completely abolished OAZ1 expression in Tsc2-RG mice (Supplementary Material, Fig. S2A and B). Unexpectedly, OAZ1 expression was also reduced in the brains of untreated Tsc2-RG mice, suggesting that loss of Tsc2 may induce antizyme inhibitor expression in order to maintain elevated ODC and putrescine levels. A similar reduction in OAZ1 expression has been reported in a Tsc2-deficient cell culture model (24).

Suppression of polyamine synthesis affects hippocampal and cortical development of Tsc2-RG mice

Comparison of the effects of genetic and pharmacologic reduction of ODC on Tsc2-RG neuropathology is summarized in Table 1. A common feature of TSC neuropathology is the presence of SENs, which may transform into SEGAs (25,26). We have previously reported that Tsc2-RG mice develop ring or nodular heterotopia in the stratum lacunosum moleculare of the hippocampus, and rarely, subependymal-like nodules along the ventricles (8,27). Both lesions develop prior to P10. To investigate the effects of dysregulated polyamine synthesis on hippocampal heterotopia and SENs in Tsc2-RG mice, we performed H&E staining of serial brain sections obtained from 21-day-old Tsc2-RG;Odc1+/− and control mice. As previously reported, we observed both hippocampal ring heterotopia and subependymal-like nodules in Tsc2-RG mice, whereas these were absent in wild-type controls (Fig. 3A, B, E, F). On an Odc1+/− background, we observed an increased number of these structures in Tsc2-RG mice (Fig. 3C, E and F), indicating that elevated ODC activity and putrescine levels in Tsc2-RG mice suppress development of hippocampal heterotopia and SENs.

Table 1.

Phenotypic summary of Tsc2-RG mice following genetic or pharmacologic reduction of ODC

Phenotype Tsc2-RG; Odc+/− Tsc2-RG (DFMO)
ODC activity ↓ ↓
Putrescine ↓ ↓
Hippocampal ring heterotopia ↑ N/A
Subependymal-like nodules ↑ N/A
Cortical thickness ↑ ↓
Lamination defects (CUX1) ↑ N/A
Cortical migration defects (BrdU) ↑ N/A
Astrogliosis (GFAP) ↑ ↑
Oxidative stress (HO-1) = ↑
Inflammation (IBA1) = =
Hypomyelination (MBP) = =
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↑ indicates increased; ↓, decreased; =, no change; relative to untreated Tsc2-RG mice; N/A, not applicable.

Figure 3.

Figure 3

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Odc1 haploinsufficiency increases hippocampal heterotopia (HET), SENs and cortical thickness in Tsc2-RG mice. (A–D) H&E staining of hippocampi revealed increased number of hippocampal HET and SENs in Tsc2-RG;Odc1+/− mice (C) compared with Tsc2-RG mice (D). White arrows indicate presence of hippocampal HET; black arrows indicate SENs. HET and SENs are quantified in (E) and (F), respectively. (G–J) H&E staining of cortex shows increased cortical thickness in Tsc2-RG;Odc1+/− mice (I) compared with Tsc2-RG mice (H). Cortical thickness is quantified in panel (K).

Tsc2-RG mice are also characterized by increased cortical thickness, a phenotype reminiscent of hemimegalencephaly reported in a subset of TSC patients (8,28). H&E staining of brain sections from Tsc2-RG;Odc1+/− mice revealed that Odc1 haploinsufficiency augmented cortical thickness of Tsc2-RG mice (Fig. 3G–I and K). Surprisingly, DFMO treatment of Tsc2-RG mice from P10 to 21 had the opposite effect, with cortical thickness of treated Tsc2-RG mice comparable with that of wild-type control animals (Fig. 3G, J and K). These data indicate a complex effect of polyamines on cortical development.

