Cre-mediated recombination of TrkB in spinal cord motor neurons and interneurons
We use the Cre-LoxP system to eliminate TrkB
from motor neurons. To avoid developmental effects that could complicate interpretation of these experiments, we chose a Cre driver line in which recombination begins around the first month of postnatal life. The vesicular acetylcholine transporter (VAChT) promoter driving Cre expression has been used previously to disable LoxP-flanked genes specifically in motor neurons (28
). Using the ROSA 26 reporter line, we confirmed that VAChT-Cre-medicated recombination begins at around 4 weeks of age and reaches its maximum by 6 months of age (Fig. A). Based on size, shape and location, it appears that Cre-mediated recombination is occurring mostly in motor neurons (~50% of all motor neurons) (29
) but also in a small population of interneurons. The LoxP-flanked TrkB
allele has been shown to readily undergo recombination (30
). We chose the G85R mutant SOD1 mice for study because in this mouse strain, the first pathological features are identified at 6 months of age (31
). Thus, motor neuron development is likely to be normal in the G85R-SOD1+/−; TrkBLoxP/LoxP; VAChT-Cre+/−
mice and any effects of TrkB signaling in mature motor neurons of mice expressing mutant SOD1 could be assessed. Through a five-step breeding strategy (see Materials and Methods), we generated mice in our prime comparison groups with genotypes: G85R-SOD1+/−; TrkBLoxP/LoxP; VAChT-Cre+/−
(deleted TrkB in motor neurons from mutant SOD1 mice) or G85R-SOD1+/−; TrkBLoxP/LoxP; VAChT-Cre−/−
(intact TrkB in motor neurons from mutant SOD1 mice). Prior to generation of trigenic mice, each primary strain was backcrossed into the C57BL/6 background for at least five generations. All animals discussed from this point forward are homozygous for the floxed TrkB
). In light of this, unless otherwise specified, we will simplify the notation of the genetic background of the mice we studied by eliding this piece of data. Thus, in this short hand, G85R-SOD1+/−; VAChT-Cre+/−
mice have the genotype G85R-SOD1+/−; TrkBLoxP/LoxP; VAChT-Cre+/−
Figure 1. Cre-mediated TrkB recombination in spinal cord motor neurons. (A) Lumbar spinal cord frozen sections from postnatal day 10, day 26 and 6-month ROSA 26 reporter mice expressing Cre under the control of the VAChT promoter were stained with X-gal and showed (more ...)
Each LoxP-flanked gene undergoes Cre-mediated recombination with variable efficiency, and effect likely related to chromatin structure. To show that TrkB was eliminated from motor neurons in G85R-SOD1+/−; VAChT-Cre+/− mice, we began with immunological approaches. None of the antibodies we used provided high-quality images in immunological assays. Immunoblots for TrkB using spinal cord lysates from G85R-SOD1+/−; VAChT-Cre+/− versus G85R-SOD1+/−; VAChT-Cre−/− animals were indistinguishable and this is likely to be due to TrkB expression in non-Cre-expressing cells in the TrkBLoxP/LoxP mice. Given these problems, we turned to laser capture microscopy. We dissected individual large neurons in the ventral horn (putative motor neurons) from frozen sections of 8-month-old G85R-SOD1+/−; VAChT-Cre+/− mice using a laser microdissector (Fig. B–D). We extracted RNA from lysates of each dissected neuron and performed RT-PCR and nested PCR for TrkB, Cre and choline acetyltransferase (ChAT, a biosynthetic enzyme enriched in motor neurons) (Fig. E).
Among 99 specimens examined, 16 specimens were ChAT-positive, Cre-positive and TrkB-negative (Fig. E, lanes 1, 3, 12 and 15; 1F), and these cells probably represent motor neurons (expressing ChAT) in which TrkB expression is eliminated when Cre recombinase is expressed. Twelve specimens were ChAT-positive, Cre-positive and TrkB-positive (Fig. E, lane 2; 1F) and these cells probably represent motor neurons in which TrkB is expressed even though Cre is also expressed. Eight specimens were ChAT-positive, Cre-negative and TrkB-negative (Fig. F) and these cells probably represent motor neurons in which neither TrkB nor Cre is expressed. Finally, 14 neurons were ChAT-positive, Cre-negative and TrkB-positive and these neurons probably represent motor neurons that express TrkB when Cre is lacking (Fig. E, lanes 4, 6, 14, 18; 1F). Assuming that the ChAT-expressing, large ventral horn cells studied by laser capture single-cell PCR are motor neurons, we estimate that ~57% of Cre (+) cells have no demonstrable TrkB mRNA expression and ~37% of Cre (−) cells have no demonstrable TrkB expression. These observations lead us to conclude that Cre expression in motor neurons promotes deletion of the LoxP-flanked TrkB allele.
