news and views
Wrong place, wrong time: ectopic progenitors cause cortical heterotopias Laura Cocas & Samuel J Pleasure
npg
© 2014 Nature America, Inc. All rights reserved.
A report reveals that giant subcortical heterotopia is caused by mutation of a microtubule-associated protein, Eml1. Defects in Eml1 lead to disruption of the radial migratory scaffolding network in mice and humans. Accidents in the precise temporal and spatial orchestration of the various steps involved in corticogenesis can lead to cortical malformations in humans and frequently result in epilepsy and intellectual disability1. The dynamic control of the timing of the onset of asymmetric divisions is fundamentally important for controlling cerebral cortical size2–4. During cortical development, the asymmetric division of neural progenitors in the ventricular zone (VZ) produces neurons and intermediate progenitors5. As cortical development proceeds, a secondary zone of progenitors forms just adjacent to the VZ called the subventricular zone (SVZ), where symmetric proliferation of the intermediate progenitors continues6,7. Neurons generated in the VZ and SVZ then migrate away from the VZ toward more superficial areas of the cortical wall, transiting through the intermediate zone to the cortical plate, where the laminated cortex forms8 (Fig. 1). Many of the genetic mutations responsible for cortical malformations in humans occur in genes such as DCX (doublecortin), LIS1 (the causative gene in Miller-Dieker type 1 lissencephaly) and TUBA1A (α1a-tubulin), which are involved in cytoskeletal stabilization in neurons, often at the level of microtubule function9. These mutations all cause cell-autonomous defects in neuronal migration, probably by impairing the microtubule-dependent movements of the cell body and nucleus that are necessary for migration9. As the mutant neurons stall en route to the cortical wall, they accumulate ectopically and form subcortical heterotopias, which accompany the other features associated with these syndromes, such as a simplified cortical gyral structure, also known as lissencephaly or agyria/pachygyria10. Thus, these cortical malformations are primarily thought of as neuronal migration disorders.
In this issue of Nature Neuroscience, Kielar et al.11 elegantly examine an extensive cortical malformation syndrome that they show is caused by mutations in mice and humans that result in the formation of massive subcortical heterotopias not because of primary defects in neuronal migration competence, but rather as a result of abnormalities in the plane of cell division, detachment of mitotic cells from the VZ and defects in the radial migratory scaffolding. The authors found that the HeCo mouse model of heterotopia12 shows cortical developmental defects that are marked by the presence of ectopic progenitors outside the ventricular zone, as well as defects in the radial glial cell scaffolding, progenitor proliferation and cortical organization. Furthermore, the mouse histology recapitulates human magnetic resonance imaging phenotypes in patients with mutations in the corresponding gene and a rare syndrome called giant subcortical heterotopia13. Kielar et al.11 began by studying adult mice with the HeCo phenotype12. These mice have massive ribbon-like subcortical heterotopias. The radial glial fibers in HeCo mice are disrupted and the formation of appropriate layers is extremely abnormal in these mice11, with intermixing of upper- and lower-layers neurons. The authors also found proliferating progenitors ectopically in the developing cortical plate, far from their normal home in the ventricular zone. In addition, neural progenitors
a
Neurogenesis
Neuronal migration
894
Cortical lamination
b Eml1 mutation leads
Pia
Pia
Cortical lamination
to scaffolding defects
Pia
Pia
CP
CP
Ectopic neurons VZ Embryonic stages
Laura Cocas and Samuel J. Pleasure are in the Department of Neurology and Samuel J. Pleasure is in the Program in Neuroscience, Program in Developmental and Stem Cell Biology, and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, USA. e-mail: [email protected]
in these mice have defects in exiting the cell cycle, as well as increased cell death. The authors further characterized these phenotypes using a series of elegant in vivo and ex vivo experiments showing that neurons from mutant mice migrate at normal speed in vitro and that, when transplanted into wild-type brains, they migrate as well as wild-type host neurons and do not form heterotopias. This suggests that the defect in cortical organization in mutant mice lies in the organization of the progenitors that make up the radial glial scaffolding rather than in migration of the postmitotic neurons. This very clear set of experiments directly shows that the HeCo phenotype is not cell autonomous. These mice have no primary defects in neuronal migration, as is common in many other causes of heterotopia or lissencephaly, but instead have defects in the function of the migratory scaffolding. The authors go on to identify the gene responsible for this defect in humans and mice by identifying a common single nucleotide polymorphism in two families with characteristic giant cortical heterotopias similar to the HeCo phenotype. This SNP occurs in the EML1 gene, which encodes a microtubule-associated protein that is conserved from echinoderms to humans. The authors then electroporated Eml1 constructs into HeCo mice and were able to rescue cortical development and prevent the ectopic localization of radial cortical
Progenitor cell
VZ Postnatal stages
VZ
VZ
Neuron
Figure 1 Schematic diagram showing aspects of normal cortical development and the point at which development is disrupted in HeCo mice. (a) Stages of normal cortical development. During normal cortical development, asymmetric division generates neurons that migrate radially on radial progenitors (radial glia) at embryonic stages. This yields the normally laminated cortex at postnatal stages. (b) Migration defects in Eml1 mutants. In HeCo mice, the radial progenitors (which serve as migratory scaffolding) are displaced, leading to disrupted migration of neurons (red cells) at embryonic stages and widespread heterotopic neurons at postnatal stages.
