HMG Advance Access published September 12, 2007
Distal axonopathy in an alsin-deficient mouse model Han-Xiang Deng 1*, Hong Zhai 1, Ronggen Fu 1, Yong Shi 1, George H.Gorrie 1, Yi Yang 1
1
, Erdong Liu 1, Mauro C. Dal Canto 2, Enrico Mugnaini 3 and Teepu Siddique 1,3,4* Davee Department of Neurology and Clinical Neurosciences, 3
Pathology, Division of Neuropathology,
2
Department of
Northwestern University Institute for
Neuroscience, Northwestern University Feinberg School of Medicine,
4
Department of
Cell and Molecular Biology, Tarry Building, Room 13-715, 303 East Chicago Avenue, Chicago, IL 60611. USA.
[email protected]) Tel: (312) 503-4737 Fax: (312) 908-0865
© 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
1
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Correspondence should be addressed to HXD (
[email protected]) or TS (t-
ABSTRACT Mutations in Alsin are associated with chronic juvenile amyotrophic lateral sclerosis (ALS2), juvenile primary lateral sclerosis and infantile-onset ascending spastic paralysis. The primary pathology and pathogenic mechanism of the disease remain largely unknown. Here we show that the alsin-deficient mice have motor impairment and degenerative pathology in the distal corticospinal tracts without apparent motor neuron pathology. Our data suggest that ALS2 is predominantly a distal axonopathy, rather than a neuronopathy in the central nervous system of the mouse model, implying that alsin plays an important role in maintaining the integrity of the corticospinal axons. Downloaded from http://hmg.oxfordjournals.org/ by guest on June 2, 2013
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INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of the upper motor neurons of the motor cortex in brain and lower motor neurons in brainstem and spinal cord, resulting in progressive wasting and paralysis of voluntary muscles 1.
The progressive paralysis in ALS usually affects respiratory
function, leading to ventilatory failure and death; 50% of patients die within three years of onset of symptoms and 90% within five years. The juvenile form of ALS usually has a prolonged course extending over two to three decades. There is no known effective treatment for this fatal disease, although marginal delay in mortality has been noted with
Most of the ALS cases are sporadic (SALS), but about 5~10% are familial (FALS). FALS can be transmitted as either a dominant or a recessive trait.
Mutations in Cu/Zn
superoxide dismutase (SOD1) gene have been shown to be associated with about 20% of FALS cases
3, 4
. Transgenic mice overexpressing ALS-associated mutant SOD1
developed ALS-like phenotype, while transgenic mice overexpressing wild type SOD1 (wtSOD1) remained unaffected 5, and SOD1 knockout mice did not show motor neuron degeneration 6, suggesting mutant SOD1 has a toxic property that triggers the motor neuron degeneration in ALS. We and others have previously shown that mutations in a novel gene, Alsin, cause juvenile ALS type 3 (ALS2), juvenile primary lateral sclerosis (JPLS) or infantile-onset ascending spastic paralysis (IAHSP), depending on the location of the mutations
7-9
.
Twelve mutations have been reported to date, among which, two (A46fsX50 and T185fsX189) lead to ALS2 and the others seem to result in upper motor neuron syndromes without the involvement of lower motor neurons 7-14. Most of these mutations lead to truncated alsin proteins. It is thus postulated that loss of normal function of alsin may trigger the pathogenic process in ALS2. It is well known that in ALS patients with SOD1 mutations and mutant SOD1 transgenic mouse models, a pathological hallmark is the presence of SOD1-containing aggregates or inclusions in neurons
15, 16
. We have
recently demonstrated that conversion of SOD1 from soluble form to insoluble aggregates by intermolecular disulfide bonds via redox processes contributes to the
3
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the drug riluzole 2.