Twenty-six specimens were ChAT-negative (putative non-motor neurons) but positive for Cre and/or TrkB. Among them, 7 cells were ChAT-negative, Cre-positive and TrkB-negative (Fig. E, lanes 16, 17 and 19), 8 cells were ChAT-negative, Cre-positive and TrkB-positive (Fig. E, lanes 10) and 11 cells were ChAT-negative, Cre-negative and TrkB-positive (Fig. E, lanes 5 and 8). We suggest that these specimens are derived from interneurons and that TrkB undergoes Cre-mediated recombination in the presence of Cre recombinase although with less than perfect efficiency. We did not detect expression of ChAT, Cre recombinase and TrkB in other 23 specimens (Fig. E, lanes 7, 9, 11 and 13). Together, these data demonstrate that Cre-mediated recombination of the LoxP-flanked TrkB allele occurs in subsets of both motor neurons and a subpopulation of interneurons in ventral horn of G85R-SOD1+/−; VAChT-Cre+/− mice.
Elimination of TrkB increases lifespan, slows disease progression and improves locomotor function in G85R SOD1 mice
We began by assessing survival and health of mice with a single genetic manipulation (i.e. VAChT-Cre+/− or TrkBLoxP/LoxP) or mice with two genetic manipulations (VAChT-Cre+/−; TrkBLoxP/LoxP). Both the uni- and bi-genic mice strains lived at least for 18 months without evidence of an adverse effect on health monitored as normal weight gain, fecundity and activity levels (data not shown). We next turned to the trigenic mice. Male G85R-SOD1+/−; VAChT-Cre+/− mice lived for an average 26 days longer than male G85R-SOD1+/−;VAChT-Cre−/− mice (384 ± 7 (n = 15) versus 358 ± 8 days (n = 15), P = 0.033, Fig. A). Female G85R-SOD1+/−; VAChT-Cre+/− mice lived for an average of 28 days longer than female G85R-SOD1+/−; VAChT-Cre−/− [363 ± 7 days (n = 21) versus 335 ± 9 days (n = 14), P = 0.037, Fig. B]. Thus, conditional deletion of TrkB from adult motor neurons, and a subpopulation of adjacent interneurons, is associated with lifespan extension of mutant SOD mice of both sexes. For the remainder of this report, we focus on male mutant SOD1 mice.
Figure 2. Elimination of TrkB in motor neurons increases lifespan, slows disease progression, delays the onset of paralysis and improves locomotor function in G85R SOD1 mice. (A) Cre-mediated deletion of TrkB in adult motor neurons increases lifespan of male G85R-SOD1 (more ...)
We monitored body weight of four groups of male animals: (i) G85R-SOD1−/−; VAChT-Cre−/−, (ii) G85R-SOD1−/−; VAChT-Cre+/−, (iii) G85R-SOD1+/−; VAChT-Cre−/− and (iv) G85R-SOD1+/−; VAChT-Cre+/− (Fig. C). By ANOVA, we found no group differences at 46, 48, 50 or 52 weeks of life. Group differences were observed at week 54 (F(3,26) = 3.888, P = 0.02) and at week 56 (F(3.25) = 5.871, P = 0.0035). At week 54, all mouse groups in which G85R SOD1 was expressed were significantly lighter than mice that did not express G85R SOD1 (post hoc analysis with significance set at P<0.05). The same was true at week 56 time point. It is noteworthy that no statistically significant difference in weight was seen between G85R-SOD1+/−; VAChT-Cre−/− and G85R-SOD1+/−; VAChT-Cre+/− mice.
