Some debate still exists on the presence of endogenous NMNAT1 in the axonal compartment in neurons and on its role in axon survival [16], but targeting NMNAT1 to axons, even at low level
Trang 1NMNAT1 does not affect the rate of Wallerian
degeneration
Laura Conforti1,2,3, Lucie Janeckova1, Diana Wagner2, Francesca Mazzola4, Lucia Cialabrini4,
Michele Di Stefano4,*, Giuseppe Orsomando4, Giulio Magni4, Caterina Bendotti5, Neil Smyth6 and Michael Coleman1,2
1 The Babraham Institute, Cambridge, UK
2 Center for Molecular Medicine, University of Cologne (ZMMK), Germany
3 School of Biomedical Sciences, University of Nottingham, UK
4 Dipartimento di Patologia Molecolare e Terapie Innovative, Universita’ Politecnica delle Marche, Ancona, Italy
5 Mario Negri Pharmacological Research Institute, Milan, Italy
6 School of Biological Sciences, University of Southampton, UK
Introduction
The essential role of NAD+ in cell metabolism and
energy production has been known for over a century
and the NAD+ synthesizing enzymes nicotinamide
mononucleotide adenylyltransferases (NMNATs) are evolutionarily ancient and present throughout evolu-tion, including archaebacteria While in prokaryotes
Keywords
axon; Cre-loxP knockout; NAD(P) + ; NMNAT;
Wallerian degeneration
Correspondence
L Conforti, School of Biomedical Sciences,
D37c, University of Nottingham, Medical
School, Queen’s Medical Centre,
Nottingham, NG7 2UH, UK
Fax: +44 (0)115 8231476
Tel: +44 (0)115 8230142
E-mail: laura.conforti@nottingham.ac.uk
*Present address
School of Biomedical Sciences, University of
Nottingham, Medical School, Queen’s
Medical Centre, Nottingham, NG7 2UH, UK
(Received 7 March 2011, revised 4 May
2011, accepted 23 May 2011)
doi:10.1111/j.1742-4658.2011.08193.x
NAD+synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein WldS, which delays axon degeneration by 10-fold NMNAT1 activity is necessary but not sufficient for WldSneuroprotection
in mice and 70 amino acids at the N-terminus of WldS, derived from poly-ubiquitination factor Ube4b, enhance axon protection by NMNAT1 NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro and partially in Drosophila However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet To address this question we disrupted the Nmnat1 locus by gene targeting Homozygous Nmnat1knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurode-generation or axon pathology Wallerian deneurode-generation after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration
Enzymes NMNAT ( EC 2.7.7.1 )
Abbreviations
ES cell, embryonic stem cell; KO, knockout; NAMPT, nicotinamide phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; PARP1, poly(ADP-ribose) polymerase 1; SCG, superior cervical ganglia; VCP ⁄ p97, valosin-containing protein;
YFP, yellow fluorescent protein.
Trang 2and in some eukaryotes such as Drosophila only one
NMNAT isoform has been found to date, in other
simple eukaryotes such as yeast and in higher
eukary-otes including mice and humans more than one
NMNAT isoform has been identified [1] In mammals
there are three NMNAT isoforms with different tissue
distribution and intracellular localization [2–4] The
location of the different isoforms could be related to
specific roles played by NAD+ and its metabolites as
second messengers in cell signalling cascades in
differ-ent environmdiffer-ents, as recdiffer-ently described [5] Higher
organisms could have evolved isoform-specific domains
mediating subcellular targeting and
post-transcrip-tional modifications responsible for NMNAT specific
functions at subcellular level [6] Alternatively, there
could be some redundancy, for example with
extranu-clear NMNAT isoforms being able to compensate for
the nuclear isoform Studies describing subcellular
localization of the three NMNAT isoforms are based
on the overexpression of fusion proteins which could
reach ectopic locations The possibility also exists that
NMNATs could localize to other compartments and
act at very low levels [7] Thus, their roles may not be
restricted to the reported locations
Nuclear NMNAT1 synthesizes NAD+ which is
required for the activity of histone deacetylase sirtuins
and as substrate of poly(ADP-ribose) polymerase 1
(PARP1) High levels of NAD+ are required for
life-span extension in yeast and this response is mediated
by the activity of sirtuin family member Sir2p [8]
Another member of this family, SIRT1, also regulates
circadian rhythm in mammals [9] Notably,
nicotin-amide phosphoribosyltransferase (NAMPT), the rate
limiting enzyme in NAD+synthesis, is correlated with
increased longevity in human cells [10] and is also
involved in the regulation of circadian rhythm [9]
NMNAT1 interacts with SIRT1 at target gene
pro-moters, regulating transcription of genes important for
neuronal function [11] Nuclear NMNAT1 also
regu-lates the activity of genotoxic stress activated nuclear
protein PARP1 by providing NAD+[12] and by
phos-phorylation-dependent association with PARP1 [13],
thus participating in cell death pathways [14,15] Some
debate still exists on the presence of endogenous
NMNAT1 in the axonal compartment in neurons and
on its role in axon survival [16], but targeting
NMNAT1 to axons, even at low levels, does confer
protection [17,18] Extranuclear NAD+, such as that
generated by Golgi-associated NMNAT2 and by
mito-chondrial NMNAT3, is mainly used for energy
pro-duction, as a redox cofactor and as substrate of
enzymes like NAD+kinase, which converts NAD+to
NADP+, and NAD+ glycohydrolases that convert