pathogenesis of mutant SOD1-mediated ALS (ALS1)
17, 18
. In addition to SOD1, these
aggregates also showed immunoreactivity to other cellular proteins
19
. It has been
proposed that one of the possible mechanisms leading to motor neuron degeneration in ALS1 is that an essential survival factor for motor neuron is trapped by the SOD1 aggregates, leading to depletion of such a factor and thus triggering the process of the motor neuron degeneration
20
. The identification of alsin mutations in ALS2 raises
several interesting questions. (1) Is alsin such an essential factor for motor neuron survival? (2) Is depletion of alsin the common signaling pathway triggering motor neuron degeneration in both ALS1 and ALS2? (3) Does loss of alsin contribute to ALS1? Kanekura et al. reported in vitro evidence that the long form of alsin (alsin LF) Rac1/PI3K/Akt3 pathway 21, 22, raising a possibility that loss of alsin is a shared upstream signal pathway of motor neuron degeneration in both ALS1 and ALS2 or loss of alsin significantly contributes to development of ALS1. Previous studies of the alsin-deficient mice showed variable phenotypes and pathology
23-26
. A moderate impairment in motor coordination, a higher level of anxiety
response, and increased susceptibility to oxidative stress were observed in one line 23. A mild loss of Purkinje cells, motor unit remodeling, and gliosis in brain and spinal cord were reported in the second line 24. Slowness and degeneration of the corticospinal axons in the dorsolateral column were shown in the third line 25. Motor behavioral abnormalities disturbances in endosome trafficking were observed in the fourth line
26
. The
discrepancies in phenotype and pathology in these three alsin-deficient lines were apparent and the reasons for these discrepancies remain unclear. To address these issues, we developed and characterized a new Alsin knockout mouse model. We show here that ALS2 is predominantly a distal axonopathy, rather than a neuronopathy in the murine central nervous system. This pathology is consistent with the observed predominance of the upper motor neuron symptoms in human subjects with Alsin mutations, implying important physiological functions of Alsin in maintaining the integrity of the corticospinal axons. RESULTS
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protected motor neuron cells from the toxicity induced by mutant SOD1 through
Development of Alsin-/- mice Human Alsin is relatively a large gene with 83kb genomic DNA in size. It has 34 exons with the first exon being non-coding. Alsin has two transcriptional forms with two distinct poly(A) signals and encodes one short and one long form of protein product, alsin. The short form of Alsin gene has 4 exons and the long form of Alsin has 34 exons. Both forms share the first 4 exons. We previously reported that a homozygous deletion mutation, A46fsX50 that interrupts both forms of alsin, leads to ALS2. However, a different homozygous deletion mutation, L623fsX647 that only interrupts the long form, leaving the short form intact, leads to JPLS 7. Mouse Alsin also has two alternatively
designed to replace exon 4 and a part of exon 3 with neo-cassette (Fig.1a). This vector was designed to target both short and long forms of Alsin, leading to a very short, truncated polypeptide consisting of only 9 amino acids (aa). Electroporation of the targeting vector into 129S6/SvEv embryonic stem (ES) cells and subsequent neomycin and gancyclovir selection, yielded 582 ES cell clones. We identified two positive ES cell clones by PCR and Southern blot analysis (Fig.1b, 1c). The positive ES cells were used for blastocyst injection. We developed nine highly chimeric male mice, four of which were used for crossbreeding with B6/SJL females to generate heterozygotes. Homozygous Alsin-knockout mice were generated by crossbreeding heterozygous mice (Fig. 1d). Targeting exons 3 and 4 resulting in skipping of exons 3 and 4 during transcription Mutations in Alsin were distributed among various exons from exons 3 to 32. We have previously shown that a 1-bp deletion in exon 3, leading to frame shift and premature stop codon (A46fsX50) causes ALS2 in humans. The mutant version of Alsin mRNA with the 1-bp was verified in the lymphoblasts of these ALS2 patients 7. To test if targeting exons 3 and 4 will generate a frame shift in Alsin mRNA, we extracted RNA from the brain of the Alsin-/- mice.
RT-PCR and sequencing analysis indicated that
targeting exons 3 and 4 resulted in a mutant Alsin mRNA with exons 3 and 4 skipped, leading to a frame shift and premature stop codon in exon 5 (Fig. 1e). This mutant mRNA is predicted to encode a polypeptide with only 9aa. We also identified alternatively
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spliced forms 24. To develop a mouse model of ALS2, we constructed a targeting vector
transcribed Alsin mRNAs with exon 4 as well as exons 3 and 4 skipped in the brain and spinal cord of normal mice (Fig. 1e). However, mRNAs with skipping of exon 4 or combined exons 3 and 4 will not be functional, because these skipping are predicted to result in frame shift mutations. As predicted by the targeting vector design, the shared exon 4 genomic DNA sequence by both forms of Alsin was not detected and alsin protein was not produced in Alsin-/- mice (Fig. 1f and 1g). Conserved reproductive fitness in Alsin-/- mice ALS2 is an early onset and slowly progressing disease. The age of the disease onset varies from 1-10 years of age based on the clinical data reported so far. The duration of