The onset of disease has been defined by the peak of bodyweight curve (32
), and the early phase of the clinical motor neuron disease has been defined as the time from the peak of bodyweight to 10% weight loss (33
). Previous work suggests that events occurring within motor neurons themselves underlie the ‘initiation phase’ of mutant SOD1 disease (33
). We next compared the disease onset and progression between male G85R-SOD1+/−; VAChT-Cre+/−
mice and G85R-SOD1+/−;VAChT-Cre−/−
= 12 in each group). When we analyzed our data this way, we found that these two groups of mice had similar disease onset (308 ± 10 versus 306 ± 6 days, G85R-SOD1+/−; VAChT-Cre+/−
, respectively, P
= 0.460, Fig. D). On the other hand, the early phase of the disease was 19 days longer in the G85R-SOD1+/−; VAChT-Cre+/−
mice versus the G85R-SOD1+/−; VAChT-Cre−/−
mice (358 ± 6 versus 339 ± 7 days, P
= 0.027, Fig. E and F). We also monitored the onset of paralysis by measuring grip strength. We found that the cumulative probability of the onset of paralysis, as defined by 30% decline of either forelimb or hindlimb grip strength, was significantly delayed by 21 days in G85R-SOD1+/−; VAChT-Cre+/−
mice in comparison with the G85R-SOD1+/−; VAChT-Cre−/−
mice (361 ± 7 versus 340 ± 6 days, P
= 0.037, Fig. G). Finally, we evaluated locomotor function using the accelerating rotarod test. When assessed at peak bodyweight, G85R-SOD1+/−; VAChT-Cre+/−
mice stayed on the device significantly longer than the G85R-SOD1+/−; VAChT-Cre−/−
mice (272 ± 19 versus 197 ± 27 seconds, P
= 0.035, Fig. H). We also assessed the locomotor function 2 weeks after the peak of bodyweight and again found that G85R-SOD1+/−; VAChT-Cre+/−
mice stayed on the device significantly longer than the G85R-SOD1+/−; VAChT-Cre−/−
mice (246 ± 25 versus 179 ± 24 s, P
= 0.033, Fig. I). Together, these observations indicate that the early phase of mutant SOD1 disease is more prolonged and milder in mice with the conditional deletion of TrkB. These observations are consistent with the notion that activation of TrkB regulates biological processes within motor neurons themselves that play a crucial role in the earliest phases of clinical mutant SOD1 disease.
Deletion of TrkB reduces the formation of mutant SOD1 inclusions and ubiquitination in G85R SOD1 mice
The presence of SOD1 immunoreactive inclusions in neuronal cell bodies and processes had been shown as early hallmarks of disease in G85R transgenic mice (31
). We next compared the development of SOD1 aggregation in G85R-SOD1+/−;VAChT-Cre+/−
and G85R-SOD1+/−; VAChT-Cre−/−
mice. We found that prior to the clinical onset, intense and diffusely localized human SOD1 immunoreactivity were seen in cell bodies of ventral motor neurons of both G85R-SOD1+/−;VAChT-Cre+/−
and G85R-SOD1+/−; VAChT-Cre−/−
mice (Fig. A and B, arrows). At this stage, more neuronal processes showing intense immunoreactivity to human SOD1 were seen in G85R-SOD1+/−; VAChT-Cre−/−
mice (Fig. A, arrowheads) than in G85R-SOD1+/−; VAChT-Cre+/−
mice (Fig. B). As disease progresses, prominent neuronal processes with intense human SOD1 immunoreactivity increased in abundance in the ventral area of G85R-SOD1+/−; VAChT-Cre−/−
(Fig. C and E, arrowheads) and G85R-SOD1+/−; VAChT-Cre+/−
mice (Fig. D and F, arrowheads). Western blot analysis showed similar expression level of human SOD1 in Triton-soluble supernatant of spinal cord lysates from presymptomatic G85R-SOD1+/−; VAChT-Cre−/−
mice (Fig. G). However, more G85R SOD1 was found in Triton-insoluble pellet in G85R-SOD1+/−;VAChT-Cre−/−
mice than in G85R-SOD1+/−;VAChT-Cre+/−
mice (Fig. H), suggesting that conditional deletion of TrkB decreases aggregation of mutant SOD1 in the early stage of mutant SOD1 disease.