NAD+ and NADP+ to ribose, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phos-phate, all of which act as second messengers in Ca2+ release from intracellular stores
The role of NAD+ in Wallerian degeneration has emerged since the discovery of the WldS gene, where the full coding sequence of Nmnat1 is fused to the 5¢ end of ubiquitination factor Ube4b giving rise to the WldS protein, a modified NMNAT1 enzyme with an extended N-terminal sequence Wallerian degenera-tion, the degeneration of axons and synapses after an injury, is delayed 10-fold by WldS both in vivo and
in vitro, in organisms as diverse as mice, rats and flies [19] NMNAT1 enzyme activity is required for the protective phenotype [20,21] but the N-terminal sequences are also necessary to achieve full protection
in vivo NMNAT1 overexpression is not sufficient to delay axon degeneration in transgenic mice [22] and does so only weakly in Drosophila [20] In dorsal root ganglia cultures, NMNAT1 confers protection when locally transduced into axons or when highly overexpressed [18,23] The critical N-terminal sequence of WldS resides within the first 16 amino acids, as their removal results in loss of neuroprotec-tive phenotype [20,21] Interestingly, the only known binding partner of the N-terminal region, AAA ATPase valosin-containing protein (VCP⁄ p97), is a very abundant cellular protein mainly localized at the surface of membranous intracellular organelles [24,25] It is possible that NMNAT1 is redistributed
to a specific location by binding to this N-terminal region and acquires a protective function by produc-ing or overproducproduc-ing NAD+at that locus As down-regulation or rapid degradation of NMNAT2 triggers spontaneous Wallerian degeneration, the NMNAT1 component of WldS is likely to substitute for endoge-nous NMNAT2 when this is degraded after an injury [26]
In order to evaluate the role of endogenous NMNAT1 in Wallerian degeneration, we inactivated the gene by homologous recombination Complete inactivation of both alleles was embryonic lethal but Nmnat1 heterozygous knockout (KO) mice were born, developed as normal and showed reduced NMNAT1 mRNA, protein and enzyme activity levels Wallerian degeneration of transected sciatic nerves proceeded at wild-type rate These data confirm that NMNAT1 is
an essential enzyme for which NMNAT2 or NMNAT3 cannot compensate and that NAD+ synthesis in the nucleus is indispensable for survival These data are also consistent with a primary role for other endoge-nous NMNAT isoforms such as NMNAT2 in main-taining axon integrity
Trang 3Targeting the Nmnat1 gene
Mouse Nmnat1 is formed by four exons and spans a
148 850 kb genomic region on distal chromosome 4 In
order to allow eventual conditional deletion, we
designed a targeting construct based on the vector
pEASYFlox (a gift from W Mu¨ller and K Rajewsky)
to insert a NEOR selection cassette flanked by loxP
sites upstream of exon 1, within the promoter region,
approximately 600 bp 5¢ of the start ATG A third
loxP site was placed within intron 2, between exons 2
and 3 (Fig S1A), so that a 2.3 kb region comprising
some 5¢UTR and exons 1 and 2 was in turn flanked by
two loxP sites After Cre-mediated recombination
between the second and the third loxP sites, part of
promoter and the first two exons of the gene would be
disrupted Even in the unlikely event that a truncated
protein lacking these two exons was expressed, it
would not be functional because important substrate
binding sites are encoded within the first two exons
After introduction of the targeting vector into
C57BL⁄ 6 embryonic stem (ES) cells we verified correct
integration of the NEOR selection cassette and of the
third loxP site by southern blotting using 5¢ and 3¢
specific probes (Fig S1) A 420 bp probe, placed 5¢ of
the targeting region, recognized a 9.5 kb wild-type
band on southern blots of EcoRI digested ES cell
genomic DNA In heterozygous targeted ES cells, in
addition to the wild-type band, another band at
approximately 3 kb was found, due to the
introduc-tion of an addiintroduc-tional EcoRI site within the NEOR
cas-sette Cointegration of the third loxP site was also
verified in southern blots of ES cell genomic DNA
digested with HindIII A 3¢, 750 bp probe recognized
an 8.7 kb band in wild-type and a 6.3 kb band in the
correctly targeted ES cells due to the introduction of a
HindIII site located immediately outside the loxP
sequence (Fig S1)
We had a success rate of 0.26% in the generation of
correctly targeted ES cell clones, with one clone where
both NEOR cassette and third loxP site were correctly
integrated out of 384 total screened We refer to the
correctly targeted allele as Nmnat1+ ⁄ 3lox
Next, we transfected Nmnat1+⁄ 3lox ES cells with a
Cre recombinase expressing vector (pPGK-Cre-bpA,
kind gift of W Mueller) Cre in vitro excised the DNA
between the loxP sites as shown in Fig 1A We
selected only the ES cell clones with a type II deletion
(Fig 1A) Those clones became again sensitive to
G418 due to the excision of the NEORcassette
South-ern blot analysis of G418 sensitive ES cell genomic
DNA digested with BamHI, using a probe located out-side the third loxP site, showed a 3.5 kb band, in addi-tion to the wild-type 13 kb band, in cells where Cre-mediated type II deletion and splicing of the first and second loxP sites had occurred (Fig 1A,B) The tar-geted allele in these ES cells had the NEOR cassette removed and only two loxP sites remaining; therefore the cells are referred to as Nmnat1+⁄ 2lox