Cortical lamination and neuronal migration defects in Tsc2-RG mice are more severe on an Odc1+/− background

Another neurodevelopmental phenotype of Tsc2-RG mice is the presence of cortical lamination defects due to abnormal neuronal migration (8). Polyamines have been implicated in cell proliferation and migration (13), prompting us to investigate if the cortical organization phenotypes in Tsc2-RG mice are modified by ODC inhibition and polyamine depletion. These phenotypes in Tsc2-RG mice develop prior to postnatal day 10, the onset of DFMO treatment in our pharmacologic study; therefore, we focused on the effect of constitutive Odc1 haploinsufficiency on cortical organization. The transcription factor CUX1, expressed primarily in cortical layers II–IV (29), was used to assess lamination defects. By IHC analysis, a significantly higher percentage of CUX1-positive ectopic cells (layers V and VI) were observed in Tsc2-RG than control cortex, as previously reported (8). Compared with Tsc2-RG brains, Tsc2-RG;Odc1+/− brains showed an additional increase in the percentage of ectopic CUX1-positive cells (Fig. 4A–D and I). We next performed BrdU pulse-labeling of cortical neurons at E15.5. Most neurons labeled at E15.5 will eventually migrate to the upper layers of the cerebral cortex. We found no significant difference in the total number or distribution of BrdU-labeled cells between control and Odc1+/− mice (Fig. 4E, F and J). As previously reported, we observed more labeled cells closer to the ventricular zone (bins 5) in Tsc2-RG mice, supporting a role for tuberin in neuronal migration (Fig. 4E, G and J). Even more BrdU-labeled neurons were observed in bin 5 in the cortex of Tsc2-RG;Odc1+/− mice (Fig. 4E–H and J). Thus, Odc1 haploinsufficiency exacerbates the neuronal migration defects due to loss of Tsc2. These results suggest that ODC activity is an important modifier of cortical lamination in a Tsc2-deficient background.

Figure 4.

Figure 4

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Cortical lamination and neuronal migration defects in Tsc2-RG mice are more severe on an Odc1+/− background. (A–D, I) CUX1 immunofluorescence in cortex of P21 mice. CUX1 labels neurons primarily in cortical layers II–IV as indicated in control mouse (A). Ectopically labeled cells in deeper cortical layers are increased in Tsc2-RG and Tsc2-RG;Odc1+/− mice (C, D, I). Cortical layers are indicated with Roman numerals. (E–H, J) BrdU pulse-labeling of cortical neurons on E15.5 reveals neuronal migration defects in Tsc2-RG and Tsc2-RG;Odc1+/− mice. Cortices were subdivided in five bins of equal thickness (bin #1 most dorsal) and fractions of BrdU-positive cells per bin were calculated. Tsc2-RG and Tsc2-Odc1+/− brains had a higher fraction of BrdU-positive cells in the ventral-most area. D indicates dorsal; V ventral.

Genetic and pharmacologic reduction of ODC activity increases reactive astrogliosis in the cortex of Tsc2-RG mice

Our previous finding that DFMO treatment dose-dependently reduced hippocampal astrogliosis in Tsc2+/− mice suggested that DFMO and/or Odc1 haploinsufficiency may have a similar effect on reducing hippocampal and cortical astrogliosis in Tsc2-RG mice. To our surprise, we observed a dramatic increase in GFAP immunofluorescence in the brains of both Tsc2-RG;Odc1+/− and DFMO-treated Tsc2-RG mice compared with untreated Tsc2-RG controls (Fig. 5A–D and I). These data indicate that elevated ODC activity and putrescine levels in Tsc2-RG mice ameliorate some of the reactive astrogliosis triggered by loss of Tsc2.

Figure 5.

Figure 5

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Genetic and pharmacologic reduction of ODC activity increases cortical astrogliosis and oxidative stress in Tsc2-RG mice. (A–D) GFAP immunofluorescence showing increased labeling of astrocytes throughout the cortex of Tsc2-RG mice (B) compared with control animals (A). GFAP signal in Tsc2-RG mice is further increased on an Odc1+/− background (C) or following DFMO treatment (D). (E–H, E′–H′) HO-1 immunofluorescence showing increased reactivity in Tsc2-RG (F, F′) cortex compared with control mice (E, E′). HO-1 is a marker of oxidative stress. Treatment of Tsc2-RG mice with DFMO (H, H′) further increased HO-1 staining, whereas Odc1 haploinsufficiency had no effect (G, G’). E′–H′ show increased magnification of boxed inset fields indicated in E–H, respectively. (I, J) Quantification of immunoreactivity for GFAP (I) and HO-1 (J).