children. It is unclear if loss of alsin has an impact on reproductive fitness. We generated the homozygous Alsin-/- mice by crossbreeding Alsin+/- mice. The Alsin-/- newborn pups were obtained with the expected Mendelian frequency, suggesting that there was no increased embryonic mortality in the mutant mice. To test if Alsin-/- mice are productive, we intercrossed Alsin-/- mice. The number of progeny was comparable to that generated by crossbreeding between wild type mice, suggesting that loss of alsin has no apparent effect on reproductive fitness. Motor deficit and distal axonopathy in Alsin-/- mice ALS2 is characterized by bilateral pyramidal syndrome, weakness with atrophy and fasciculation of the hands and/or legs, but without sensory disturbance. ALS2 has an early onset at an infantile and a juvenile age with very slow progression 27. We carefully monitored the Alsin-/- mice for potential phenotype similar to human ALS2. However, we did not find any apparent neurological phenotype even in mice over 400 days old. Then, we tested the Alsin-/- mice on a Rota-Rod (Cat# 7650, Ugo Basile, Italy). The mice were tested on a mode starting from 2 rpm to a maximum speed of 20 rpm for a maximum time of 300 seconds, accelerating at a rate of 4 rpm/30 seconds. We could not observe any significant difference between Alsin-/- mice and control mice, because mice from both groups were able to stay on the drum for a maximum time. When an accelerating mode was applied at a rate of 4 rpm/30 seconds, starting from 4 rpm to a maximum speed of 40 rpm for a maximum of 400 seconds, we found a significantly reduced motor activity in
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ALS2 may be as long as 50 years. No patients with ALS2 were reported to give birth to
the Alsin-/- mice [199.61 ± 63.1 seconds in Alsin-/- mice (n=19) versus 296.73 ± 21.5 seconds in controls (n=5) in the age-matched litter controls, P=0.0048]. No significant difference was observed between Alsin-/- males and females [173.4 ± 73.3 seconds (n=8) versus 218.2 ± 46.1 seconds (n=11), P=0.11]. The pathology of the ALS2 in humans has never been documented, although clinical presentations in ALS2 patients suggest that the major pathology might be in the upper motor neuron system, including upper motor neurons in the cortex, corticobulbar and corticospinal tracts, with lesser involvement of the low motor neuron system. To uncover pathological changes in the Alsin-/- mice, we analyzed the central nervous system with
motor neuron loss nor a gross atrophy was found in motor cortex and spinal cord (Fig. 2). We then applied Amino-Cupric-Silver staining, a very sensitive method for detection of the degenerating or degenerated neuronal cell bodies, dendrites, synaptic terminals, and axons 28, 29. We used MultiBrain Technology (NeuroScience Associates, Knoxville, TN), so that multi-brains and spinal cords from the Alsin-/- and control mice could be processed in the same sections at the same time. We found consistent deposition of silver particles in the corticospinal tract in the dorsal column (dorsal CST) of the spinal cord in the Alsin/-
mice (Fig. 3b and 3c) compared with matched control (Fig. 3a), with the low thoracic
and lumbar segments being most severely affected. By contrast, substantial deposition of the silver particles was absent in the corticospinal tract around and above pyramidal decussation (Fig. 3d and 3e). Although we observed silver deposition in neurons and their processes in the brain and spinal cord of Alsin-/- mice, this staining was quite focal and rare. We only observed less than 40 such cells, mostly from the spinal cord, in each mouse from 100 out of a total of 700 sections with a thickness of 35um. A similar moderate neuronal degeneration could also be found in age-matched controls with similar frequency (Fig. 4). Compared to dorsal CST, the silver deposition in dorsolateral CST was less apparent (Fig. 3a-c). We also observed that Alsin-/- mice showed a slightly higher silver staining background in spinal cord (Fig. 3a-c), but not in the brain compared with control mice. alsin is not a major component in SOD1-containing aggregates
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routine pathological methods such as H&E and thionine Nissl stains. Neither an apparent
We previously produced transgenic mice overexpressing ALS1-associated mutants, such as SOD1G93A
(5)
and SOD1L126Z
(17)
, that developed ALS phenotype and pathology.
Immunohistochemistry shows massive SOD1-containing aggregates which are also ubiquitin-immunoreactive in neurons and their neuritic processes in the spinal cord sections (Fig. 5a-c). To examine if alsin is trapped in these aggregates, we stained the spinal cord sections of the affected mice using anti-alsin antibodies. We found that alsin is highly expressed in large neurons in cortex, brain stem, and spinal cord. It is mainly distributed in cytoplasm and neuronal processes. However, we did not find apparent aggregates that are positive either alone or together with ubiquitin (Fig. 5d-f), suggesting that alsin is not a major component in SOD1/ubiquitin-immunoreactive aggregates.