Generation of heterozygous Nmnat1 knockout mice
Nmnat1+⁄ 2lox ES cells were injected into the blast-ocysts of 129⁄ J mice and chimeric mice identified by coat colour and bred to obtain a germline transmission
of the mutant floxed allele Germline transmission events were confirmed by both southern blotting and PCR of tail DNA (Fig 1B) For PCR, primer pairs Pr1 + Pr2 and Pr3 + Pr4 were designed to amplify across the two loxP sites, and detected a 32 or 39 bp wild-type band respectively that increased to 66 and
73 bp when the loxP sites were also present
For constitutive Nmnat1 gene inactivation, we crossed Nmnat1+⁄ 2lox male mice with C57⁄ BL6 K14 Cre female mice to produce heterozygous null mice on
a black background The K14 Cre induces a full dele-tion when bred from the female as K14 is expressed in the oocyte [27] The offspring of this cross had recom-bination between the two loxP sites; therefore the 2.3 kb floxed region had been removed and only one
of the loxP sites was left behind (Fig 1A,C) Identifi-cation of heterozygous KO (Nmnat1+⁄)) mice was done by southern blot analysis and PCR using primers Pr1 and Pr4 (see Fig 1A) The BamHI band shifted from 3.5 kb in the Nmnat1 floxed mice to 1.2 kb in Nmnat1+⁄) mice The wild-type band of 13 kb was still present (Fig 1C) PCR with primers Pr1 + Pr4 gave a 2.4 kb PCR product in wild-type (not shown), shortened to 80 bp if Cre-mediated recombination between the two loxP sites had occurred (Fig 1C) When we intercrossed Nmnat1+⁄) mice to produce homozygous knockouts, we found no live homozygous nulls from a total of 88 offspring that were genotyped Heterozygotes were born at the expected Mendelian ratio given the absence of homozygotes (two-thirds of total births) The remaining offspring were wild-type Thus, Nmnat1 is essential for embryo development
Protein and mRNA expression analysis in Nmnat1+ ⁄ )mice
We tested whether NMNAT1 expression and enzyme activity were decreased in Nmnat1+⁄) mice Because
Trang 4NMNAT1 is not abundantly expressed in brain, we
first assessed protein levels in skeletal muscle, where
the protein is expressed in higher amounts [28,29]
NMNAT1 expression was significantly reduced in
het-erozygous KO mice, as shown in western blots of
skel-etal muscle homogenates probed with antibody 183
[28] (Fig 2A) Although more difficult to visualize,
NMNAT1 band intensity was also reduced in western
blots of Nmnat1+⁄) brain homogenates probed with
antibody 183 relative to wild-type (Fig S2A)
North-ern blots from total brain RNA of Nmnat1+⁄) and
wild-type mice probed with an Nmnat1 cDNA probe
also showed a reduced band intensity of Nmnat1
tran-script (Fig S2B) In agreement with expression data,
total NMNAT enzyme activity was significantly
reduced in brain homogenates of Nmnat1+⁄)mice
rel-ative to wild-types (Fig 2B) Despite the reduction in
protein levels and enzyme activity, NAD+levels were not reduced in Nmnat1+⁄)mouse brains (Fig 2C)
In order to test whether NMNAT1 partial deletion had any influence on the expression of the other two NMNAT isoforms and to investigate any compensa-tory mechanisms, we assayed isozyme-specific mRNA expression levels in brain homogenates by real time RT-PCR (Fig 2D) As expected, we found that NMNAT1 mRNA was greatly reduced in Nmnat1+⁄) brain homogenates (Fig 2D, left panel) However, no significant differences were observed in NMNAT2 and NMNAT3 mRNA relative expression levels in Nmnat1+⁄) brain compared with wild-type (Fig 2D, right panel)
We also determined the enzyme activity of each NMNAT isoform in order to evaluate their relative contribution to total NAD+ formation
Isoform-spe-HindIII (18 670)
EcoRI
(7282)
15 000
14 000 16 000 17 187
HindIII (10 138)
BamHI (13 390)
EcoRI (16 760)
Sal1 Sal1 HindIII
BamHI
(1)
1
BamHI
11 000 8000
7000
Probe 4
Wild-type (BamHI band ca 13 kb)
Type II del (BamHI band 3.5 kb)
Type I del or Cre-mediated recombination from type II del (BamHI band 1.2 kb)
1° loxP 3° loxP
Wild-type allele LoxP insertion
Wild-type allele
(13 kb)
Floxed allele
(3.5 kb)
60 61
Wild-type allele (13 kb)
KO allele (1.2 kb)
Nmnat1+/2lox (Floxed Nmnat1 het) Nmnat1+/– (KO Nmnat1 het)
NEO
BamHI band
BamHI band
A
Fig 1 Generation of heterozygous targeted mice (A) Representation and map of Nmnat1 targeted allele and the deletion events after Cre transfection of Nmnat1 + ⁄ 3lox ES cells The expected change in the size of a BamHI band in genomic southern blots is shown in the diagram (B) Southern blot and PCR analysis of genomic DNA of ES cell clones after Cre-mediated recombination (type II deletion according to the dia-gram in A) The genomic DNA was digested with BamHI and probed with probe 4 The wild-type and the recombinant band are the expected size PCR with primer pairs Pr1 + Pr2 and Pr3 + Pr4 shows the correct placement of the two remaining loxP sites Exactly the same result was shown in southern blot and PCR analysis of Nmnat1 + ⁄ 2lox mouse tail DNA, after blastocyst injection and coat colour screen-ing of mice (C) PCR and southern blot analysis of tail DNA from Nmnat1+⁄ 2lox· C57BL ⁄ 6 K14 Cre offspring PCR was performed with prim-ers Pr1 + Pr4 to demonstrate the correct deletion of the genomic region between the first and the third loxP site as shown by the 80-bp product formed The 2.4 kb wild-type PCR product cannot be distinguished on this high percentage agarose gel (left panel) The fact that the new Cre-mediated recombination leaves only one loxP site is also demonstrated by the 1.2 kb BamHI specific band on a southern blot (right panel).