DFMO treatment of Tsc2-RG mice increases cortical oxidative stress

Earlier work established a function of polyamines in protection against oxidative stress, in part by acting directly as free radical scavengers (30). To determine if the increased astrogliosis we observed in Tsc2-RG;Odc1+/− and DFMO-treated Tsc2-RG brains is associated with increased oxidative stress, we performed immunofluorescence experiments using anti-heme oxygenase 1 (HO-1) antibody. HO-1 catalyzes the degradation of heme to biliverdin, ferrous iron and carbon monoxide, and its expression is induced by oxidative stress (31). We observed increased HO-1 immunoreactivity in the brains of Tsc2-RG mice compared with control animals, consistent with previous studies (32–34), and a further increase in HO-1 immunoreactivity upon DFMO treatment of Tsc2-RG mice. HO-1 expression appeared unchanged in Tsc2-RG mice on an Odc1+/− background (Fig. 5E–H, E′–H′ and J). Colocalization of HO-1 and vimentin, an astrocytic marker, confirmed that the increased HO-1 expression was primarily restricted to astrocytes (Supplementary Material, Fig. S3), indicating that the ODC inhibition in Tsc2-RG mice increases oxidative stress within astrocytes.

Reduction of ODC activity does not affect neuroinflammation or myelination in Tsc2-RG mice

Microglia, the resident immune cells within the CNS, are reported to infiltrate cortical tubers and SEGAs from TSC patients and are increased in the brains of other TSC mouse models (35–37). Furthermore, polyamines may exert anti-inflammatory effects (38), suggesting that ODC inhibition may increase inflammation in the brains of Tsc2-RG mice. To test this possibility, we performed immunofluorescence of control, Tsc2-RG, Tsc2-RG;Odc1+/− and DFMO-treated Tsc2-RG brains using an anti-IBA1 antibody, a marker of microglia and macrophages. Consistent with previous work, IBA1 brain immunoreactivity was markedly increased in Tsc2-RG mice (Supplementary Material, Fig. S4), indicating an inflammatory response. Reduction of ODC activity, either genetically or pharmacologically, did not appear to affect IBA1 immunoreactivity (Supplementary Material, Fig. S4) in Tsc2-RG brains. Although we did not directly assess the expression of specific pro- or anti-inflammatory genes in our model, our data suggest that ODC inhibition has little or no effect on neuroinflammation in Tsc2-RG mice.

Hypomyelination has been described as a clinical feature of human TSC lesions and has been observed in mouse models of TSC (8,39–41). To assess the effects of ODC reduction on hypomyelination in Tsc2-RG mice, we performed immunofluorescence of control, Tsc2-RG, Tsc2-RG;Odc1+/− and DFMO-treated Tsc2-RG brains using an anti-MBP antibody, a marker of myelin basic protein. As previously reported, we observed decreased MBP immunoreactivity in Tsc2-RG brain compared with controls (Supplementary Material, Fig. S5A and B). Neither genetic nor pharmacologic reduction of ODC activity appeared to affect MBP staining in Tsc2-RG mice (Supplementary Material, Fig. S5C and D), indicating that ODC inhibition does not grossly alter myelination in our model.

Discussion

Using complementary genetic and pharmacologic means, we reduced cortical ODC activity and putrescine in Tsc2-RG mice to comparable levels without affecting spermine or spermidine. While we did observe differences in resulting phenotypes (Table 1), the general outcome was a worsening of TSC-related pathology. These data support the hypothesis that elevated putrescine and/or its derivatives are a protective mechanism to counteract multiple mTORC1-induced neurodevelopmental defects in Tsc2-RG mice. Recently, dysregulated polyamine metabolism has also been reported upon rapalog treatment of lymphangioleiomyomatosis, a TSC-associated cystic lung disease, implicating polyamines in TSC pathogenesis of multiple organ systems (42).

