Chronic JM4 treatment reduced neurological deficit and prolonged lifespan in PS19 mice
A cohort of PS19 mice was treated with JM4 (10 μg for 5 days/week, subcutaneously) initiated at 2 months of age, before the onset of neurological deficit, to assess the effect of JM4 treatment on the onset of disease and the lifespan of mice. Neurological deficit was assessed daily on a scale of 0 (no deficit) to 5 (severe deficit). The onset of neurological disease was defined as the age at which a mouse reached a clinical score of 2. A clinical score of 2 corresponds to a mouse displaying clasping of at least one hind limb for 30 s when suspended by its tail. JM4 treatment (n = 15) significantly delayed the onset of neurological disease compared to the PBS sham-treated group (n = 22) (median onset of disease = 348 days for JM4-treated group vs. 305 days for PBS sham-treated group) (Fig. 1a). This cohort of mice was then monitored until end of life. The end of life data allowed us to develop Kaplan-Meier survival curves, which showed that JM4 treatment prolonged survival of PS19 mice compared to the PBS sham-treated group (median lifespan 447 days, for JM4-treated group vs. 348 days for PBS sham-treated group) (Fig. 1b). Note that this cohort of PS19 mice had a longer lifespan compared to the median survival of about nine months of PS19 mice reported initially by Yoshiyama et al. [18]. However, it has been reported that the onset and severity of tau pathology in PS19 mice are rather variable [38].
Another cohort of PS19 mice was treated with JM4 (10 μg for 5 days/week, subcutaneously) initiated at 1 month of age before the onset of neurological deficits. Every half month, neurological deficit was scored on a scale of 0 (no deficit) to 5 (severe deficit). We found JM4 treatment significantly reduced neurological deficit in PS19 mice compared to a group sham-treated with PBS (n = 12 for each group) (Fig. 1c). Because neurological deficit in PS19 mice was minimum up to 5 months of age, the difference in clinical scores between JM4-treated group and sham-treated group only became significant from 5.5 months of age onward. This cohort of mice was subjected to behavioral testing and subsequent neuropathological assessment.
Chronic JM4 treatment rescued behavioral abnormalities in PS19 mice
PS19 mice display cognitive impairment, including selective deficits in spatial learning and memory at 6 months of age [35]. A subset of mice that were treated with JM4 from 1 month old underwent several behavioral tests to assess the effect of JM4 treatment on cognition of these animals.
For assessing spatial memory, we used the Barnes maze test, which relies on the innate preference of mice for dark, enclosed spaces [39]. At 7 months of age, JM4-treated PS19 mice took less time than PBS sham-treated PS19 mice to find a target hole (JM4-treated group 33.0 ± 6.6 s, PBS sham-treated group 61.6 ± 10.7 s, n = 9 for each group) suggesting that JM4 treatment can rescue memory impairment in PS19 mice (Fig. 2a). The PBS sham-treated PS19 mice also traveled a longer distance to find a target hole (JM4-treated group 325.5 ± 37.5 cm, PBS sham-treated group 593.2 ± 98.7 cm, n = 9 for each group), suggesting that PBS sham-treated PS19 mice took more time to find a target hole due to memory impairment rather than due to motor deficit (Fig. 2b).
Next, we carried out elevated plus maze test to assess anxiety level since previous studies found PS19 mice had reduced anxiety-like behavior [35]. We found that PS19 mice spent more time in the open arm than wild-type mice at 8 months of age. This result is consistent with reduced anxiety-like behavior in PS19 mice compared to wild-type mice. Importantly, chronic JM4 treatment significantly reduced this behavioral deficit in PS19 mice (percentage of time spent in the open arms: JM4-treated group 47 ± 3.3%, PBS sham-treated group 60 ± 3.4%, wild-type control 31 ± 2.7%, n = 7 for each group) (Fig. 2c). Again, motor deficit of PS19 mice at this age did not interfere with test performance, as there were no significant differences in the distance traveled among different groups (JM4-treated group 3543 ± 300 cm, PBS sham-treated group 4099 ± 689 cm, wild-type control 2784 ± 126 cm, n = 7 for each group) (Fig. 2d). Instead, there is a trend that the total distance traveled by PBS sham-treated mice is greater than that by wild-type mice, which is consistent with previous findings that PS19 mice are hyperactive [35].