Trang 5cific NMNAT enzyme activity was determined with a
biochemical discrimination assay based on the
dis-tinctive metal ion sensitivity of the three isoforms
(Orsomando G, Cialabrini L, Amici A, Agostinelli S,
Janeckova L, Di Stefano M, Conforti L, Coleman M,
Magni G, manuscript in preparation, adapted from
[30,31]) In agreement with mRNA and protein
expression analysis, NMNAT1 enzyme activity in
Nmnat1+⁄) mouse brain was about half that in wild-type (Fig 2E) In contrast, no significant differences were observed in NMNAT2 and NMNAT3 activity (Fig 2E) Despite the high brain mitochondrial content and energy demand, NMNAT3 enzyme activity is very low This result was obtained in brain extracts after dis-ruption of mitochondrial membranes, excluding the possibility of an underestimation of NMNAT3 activity
0.30
0.25
0.20
0.15
*(P = 0.046)
3 2
A
E
D
NMNAT1 (31.5 kDa)
β-actin
(42 kDa)
+ levels
0 50 100 150 200 250 300 350
N.S.
C57BL/6 Nmnat1+/–
NMNAT1
**(P = 0.0054)
0 20 40 60 80 100 120 140 160
0 0.02 0.04 0.06 0.08 0.1
NMNAT enzyme activity (m
N.S.
N.S.
*(P = 0.0212)
Nmnat1+/–
Wild-type NMNAT2
N.S.
NMNAT3
0 20 40 60 80 100 120
N.S.
**
NMNAT1
Nmnat1+/–
Wild-type
C57BL/6 Nmnat1+/–
C57BL/6 Nmnat1+/–
0.40
0.30
0.20
0.10
0.00
C57BL/6 Nmnat1+/–
*(P = 0.013)
Fig 2 NMNAT isoform expression and enzyme activity in Nmnat1 + ⁄ ) mice (A) Western blots of skeletal muscle homogenates from Nmnat1+⁄)and C57BL ⁄ 6 mice probed with antibody 183 (the antibody also reveals a non-specific upper band) The histogram represents the integrated band intensity of the NMNAT1 band normalized to the b-actin control (n = 3, Mann–Whitney test, P = 0.046) (B) Total NMNAT activity of brain homogenates from Nmnat1+⁄)and C57BL ⁄ 6 mice The enzyme activity is strongly reduced in the heterozygous mice with respect to wild-types (n = 9, Student’s t-test, P = 0.013) (C) NAD+levels in wild-type and Nmnat1+⁄) total brain homogenate (n = 5, Student’s t-test) (D) Left panel: NMNAT1 mRNA relative expression in Nmnat1 + ⁄ )and wild-type brains showing strong reduction of NMNAT1 mRNA in heterozygous KO mice Right panel: Relative mRNA expression of each NMNAT isoform in Nmnat1 + ⁄ )compared with wild-type, showing that while NMNAT1 mRNA expression is reduced, NMNAT2 and 3 mRNA relative expression is not changed Normaliza-tion was performed for each isoform by calculating the ratio between the expression of an individual NMNAT isoform and that of the refer-ence gene (b-actin) in wild-type samples The arbitrary number of 100% was assigned to this ratio for one control, and NMNAT expression
of the same isoform in the remaining controls and in Nmnat1+⁄)brains relative to the reference gene was compared with this number Therefore relative mRNA expression levels can be compared between wild-type and Nmnat1 + ⁄ )(n = 3, Student’s t-test, **P = 0.0054) (E) Determination of NMNAT isozyme activity in wild-type and Nmnat1 + ⁄ )total brain homogenates reveals highly reduced NMNAT1 activity in heterozygous KO tissue compared with wild-type but no change in the activity of the other two isoforms Note the very low activity of NMNAT3 in mouse brain (n = 3, Student’s t-test, *P = 0.0212).
