Sci. Rep. Kanazawa Univ.
Vol.59, pp.51–60, 2015

A cell-autonomous role for JSAP1 and JLP inmouse cerebellar Purkinje cell survival
Momoe I SHIKAWA1,∗, Tokiharu S ATO1 , Hiroshi K IYONARI2,3 ,and Katsuji YOSHIOKA1,∗
1 Division of Molecular Cell Signaling, Cancer Research Institute, Kanazawa University,

Kakuma-machi, Kanazawa 920–1192, Japan
2 Animal Resource Development Unit and 3 Genetic Engineering Team,RIKEN Center for Life Science Technologies,
2–2–3 Minatojima Minami-machi, Chuou-ku, Kobe 650–0047, Japan.
(Received November 26, 2015 and accepted in revised form December 21, 2015)

Abstract We previously reported that the structurally related JNK scaffolding proteins
JSAP1 and JLP play functionally redundant and key roles in maintaining mouse cerebellar Purkinje cell (PC) survival, which may be related to their regulation of axonaltransport. The JSAP1 and JLP proteins are also widely expressed in other cell types
throughout the brain. Notably, in the cerebellum, JSAP1 is abundantly expressed in thepinceau, a cluster of basket cell (BC) axons and termini that surround the PC axon initial
segment. Thus, it is possible that BC-expressed JSAP1 plays a role in PC survival. To
investigate this possibility, we generated and analyzed mice containing a PC/BC-speciﬁcdouble knockout (DKO) of Jsap1 and Jlp (Jsap1:Jlp cDKO mice). These mice exhibited
PC axonal dystrophy, followed by progressive PC loss, consistent with the phenotypes
previously reported for PC-speciﬁc Jsap1 and Jlp DKO mice. Furthermore, we foundthat the phenotypes of Jsap1:Jlp cDKO PCs were rescued by PC-speciﬁc transgenic
expression of JSAP1. Taken together, these results indicate that BC-expressed JSAP1
plays little or no role in PC survival, and that PC-expressed JSAP1 and JLP play cellautonomous roles in preventing PC degeneration.
Keywords: Axonal swelling; Knockout mice; Neuronal degeneration

1

Introduction

The mitogen-activated protein kinase (MAPK) intracellular signaling pathway, consisting of MAPK, MAPK kinase (MAPKK), and MAPKK kinase, is well conserved
from yeast to humans (Cano and Mahadevan, 1995; Herskowitz, 1995; Waskiewicz∗ Corresponding authors: momonomi@stu.kanazawa-u.ac.jp and katsuji@staff.kanazawa-u.ac.jp

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A cell-autonomous role for JSAP1 and JLP in mouse cerebellar Purkinje cell survival

and Cooper, 1995). Scaffolding proteins associated with MAPK modules contribute,at least in part, to the speciﬁcity and efﬁciency of these signaling cascades (Morrison
and Davis, 2003; Yoshioka, 2004; Dhanasekaran et al., 2007). JNK/stress-activatedprotein kinase-associated protein 1 (JSAP1, also known as JIP3) and JNK-associatedleucine zipper protein (JLP, also known as JIP4) are structurally related scaffolding
proteins associated with mammalian JNK and p38 MAPK signaling modules (Ito etal., 1999; Kelkar et al., 2000; Lee et al., 2002; Kelkar et al, 2005) that can also interact
with kinesin-1 light and heavy chains (KLC and KHC, respectively) through speciﬁc
binding domains. JSAP1 and JLP are reported to function as adaptor proteins thatlink cell cargoes to kinesin-1 (Verhey et al., 2001; Nguyen et al., 2005), and regulatekinesin-1 activity and motility (Sun et al., 2011; Watt et al., 2015). Studies of JSAP1
loss-of-function mice indicate that JSAP1 is required during early development of the
corpus callosum (Kelkar et al., 2003; Ha et al., 2005), and Jsap1 knockout (KO) micedie shortly after birth due to neuronal defects (Iwanaga et al., 2007). In contrast, Jlp
KO mice are viable and grow normally (Iwanaga et al., 2008). Recently, however,
JSAP1 and JLP were reported to have crucial and functionally redundant roles in thedeveloping mouse brain and in the survival of Purkinje cells (PCs), both of which areassociated with their regulation of kinesin-1-dependent axonal transport (Sato et al,2015a and 2015b).
JSAP1 and JLP are widely expressed in the mouse brain. Notably, in the cerebellum, JSAP1 is highly expressed in the pinceau, a cluster of basket cell (BC) axons andtermini that surround the PC axon initial segment (Miura et al., 2006). Thus, although
we concluded from our previous study that PC-expressed JSAP1 and JLP play key,functionally redundant roles in PC survival (Sato et al, 2015b), it remains possible that
BC-expressed JSAP1 and JLP are also involved in maintaining PC survival. To investigate this possibility, we generated and analyzed mice containing a PC/BC-speciﬁcdouble KO (DKO) in Jsap1 and Jlp (Jsap1:Jlp cDKO), and then evaluated the effectof transgenic rescue with PC-expressed JSAP1.