Chronic JM4 treatment blocks neuronal loss in PS19 mice
A subset of PS19 mice that were treated with JM4 from 1 month old and underwent behavioral testing was sacrificed at 10 months old and used for immunohistochemical analysis (Figs. 3, 4, 5).
PS19 mice develop neuronal loss and brain atrophy by 8 months of age, mainly in the hippocampus, and then neuronal loss becomes apparent in other brain regions including neocortex by 12 months of age [18]. Thus, the brain sections of 10-month-old PS19 mice were examined for the effects of JM4 treatment on neuronal loss by quantifying neuronal nuclei (NeuN) staining in hippocampal and cortical sections. JM4 treatment led to a significantly greater area of NeuN immunoreactivity in the hippocampus CA1 region compared to PBS sham-treatment in PS19 mice (relative NeuN immunoreactive area: PBS sham-treated PS19 mice 0.49 ± 0.02, JM4-treated PS19 mice 0.97 ± 0.04, wild-type mice 1.00 ± 0.04. n = 5 for each group) (Fig. 3a–c). To exclude the possibility that the change in the area of NeuN immunoreactivity was due to a change in size or in morphology of neurons, we also counted the number of NeuN positive neurons and found similar results (the number of NeuN positive neurons per a field of view: PBS sham-treated PS19 mice 110.2 ± 17.8, JM4-treated PS19 mice 176.9 ± 6.4, wild-type mice 213.6 ± 13.2, n = 5 for each group) (Fig. 3d). These results suggest that chronic JM4 treatment prevented neuronal loss in the hippocampus of PS19 mice. There was no significant difference in the area of NeuN immunoreactivity or the number of NeuN positive neurons in the frontal cortical sections of 10 months old PS19 mice vs. 10 months old wild-type mice (relative NeuN immunoreactive area: PBS sham-treated PS19 mice 0.95 ± 0.10, JM4-treated PS19 mice 1.04 ± 0.04, wild-type mice 1.00 ± 0.04; the number of NeuN positive neurons per a field of view: PBS sham-treated PS19 mice 737.4 ± 51.9, JM4-treated PS19 mice 767.5 ± 32.1, wild-type mice 799.5 ± 57.4, n = 5 for each group) (Fig. 3e–g). This finding is consistent with a published report that neuronal loss occurs at later time in the neocortex compared to the hippocampus in PS19 mice [18].
Chronic JM4 treatment reduced phosphorylated tau aggregates in PS19 mice
Accumulation of hyperphosphorylated tau aggregates, in addition to neuronal loss, is a pathologic hallmark of PS19 mice [18]. Thus, the brain sections of 10-month-old mice were also evaluated for tau aggregates by immunohistochemistry. We used AT8 tau antibody, which recognizes tau protein phosphorylated at both serine 202 and threonine 205, but lacks cross-reactivity with unphosphorylated tau [40]. JM4 treatment significantly reduced AT8 immunoreactivity per unit area in the hippocampus compared to PBS sham-treatment in PS19 mice, while brains of wild-type mice showed essentially no AT8 immunoreactivity (percentage of AT8 immunoreactive area: PBS sham-treated PS19 mice 3.86 ± 0.58, n = 8, JM4-treated PS19 mice 1.93 ± 0.64, n = 8, wild-type mice 0.11 ± 0.06, n = 4) (Fig. 4a, b). Qualitatively similar results are seen in the cortex, but we focused the analysis of tau immunoreactivity and subsequent analysis of neuroinflammation on the hippocampus because the hippocampus was the main brain region with neuronal loss in 10-month-old PS19 mice. We also carried out Western blot analysis of total brain homogenates of these mice. Similar to immunohistochemistry of brain sections, we found that chronic JM4 treatment decreased phosphorylated tau level in soluble RAB fraction, RIPA fraction, and insoluble urea fraction of mouse brain homogenates (Fig. 4c).
To exclude the possibility that JM4 treatment reduced AT8 immunoreactivity by suppression of human tau transgene expression in PS19 mice, we also immunostained the hippocampal sections with TAU-5 antibody, which stains total tau—both unphosphorylated and phosphorylated tau species [41]. Unlike the finding with AT8 tau antibody, there was no significant difference in TAU-5 immunoreactivity between the hippocampal sections of PS19 mice treated with JM4 and sham-treated with PBS (percentage of TAU-5 immunoreactive area: PBS sham-treated PS19 mice 6.53 ± 1.14, JM4-treated PS19 mice 5.29 ± 0.48. n = 6 for each group) (Fig. 4d).