Figure 3. Decreased abundance of mutant SOD1 immunoreactive inclusions in presymptomatic G85R SOD1/TrkB KO mice. (A and B) Immunostaining of presymptomatic spinal cord sections using human SOD1 antibody showed diffuse SOD1 immunoreactivity in ventral horn motor (more ...)
Another pathological hallmark of ALS is the presence of ubiquitinated inclusions in the perikaryon and proximal axons of surviving spinal motor neurons (34
). Next we set out to assess the effects of elimination of motor neuron TrkB on deposition of ubiquitinated proteins in spinal motor neurons. Immunohistochemical staining of lumbar spinal cord sections from mice at different disease stages with ubiquitin antibodies showed that G85R-SOD1−/−; VAChT-Cre−/−
control mice had diffuse distribution of ubiquitin immunoreactivity in motor neurons at all ages examined (Fig. A–C, D–F, arrows). In presymptomatic G85R-SOD1+/−; VAChT-Cre−/−
mice, most ventral horn motor neurons show diffuse ubiquitin staining (Fig. G, arrows). Intense ubiquitin inclusions were readily found in some neurons and neuronal processes (Fig. G, arrowheads). In presymptomatic G85R-SOD1+/−; VAChT-Cre+/−
mice, diffuse ubiquitin staining was seen in spinal motor neurons (Fig. J, arrows) with fewer neuronal processes containing ubiquitin inclusions (Fig. J, arrowheads). At later disease stage, more neuronal processes containing ubiquitin inclusions were seen in ventral horn area of both G85R-SOD1+/−; VAChT-Cre−/−
mice (Fig. H and I, K and L, arrowheads).
Figure 4. Ubiquitin immunoreactive inclusions in G85R SOD1/TrkB KO mice. (A–F) Immunohistochemical staining of spinal cord sections showed diffuse ubiquitin immunoreactivity in ventral motor neurons from all age groups of G85R-SOD1−/−; VAChT-Cre (more ...)
Elimination of TrkB in motor neurons reduces inflammation in G85R SOD1 mice
A number of potential processes could count for the effects of conditional deletion of TrkB on the early phase of mutant SOD1 disease. The cell non-autonomous features of mutant SOD1-induced motor neuron disease have received increasing attention recently. During the clinically manifest phase of the disease, reactive astrocytosis and inflammatory responses are evident. Does the elimination of TrkB from motor neurons influence these cellular responses? To assess this, we immunohistologically examined spinal cords from mice in presymptomatic phase (8-month-old), symptomatic (11-month-old) and end stage of disease for glial fibrillary astrocytic protein (GFAP—a marker for reactive astrocytosis) as an indicator of astroglial response. In both the presymptomatic and symptomatic phase of the disease, the G85R-SOD1+/−;VAChT-Cre+/− mice displayed significantly less GFAP-immunopositive astrocytes in the spinal cord compared with G85R-SOD1+/−; VAChT-Cre−/− mice (Fig. A–C, P = 0.0062; Fig. D–F, P = 0.026). This was evident by lower power viewing of spinal cord sections and was confirmed by counting the number of GFAP+ cells in the two study groups. Western blot analysis of spinal cord lysates from presymptomatic 8-month-old mice further showed higher expression level of GFAP in G85R-SOD1+/−;VAChT-Cre−/− mice (Fig. I).
Figure 5. Elimination of TrkB in motor neurons reduces mutant SOD1-associated gliosis. (A–H) Immunostaining of spinal cord sections using GFAP antibody indicated reduced astrocytosis in presymptomatic (B) and symptomatic (D) G85R-SOD1+/−; VAChT-Cre (more ...)
We used immunoreactivity against ionized calcium-binding adaptor molecule (Iba)-1, a specific marker for activated microglial cells, to detect microglia in the spinal cord. We found that the numbers of Iba1-immunopositive cells were significantly decreased in spinal cords of presymptomatic (8-month-old) (Fig. B and C, P = 0.009) and symptomatic G85R-SOD1+/−;VAChT-Cre+/− mice (Fig. E and F, P = 0.016) compared with G85R-SOD1+/−; VAChT-Cre−/− mice (Fig. A and D). In agreement with this observation, western blot analysis of presymptomatic spinal cord lysates showed lower expression level of Iba1 in G85R-SOD1+/−; VAChT-Cre+/− mice compared with G85R-SOD1+/−; VAChT-Cre−/− mice (Fig. I). This result probably indicates that mutant SOD1 expressing motor neurons that lack TrkB survive longer than those that express TrkB, and as a by product of this, cellular responses within the spinal cord parenchyma to damaged motor neurons are delayed.
