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PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment

View Article: PubMed Central - PubMed

ABSTRACT

Zollo et al. report that mutations in PRUNE1, a phosphoesterase superfamily molecule, underlie primary microcephaly and profound global developmental delay in four unrelated families from Oman, India, Iran and Italy. The study highlights a potential role for prune during microtubule polymerization, suggesting that prune syndrome may be a tubulinopathy.

No MeSH data available.


Related in: MedlinePlus

Impact of PRUNE mutations on known PRUNE functions. (A) Graphs showing normalized cell index as a measure of proliferation of AdV-sh-Prune treated, PRUNE FLAG, PRUNE D30N-FLAG and PRUNE R297W-FLAG cells, stimulated with doxycycline (DOX). Proliferation of Ad-sh-UNR treated PRUNE-FLAG cells was followed as control (light blue circle). Cell proliferation is shown as cell index after normalization to the last cell index recorded before the addition of doxycycline. Data are expressed as mean ± SD of samples assayed in triplicate. (B) Graphs showing cell index as a measure of migration of HEK293 cells transfected with plasmids encoding wild-type D30N or R297W PRUNE generated by xCELLigence RICA. Migration kinetics, shown as cell index, were monitored in response to 10% FBS (oval colours) and to 0% FBS (circle colours) as negative control. Data are expressed as mean ± SD of samples assayed in triplicate. (C) mRNA expression levels of TUBB3 (TuJ1) in SH-SY5Y PRUNE-wild-type, PRUNE-D30N and PRUNE-R297W cells treated with doxycycline and all-trans retinoic acid (ATRA) for 7 days as determined by RT-PCR. The levels of mRNA expression are represented as fold-multiples of 2−dCt values relative to untreated expression. Data are means (mRNA expression 2−dCt) ± SD (n = 3) (**P < 0.005). EV = empty vector; NT = not treated; TET = tetracycline (or DOX); AR = all Trans retinoic acid. (D) The biochemical activity of both wild-type and mutated (D30N and R297W) PRUNE on tetraphosphates (P4) substrate was determined with a fixed-time assay using BIOMOL® Green phosphate reagent. The increase in the absorbance at 620 nm was measured. Kinetic parameters were fitted by non-linear regression with GraphPad Prism 4Project. Both D30N (orange curve) and R297W (green curve) PRUNE proteins show a higher biochemical activity compared to that of wild-type PRUNE (black curve). WT = wild-type.
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awx014-F2: Impact of PRUNE mutations on known PRUNE functions. (A) Graphs showing normalized cell index as a measure of proliferation of AdV-sh-Prune treated, PRUNE FLAG, PRUNE D30N-FLAG and PRUNE R297W-FLAG cells, stimulated with doxycycline (DOX). Proliferation of Ad-sh-UNR treated PRUNE-FLAG cells was followed as control (light blue circle). Cell proliferation is shown as cell index after normalization to the last cell index recorded before the addition of doxycycline. Data are expressed as mean ± SD of samples assayed in triplicate. (B) Graphs showing cell index as a measure of migration of HEK293 cells transfected with plasmids encoding wild-type D30N or R297W PRUNE generated by xCELLigence RICA. Migration kinetics, shown as cell index, were monitored in response to 10% FBS (oval colours) and to 0% FBS (circle colours) as negative control. Data are expressed as mean ± SD of samples assayed in triplicate. (C) mRNA expression levels of TUBB3 (TuJ1) in SH-SY5Y PRUNE-wild-type, PRUNE-D30N and PRUNE-R297W cells treated with doxycycline and all-trans retinoic acid (ATRA) for 7 days as determined by RT-PCR. The levels of mRNA expression are represented as fold-multiples of 2−dCt values relative to untreated expression. Data are means (mRNA expression 2−dCt) ± SD (n = 3) (**P < 0.005). EV = empty vector; NT = not treated; TET = tetracycline (or DOX); AR = all Trans retinoic acid. (D) The biochemical activity of both wild-type and mutated (D30N and R297W) PRUNE on tetraphosphates (P4) substrate was determined with a fixed-time assay using BIOMOL® Green phosphate reagent. The increase in the absorbance at 620 nm was measured. Kinetic parameters were fitted by non-linear regression with GraphPad Prism 4Project. Both D30N (orange curve) and R297W (green curve) PRUNE proteins show a higher biochemical activity compared to that of wild-type PRUNE (black curve). WT = wild-type.