Gain-of-function ODC1 mutations have been identified in humans, resulting in macrocephaly, white matter abnormalities, developmental delay and additional phenotypes (43,44). In contrast, overexpression of Odc1 in mice does not appear to affect normal, gross brain morphology (45,46), underscoring fundamental differences in ODC and polyamine function in brain development between humans and mice. Interestingly, Odc1 overexpressing mice are resistant to chemically and electrically induced seizures (46), indicating that increased putrescine and its derivatives may be protective for TSC-related epilepsy. Apart from the general effects of polyamines on transcription and translation, studies suggest that polyamines may exert anticonvulsant effects via conversion of putrescine to GABA (47,48) and inhibition of voltage-gated sodium channels (49). Although we did not directly assess epileptiform/seizure activity in the present study, we observed that Tsc2-RG;Odc1+/− and DFMO-treated Tsc2-RG mice die earlier than Tsc2-RG mice, suggesting that seizure activity may be enhanced by ODC inhibition in our model.

Cortical lamination, HC heterotopia, SENs and cortical thickness

ODC and polyamines play a role in the aberrant cortical development resulting from Tsc2 deletion. Prenatal reduction of ODC in Tsc2-RG; Odc1+/− mice resulted in an increased number of ectopic neurons in deep cortical layers, indicative of a worsened cortical lamination defect. We also observed an increased incidence of hippocampal heterotopia in Tsc2-RG mice on an Odc1+/− background. We did not analyze these lesions in the DFMO-treated mice as these defects have been shown to occur before P10 when DFMO administration was started in the pharmacologic group (8,27). We tried to treat animals with DFMO starting from birth, but the dose used made the animals extremely sick, underscoring the importance of ODC1 during cell growth and proliferation. Although the functional significance of these lesions is unclear, it has been hypothesized that they may result from abnormal migration of neural stem cells from the subventricular zone (SVZ) (8). One major clinical feature of TSC that has been inconsistently recapitulated in mouse models is the development of periventricular nodular lesions, or SENs. SENs are present in 80–90% of TSC patients and are considered precursors of SEGAs. We and other investigators have reported the presence of SEN-like lesions in Tsc1-deficient mice, and less frequently, in Tsc2-RG mice (27,50,51). In the present study, we consistently observed SENs in Tsc2-RG;Odc1+/− mice (4/4 animals), representing a reliable model for future studies of SEN development. Like hippocampal heterotopia, it has been proposed that SENs result from abnormal migration of neural stem/progenitor cells within the SVZ (50). These observations suggest that dysregulated polyamine-dependent expression of cell migration/guidance cues in the CNS may underlie these malformations of cortical development.

We did observe reduced cortical thickness in the DFMO-treated mice. DFMO is a potent inhibitor of cell proliferation (16). We administered DFMO from P10 to P21, timing that precludes cell fate perturbations. Nonetheless, DFMO could have inhibited glial proliferation leading to the reduced cortical thickness we observed in DFMO-treated animals compared with Tsc2-RG;Odc1+/− animals. DFMO can also inhibit arginase affecting the flow of nitrogen through the urea cycle and the flux of ornithine (52). This off-target effect may also explain the difference in cortical thickness observed. Lastly, DFMO is a suicide substrate, binding irreversibly to ODC1 after decarboxylation. The dead enzyme might exert some unknown toxic effects. Both DFMO treatment and Odc1 haploinsufficiency worsened reactive astrogliosis, though astrogliosis was associated with increased oxidative stress only in DFMO-treated mice.