volume 17 | number 7 | july 2014 nature neuroscience
news and views
npg
© 2014 Nature America, Inc. All rights reserved.
progenitors in the intermediate zone and SVZ. Through knockdown of Eml1 synthesis using short hairpin RNA, Kielar et al.11 also show that Eml1 deficiency alone is able to cause disorganized migration and that reintroduction of wild-type Eml1 rescues this defect. These data indicate that lack of Eml1 is necessary and sufficient for the cortical heterotopia in HeCo mice and that loss of function leads to misplacement of upper-layer progenitors and cortical disorganization. The results of this study reveal that the mutation of a single gene, encoding the Eml1 protein, can cause subcortical heterotopias in mice and is responsible for some fraction of cases of giant subcortical heterotopia. These defects, unlike classic DCX- or LIS1-associated lissencephalies, are not the result of a primary
neuronal migration defect, but are rather a result of the disruption of Eml1’s role in the maintenance and organization of progenitors in the ventricular zone. All indications are that ectopic progenitors are the primary cause of the cortical heterotopias in this syndrome, and it seems likely that this will be the first of a new set of similar disorders that are identified as resulting from disruption of cortical development in humans because of scaffolding defects that secondarily impair neuronal migration. Previous studies have hinted at the possibility that cytoskeletal disruption by manipulating RhoA GTPase function in the radial glial scaffolding can secondarily cause cortical heterotopias in experimental animals14, and Kielar et al.11 bring these previous suspicions to fruition by identifying the cause of this human syndrome.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Geschwind, D.H. & Rakic, P. Neuron 80, 633–647 (2013). Chenn, A. & Walsh, C.A. Science 297, 365–369 (2002). Siegenthaler, J.A. et al. Cell 139, 597–609 (2009). Asami, M. et al. Development 138, 5067–5078 (2011). 5. Konno, D. et al. Nat. Cell Biol. 10, 93–101 (2008). 6. Javaherian, A. & Kriegstein, A. Cereb. Cortex 19 (suppl. 1), i70–i77 (2009). 7. Noctor, S.C., Martinez-Cerdeno, V. & Kriegstein, A.R. J. Comp. Neurol. 508, 28–44 (2008). 8. Molnár, Z. & Clowry, G. Prog. Brain Res. 195, 45–70 (2012). 9. Moon, H.M. & Wynshaw-Boris, A. Wiley Interdiscip. Rev. Dev. Biol. 2, 229–245 (2013). 10. Barkovich, A.J., Kuzniecky, R.I., Jackson, G.D., Guerrini, R. & Dobyns, W.B. Neurology 65, 1873–1887 (2005). 11. Kielar, M. et al. Nat. Neurosci. 17, 923–933 (2014). 12. Croquelois, A. et al. Cereb. Cortex 19, 563–575 (2009). 13. Novegno, F. et al. Epilepsy Res. 87, 88–94 (2009). 14. Cappello, S. et al. Neuron 73, 911–924 (2012). 1. 2. 3. 4.