Degeneration of the motor neuron system is a shared pathology in both ALS1 and ALS2. The upstream signaling pathways triggering the degeneration in ALS1 and ALS2 are unknown. ALS1 is a dominant disease caused by mutations in SOD1 through a gain of function mechanism. ALS2 is a recessive disease that is likely linked to loss of alsin. It was reported that alsin protected motor neurons from the toxicity induced by mutant SOD1 through RAC/PI3K/Akt3 pathway 21,
22
. To test if loss of alsin contributes to the
pathogenesis of ALS1, we crossbred the SOD1G93A mice with Alsin-/- mice to generate SOD1G93A/ Alsin-/- mice. We found that SOD1G93A/ Alsin-/- mice had the similar phenotype and pathology to those of the SOD1G93A mice. The age of disease onset and the lifespan of the SOD1G93A/ Alsin-/- mice were not significantly altered compared with SOD1G93A mice (Fig. 5g and h). DISCUSSION Our Alsin-/- mice showed motor deficits, confirming alsin’s function in motor activity in mammals. Motor deficit on Rotarod test was observed in two of the previously reported three Alsin-/- mouse lines
23, 25
, but not in another line
24
. We observed that the Rotarod
performance could vary substantially from day to day when the mice were not well trained. The performance could be improved and become relative stable after a 10 minutes training every day for 3-4 weeks. We also observed that the well-trained Alsin-/mice remained on the Rotarod at a speed of 20 rpm for over 300 seconds, as the control
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Loss of alsin does not change the disease course of ALS1 in SOD1G93A mice
mice did. The difference became apparent when the Rotarod speed was increased to 40rpm for 400 seconds, indicating that the motor deficit in the Alsin-/- mice was mild. The pathology of ALS2 in humans has not been elucidated. In our Alsin-/- mice, no substantial neuron loss was found in cortex and spinal cord judging from the H&E and thionine Nissl stained sections. However, using Amino-Cupric-silver staining
28, 29
, we
were able to identify consistent deposition of silver particles in the corticospinal tract in the dorsal column of the spinal cord in the Alsin-/- mice, with the thoracic and lumbar segments being most severely affected. Although we also found silver deposition in neurons and their processes in brain and spinal cord, this pathology was quite focal and
total of 700 sections. This indicates that there may be no more than a few hundred such degenerating neurons in the central nervous system of each Alsin-/- mouse. Importantly, we also observed such silver staining-positive neurons in age-matched controls with similar frequency, suggesting that a moderate degree of the neuronal degeneration in Alsin-/- mice is likely a non-specific degeneration related to the aging process. Our finding suggests that the degeneration of the motor neuron itself may not be the major pathological event in ALS2 and alsin may not be indispensable for motor neuron survival. Compared to the corticospinal tract in spinal cord, no substantial deposition of silver particles was found in the corticospinal tract around and above pyramidal decussation. Based on these findings, we propose that alsin–mediated ALS2 is primarily a distal axonopathy rather than a neuronopathy. In one of the previously reported Alsin-/mouse line, mild loss of Purkinje cells was reported in 18 months old mice based on cell counting
24
. But such a loss was not detected in another line
25
. We used the Amino-
Cupric-Silver staining which can detect degeneration of the surviving cells and their neuritic processes
28, 29
. We detected non-disease-specific neuronal cell degeneration in
both control and alsin-deficient mice, but we did not found degeneration of Purkinje cells in our Alsin-/- mice, suggesting that the degeneration of Purkinje cells may not be a common feature in alsin-deficient mouse models. Recently, an increased susceptibility to glutamate receptor-mediated excitotoxicity was observed in the cultured spinal cord slice of alsin-deficient mice
30
. Since we did not find apparent increase in motor neuron
degeneration in our alsin-deficient mice compared with controls, it remains to be further 9
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rare. Only less than 40 such cells in each mouse were observed in 100 sections from a
addressed if this increased susceptibility has significant contribution to the pathogenesis of ALS2 in in vivo system. Progressive degeneration of spinal cord axons was reported predominantly in the dorsolateral column, rather than dorsal column in the third alsindeficient mouse line using conventional toluidine blue staining 25. We also observed the degeneration of dorsolateral CST axons, but the degeneration in the dorsal CST seemed more apparent using Amino-Cupric-Silver staining in our mouse model. These discrepancies in phenotype and pathology among different alsin-deficient mouse models may be attributed in part to the differences in mouse genetic backgrounds, regions of alsin to be targeted and approaches for evaluation.