Astrogliosis

One of the most striking and unexpected results of this study was the observation that ODC inhibition dramatically increased reactive astrogliosis in Tsc2-RG mice. We have previously shown that DFMO dose-dependently reduced hippocampal astrogliosis in Tsc2+/− mice. Why ODC inhibition has opposite effects in these two models is unclear. In experiments using cultured astrocytes, we observed that DFMO inhibited mTORC1 signaling in Tsc2+/+ and Tsc2+/− but not Tsc2−/− cells (Supplementary Material, Fig. S1). However, we failed to detect an effect of DFMO on mTORC1 signaling in vivo in Tsc2+/− or Tsc2-RG mice (Supplementary Material, Fig. S1), perhaps due to sampling mixed cell populations (whole cortex), in vivo drug bioavailability/turnover or some unidentified reason. Previous studies have shown regulation of ODC expression by mTORC1 activity (53,54); however, to our knowledge, a feedback mechanism has not been described. This apparent Tsc2-dependent negative feedback of DFMO on mTORC1 activity in culture might underlie the seemingly contradictory effects of DFMO on astrogliosis in Tsc2+/− and Tsc2-RG mice, and if confirmed, could be useful in differentially targeting Tsc2-expressing versus Tsc2 null cells.

Similar to Tsc2-RG mice (8), other TSC mouse models (9,27,55–57), cell culture models in which the Tsc1 or Tsc2 genes have been deleted (58–60), and tubers from TSC patients (61) exhibit an increase in astrocyte number. It has been proposed that TSC1/2 loss causes a shift in cell fate from neurons to astrocytes (59). Our data suggest that this cell fate specification may be further modulated by polyamines. Additionally, reactive astrogliosis may result from seizure activity or other insults (62), consistent with the hypothesis that elevated putrescine/derivatives in Tsc2-RG mice may have anticonvulsant effects.

Increased oxidative stress

We also observed that DFMO increased HO-1expression in Tsc2-RG cortex, primarily within astrocytes. Hmox1 is upregulated in response to oxidative stress, converting heme to the antioxidants bilirubin and biliverdin, thus exerting a protective effect (31). Additionally, this degradation of heme generates carbon monoxide and iron, which may exacerbate cellular stress under chronically upregulated HO-1 conditions. Thus, the consequences of increased HO-1 expression are complex and have been associated with neurodegeneration in Parkinson’s and Alzheimer’s diseases (63,64). In epilepsy models, an increase in HO-1 expression post-seizure has been associated with overexpression of the upstream transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) (65). RNAseq analysis of Tsc2-RG cortical tissue similarly shows increased expression of both Nrf2 and Hmox1 (data in preparation). Observations that polyamines act as free radical scavengers (66,67) are consistent with the increased oxidative stress and HO-1 expression in Tsc2-RG brains upon ODC inhibition/putrescine depletion.

ODC1 cellular localization

One surprising observation in our study is the apparent enrichment of nuclear ODC1 protein in Tsc2-RG cortex and hippocampus and partial reversal by DFMO treatment or Odc1 haploinsufficiency. Within the hippocampal CA1 pyramidal layer of control mice we observed primarily cytoplasmic ODC1 immunoreactivity, consistent with previous work (46,68). In these studies, neither ODC1 overexpression nor ischemia grossly altered ODC1 subcellular localization, suggesting that the changes we observe in Tsc2-RG mice may be TSC and/or seizure-specific. The functional implications of these changes in ODC1 subcellular localization are unclear. Factors affecting the ratio of nuclear to cystosolic ODC1 include cell type, cell cycle, disease state and other physiologic factors, with the caveat that the methodology used to measure ODC1 may confound the results (69,70). In the present study, we used immunohistochemical analysis, which may not differentiate between active and inactive ODC1 enzyme, complicating interpretation of the data. In cell fractionation studies, it has been reported that nuclear ODC1 in multiple brain regions is more likely to be enzymatically active than cytoplasmic ODC1, resulting in increased nuclear putrescine (71). One might postulate that altering nuclear ODC1/putrescine may directly impact gene transcription and RNA processing/stability; however, additional work will be required to determine the functional consequences of increased nuclear ODC1 in Tsc2-RG mice and associated neuropathology.