Unlocking the constraints on memory formation Dina P Matheos & Marcelo A Wood A leading therapeutic molecule for multiple sclerosis, FTY720, is shown to mimic a key component of sphingolipid signaling, resulting in specific manipulation of histone deacetylases and the extinction of memory. It is well known that genomic DNA undergoes a nearly incomprehensible level of compaction so that all of our genetic information fits into a tiny nucleus. This compaction, carried out by specialized chromatin modification and chromatin remodeling complexes, solves a storage problem, but in turn creates a DNA access problem. How, then, is access achieved to turn on and off specific genes required for long-term memory? One mechanism involves the same chromatin-modifying enzymes that package genomic DNA: histone deacetylases (HDACs). Endogenous regulators of HDACs remain elusive. However, a relatively recent discovery demonstrated that sphingosine-1-phosphate (S1P) is an endogenous HDAC inhibitor1. Hait et al.2 report in this issue of Nature Neuroscience that a synthetic analog of sphingosine, fingolimod (referred to here as FTY720), is recognized by intracellular machinery in a similar fashion to S1P, including the inhibition of HDACs. This opens a line of investigation into the signaling mechanisms that regulate HDAC activity, as well as a potential avenue for therapeutic development of cognitive enhancers. Fingolimod (Gilenya) is US Food and Drug Administration approved for the treatment of Dina P. Matheos and Marcelo A. Wood are in the Department of Neurobiology and Behavior, Center for the Neurobiology of Learning and Memory, University of California Irvine, Irvine, California, USA. e-mail: [email protected] or [email protected]
relapsing-remitting multiple sclerosis (RRMS; see ref. 3 for a review). In earlier studies examining the action of FTY720, researchers found that the phosphorylated form of FTY720 (FTY720-P) was structurally similar to sphingosine and was likewise a substrate of sphingosine kinase 2 (SphK2)4,5. As a mimetic of S1P, FTY720 acts initially as an agonist of the S1P receptors on the cell membrane, which are then internalized, thereby inhibiting S1P receptor function. For its treatment of RRMS, FTY720 acts by binding of S1P receptors at the plasma membrane. This binding inhibits egress of a certain subset of T cells from lymph nodes, thereby reducing the destructive inflammatory response in the CNS associated with RRMS3. However, S1P has an intriguing second mode of action that is independent of binding to S1P receptors on the cell membrane. S1P is an endogenous inhibitor of HDACs, and this inhibition occurs through an interaction with HDACs in the nucleus1. This finding represents a paradigm shift in thinking about the function of sphingolipid signaling because the nuclear actions of S1P are independent of its cell surface receptor signaling mechanisms. Considering the structural similarity between S1P and FTY720, it might be possible for FTY720 to hijack the pathways involving SphK2, ultimately leading to the regulation of HDACs and even memory (Fig. 1). This is the focus of the study by Hait et al.2. The authors first asked whether FTY720 would function similarly to S1P in neurons.
nature neuroscience volume 17 | number 7 | july 2014
They demonstrated that FTY720 is taken up by neurons and phosphorylated by SphK2 (the main isoform of this kinase found in the CNS), and that this phosphorylation event leads to retention in the nucleus. Furthermore, in hippocampal neurons treated with FTY720, accumulation of nuclear FTY720-P resulted in the decrease of endogenous nuclear S1P, suggesting direct competition between the similar molecules. Functionally, FTY720-P led to the increase of the same histone acetylation marks that S1P affects in vivo. The phosphorylation of FTY720 was blocked by short interfering RNA against SphK2, suggesting that SphK2 is indeed the kinase directly phosphorylating FTY720. To show that the increases in acetylation by FTY720-P are independent of potential interaction between FTY720-P and S1P receptors on the cell surface, Hait et al.2 treated purified nuclei with FTY720. This led to increased histone acetylation. Thus, FTY720 is sufficient to drive histone acetylation in purified nuclei. The authors also demonstrated that FTY720-P added to intact cells had no effect on acetylation. Together, these and additional results suggest that FTY720 is phosphorylated in the nucleus by SphK2 and that nuclear FTY720-P regulates specific histone acetylation. How does FTY720-P lead to the increase in histone acetylation? Modeling the docking of FTY720-P to the catalytic domain of HDAC2 using the crystal structure of HDAC2 suggests that FTY720-P has interactions in the catalytic 895
Wrong place, wrong time: ectopic progenitors cause cortical heterotopias Laura Cocas & Samuel J Pleasure
npg
© 2014 Nature America, Inc. All rights reserved.