Trang 6due to lack of solubilization of mitochondria during
the extraction procedure The absence of compensatory
changes in NMNAT2 and NMNAT3 when NMNAT1
is depleted supports the model of non-redundant
functions for these isoforms
Despite the reduction in NMNAT1 protein levels
and enzyme activity, Nmnat1+ ⁄ ) mice are healthy,
indistinguishable from their wild-type littermates and
have a normal lifespan, suggesting that downregulation
of NMNAT1 is compatible with normal life and a
healthy nervous system, although complete inactivation
is lethal
Wallerian degeneration rate in Nmnat1+⁄)mice
WldS neuroprotective protein contains NMNAT1 and
requires its enzyme activity to delay axon degeneration
after injury, but NMNAT1 overexpression in vivo is
not neuroprotective [21,22] However, the role of
endogenous NMNAT1 on the rate of Wallerian
degen-eration has never been determined To test this, we
lesioned sciatic nerves of Nmnat1+ ⁄ ) mice and their
wild-type littermates after crossing them with YFP-H
mice [32] where some axons are labelled with the
yel-low fluorescent protein (YFP) In YFP-H positive mice
it is easy to follow axon degeneration in longitudinal
sections of lesioned sciatic nerves observed under a
fluorescent microscope [22,33] Wallerian degeneration
of the distal stump of a sciatic nerve after an injury
follows a precise time course in wild-type mice Axon
fragmentation begins at around 36 h, then proceeds
quickly and is complete 42 h after the lesion In
spon-taneous mutant WldS, however, Wallerian
degenera-tion is highly delayed and axon continuity is preserved
up to 3 weeks from injury [33,34] Thus we studied
Wallerian degeneration in Nmnat1+⁄)mice with sciatic
nerves lesioned for 30 h as a non-stringent test for
accelerated Wallerian degeneration, and for 72 h as a
non-stringent test for any delay in Wallerian
degenera-tion Nmnat1+⁄) X YFP-H nerves fully maintained
axon integrity 30 h after sciatic nerve lesions, similar
to wild-type nerves [Fig 3A(a,b)] All axons were
com-pletely fragmented 72 h after lesion, in the same way
as wild-types [Fig 3A(d,e)] In great contrast, axons
from WldSheterozygous mice are completely preserved
at this time point [Fig 3A(f),B] In order to exclude an
effect on the time of onset of the degenerative process,
we also analysed axon degeneration in wild-type and
Nmnat1+⁄)mice 36 h after sciatic nerve lesion At this
time, axon degeneration has just begun to occur in
wild-types [34] However, even at this time point, we
could not detect any significant difference in the
num-ber of degenerated Nmnat1+⁄) axons compared with
wild-types (Fig 3B) We conclude that NMNAT1 downregulation neither accelerates nor delays axon degeneration after sciatic nerve lesion
We tested the rate of neurite degeneration after cut also in vitro, in superior cervical ganglia (SCG) cultures obtained from Nmnat1+ ⁄ ) and wild-type pups SCG explants were allowed to extend neurites in culture for
7 days The neurites were then cut with a scalpel per-pendicular to the direction of growth and observed at different times Axons in wild-type SCGs remain intact
3 h after cutting, but start degenerating at 6–9 h, with degeneration complete by 24 h Axon fragmentation in Nmnat1+⁄) SCG explants followed an identical time course (Fig 4A,B)
We determined NMNAT1 specific enzyme activity
in SCG explant extracts from wild-type and Nmnat1+⁄)mice (Orsomando G, Cialabrini L, Amici
A, Agostinelli S, Janeckova L, Di Stefano M, Conforti
L, Coleman M and Magni G, manuscript in prepara-tion, adapted from [30,31]) Similarly to what was detected in brain, NMNAT1 activity in Nmnat1+⁄) SCG explants (0.015 mUÆmg)1) was half that in wild-types (0.033 mUÆmg)1) NAD(P)+levels in SGC whole cell extracts showed a non-significant trend towards lower levels in heterozygous null mice relative to wild-types (Fig 4C) This could reflect a reduced level of nuclear NAD+that is masked by the activity of extra-nuclear NMNAT isoforms synthesizing high levels of NAD+in neurites Indeed, neurite density in these cul-tures is very high, and NAD(P)+ levels in wild-type SCG neurites are around double those of their corre-sponding cell bodies (L Conforti, L Janeckova and
M Coleman, unpublished results) Thus reduction of NAD+ within nuclei remains possible However, in agreement with the result in vivo, dowregulation of NMNAT1 expression does not affect the rate of axon degeneration in vitro
Discussion These data indicate that complete NMNAT1 gene inactivation is incompatible with the normal develop-ment of embryos, as the extranuclear isoforms NMNAT2 and NMNAT3 cannot compensate for complete loss of NMNAT1 Nmnat1+⁄) mice have reduced NMNAT1 expression and enzyme activity; however, they develop normally and their lifespan is not altered We show that the rate of Wallerian degen-eration in vivo and in vitro in sciatic nerves and
in SCG explant cultures from Nmnat1+⁄)mice is not different from wild-type
NMNAT1-generated NAD+ in the nucleus is used
as substrate of histone deacetylase sirtuins and
Trang 7PARP1 Sirtuins have been implicated in cellular
pro-cesses such as ageing, transcription, apoptosis and
stress resistance Yeast Sir2 and its mammalian
homo-logue SIRT1 are upregulated upon caloric restriction
and this is associated with increased lifespan [8]
SIRT1 controls the activity of genes that regulate
circadian rhythm and promotes the transcription of
NAMPT, the rate limiting enzyme in NAD+synthesis,
in a feedback loop that has been recently described
[35,36] NAD+ is substrate also for nuclear PARP1,
whose overactivation consequent to genotoxic stress
leads to NAD+ depletion in the cytoplasm and cell
necrosis, demonstrating a communication between the
nuclear and the cytoplasmic NAD+pool [37]
Thus, the failure of Nmnat1 homozygous null
embryos to survive and develop may reflect
perturba-tions in gene transcription, especially sirtuin targets, or
PARP1-mediated NAD+ depletion that cannot be
replenished locally within the nucleus Indeed,
NMNAT1 downregulation in cell lines by small
inter-fering RNA has a profound effect on transcription of
a number of genes, some of which are important for neuronal maintenance and normal neuronal function [11] Conditional homozygous inactivation of Nmnat1in neurons in the adult mouse will be essential
to understand whether and how transcriptional regula-tion affects neuronal maintenance and survival NMNAT1 is also part of the neuroprotective protein WldSand its enzyme activity is necessary but not suffi-cient for this protein to delay degeneration of axons after an injury in vivo [20–22] However, in cell cultures and in Drosophila NMNAT1 overexpression is par-tially neuroprotective [20,23] Moreover, in Drosophila, targeted disruption of NMNAT causes spontaneous axon degeneration via a chaperone activity [38,39] We investigated the role of endogenous NMNAT1 in axon protection in heterozygous null mice where we found a strong reduction in NMNAT1 protein expression and enzyme activity, while the other two isoforms were expressed at wild-type levels and their enzyme activity
Nmnat1+/–
cut t = 72h
WT cut
t = 72 h
UNCUT
WT cut
t = 30 h
Wld S het
cut t = 72 h
Nmnat1+/–
cut t = 30 h 50 µm
(f) (e)
(d)
0
20
40
60
80
100
120
Wild-type
Nmnat1+/–
Wld S het
N.S.