together with degeneration of the distal corticospinal tract in spinal cord of our Alsin-/mice may reflect a defect of axonal trafficking, as suggested by in vitro studies
31-33
. We
also noticed that the background of the Amino-Cupric-Silver-staining on spinal cord sections of Alsin-/- mice was slightly higher than that of control mice, especially in the white matter. This difference is unlikely to be caused by differential staining of the spinal cord sections, because the MultiBrain Technology was applied and we did not find such difference in brain sections. Thus, it may suggest that alsin also play a role in maintaining the integrity of the other spinal cord axons, although CST axons are the most vulnerable to its loss. Previous studies suggest that loss of alsin may not only be the signaling pathway leading to ALS2, but also play a role in mutant SOD1-mediated ALS1
21, 22
. One possible
mechanism leading to loss of alsin in ALS1 is that alsin may be trapped by the SOD1containing aggregates. If loss of alsin underlies or significantly contributes to the pathogenesis of ALS1, we may expect that in the alsin-deficient mouse model, ALS phenotype appears earlier than in any transgenic mouse models overexpressing mutant SOD1, or mutant SOD1 transgenic mice develop disease earlier or severer on the Alsin-/background than those with Alsin intact. The age of disease onset in SOD1 transgenic mice depends on the expression level of the mutant SOD1. The transgenic lines with high expression of mutant SOD1G93A developed ALS around 100 days 5. However, we found that the Alsin-/- mice did not show apparent motor abnormality by the age of one year in our model and the other four alsin10
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The physiological function of alsin remains unknown. The deficit of motor function
deficient mouse models
23-26
. This suggests loss of alsin is not responsible for ALS1.
To test if alsin has a protective role in mutant SOD1-mediated ALS1, or loss of alsin contributes to the development of ALS1, as suggested by in vitro studies crossbred the SOD1
G93A
-/-
G93A
mice with Alsin mice to generate SOD1
21, 22
, we
mice on the Alsin-/-
background. We found the disease course is not significantly changed in SOD1G93A/Alsin/-
mice compared with SOD1G93A siblings. This observation is consistent with a recent
report
34
, suggesting that loss of alsin is neither responsible for, nor significantly
contributes to, the development of ALS1. Thus, loss of alsin is not the signaling pathway triggering motor neuron degeneration in ALS1. Therefore, it seems likely that the signaling pathways triggering motor neuron degeneration in ALS1 and ALS2 are
Compared to ALS1, ALS2 and JPLS have earlier onset of the disease. The time of onset of ALS2 varies from 1-10 years of age. The patients with 138delA mutation of Alsin gene became bedridden at around age 59. However, Alsin-/- mice do not show apparent overall locomotion deficit in their lifetime, although mild motor deficit could be detected in Rotarod test. The reasons why alsin-mediated, early onset disease in humans is not well replicated in mice are not well understood. It may be partly due to the anatomical and functional differences between humans and mice. In humans, a substantial proportion of the corticospinal tract (CST) axons are located in the middle portion of the lateral column, with a small contingent of CST axons descending in the ventral columns. In contrast, the main contingent of the CST axons in mice is located in the ventral part of the dorsal column (dorsal CST) and a small portion of the CST descends in the dorsal portion of the lateral column, termed the “dorsolateral CST” 35, 36. Importantly, unlike in humans, the input from the CST seems not essential for normal overground locomotion in rodents 37, possibly due to lack of direct cortico-motoneuronal synaptic connections between corticospinal axon boutons and motor neurons in anterior horns 38. In addition to mice, other mammals and primates such as cats and monkeys are also able to locomote overground after transection of the CST with only a brief recovery period
39-41
. The overall body and limb movements are almost indistinguishable from
those of the unlesioned animals, but the dedicated independent movements of the individual fingers were incapacitated in the lesioned animals
11
41
. This notion is also
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independent.
supported by the facts that targeting mouse spastin and paraplegin genes, mutations of which cause uncomplicated hereditary spastic paraplegias SPG4 and SPG7, respectively, did not robustly replicate human clinical manifestations of SPG4 and SPG7, although mild abnormal phenotypes and pathology were observed
42
.
Furthermore, we also
noticed that in SOD1G93A and SOD1L126Z transgenic mice, the pathology mainly occurs in brain stem and spinal cord, but the motor cortex and CST are much less involved, even though both upper and lower motor neuron are involved in humans in SOD1-mediated ALS1. This difference suggests that the upper motor neurons and CST may be less vulnerable in mice than in humans. Because of this lesser vulnerability and lesser functional role of the upper motor neuron system, a mouse model may not be an ideal
neuron symptoms.