In conclusion, we report that reduction of ODC activity and downstream polyamine metabolism modifies many of the neurodevelopmental phenotypes in a mouse model of TSC. Differential ODC activity may contribute to the variable disease severity of TSC patients. These data underscore the functional significance of polyamines in regulating neuronal migration, reactive astrogliosis and oxidative stress and define a biological pathway that may be targeted by pharmacologic and/or dietary means to improve TSC disease outcome. To this end, future studies will focus on increasing ODC activity/putrescine production as a means to improve neurodevelopmental phenotypes in Tsc2-RG mice, as well as investigating whether modifying polyamine metabolism might affect post-developmental defects associated with TSC.

Materials and Methods

Animals and drug treatment

All animal experiments were approved by the Emory University Institutional Animal Care and Use Committee and were carried out in accordance with the Guide for the Humane Use and Care of Laboratory Animals. We generated and genotyped Tsc2-RG mice (Tsc2flox/−; hGFAP-Cre) and controls (Tsc2flox/+) as previously described (8). To generate Tsc2-RG;Odc1+/− and control mice, we crossed Odc1+/−; Tsc2+/−; hGFAP-Cre X Tsc2flox/flox mice. Mice were genotyped for Odc1-deleted and wild-type alleles by multiplexing three primers in one PCR: Odc35: 5′-CTCTGTAAGTACGGGAAGCCC-3′, Odc43: 5′-CGAGGTCCGCAACATAGAACG-3′ and OdcNeo: 5′-CCCACACCTCCCCCTGAACC-3′. Band sizes were 270 bp for wild-type and 470 bp for knockout alleles. Odc1+/− mice were a generous gift from Dr John Cleveland, Moffitt Cancer Center. For DFMO experiments, mice were treated intra-peritoneally with a single daily dose of 250 mg/kg DFMO diluted in sterile PBS, from P10 to P21. DFMO was a generous gift from Dr Patrick M. Woster, MUSC. Using timed matings, pregnant dams were injected at E15.5 with 50 mg/kg BrdU, ip, dissolved in 7 mM NaOH/PBS.

ODC activity and determination of intracellular polyamines

Tissue homogenates were prepared on ice using a tissue tearor in buffer containing 25 mM TrisCl, pH 7.5, 0.1 mM EDTA and 2.5 mM DTT. ODC activity was measured as the release of radiolabeled CO2 following incubation with [14C]-ornithine, as previously described (72). Aliquots of these lysates were also used for acid extraction and precolumn dansylation followed by HPLC analysis to determine intracellular concentrations of the individual polyamines (73). Both ODC activity and polyamine concentration are presented relative to total cellular protein, which was measured by the method of Bradford using interpolation on a bovine serum albumin standard curve (74).

Human tissue samples

Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD. The NICHD Brain and Tissue Bank fixed the right side of the patient’s brain in 10% formalin. The tuber sample and control sample were from the same TSC patient, a 56-year-old female. In addition to TSC, she had diffused interstitial lung disease. Her medications were valsartan, hydrochlorothiazide, prednisone, vitamin D, calcium, moxifloxacin, inhaled tiotropium, ipratropium and albuterol.

Primary astrocyte cultures

Neocortical astrocyte cultures were generated from individual P0-2 mice as described previously (75) and genotyped. Purity of cultures was assessed by immunofluorescence using astrocyte-specific markers with anti-GFAP and anti-vimentin antibodies (see below). Experiments were performed at passages 2–4. Briefly, astrocytes were seeded in 9.6 cm2 wells and grown to 75% confluence. On the day of treatment, growth medium [DMEM/F12 (1:1), 10% fetal bovine serum, 4.6 g/L glucose, 50 μg/ml penicillin, 50 μg/ml streptomycin] was removed and replaced with fresh medium with or without DFMO (15 mM) for 2 h. Following treatment, cells were washed with PBS and trypsinized and protein was extracted for immunoblotting. Experiments were performed in triplicate.