N.S.
N.S.
A
B
Fig 3 Wallerian degeneration rate in Nmnat1 +⁄ )mice (A) Tibial nerves from Nmnat1+⁄)mice crossed to YFP-H with sciatic nerves lesioned for the indicated time show a wild-type rate of Wallerian degeneration with intact axons 30 h after the lesion (a, b) and completely degenerated axons 72 h after the lesion (d, e) At this time point, Wld S heterozygous axons are still completely preserved (f) Bar, 50 lm (B) Quantification of axon degeneration at the indicated time points after sciatic nerve lesions Note that at 36 h post-lesion, when Wallerian degeneration normally begins, the number of degenerated axons is similar in wild-type and Nmnat1+⁄) (n = 4, Student’s t-test).
Trang 8was unchanged Since NMNAT1 activity is
predomi-nant in brain (Fig 2E) and NMNAT1 is also the most
catalytically efficient isoform [31], its downregulation
determines a significant reduction in total NMNAT
activity in Nmnat1+⁄) mice that cannot be
compen-sated by NMNAT2 and⁄ or NMNAT3 Sorci et al [31]
reported that NMNAT2 is the predominant activity in
human brain However, these authors used human
per-itumoural tissue for their determination of
isoform-specific NMNAT activity, whereas we used mouse half
brain homogenates Brain has a heterogeneous cellular
composition that could influence relative abundance of
this enzyme activity; therefore our result is neither
directly comparable nor in conflict with that described
by Sorci et al [31]
Despite NMNAT1 strong downregulation,
Nmnat1+⁄) mice do not show any unusual phenotype
and the rate of Wallerian degeneration in these mice
or in primary neurons derived from them is unaltered
It is possible that the maintenance of normal NAD+ steady state levels despite the decrease in NMNAT activity in our mutant mice underlines the lack of any adverse phenotype The embryonic lethality of NMNAT1 full inactivation precludes the possibility of testing the rate of Wallerian degeneration in the com-plete absence of NMNAT1 However, the result obtained in heterozygous NMNAT1 KO mice suggests that extranuclear NMNAT activities predominantly control the rate of Wallerian degeneration Accord-ingly, the two extranuclear NAD+-synthesizing iso-zymes, NMNAT2 and NMNAT3, maintain wild-type expression levels and enzyme activities in Nmnat1+⁄) mice where Wallerian degeneration after injury pro-ceeds at a wild-type rate
0.60
Wild-type t = 0
Wild-type t = 3 h Wild-type t = 6 h Wild-type t = 9 h Wild-type t = 24 h
0.50
0.40
0.30
0.20
0.10
0.00
N.S.
–1 pr
A
Fig 4 In vitro degeneration of injured axons in Nmnat1 +⁄ )SCG cultures (A) SCG explants from C57BL⁄ 6 and Nmnat1 + ⁄ )mice were cul-tured for 7 days and the extended neurites were separated from the cell body mass using a scalpel Neurites were imaged after the cut at the time points indicated Bar, 10 lm (B) Quantification of axon degeneration in SCG explant cultures after cut The results show that there
is a time effect (P < 0.0001) but no difference between wild-type and Nmnat1 + ⁄ )(n = 6, two-way repeated measures ANOVA, P = 0.808). (C) NAD(P) + levels in whole SCG explant cultures from C57BL ⁄ 6 and Nmnat1+⁄) mice are similar (n = 7, independent samples t-test,
P = 0.492).