2

Materials and Methods

2.1

Animals

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committees of Kanazawa University and RIKEN Kobe, respectively. GluRδ 2-Cre mice (Yamasaki et al., 2011), in which Cre is driven by GluRδ 2
promoter and thus is expressed speciﬁcally in BC and PC, were provided by Dr. Kenji
Sakimura (Niigata University), and were crossed with Jsap1 f / f (Iwanaga et al., 2007)
and Jlp f / f (Sato et al., 2015a) mice. The green ﬂuorescent protein (GFP) coding
region in pBS-L7-GFP, a gift from Dr. Hirokazu Hirai (Gunma University), and a

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53

derivative of the pL7ΔAUG vector containing the PS-speciﬁc L7 promoter but disrupting the translational start site (ATG) of the L7 gene (Oberdick et al, 1990; Tomomura et al., 2001), was replaced with the entire coding region of the mouse wild-typeJsap1 gene. The L7-Jsap1 transgene was then injected into fertilized eggs to generate
L7-Jsap1 transgenic mice (Accession No. CDB0513T: http://www2.clst.riken.jp/arg/TG%20mutant%20mice%20list.html). Genotyping of L7-Jsap1 transgenic mice was
performed using the following two primer sets:
5’-TGTTTGGAGGCACTTCTGACTTGC-3’ and5’-CTCGTCCATCTGGATCTCCATCAT-3’ (primer set 1) and5’-CGGAGAGGATGATGAAACTGAGGA-3’ and5’-CACTCAACTCTTTGTTGCTAGTGCC-3’ (primer set 2) giving PCR products of
156-bp and 261-bp, respectively.

2.2

Antibodies (Abs)The following primary Abs were used in this study: anti-JSAP1 (Miura et al, 2006),
anti-calbindin D-28K (C9848, Sigma-Aldrich or AB1778, Millipore), anti-synaptotagmin 1 (SYT1) (105002, Synaptic Systems), anti-SNAP25 (S5187, Sigma-Aldrich),anti-glial ﬁbrillary acidic protein (GFAP) (G3893, Sigma-Aldrich), anti-phospho-JNK(9251, Cell Signaling), and anti-α-tubulin (T5168, Sigma-Aldrich) Abs. The following secondary Abs were used: Alexa 488- and 568-conjugated anti-mouse or antirabbit IgG (Invitrogen) and horseradish peroxidase (HRP)-conjugated anti-mouse oranti-rabbit IgG (GE Healthcare) Abs.

2.3

Western blotting (WB), immunohistochemistry (IHC), and PC quantiﬁcation

WB, IHC, and PC quantiﬁcation were performed as described previously (Sato et al.,2008 and 2015b). IHC images were acquired with a confocal laser scanning microscope (LSM510 META, Carl Zeiss) using a 20x or 40x objective lens.