A report reveals that giant subcortical heterotopia is caused by mutation of a microtubule-associated protein, Eml1. Defects in Eml1 lead to disruption of the radial migratory scaffolding network in mice and humans. Accidents in the precise temporal and spatial orchestration of the various steps involved in corticogenesis can lead to cortical malformations in humans and frequently result in epilepsy and intellectual disability1. The dynamic control of the timing of the onset of asymmetric divisions is fundamentally important for controlling cerebral cortical size2–4. During cortical development, the asymmetric division of neural progenitors in the ventricular zone (VZ) produces neurons and intermediate progenitors5. As cortical development proceeds, a secondary zone of progenitors forms just adjacent to the VZ called the subventricular zone (SVZ), where symmetric proliferation of the intermediate progenitors continues6,7. Neurons generated in the VZ and SVZ then migrate away from the VZ toward more superficial areas of the cortical wall, transiting through the intermediate zone to the cortical plate, where the laminated cortex forms8 (Fig. 1). Many of the genetic mutations responsible for cortical malformations in humans occur in genes such as DCX (doublecortin), LIS1 (the causative gene in Miller-Dieker type 1 lissencephaly) and TUBA1A (α1a-tubulin), which are involved in cytoskeletal stabilization in neurons, often at the level of microtubule function9. These mutations all cause cell-autonomous defects in neuronal migration, probably by impairing the microtubule-dependent movements of the cell body and nucleus that are necessary for migration9. As the mutant neurons stall en route to the cortical wall, they accumulate ectopically and form subcortical heterotopias, which accompany the other features associated with these syndromes, such as a simplified cortical gyral structure, also known as lissencephaly or agyria/pachygyria10. Thus, these cortical malformations are primarily thought of as neuronal migration disorders.
In this issue of Nature Neuroscience, Kielar et al.11 elegantly examine an extensive cortical malformation syndrome that they show is caused by mutations in mice and humans that result in the formation of massive subcortical heterotopias not because of primary defects in neuronal migration competence, but rather as a result of abnormalities in the plane of cell division, detachment of mitotic cells from the VZ and defects in the radial migratory scaffolding. The authors found that the HeCo mouse model of heterotopia12 shows cortical developmental defects that are marked by the presence of ectopic progenitors outside the ventricular zone, as well as defects in the radial glial cell scaffolding, progenitor proliferation and cortical organization. Furthermore, the mouse histology recapitulates human magnetic resonance imaging phenotypes in patients with mutations in the corresponding gene and a rare syndrome called giant subcortical heterotopia13. Kielar et al.11 began by studying adult mice with the HeCo phenotype12. These mice have massive ribbon-like subcortical heterotopias. The radial glial fibers in HeCo mice are disrupted and the formation of appropriate layers is extremely abnormal in these mice11, with intermixing of upper- and lower-layers neurons. The authors also found proliferating progenitors ectopically in the developing cortical plate, far from their normal home in the ventricular zone. In addition, neural progenitors
a
Neurogenesis
Neuronal migration
894
Cortical lamination
b Eml1 mutation leads
Pia
Pia
Cortical lamination
to scaffolding defects
Pia
Pia
CP
CP
Ectopic neurons VZ Embryonic stages
Laura Cocas and Samuel J. Pleasure are in the Department of Neurology and Samuel J. Pleasure is in the Program in Neuroscience, Program in Developmental and Stem Cell Biology, and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, USA. e-mail: [email protected]
in these mice have defects in exiting the cell cycle, as well as increased cell death. The authors further characterized these phenotypes using a series of elegant in vivo and ex vivo experiments showing that neurons from mutant mice migrate at normal speed in vitro and that, when transplanted into wild-type brains, they migrate as well as wild-type host neurons and do not form heterotopias. This suggests that the defect in cortical organization in mutant mice lies in the organization of the progenitors that make up the radial glial scaffolding rather than in migration of the postmitotic neurons. This very clear set of experiments directly shows that the HeCo phenotype is not cell autonomous. These mice have no primary defects in neuronal migration, as is common in many other causes of heterotopia or lissencephaly, but instead have defects in the function of the migratory scaffolding. The authors go on to identify the gene responsible for this defect in humans and mice by identifying a common single nucleotide polymorphism in two families with characteristic giant cortical heterotopias similar to the HeCo phenotype. This SNP occurs in the EML1 gene, which encodes a microtubule-associated protein that is conserved from echinoderms to humans. The authors then electroporated Eml1 constructs into HeCo mice and were able to rescue cortical development and prevent the ectopic localization of radial cortical
Progenitor cell
VZ Postnatal stages
VZ
VZ
Neuron
Figure 1 Schematic diagram showing aspects of normal cortical development and the point at which development is disrupted in HeCo mice. (a) Stages of normal cortical development. During normal cortical development, asymmetric division generates neurons that migrate radially on radial progenitors (radial glia) at embryonic stages. This yields the normally laminated cortex at postnatal stages. (b) Migration defects in Eml1 mutants. In HeCo mice, the radial progenitors (which serve as migratory scaffolding) are displaced, leading to disrupted migration of neurons (red cells) at embryonic stages and widespread heterotopic neurons at postnatal stages.