Figure 6. Decreased mutant SOD1-associated neuroinflammation in G85R SOD1/TrkB KO mice. (A–F) Immunostaining of spinal cord sections using Iba-1 antibody showed reduced inflammation in presymptomatic (B) and symptomatic (E) G85R-SOD1+/−; VAChT-Cre (more ...)
Elimination of TrkB delays motor axon degeneration in G85R SOD1 mice
To assess the effects of these manipulations on progression of ventral motor neuron axonal degeneration, we examined and counted axons in the L5 ventral roots. At the 11-month time point, we found obvious loss of axons and degenerating profiles in G85R-SOD1+/−; VAChT-Cre−/− mice (Fig. C) in comparison with control groups (Fig. A and B). The predominant degenerative features were increased interaxonal space, presence of macrophages and greater variation in axon size (Fig. C). In marked distinction, analysis of L5 ventral root from G85R-SOD1+/−; VAChT-Cre+/− mice showed a substantial retention of large-diameter axons (Fig. D). Their axons are tightly packed, similar to control G85R-SOD1−/−;VAChT-Cre−/− (Fig. A) and G85R-SOD1−/−;VAChT-Cre+/− mice (Fig. B). We measured the diameter of axons and then generated size distribution histograms by plotting the number of axons against diameter of axon subdivided into bins. L5 roots from G85R-SOD1−/−; VAChT-Cre−/− animals showed a clear separation of the histograms into two peaks (Fig. E). One peak of myelinated fibers is at between 1 and 2 μm diameter (likely represents the axons from γ-motor neurons) and a second peak is in the range of 5–10 μm (likely represents α-motor neuron axons) (Fig. E). Elimination of TrkB alone has no effect on axon morphology (Fig. B and F). The histograms were shifted to the left, and the total number of large myelinated axons was reduced in G85R-SOD1+/−; VAChT-Cre−/− mice (Fig. C and G). In G85R-SOD1+/−; VAChT-Cre+/− mice, there were two clear peaks with size range similar to that of G85R-SOD1−/−;VAChT-Cre−/− animal (Fig. H). We counted the remaining axons from L5 ventral roots in three G85R-SOD1−/−;VAChT-Cre−/− mice, three G85R-SOD1+/−; VAChT-Cre−/− mice and six G85R-SOD1+/−; VAChT-Cre+/− mice, and found that there was a significant reduction of large axons (diameter >4 μm) in G85R-SOD1+/−; VAChT-Cre−/− mice (Fig. I, 345 ± 19 for G85R-SOD1+/−; VAChT-Cre−/−, 717 ± 14 for G85R-SOD1−/−; VAChT-Cre−/− mice, P < 0.001). Significant preservation of large axons (diameter >4 μm) in L5 ventral roots was seen in G85R-SOD1+/−; VAChT-Cre+/− mice in comparison with G85R-SOD1+/−; VAChT-Cre−/− mice (Fig. I, 552 ± 104, P = 0.0039). There is no alteration in the number of small-diameter axons (<4 μm) in L5 ventral roots (Fig. J). Examination of L5 ventral roots from 9-month-old animals showed that <1% of large-diameter axons underwent degeneration in both G85R-SOD1+/−; VAChT-Cre−/− and G85R-SOD1+/−; VAChT-Cre+/− mice (Fig. K and L). These results indicate that elimination of TrkB from motor neurons in mutant SOD1 mice delays large-diameter putative α-motor neuron axon degeneration.
Figure 7. Delayed motor axon degeneration in G85R SOD1/TrkB KO mice. (A–D) Toluidine blue staining of thin sections of L5 ventral root axons from 11-month-old animals showed substantial retention of large-diameter axons in G85R-SOD1+/−; VAChT-Cre (more ...)