2.4

Reverse transcription (RT)-polymerase chain reaction (PCR)

RT-PCR was carried out as previously described (Sato et al, 2004), using the following
primer pairs: L7-Jsap1, 5’-CGTGTAACAGTTAATTCCCTGCCTG-3’ (L7) and5’-CTCGTCCATCTGGATCTCCATCAT-3’ (Jsap1); Gapdh,
5’-AAATGGTGAAGGTCGGTGTG-3’ and 5’-TGAAGGGGTCGTTGATGG-3’.

2.5

Statistical analysis

Signiﬁcance was determined using the two-tailed unpaired Student’s t-test. P values< 0.05 were considered to be statistically signiﬁcant.

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A cell-autonomous role for JSAP1 and JLP in mouse cerebellar Purkinje cell survival

3

Results and Discussion

3.1

PC/BC-speciﬁc deletions in Jsap1 and Jlp cause PC axonal swellingand progressive cell loss

To investigate the roles of JSAP1 and JLP in BCs, we generated mice with PC/BCtargeted conditional deletions in Jsap1, Jlp, or both, using loxP-ﬂanked (ﬂoxed) alleles of these genes (Iwanaga et al., 2007; Sato et al., 2015a), in combination with the
GluRδ 2-Cre transgene (Yamasaki et al., 2011). We ﬁrst performed IHC of the midsagittal brain sections of 24-week-old control (Jsap1 f / f :Jlp f / f ), single Jsap1 or Jlp
cKO (Jsap1 f / f :Jlp f /+ :GluRδ 2-Cre or Jsap1 f /+ :Jlp f / f :GluRδ 2-Cre), and Jsap1:Jlp
cDKO (Jsap1 f / f :Jlp f / f :GluRδ 2-Cre) mice using an Ab against calbindin, a PC
marker. The results showed the presence of swollen PC axons associated with PC loss
in the Jsap1:Jlp cDKO mice, but not in the single Jsap1 or Jlp cKO mice (Fig. 1A).
We quantiﬁed the PCs by Niss1 staining of the sections and found that the PC numbersin the cDKO mice were signiﬁcantly reduced compared to those in the cKO or control mice (Fig. 1B). Taken together, these results indicated that JSAP1 and JLP playfunctionally redundant roles in PC survival, which may be related to the regulation ofaxonal transport in PCs.
Next, we used anti-calbindin Ab IHC to examine the PC axons of Jsap1:Jlp cDKO
mice at different ages (4, 8, 16, and 24 weeks of age; Fig. 2A). Many swollen axons
were observed in regions near the PC bodies of the cDKO mice at each of the ages
examined. We also quantiﬁed the PCs in Jsap1:Jlp cDKO mice at those ages, and at
40 weeks of age. The PC numbers were comparable between the control and Jsap1:Jlp
cDKO mice at 4 and 8 weeks of age (Fig. 2B). However, the cDKO mice at16 weeks
of age showed signiﬁcantly decreased PC numbers compared to control mice, and thedegree of PC loss increased with age (Fig. 2B). In addition, intense immunosignals for
GFAP, a hallmark of reactive gliosis, were detected in association with PC death in
the Jsap1:Jlp cDKO mice at 24 weeks of age (Fig. 2C). These results are consistent
with those previously obtained using PC-speciﬁc Jsap1:Jlp DKO mice (Sato et al.,2015b), suggesting that JSAP1 and JLP expressed in BCs have little or no effect on
PC survival.