volume 17 | number 7 | july 2014 nature neuroscience
news and views
npg
© 2014 Nature America, Inc. All rights reserved.
progenitors in the intermediate zone and SVZ. Through knockdown of Eml1 synthesis using short hairpin RNA, Kielar et al.11 also show that Eml1 deficiency alone is able to cause disorganized migration and that reintroduction of wild-type Eml1 rescues this defect. These data indicate that lack of Eml1 is necessary and sufficient for the cortical heterotopia in HeCo mice and that loss of function leads to misplacement of upper-layer progenitors and cortical disorganization. The results of this study reveal that the mutation of a single gene, encoding the Eml1 protein, can cause subcortical heterotopias in mice and is responsible for some fraction of cases of giant subcortical heterotopia. These defects, unlike classic DCX- or LIS1-associated lissencephalies, are not the result of a primary
neuronal migration defect, but are rather a result of the disruption of Eml1’s role in the maintenance and organization of progenitors in the ventricular zone. All indications are that ectopic progenitors are the primary cause of the cortical heterotopias in this syndrome, and it seems likely that this will be the first of a new set of similar disorders that are identified as resulting from disruption of cortical development in humans because of scaffolding defects that secondarily impair neuronal migration. Previous studies have hinted at the possibility that cytoskeletal disruption by manipulating RhoA GTPase function in the radial glial scaffolding can secondarily cause cortical heterotopias in experimental animals14, and Kielar et al.11 bring these previous suspicions to fruition by identifying the cause of this human syndrome.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Geschwind, D.H. & Rakic, P. Neuron 80, 633–647 (2013). Chenn, A. & Walsh, C.A. Science 297, 365–369 (2002). Siegenthaler, J.A. et al. Cell 139, 597–609 (2009). Asami, M. et al. Development 138, 5067–5078 (2011). 5. Konno, D. et al. Nat. Cell Biol. 10, 93–101 (2008). 6. Javaherian, A. & Kriegstein, A. Cereb. Cortex 19 (suppl. 1), i70–i77 (2009). 7. Noctor, S.C., Martinez-Cerdeno, V. & Kriegstein, A.R. J. Comp. Neurol. 508, 28–44 (2008). 8. Molnár, Z. & Clowry, G. Prog. Brain Res. 195, 45–70 (2012). 9. Moon, H.M. & Wynshaw-Boris, A. Wiley Interdiscip. Rev. Dev. Biol. 2, 229–245 (2013). 10. Barkovich, A.J., Kuzniecky, R.I., Jackson, G.D., Guerrini, R. & Dobyns, W.B. Neurology 65, 1873–1887 (2005). 11. Kielar, M. et al. Nat. Neurosci. 17, 923–933 (2014). 12. Croquelois, A. et al. Cereb. Cortex 19, 563–575 (2009). 13. Novegno, F. et al. Epilepsy Res. 87, 88–94 (2009). 14. Cappello, S. et al. Neuron 73, 911–924 (2012). 1. 2. 3. 4.