Protein analysis

Antibodies used for western blot analysis were as follows: mouse anti-β-actin and mouse anti-GFAP (1:2000, Sigma-Aldrich, St. Louis, MO), rabbit anti-tuberin, total rabbit anti-S6, phosphorylated (S240/244) rabbit anti-S6 and rabbit anti-α-tubulin (1:1000, Cell Signaling Technology, Bedford, MA), mouse anti-HO-1 (1:1000, Enzo Life Sciences, Farmingdale, NY), rabbit anti-IBA1 (1:1000, Fujifilm Wako Pure Chemical Corp., Osaka, Japan) and rabbit anti-OAZ1 (1:2000, a generous gift from Dr Senya Matsufuji at The Jikei University School of Medicine, Minato-ku, Japan). Experiments were performed with a minimum of three biological replicates. Whole cell lysates were made from day P21 cerebral cortex that was quick-frozen in liquid nitrogen. Samples were homogenized in a Dounce homogenizer with 10 volumes of RIPA buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Lysates were centrifuged at 4°C, sonicated and frozen until use. Protein concentrations were determined with a Pierce BCA reagent kit (ThermoFisher Scientific, Rockford, IL). Equal amounts of protein were separated on a denaturing 4–12% gradient gel (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes (Immobilon, Sigma-Aldrich). Secondary antibodies were horseradish peroxidase-conjugated. Visualization was conducted with an ECL kit (Amersham, Piscataway, NJ, USA) and a ChemiDoc™ Imaging System (Bio-Rad, Hercules, CA). Protein band intensities were quantified with ImageJ software and normalized to β-actin or α-tubulin control bands.

Histology

Histological analysis was performed with a minimum of three animals per experimental group. P21 mice were deeply anesthetized before undergoing transcardiac perfusion with PBS followed by 4% paraformaldehyde (PFA). Mouse brains were post-fixed in PFA overnight and stored in 70% ethanol prior to embedding in paraffin. Paraffin blocks were sectioned at 8 μm and slide-mounted. Slides were rehydrated, stained with routine H&E and coverslipped. For DAB immunohistochemistry, slides were incubated with 0.3% H2O2 in methanol for 20 min before the application of primary antibody [rabbit anti-ODC1 (1:100, Proteintech Group Inc., Rosemont, IL)]. After biotinylated secondary antibody incubation, the slides were washed and incubated in Vectastain ABC working solution (Vector Laboratories, Burlingame, CA). DAB (Cell Signaling, #8059) was used for visualization. Immunofluorescence was performed as previously described (8) using mouse anti-GFAP antibody (1:400, Sigma-Aldrich), rabbit anti-CUX1 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-HO-1 (1:100, Enzo Life Sciences), rabbit anti-IBA1 (Fujifilm Wako Pure Chemical Corp.), rabbit anti-vimentin (1:200, Cell Signaling Technology) and anti-BrdU (1:50, Becton Dickinson). Secondary antibodies (1:250; Invitrogen) were Alexa Fluor 488 (anti-rabbit IgG) (anti-mouse IgG1) and Alexa Fluor 555 (anti-rabbit IgG) (anti-mouse IgG1) (anti-mouse IgG2a). Tissue images were examined using a Leica DM6000 and captured with a QImaging RETIGA-2000RV digital camera. Digital images were then processed using Adobe Photoshop CS6 (San Jose, CA, USA). Quantification of immunofluorescence was performed using ImageJ software by calculating the number of pixels above the intensity of background signal determined for each subject and expressed as a percentage of total pixels in the field of analysis.

Supplementary Material

Supplemental_Figure_1_ddaa121
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Supplemental_Figure_2_ddaa121
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Supplemental_Figure_3_ddaa121
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Supplemental_Figure_4_ddaa121
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Supplemental_Figure_5_ddaa121
Click here for additional data file. (3.4MB, png)

Acknowledgements

Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD. ‘The role of the NICHD Brain and Tissue Bank is to distribute tissue, and, therefore, cannot endorse the studies performed or the interpretation of results.’

Conflict of Interest statement. None declared.

Funding

National Institutes of Health, National Institute of of Neurological Diseases and Stroke (R21 NS104410 to M.J.G.); National Institutes of Health, National Cancer Institute (RO1 CA204345 to R.A.C.); University of Pennsylvania Orphan Disease Center in partnership with the Snyder-Robinson Foundation (to R.A.C. and T.M.S.).

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