3.2

Selective accumulation of the kinesin-1 cargo SNAP25 in the swollenaxons of JSAP1- and JLP-deﬁcient PCs

Axonal dystrophy is thought to reﬂect defective axonal transport. Since JSAP1 and
JLP are known to interact with kinesin-1, a motor protein involved in axonal transport,
the axonal dystrophy phenotype of Jsap1:Jlp cDKO may be due to defects in kinesin1-dependent axonal transport. To examine this possibility, we analyzed the swollenaxons of PCs in 16-week-old Jsap1:Jlp cDKO mice by double-immunostaining, using

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55

Figure 1. PC/BC-speciﬁc deletion of
Jsap1 and Jlp causes PC axonal swellingand cell loss.
(A) IHC analysis of
Jsap1 f / f :Jlp f / f (control), single Jsap1 orJlp cKO, and Jsap1:Jlp cDKO mice at 24
weeks of age. Midsagittal sections fromthe indicated mice were stained with ananti-calbindin Ab. PC axonal swelling
and loss were seen in the Jsap1:Jlp cDKO
mice, but not in the single Jsap1 or Jlp
cKO mice. (B) PC quantiﬁcation. Midsagittal sections from the indicated mice at
24 weeks of age were stained with Nissl,
and the PCs per μm in lobules II to IX
were counted (n = 3 mice per group).
PC numbers in the Jsap1:Jlp cDKO mice
were signiﬁcantly decreased compared tothose in the control mice. Scale bar,
50 μm. ***P < 0.001; n.s., not signiﬁcant.

Abs against calbindin and either SNAP25, a kinesin-1 cargo (Hirokawa et al., 2009),
or SYT1, a kinesin-3 cargo (Hirokawa et al., 2009). Strong SNAP25 immunosignals were detected throughout almost all of the swollen PC axons, whereas the SYT1
immunosignals were very low and limited in distribution (Fig. 3A). These results, together with the earlier ﬁnding of progressive PC loss in these mice, suggested that
JSAP1 and JLP play important roles in the kinesin-1-dependent axonal transport inPCs, and that their ablation causes PC axonal dystrophy, leading to neuronal loss.
In addition, we analyzed JNK activation in the axons using a phospho-JNK Ab. The
results showed substantial JNK activation in the swollen axons of PCs in the Jsap1:Jlp
cDKO mice (Fig. 3B), consistent with that observed in the PC-speciﬁc Jsap1:Jlp DKO

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A cell-autonomous role for JSAP1 and JLP in mouse cerebellar Purkinje cell survival

Figure 2. PC axonal dystrophy, progressive PC loss, and reactive gliosis in
Jsap1:Jlp cDKO mice. (A) Midsagittal sections of control and Jsap1:Jlp cDKO miceat 4, 8, 16, and 24 weeks of age were subjected to IHC with an anti-calbindin Ab.
Higher magniﬁcations of the boxed areas are shown in the right-most panels. (B) Midsagittal sections from control and Jsap1:Jlp cDKO mice at the indicated ages were
stained with Nissl, and the PCs were counted as in Fig. 1B (n = 4 mice per group).
PC numbers were signiﬁcantly decreased in Jsap1:Jlp cDKO mice at 16 weeks of age,compared to control mice, and the degree of PC loss increased with age. (C) Sectionsof the PC layer in control and Jsap1:Jlp cDKO mice at 24 weeks of age were examined
by IHC with an anti-GFAP Ab and 4,6-diamidino-2-phenylindole (DAPI). Scale bars,
50 μm. ***P < 0.001; n.s., not signiﬁcant.

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57

Figure 3. Selective accumulation of
SNAP25 kinesin-1 cargo and JNK
activation in the swollen PC axonsof Jsap1:Jlp cDKO mice. Midsagittal sections from 16-week-old control and Jsap1:Jlp cDKO mice were
examined by double-immunostaining
for calbindin and either SNAP25 (akinesin-1 cargo) or SYT1 (a kinesin3 cargo) (A), and for calbindin andPhospho-JNK (B), as indicated. Boxed
areas are magniﬁed in the insets. Scalebars, 50 μm.
mice (Sato et al., 2015b). At present, the precise mechanism underlying the PC death
induced by the ablation of JSAP1 and JLP remains unknown. However, these results,
together with the results of our previous study using Jsap1:Jlp DKO primary cultured
neurons (Sato et al., 2015a), suggest that JNK activation may be involved in mediating
the neuronal death.