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animal model for replicating the human diseases that have predominantly upper motor
MATERIALS and METHODS Determination of mouse genomic DNA structure. To determine the mouse genomic DNA sequence of Alsin gene, especially the genomic DNA sequence containing the first seven exons required to construct the targeting vector, we used a PCR DNA fragment amplified from human exon 4 as a probe to screen mouse genomic DNA BAC library (RPCI-21). From eight positive clones, we identified two clones containing the 5’ end and at least the first 10 exons of mouse Alsin gene. Directly using these BAC DNAs as templates, we sequenced more than 20 kb of mouse DNA fragment containing exons 2-8 (exon 1 is a non-coding exon) of mouse Alsin gene. A restriction map was established
Modification of targeting vector. We obtained a basic targeting vector pPNT from Dr. R.C. Mulligan of Whitehead Institute for Biomedical Research, Cambridge, Massachusetts. This vector contains both neo- gene and HSV-tk gene driven by mouse phosphoglycerate kinase-1 (PGK-1) promoter with mouse PGK-1 poly (A) addition site. We modified this vector by adding a 9bp sequence (ACATGTATC) between NotI and XhoI sites by site-directed mutagenesis, so that both NotI and XhoI can be conveniently used for construction of the targeting vector. The modified targeting vector sequence was verified by direct sequencing. Construction of targeting vector for the mouse Alsin gene. Exon 3 of mouse Alsin gene encodes the first RCC1-like domain (RLD). Exon 4 encodes the RLDs 2, 3 and 4. Exon 4 is the major coding exon. The mutation identified in our ALS family occurs in exon 3, leading to a truncated alsin without any RLD. To generate a mouse model that functionally mimics the mutation in human ALS2, we constructed a targeting vector to replace a 3.7 kb fragment containing a part of exon 3, entire exon 4, entire intron 3 and a part of intron 4 of Alsin gene with a neomycin cassette, so that both forms of alsin can be inactivated. A 6.5 kb KpnI/BamH1 fragment (KpnI site was introduced by PCR) extending from intron 1, exon 2, intron 2 and a part of exon 3 was subcloned into KpnI and BamHI sites of the modified vector pPNT (mpPNT). A 2.0 kb XhoI/NotI fragment from intron 4 (XhoI and NotI sites were introduced by PCR) was then subcloned into XhoI and NotI sites of this vector. The sequence and orientation were verified by direct sequencing using this constructed targeting vector as a template. The orientations of the 13
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based on computational analysis using the computer program Vector NTI Suite 6.
two Alsin fragments were chosen so that they were both in the opposite transcriptional orientation from the PGKneo cassette. This targeting vector was linearized by NotI digestion and used for electroporation of ES cells (129/Sv) using standard protocols. Positive and negative selection of ES cells. The electroporated ES cells were selected with 220ug/ml of G418 48 hours after electroporation and selected with 2uM Gancyclovir for 14 days. The ES cells surviving drug selections were harvested individually and replated in 96-well culture plates. The cells were cultured without selection until the cells became 50-75% confluent and the cells were harvested by trypsinization. DNA from each ES cell was extracted. The extracted DNA was subjected
Identification of positive ES cells by PCR and sequencing analysis. Two primers, one in neor (5’-gagaacctgcgtgcaatccatcttgttc-3’) and the other one in the region outside of the short arm of the targeting vector (5’-cctaacctactacccctcaactac-3’) were used for PCR amplification of the recombinant fragment.
The expected product size of the PCR
amplification was 2.6kb in the targeted ES cells. We employed a long-range PCR system (TaKaRa LA Taq, Panvera, Madison, WI) to amplify this fragment. Briefly, the PCR reaction is carried out in a microcentrifuge tube containing 3ul of 10x reaction buffer containing 15mM MgCl2, 50ng of template DNA, 125ng of each primer, 1ul of 10mM dNTP mixture (2.5mM each dNTP), 10% DMSO and 1.5U Takara LA Taq DNA polymerase. The total volume is brought to 30ul by addition of distilled water. The PCR reaction is performed under the following conditions: initial denaturing segment is 94ºC for 4 minutes for one cycle; the cycling segment is 98ºC for 25 seconds, 60ºC for 30 seconds, extension at 70ºC for 5 minutes for 30 cycles. Of the 345 ES cell clones, two clones (#1A5 and #3G11) were amplified and supplied the expected size of the amplified DNA, suggesting a homologous recombination event occurred in these clones. To confirm that these were the specific DNA fragments produced as a result of homologous recombination, we further sequenced these PCR fragments. Sequencing results verified the expected event. Identification of positive ES cells by Southern blot analysis. We further verified the PCR positive clones by Southern blot. Approximately five micrograms of genomic DNA isolated from ES cells were digested with SpeI to completion, separated on a 0.8% 14
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to PCR sequencing and Southern blot analyses.