Elimination of TrkB reduces denervation of NMJs in G85R SOD1 mice
Finally, we examined the NMJs in gastrocnemius, extensor digitorum longus (EDL) and soleus from 10-month-old animals by immunofluorescence labeling. We found that >95% of endplates were innervated in gastrocnemius, EDL and soleus of G85R-SOD1−/−; VAChT-Cre−/− control mice (Fig. A–C, J–L). There was no evidence of the NMJ denervation in G85R-SOD1−/−; VAChT-Cre+/− animals (data not shown). At this age, we found that denervation of endplates was 45.9% ± 7.7, 48.7% ± 3.8 and 39.4% ± 8.2 for gastrocnemius, EDL and soleus, respectively, in G85R-SOD1+/−;VAChT-Cre−/− mice (Fig. D–F, J–L), and in G85R-SOD1+/−; VAChT-Cre+/− mice, denervation of endplates in gastrocnemius, EDL and soleus was significantly reduced (24% ± 6.2 for gastrocnemius, 32% ± 2% for EDL and 19% ± 5 for soleus) (Fig. G–I, J–L). These results indicate that elimination of TrkB from adult motor neurons in mutant SOD1 mice is associated with preservation of the NMJ structure.
Figure 8. Reduced denervation of NMJs in G85R-SOD1+/−; VAChT-Cre+/− mice. (A–I) Representative images of NMJs from gastrocnemius of 10-month-old animals showed three innervated junctions in wild-type control (A–C), two denervated/one (more ...)
VAChT-Cre reduces TrkB in pre-ganglionic sympathetic neurons
In addition to motor neurons, the pre-ganglionic sympathetic neurons in the intermediolateral (IML) column of the thoracic spinal cord are cholinergic. The sympathetic nervous system controls many biological processes, including energy expenditure. This is of interest because mutant SOD1 mice have a hypermetabolic phenotype (35
) and part of the control of resting energy expenditure (REE) is mediated by the sympathetic nervous system (36
). Pre-ganglionic sympathetic neurons regulate REE through the melanocortin 4 receptor (MC4R) (37
), and BDNF/TrkB can be a downstream effector of MC4R signaling (38
). These observations raise the possibility that VAChT-Cre-mediated elimination of TrkB from pre-ganglionic sympathetic neurons might correct the metabolic defect in the mutant SOD1 mice and be the basis of the beneficial effects we have described above. In light of this, we asked whether VAChT-Cre was expressed in pre-ganglionic sympathetic neurons. We bred VAChT-Cre
mice to the ROSA26-LacZ
reporter mice, and performed X-gal histochemistry on slices of the thoracic spinal cord. In 2-month-old animals, we found no cell labeling in the IML (data not shown)—this is consistent with the original observations of Misawa et al
). However, when we looked at 8-month-old animals, intense labeling of cells was evident in the IML of the thoracic spinal cord (Fig. A). Based on location and polygonal shape, these cells are putative pre-ganglionic sympathetic neurons.
Figure 9. VAChT-Cre reduces TrkB in pre-ganglionic sympathetic neurons. (A) Thoracic spinal cord frozen sections from 8-month-old ROSA 26 reporter mice expressing Cre under the control of the VAChT promoter were stained with X-gal, which showed intense labeling (more ...)
To determine whether pre-ganglionic sympathetic neurons express TrkB and whether Cre recombinase can lead to its ablation, we performed laser capture single-cell RT-PCR of cells in the IML column (Fig. B–D). Analyses of 60 cells led to the identification of 6 that were cholinergic (i.e. they expressed ChAT) (Fig. D, lanes 2, 4, 5, 6, 7, 10). Of these six cells, five expressed Cre (Fig. D, lanes 2, 5, 6, 7, 10) and four-fifths did not express TrkB (Fig. D, lanes 2, 5, 6, 7). A single ChAT(+) Cre(−) cell in the IML column was TrkB(+) (Fig. D, lane 4). These observations support the view that pre-ganglionic sympathetic neurons of the IML column are depleted of TrkB in our compound mice. To the extent that the hypermetabolic phenotype of mutant SOD1 mice contributes to motor neuron degeneration, it is possible that the beneficial action of eliminating TrkB from cholinergic neurons is mediated, in part, by the modulatory actions of sympathetic neurons on REE.