3.3

Transgenic expression of JSAP1 in PCs rescues the PC degenerationin Jsap1:Jlp cDKO mice

To examine whether the JSAP1 expressed in BCs affects PC survival, we generated
transgenic mice expressing the PC L7 promoter-driven Jsap1 transgene (L7-Jsap1,
Fig. 4A), and performed rescue experiments by crossing Jsap1:Jlp cDKO mice withthe transgenic mice. We ﬁrst conﬁrmed that the exogenous Jsap1 mRNA was specifically expressed in the cerebellum, and not the cerebral cortex (Fig. 4B). We then analyzed the compound mice (Jsap1:Jlp cDKO/L7-Jsap1) by WB with an anti-JSAP1

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A cell-autonomous role for JSAP1 and JLP in mouse cerebellar Purkinje cell survival

Figure 4. Rescue of PC degeneration in Jsap1:Jlp cDKO mice by the PC-speciﬁctransgenic expression of JSAP1. (A) Schematic illustration of DNA construct used
to generate transgenic mice that express L7 promoter-driven Jsap1. Arrows indicate
primers for RT-PCR in B. (B) Total RNAs were extracted from the cerebellum (Ce) andcerebral cortex (Cx) of 8-week-old control and L7-Jsap1 transgenic mice, as indicated,
and RT-PCR was performed using speciﬁc primers (indicated as arrows in A) for the
L7-Jsap1 transgene, with (lanes 1-4), or without (lanes 5-8) reverse transcriptase (RT).
Gapdh RT-PCR was used as a control. (C) WB analysis of JSAP1 in the cerebellumof the indicated mice at 8 weeks of age. α-tubulin was used as a loading control.
(D) Midsagittal sections from the indicated mice at 8 weeks of age were examined bydouble-immunostaining for JSAP1 and calbindin. Asterisks and arrowheads denote
JSAP1-labeled pinceaus and swollen PC axons, respectively. Higher magniﬁcations of
the boxed regions are shown in the panels at right. (E) Rescue of PC loss in Jsap1:Jlp
cDKO mice by the transgenic expression of JSAP1. PC numbers in the indicated miceat 24 weeks of age were quantiﬁed as in Fig. 1B (n = 4 mice per group). Scale bars,
25 μm. ***P < 0.001.

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59

Ab (Fig. 4C), and by IHC using Abs against calbindin and JSAP1 (Fig. 4D). The expression levels of JSAP1 in the cerebellum were comparable between the control andJsap1:Jlp cDKO/L7-Jsap1 mice (Fig. 4C). JSAP1 immunosignals were detected in thepinceau of the control and transgenic mice, but not in that of the Jsap1:Jlp cDKO andJsap1:Jlp cDKO/L7-Jsap1 mice (Fig. 4D), indicating that the exogenous JSAP1 was
expressed speciﬁcally in the PCs. Although swollen PC axons were observed in theJsap1:Jlp cDKO mice at 8 weeks of age (Fig. 4D lower left panels, see also Fig. 2A),
no axonal dystrophy was detected in the Jsap1:Jlp cDKO/L7-Jsap1 mice at the sameage (Fig. 4D lower right panels). Furthermore, the decreased number of PCs in theJsap1:Jlp cDKO mice at 24 weeks of age was rescued in the Jsap1:Jlp cDKO/L7Jsap1 mice of the same age (Fig. 4E). Taken together, these results indicated that the
JSAP1expressed in BCs is not required for PC survival, and that intrinsically expressed
JSAP1 and JLP play functionally redundant roles in PC survival.
In conclusion, our molecular genetic and immunohistological analyses highlighted
the key roles of JSAP1 and JLP in mouse PC survival, and strongly suggested that
JSAP1 and JLP function cell autonomously to prevent PC degeneration. At present,
the role of JSAP1 in the pinceau remains unknown. Further studies are required toclarify this issue.

Acknowledgements
We thank Drs. Kenji Sakimura (Niigata University) and Hirokazu Hirai (Gunma University) for providing the GluRδ 2-Cre mice and pBS-L7-GFP vector, respectively.

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