Trang 9This is also consistent with our observation of an
increased WldSprotective potency when this protein is
redistributed outside the nucleus [7,17] Moreover, we
showed lack of protection in transgenic mice
overex-pressing NMNAT1 alone and in variant-WldS
trans-genic mice where an N-terminal 16 (N-16) amino acid
sequence derived from Ube4b had been removed
[21,22] Interestingly, the only known property of the
N-16 amino acid sequence indispensable for WldS
action is its ability to bind the abundant cellular
pro-tein VCP⁄ p97 This protein is involved in many
cellu-lar activities and is particucellu-larly enriched at the surface
of membranous organelles [24,25] NMNAT activity
in mammals has become more specialized by evolving
several isoforms, each of them playing a particular
role according to its most abundant location within
the cell WldS protection may be the result of a fine
redistribution of NMNAT1, potentially via VCP
bind-ing, at a specific location inside the cell, where its
enzyme activity leads to downstream events finally
resulting in axon protection Accordingly, cytoplasmic
WldSand cytoplasmic or axonally targeted NMNAT1
are all neuroprotective [7,17,18,40] This location
could match that of the endogenous extranuclear
NMNAT isoform NMNAT2 NMNAT2
downregula-tion triggers spontaneous axon degeneradownregula-tion in
pri-mary SCG neurons [26], suggesting this may be the
endogenous NMNAT activity that normally controls
Wallerian degeneration NMNAT3 could also be
responsible for controlling injury-induced axon
degen-eration However, the low level of NMNAT3 activity
we detect in the nervous system and the lack of a
phe-notype when this isoform is downregulated in neuronal
cultures [26] makes it a weaker candidate NMNAT2
is rapidly degraded after an injury and its rapid
degra-dation could trigger axon degeneration However, the
more stable WldS protein, when present, or an
abnor-mal targeting of NMNAT1 itself [17] could preserve
the injured axons by substituting for NMNAT2 [26]
The results presented here argue against functional
redundancy of the three mammalian NMNAT
iso-forms NMNAT2 and 3 cannot compensate for loss of
NMNAT1 when this isozyme is completely inactivated,
leading to the lack of viability of null NMNAT1 KO
mice In addition, there is no upregulation of
NMNAT2 or 3 in Nmnat1+⁄) mice, where NMNAT1
is highly downregulated In cultured SCGs, NMNAT1
and 3 cannot compensate for loss of NMNAT2
trig-gered by RNA interference or by axon injury [26]
Indeed, the low level of NMNAT3 activity in brain
suggests its main functions may be predominant in
other tissues [30] However, the various isoforms could
compensate for each other when redistributed to a
dif-ferent location For instance NMNAT1 appears to compensate for loss of NMNAT2 when it reaches ectopic location by high overexpression or by re-target-ing, therefore conferring protection to axons after cut [17,18,22,26]
Despite the role for other NMNAT isoforms such as NMNAT2 in controlling axonal integrity, a related role for NMNAT1 remains possible in the absence of data from homozygous null mice In particular, it is possible that the level of this enzyme activity in heterozygous
KO could remain above a threshold level needed to significantly modify axon degeneration after an injury The availability of NMNAT1 floxed mice will enable
us to address this question in a future study by generat-ing conditional KOs where the NMNAT1 gene is inac-tivated only in neurons at postnatal stages, overcoming the embryonic lethality of a complete null mutant
In conclusion, NMNAT1 is indispensable for the normal development of the embryo and NMNAT2 and 3 cannot compensate for its loss Decreased NMNAT1 activity in heterozygous null mice, however, does not affect the rate of Wallerian degeneration, sug-gesting that endogenous NMNAT1 does not have a primary role in axon maintenance
Materials and methods Construction of the targeting vector
We determined the genomic sequence of the entire mouse Nmnat1 coding region and used this to design a targeting vector based on the plasmid pEASYFlox (a gift from W Mu¨ller and K Rajewsky) The positive selection marker, G418⁄ neomycin (NEOR), is flanked by two loxP sites To maximize the likelihood of achieving complete gene inacti-vation, we chose to delete a region comprising the first and second exons, including some 5¢ UTR where the promoter
is located This region was amplified by PCR with SalI tagged primers and cloned into the SalI site of the targeting vector Two additional homology regions, a 5¢ 2.3 kb region and a 3¢ 4.6 kb region, were then obtained by PCR using primers tagged with NotI⁄ BamHI and HindIII sites respectively and cloned into the respective restriction sites
of pEASYFlox We confirmed the absence of PCR and cloning artefacts by sequencing all coding regions, the loxP sites and most non-coding regions The genomic locus, the completed targeting vector and the recombination events are shown in Fig S1
The primer pair sequence was as follows: 5¢ homology arm (NotI and BamHI site underlined and italics) 5¢-AGGAAAAAAGCGGCCGCACACTTACAGCCTGAG GCG-3¢, 5¢-CGCGGATCCACTCCAAGGATACACTCC GA-3¢; 3¢ homology arm (HindIII site underlined and ital-ics) 5¢-GGCCCAAGCTTATATATTTGCCTAGGAGGGT
Trang 10C-3¢, 5¢-GGCCCAAGCTTAAGACAGTGTGGAGGAGA