Unlocking the constraints on memory formation Dina P Matheos & Marcelo A Wood A leading therapeutic molecule for multiple sclerosis, FTY720, is shown to mimic a key component of sphingolipid signaling, resulting in specific manipulation of histone deacetylases and the extinction of memory. It is well known that genomic DNA undergoes a nearly incomprehensible level of compaction so that all of our genetic information fits into a tiny nucleus. This compaction, carried out by specialized chromatin modification and chromatin remodeling complexes, solves a storage problem, but in turn creates a DNA access problem. How, then, is access achieved to turn on and off specific genes required for long-term memory? One mechanism involves the same chromatin-modifying enzymes that package genomic DNA: histone deacetylases (HDACs). Endogenous regulators of HDACs remain elusive. However, a relatively recent discovery demonstrated that sphingosine-1-phosphate (S1P) is an endogenous HDAC inhibitor1. Hait et al.2 report in this issue of Nature Neuroscience that a synthetic analog of sphingosine, fingolimod (referred to here as FTY720), is recognized by intracellular machinery in a similar fashion to S1P, including the inhibition of HDACs. This opens a line of investigation into the signaling mechanisms that regulate HDAC activity, as well as a potential avenue for therapeutic development of cognitive enhancers. Fingolimod (Gilenya) is US Food and Drug Administration approved for the treatment of Dina P. Matheos and Marcelo A. Wood are in the Department of Neurobiology and Behavior, Center for the Neurobiology of Learning and Memory, University of California Irvine, Irvine, California, USA. e-mail: [email protected] or [email protected]
relapsing-remitting multiple sclerosis (RRMS; see ref. 3 for a review). In earlier studies examining the action of FTY720, researchers found that the phosphorylated form of FTY720 (FTY720-P) was structurally similar to sphingosine and was likewise a substrate of sphingosine kinase 2 (SphK2)4,5. As a mimetic of S1P, FTY720 acts initially as an agonist of the S1P receptors on the cell membrane, which are then internalized, thereby inhibiting S1P receptor function. For its treatment of RRMS, FTY720 acts by binding of S1P receptors at the plasma membrane. This binding inhibits egress of a certain subset of T cells from lymph nodes, thereby reducing the destructive inflammatory response in the CNS associated with RRMS3. However, S1P has an intriguing second mode of action that is independent of binding to S1P receptors on the cell membrane. S1P is an endogenous inhibitor of HDACs, and this inhibition occurs through an interaction with HDACs in the nucleus1. This finding represents a paradigm shift in thinking about the function of sphingolipid signaling because the nuclear actions of S1P are independent of its cell surface receptor signaling mechanisms. Considering the structural similarity between S1P and FTY720, it might be possible for FTY720 to hijack the pathways involving SphK2, ultimately leading to the regulation of HDACs and even memory (Fig. 1). This is the focus of the study by Hait et al.2. The authors first asked whether FTY720 would function similarly to S1P in neurons.
nature neuroscience volume 17 | number 7 | july 2014
They demonstrated that FTY720 is taken up by neurons and phosphorylated by SphK2 (the main isoform of this kinase found in the CNS), and that this phosphorylation event leads to retention in the nucleus. Furthermore, in hippocampal neurons treated with FTY720, accumulation of nuclear FTY720-P resulted in the decrease of endogenous nuclear S1P, suggesting direct competition between the similar molecules. Functionally, FTY720-P led to the increase of the same histone acetylation marks that S1P affects in vivo. The phosphorylation of FTY720 was blocked by short interfering RNA against SphK2, suggesting that SphK2 is indeed the kinase directly phosphorylating FTY720. To show that the increases in acetylation by FTY720-P are independent of potential interaction between FTY720-P and S1P receptors on the cell surface, Hait et al.2 treated purified nuclei with FTY720. This led to increased histone acetylation. Thus, FTY720 is sufficient to drive histone acetylation in purified nuclei. The authors also demonstrated that FTY720-P added to intact cells had no effect on acetylation. Together, these and additional results suggest that FTY720 is phosphorylated in the nucleus by SphK2 and that nuclear FTY720-P regulates specific histone acetylation. How does FTY720-P lead to the increase in histone acetylation? Modeling the docking of FTY720-P to the catalytic domain of HDAC2 using the crystal structure of HDAC2 suggests that FTY720-P has interactions in the catalytic 895