Mentions: PRUNE1 encodes a 453 amino acid protein that is highly conserved across many species and shares functional properties with the phosphoesterases and the exopolyphosphatase family of proteins due to the presence of the DHH motif (D’Angelo et al., 2004; Tammenkoski et al., 2008). To determine the molecular mechanism by which PRUNE may regulate neurogenesis, we investigated the outcome of PRUNE mutation on its’ previously documented functional roles in cell migration, proliferation and differentiation (D’Angelo et al., 2004; Carotenuto et al., 2006). We investigated two of the mutations identified located in distinct regions of PRUNE, affecting amino acid residues located in separate functional domains so as to define and compare functional outcomes on molecular function; p.D30N identified in this study and the study by Karaca et al. (2015) (located in the DHH motif), as well as p.R297W identified in this study (located in the DHHA2 domain). As has been shown previously, our studies here determined that PRUNE silencing profoundly decreased cell proliferation (Fig. 2A and Supplementary Fig. 3D). However, while treatment with wild-type PRUNE returned cellular proliferation rates to normal, treatment with either p.D30N or p.R297W mutant proteins did not (Fig. 2A). In parallel with this we also assessed another known property of PRUNE in enhancing cell migration using HEK293 cells, which display negligible levels of endogenous PRUNE expression (Carotenuto et al., 2014), with expression levels assessed by western blotting (Supplementary Fig. 3E). Unlike wild-type PRUNE, which as expected was found to substantially promote cellular migration, both p.D30N and p.R297W mutants displayed negligible migration promoting activity and were comparable to empty vector controls (Fig. 2B). Next, we also examined the effect of both mutations on cell differentiation in SH-SY5Y cells by measuring the expression level of TuJ1 after treatment with retinoic acid. While wild-type PRUNE promoted a 2-fold increase in neuronal cell differentiation levels, no cellular differentiation was promoted by the PRUNE mutants (Fig. 2C). Finally, the exopolyphosphatase activity assay (Tammenkoski et al., 2008) measuring Kcat/Km ratios versus P4-tetraphosphates substrates, show that both mutant PRUNE (p.D30N and p.R297W) proteins retain a higher exopolyphosphatase activity compared to the wild-type protein (Kcat/Km values wild-type: 0.014 μM/s; D30N: 0.312 μM/s; R297W: 0 064 μM s−1; Fig. 2D and Supplementary Table 2).Figure 2


PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment
Impact of PRUNE mutations on known PRUNE functions. (A) Graphs showing normalized cell index as a measure of proliferation of AdV-sh-Prune treated, PRUNE FLAG, PRUNE D30N-FLAG and PRUNE R297W-FLAG cells, stimulated with doxycycline (DOX). Proliferation of Ad-sh-UNR treated PRUNE-FLAG cells was followed as control (light blue circle). Cell proliferation is shown as cell index after normalization to the last cell index recorded before the addition of doxycycline. Data are expressed as mean ± SD of samples assayed in triplicate. (B) Graphs showing cell index as a measure of migration of HEK293 cells transfected with plasmids encoding wild-type D30N or R297W PRUNE generated by xCELLigence RICA. Migration kinetics, shown as cell index, were monitored in response to 10% FBS (oval colours) and to 0% FBS (circle colours) as negative control. Data are expressed as mean ± SD of samples assayed in triplicate. (C) mRNA expression levels of TUBB3 (TuJ1) in SH-SY5Y PRUNE-wild-type, PRUNE-D30N and PRUNE-R297W cells treated with doxycycline and all-trans retinoic acid (ATRA) for 7 days as determined by RT-PCR. The levels of mRNA expression are represented as fold-multiples of 2−dCt values relative to untreated expression. Data are means (mRNA expression 2−dCt) ± SD (n = 3) (**P < 0.005). EV = empty vector; NT = not treated; TET = tetracycline (or DOX); AR = all Trans retinoic acid. (D) The biochemical activity of both wild-type and mutated (D30N and R297W) PRUNE on tetraphosphates (P4) substrate was determined with a fixed-time assay using BIOMOL® Green phosphate reagent. The increase in the absorbance at 620 nm was measured. Kinetic parameters were fitted by non-linear regression with GraphPad Prism 4Project. Both D30N (orange curve) and R297W (green curve) PRUNE proteins show a higher biochemical activity compared to that of wild-type PRUNE (black curve). WT = wild-type.
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awx014-F2: Impact of PRUNE mutations on known PRUNE functions. (A) Graphs showing normalized cell index as a measure of proliferation of AdV-sh-Prune treated, PRUNE FLAG, PRUNE D30N-FLAG and PRUNE R297W-FLAG cells, stimulated with doxycycline (DOX). Proliferation of Ad-sh-UNR treated PRUNE-FLAG cells was followed as control (light blue circle). Cell proliferation is shown as cell index after normalization to the last cell index recorded before the addition of doxycycline. Data are expressed as mean ± SD of samples assayed in triplicate. (B) Graphs showing cell index as a measure of migration of HEK293 cells transfected with plasmids encoding wild-type D30N or R297W PRUNE generated by xCELLigence RICA. Migration kinetics, shown as cell index, were monitored in response to 10% FBS (oval colours) and to 0% FBS (circle colours) as negative control. Data are expressed as mean ± SD of samples assayed in triplicate. (C) mRNA expression levels of TUBB3 (TuJ1) in SH-SY5Y PRUNE-wild-type, PRUNE-D30N and PRUNE-R297W cells treated with doxycycline and all-trans retinoic acid (ATRA) for 7 days as determined by RT-PCR. The levels of mRNA expression are represented as fold-multiples of 2−dCt values relative to untreated expression. Data are means (mRNA expression 2−dCt) ± SD (n = 3) (**P < 0.005). EV = empty vector; NT = not treated; TET = tetracycline (or DOX); AR = all Trans retinoic acid. (D) The biochemical activity of both wild-type and mutated (D30N and R297W) PRUNE on tetraphosphates (P4) substrate was determined with a fixed-time assay using BIOMOL® Green phosphate reagent. The increase in the absorbance at 620 nm was measured. Kinetic parameters were fitted by non-linear regression with GraphPad Prism 4Project. Both D30N (orange curve) and R297W (green curve) PRUNE proteins show a higher biochemical activity compared to that of wild-type PRUNE (black curve). WT = wild-type.
Mentions: PRUNE1 encodes a 453 amino acid protein that is highly conserved across many species and shares functional properties with the phosphoesterases and the exopolyphosphatase family of proteins due to the presence of the DHH motif (D’Angelo et al., 2004; Tammenkoski et al., 2008). To determine the molecular mechanism by which PRUNE may regulate neurogenesis, we investigated the outcome of PRUNE mutation on its’ previously documented functional roles in cell migration, proliferation and differentiation (D’Angelo et al., 2004; Carotenuto et al., 2006). We investigated two of the mutations identified located in distinct regions of PRUNE, affecting amino acid residues located in separate functional domains so as to define and compare functional outcomes on molecular function; p.D30N identified in this study and the study by Karaca et al. (2015) (located in the DHH motif), as well as p.R297W identified in this study (located in the DHHA2 domain). As has been shown previously, our studies here determined that PRUNE silencing profoundly decreased cell proliferation (Fig. 2A and Supplementary Fig. 3D). However, while treatment with wild-type PRUNE returned cellular proliferation rates to normal, treatment with either p.D30N or p.R297W mutant proteins did not (Fig. 2A). In parallel with this we also assessed another known property of PRUNE in enhancing cell migration using HEK293 cells, which display negligible levels of endogenous PRUNE expression (Carotenuto et al., 2014), with expression levels assessed by western blotting (Supplementary Fig. 3E). Unlike wild-type PRUNE, which as expected was found to substantially promote cellular migration, both p.D30N and p.R297W mutants displayed negligible migration promoting activity and were comparable to empty vector controls (Fig. 2B). Next, we also examined the effect of both mutations on cell differentiation in SH-SY5Y cells by measuring the expression level of TuJ1 after treatment with retinoic acid. While wild-type PRUNE promoted a 2-fold increase in neuronal cell differentiation levels, no cellular differentiation was promoted by the PRUNE mutants (Fig. 2C). Finally, the exopolyphosphatase activity assay (Tammenkoski et al., 2008) measuring Kcat/Km ratios versus P4-tetraphosphates substrates, show that both mutant PRUNE (p.D30N and p.R297W) proteins retain a higher exopolyphosphatase activity compared to the wild-type protein (Kcat/Km values wild-type: 0.014 μM/s; D30N: 0.312 μM/s; R297W: 0 064 μM s−1; Fig. 2D and Supplementary Table 2).Figure 2

View Article: PubMed Central - PubMed

ABSTRACT

Zollo et al. report that mutations in PRUNE1, a phosphoesterase superfamily molecule, underlie primary microcephaly and profound global developmental delay in four unrelated families from Oman, India, Iran and Italy. The study highlights a potential role for prune during microtubule polymerization, suggesting that prune syndrome may be a tubulinopathy.

No MeSH data available.


Related in: MedlinePlus