CCT-3¢ The targeted region (SalI site underlined and
ital-ics) was 5¢-CAACGCGTCGACCCATGTGCTGAAAGCT
TGGT-3¢, 5¢-ACTGGCGTCGACTTGAATGTCTTAGTG
ACTGGG-3¢ All primers were purchased by
Sigma-Genosys, Haverhill, UK All chemicals were obtained by
Sigma-Aldrich, Gillingham, UK, unless otherwise stated
ES cell electroporation and isolation of a double
recombinant clone for blastocyst injection
The 18 kb targeting vector was linearized with NotI and
elec-troporated into Bruce 4 ES cells (from C57BL⁄ 6 strain, kind
gift of K Rajewsky and A Egert) ES cell clones were
posi-tively selected 24 h post electroporation with 0.2 mgÆmL)1
G418 Negative selection of random integration was
per-formed by addition of 2· 10)6mganciclovir to the medium
We picked 384 clones among the ones that were resistant to
both selection agents Southern blot analysis showed that
only one clone contained the entire targeting vector correctly
integrated at both homology arms of the genomic locus This
clone was electroporated again in vitro with a Cre expression
vector (pPGK-Cre-bpA, kind gift of K Rajewsky and
W Mu¨ller) This allowed us to delete the NEORgene and
leave a loxP flanked region amenable to conditional or
con-ventional deletion One subclone was then isolated that had
lost the NEOR cassette and contained a ‘floxed’ targeted
locus (Fig 1A) We designed primers spanning the two loxP
sites (Pr1, Pr2, Pr3, Pr4, see Fig 1A) to confirm the presence
and the integrity of the loxP sites in the floxed clone after
Cre-mediated deletion The PCR across the loxP sites
con-firmed the presence of both loxP sites in the targeted clone
(Fig 1B) Furthermore, sequencing of the PCR products
confirmed that the loxP sites were correct
Generation of targeted mice
The Bruce 4 targeted ES cell clone containing the floxed
locus was used for injection into BALB⁄ c derived
blast-ocysts Chimeric mice, originally identified by coat colour,
were then confirmed by southern blotting (see Fig 1B)
Chimeric mice were backcrossed to C57BL⁄ 6 mice and the
transmission of the mutant allele to the progeny was
revealed by coat colour analysis and southern blotting
Nmnat1+ ⁄) mice were obtained by crossing the floxed
Nmnat1 male chimerics to female C57⁄ BL6 K14 Cre mice
to produce heterozygous null mice on a black background
[27] Southern blot analysis demonstrated that about 50%
of the offspring are heterozygous for the full deletion allele
The heterozygous mice were then intercrossed in an attempt
to generate homozygous null mutants Animal work was
performed in accordance with the relevant German and
UK government animal welfare legislation under licenses
K13, 11⁄ 00 (Cologne, Germany) and 80 ⁄ 1778 and 80 ⁄ 2254
(Cambridge, UK)
Preparation and analysis of DNA from ES cells, mice and embryos
Genomic DNA was isolated using standard protocols [21] For southern blot analysis, genomic DNA from ES cells was digested with EcoRI or HindIII and analysed with a 420 bp 5¢ probe and a 750 bp 3¢ probe located outside the targeted region (Fig S1) and generated by PCR from genomic DNA with the following primer pairs: 3¢ probe, 5¢-AATATTTGGAATTAGGTAAGTGT-3¢, 5¢-GTGTAAAAGACACTGTGATG-3¢; 5¢ probe, 5¢-TGT CTTAAAATGCACTTCAAAC-3¢, 5¢-GTCGAGTTGCCA TGCAGAG-3¢ Another 450 bp probe (called probe 4, Fig 1A) obtained by mouse genomic DNA PCR with the primers 5¢-GGCCCAAGCTTATATATTTGCCTAG GAGGGTC-3¢ and 5¢-TCAGACATTTATAAGTTTCG GG-3¢ was used on southern blots of tail genomic DNA digested with BamHI to identify both Nmnat1 floxed mice and Nmnat1 heterozygous KO mice PCR screening
of those mice used the following primers spanning loxP site 1 and loxP site 2: Pr1, 5¢-TCGGAGTGTATCCTTG GAGT-3¢; Pr2, 5¢-ACCAAGCTTTCAGCACATGG-3¢; Pr3, 5¢-CCCAGTCACTAAGACATTCAA-3¢; Pr4, 5¢-GA CCCTCCTAGGCAAATATA-3¢
Western blotting, NMNAT enzyme activity assay and NAD(P)+level determination
Western blotting of sagittally divided half brains was per-formed as described previously [22] Sagittally divided half brains were homogenized in five volumes of RIPA buffer [phosphate-buffered saline (PBS) containing 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecylsulphate] High-speed supernatant was diluted to approximately 0.5 mgÆmL)1total protein according to the Bradford assay (BioRad, Hemel Hempstead, UK) and fractionated by standard SDS⁄ PAGE After semidry blotting (BioRad, Hemel Hempstead, UK), nitrocellulose membranes (Bio-Rad) were blocked in PBS plus 0.02% Tween-20 and 5% low-fat milk powder before incubation with primary anti-body and then horseradish peroxidase conjugated second-ary antibody (1 : 3000; Amersham Biosciences, Little Chalfont, UK) Proteins were visualized using the ECL detection kit (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions For quantification, western blot band intensities were deter-mined with image j software and normalized to b-actin NAD+ and NAD(P)+ levels were determined in brain or whole cell extracts by HPLC identification or by a fluori-metric cyclic reaction as described previously [41,42] Total NMNAT enzyme activity was determined as described earlier [41] Tissue was suspended in six volumes
of 50 mm Hepes, pH 7.4, 0.5 mm EDTA, 1 mm MgCl2,
1 mm phenylmethylsulphonyl fluoride and 0.02 mgÆmL)1 each of leupeptin, antipain, chymostatin and pepstatin,