agarose gel, and transferred to a nylon membrane. The membrane was dried for 2 hours at 800C. For Southern blot hybridization to verify the homologous recombination in the short arm and long arms, two external probes (probe 1 for the long arm and probe 2 for the short arm) were used, respectively. Probes were labeled with dGTP-32P by PCR. Hybridization was carried out in a hybridization solution (0.125MNa2HPO3/ 0.25M NaCl / 7%SDS/ 1mM EDTA/ 10%PEG) at 65ºC for 18 hours. The hybridized membrane was washed with 2X SSC/0.1%SDS for 30 minutes and 0.5X SSC/0.1%SDS for 30 minutes, and then exposed to X-ray film. Antibody production. Anti-alsin antibodies were raised by immunizing either chickens
alsin 2-21aa, DSKKKSSTEAEGSKERGLVH), Malsin-336 (mouse alsin 336-361aa, THAVTAYLQKLSEHSMRENHEPGEKP), Hmalsin-1574 (mouse alsin 1574-1595aa, EEISQSVLASLHEDFLW),
and
Hmalsin-1603
(mouse
alsin
1603-1623,
VLRARIRNLGSEVHLIEDLMD). Hmalsin-1574 was raised in chickens and the others were raised in rabbits. The peptides were conjugated with Keyhole Limpet Hemocyanin (KLH) via an amino-terminal cysteine residue (Bio-Synthesis Inc., Dallas, TX). Each antibody was affinity-purified using an antigen-coupled sepharose column. Confocal microscopy. The basic procedures for confocal microscopy were previously described 17. Amino cupric silver staining. Mice were anesthetized with sodium pentobarbital (150mg/kg) and perfused through the heart with a rinsing solution of 0.8% sodium chloride, 0.4% dextrose, 0.8% sucrose, 0.023% calcium chloride and 0.034% sodium cacodylate followed by a perfusion fixative of 4% paraformaldehyde, 4% sucrose and 1.43% sodium cacodylate. The pH of both perfusion fluids was adjusted to 7.2-7.4 with hydrochloride acid. The brain and spinal cord were allowed to harden in fixative for at least 1 day before they were dissected out and cryoprotected in a solution of 20% glycerol and 2% dimethylsulfoxide for 6-8 hours prior to sectioning. Mouse brains were cast collectively in 5X5 array in a large gelatin block by positioning upright in identical rostro-caudal orientation. Mouse spinal cords were cut into two segments and cast together with the brains in the same orientation. Gelatin blocks were hardened in a 4% formaldehyde solution for two days, then frozen on dry ice for frozen sectioning. Sheets 15
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or rabbits with different peptides. The anti-alsin antibodies include Hmalsin-2 (mouse
of 35um-thick sections were cut from the olfactory bulb to the medulla for brain, or from cervical to thoracic segments and thoracic to sacral segments for spinal cord. These section sheets were collected into containers filled with 10% formaldehyde buffered with 0.2M sodium cacodylate. Since all the brains and spinal cords had similar orientation in each gelatin block, every composite frozen section provided a set of multiple individual cross-sections, all at approximately the same level of the brain and spinal cord. A serial set of every sixth section (a 240um interval) was selected for staining with the amino-cupric-silver stain of de Olmos to reveal disintegrative degeneration
28
and
Thionine Nissl staining.
preimpregnation, impregnation, reduction, bleaching, and fixing. The sections were then rinsed in deionized water, mounted on subbed glass slides, and counterstained with neutral red to reveal normal cell bodies. Rotarod test. Beginning at age 360 days, alsin knockout homozygotes and controls were tested three times a week for their ability to maintain balance on a Rotarod apparatus. The mice were first trained on the apparatus daily for 30 days prior to the test. An accelerating paradigm was applied at a rate 4 rpm per 30 seconds, starting from 4 rpm to a maximum speed of 40 rpm, then the rotation speed was kept constant at 40 rpm for a maximum of 400 seconds. The time each mouse managed to remain on the revolving drum was recorded. Time recording was initiated at the beginning of the acceleration phase.
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The free-floating sections were taken through the following major steps:
Acknowledgements This study is supported by The National Institutes of Health (NS40308 to HXD, NS050641 to TS, NS046535 to TS), Les Turner ALS Foundation, National Organization for Rare Disorders, Vena E. Schaff ALS Research Fund, Harold Post Research Professorship, Herbert and Florence C. Wenske Foundation, Ralph and Marian Falk Medical Research Trust, Abbott Labs Duane and Susan Burnham Professorship and The David C. Asselin MD Memorial Fund (to TS).
TS and H.-X.D conceived this project. H.-X.D., H.Z., and R.F. developed the alsin knockout mice and performed genetic analysis; H.-X.D., H.Z., Y.S., and G.H.G. performed immunohistochemistry and confocal microscopy; Y. S., and Y.Y did biochemical analysis. R.F did Rotarod test. M.C.D.C., E.M., E.L., H.-X.D., and T.S did pathological analysis; H.-X.D., Y.S. and G.H.G prepared the figures; H.-X.D., E.M and T.S. analyzed data and wrote the paper.
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Author contributions
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Figure Legends Figure 1. Construction of targeting vector and development of Alsin-/- mice. (a) The top line represents the genomic DNA structure of exons 2-7 of mouse Alsin gene and the low line the targeting vector. Exons 2 to 7 are labeled by E2 to E7. Long and short arm external probes for Southern blot are marked by P1 and P2. Restriction sites used for construction of the targeting vector and identification of the recombinant ES cells are labeled as follows: B (BamHI), Bs (BspHI), E (EcoRI), N (NotI), S (SpeI) and X (XhoI). The total genomic DNA size is 23kb (diagrams are not drawn to scale for clarity). (b-d) Southern blot analysis. Southern blot of SpeI-digested genomic DNA from ES cells with
homologous recombination events in short arm (3.6kb in b) and long arm (11.4kb in c), respectively. Homozygous mice were identified by Southern blot using restriction enzyme SpeI and verified by Southern blot using another restriction BspHI (d). The wild type allele is 12kb and targeted allele is 7.3kb when hybridized with the short arm external probe P2 (d). (e) RT-PCR by primer sequences derived from exon 1 (5’gagaagagcgcggagctgcgggagcgt-3’) and exon 9 (caacccaggctggaagtcttccgt-3’), and subsequent sequencing analysis demonstrated that the targeted allele generated an mRNA transcript with exons 3 and 4 missing (898bp fragment). The main RT-PCR product (1973bp) containing exons 3 and 4 was detected in the wild-type mice, but not in the Alsin-/- mice (e). In addition, a small amount of the alternatively spliced products was also detected. Sequencing analysis indicated that the 1053bp fragment lacked exon 4, and 898bp fragment lacked exons 3 and 4 (e). (f) Exon 4 genomic DNA sequence was not detected in Alsin-/- mice by PCR using exon 4 primers (5’-cgtcattagcagttaggattct-3’ and 5’-ccacctggagcaccagccgtcca-3’). (g) Deletion of exons 3 and 4 resulted in a frameshift mutation and loss of alsin protein in the Alsin-/- mice. Figure 2. Thionine Nissl Staining of the 35µm sections of the mouse brains and spinal cords. The images of the brain (a and b) and spinal cord (c and d) of the Alsin-/- (b and d) and control (a and c) mice. No apparent atrophy and loss of large neurons in motor cortex and spinal cord were found in Alsin-/- mice at age of 15 months. The motor cortex regions of the mice were indicated as MC flanked by arrows (a and b). Bars = 600 µm (a and b); 200 µm (c and d). 23
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a short arm external probe (P2) (b) and a long arm external probe (P1) (c) shows
Figure 3. Amino-Cupric–Silver staining of 35um sections of mouse spinal cord and medulla oblongata. Marked silver staining was apparent in the mouse corticospinal tract (arrows) in the ventral portion of the dorsal column of the spinal cord in Alsin-/- mice (b and c) compared to the control mice (a) at age of 15 month. The top panels are magnified from the middle panels. Although silver deposition was also observed in dorsolateral CST axons (small arrows in b and c), the deposition was much less compared with that in dorsal CST axons. Silver staining was not apparent in the corticospinal tract around and above pyramidal decussation in the Alsin-/- mice (e) compared to controls (d). The mouse corticospinal tracts (CST) in the region of medulla oblongata were indicated by arrows (d and e). Bars = 200 µm (a-c); 150 µm (d and e).
Amino-Cupric–Silver staining of 35µm sections. Degeneration of the large neurons and their processes can occasionally be found in spinal cord with similar frequencies in Alsin/-
mice (a-e) and controls mice (f and g). Bar = 15 µm.
Figure 5. No significant contribution of alsin to mutant SOD1-mediated ALS in mouse model. (a-f) Laser confocal microscopy of immunofluorescence staining in spinal cord sections of mutant SOD1-transgenic mice using anti-SOD1 (a, green), anti-alsin (d, green) antibodies and anti-ubiquitin antibody (c and f, red), and overlay image (b and e). Arrow in (b) points to a representative neuron and its processes containing aggregates that are positive with both SOD1 and ubiquitin antibodies. Arrow in (d) points to the diffused staining of alsin in a large neuron. Arrow in (f) points to a representative ubiquitin-positive aggregate that does not show apparent alsin signal (Bars = 30 µm). (g and h) Kaplan-Meier plots showing the age of onset and cumulative survival of the mice. The age of the disease onset (left) and lifespan (right) are not significantly altered in the SOD1G93A/Alsin-/- mice (red line, onset: 104.5 ± 5.0 days, n=8; lifespan: 127.3 ± 7.5 days, n=8) compared to the control SOD1G93A mice (blue line, onset: 103.7 ± 11.7 days, n=27; lifespan: 128.6 ± 9.4, n=24; P=0.508 and P=0.633, respectively).
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Figure 4. Degeneration of the spinal cord neurons in the Alsin-/- and control mice.
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