We also carried out quantitative RT-PCR of the PS19 mouse brain lysate to assess further whether chronic JM4 treatment altered human tau transgene expression level. There was no significant difference in human MAPT mRNA expression between PS19 mice treated with JM4 and sham-treated with PBS (normalized ratio of human MAPT/mouse GAPDH expression: PBS sham-treated PS19 mice 1.00 ± 0.16, JM4-treated PS19 mice 1.04 ± 0.12, n = 6 for each group) (Fig. 4e). Taken together, these results show that JM4 treatment leads to a reduction of hyperphosphorylated tau aggregates without affecting expression of tau transgene in PS19 mice.
Chronic JM4 treatment reduced microglial activation in PS19 mice
Since we hypothesized that the beneficial effect of JM4 was, at least in part, due to its anti-inflammatory/immunomodulatory properties leading to downregulation of microglial activation, we first assessed effects of chronic JM4 treatment on a microglial marker, IBA-1, in brain sections of these mice using immunohistochemistry. We found that IBA-1 immunoreactivity per unit area in the hippocampus was elevated in PS19 mice compared to wild-type control; more importantly, JM4 treatment led to a reduction in IBA-1 immunoreactivity compared to PBS sham-treated group in PS19 mice (percentage of IBA-1 immunoreactive area: PBS sham-treated PS19 mice 9.24 ± 2.71, n = 6, JM4-treated PS19 mice 3.59 ± 1.05, n = 6, wild-type mice 1.04 ± 0.31, n = 4) (Fig. 5a, d).
Because IBA-1 is thought to be a pan microglia marker identifying all microglia [42], we next assessed a more specific marker of microglial activation, the major histocompatibility complex II (MHC II) proteins in the hippocampus. Microglia normally express low levels of MHC II; however, in inflammatory or neurodegenerative conditions, activated microglia highly upregulate MHC II [43]. Chronic JM4 treatment led to a lower level of MHC II immunoreactivity per unit area in the PS19 mouse hippocampus compared to PBS sham-treated PS 19 mouse hippocampus, suggesting JM4 treatment led to reduced microglial activation (percentage of MHC II immunoreactive area: PBS sham-treated PS19 mice 2.54 ± 0.74, JM4-treated PS19 mice 0.61 ± 0.22. n = 8 for each group) (Fig. 5b, e).
Recently, the complement cascade, which is a major effector pathway of microglial activation, has been shown to play an important role in tau pathology in AD brain as well as the mouse model of tauopathy [7,8,9,10]. The complement cascade converges on a central component C3, whose fragments interact with their receptors, C3aR and CR3, to enact downstream immune effects such as phagocytosis [44]. For this reason, we carried out immunohistochemistry of these brain sections with an antibody against C3. Similar to the results with MHC II, we found that chronic JM4 treatment led to lower levels of C3 immunoreactivity in the PS19 mouse hippocampus compared to PBS sham-treated PS 19 mouse hippocampus (percentage of C3 immunoreactive area: PBS sham-treated PS19 mice, n = 5: 1.96 ± 0.65, JM4-treated PS19 mice, n = 6: 0.30 ± 0.20) (Fig. 5c, f).
We also carried out quantitative RT-PCR of the PS19 mouse brain lysate to assess further whether chronic JM4 treatment reduced expression levels of microglial markers. Consistent with immunohistochemistry data, we found that JM4 treatment led to a reduction in IBA-1 mRNA expression compared to PBS sham-treated group in PS19 mice (normalized IBA-1 mRNA expression level: PBS sham-treated PS19 mice 2.08 ± 0.22, JM4-treated PS19 mice 1.10 ± 0.13, wild-type mice 1.00 ± 0.12, n = 5–6) (Fig. 5g). We also assessed mRNA level of CD68 (cluster of differentiation 68), a phagocytic microglia marker that has been found to be elevated in PS19 mouse brains [8, 9]. We found that CD68 mRNA expression was elevated in PS19 mouse brains compared to wild-type mouse brain, and JM4 treatment led to a reduction in CD68 mRNA expression compared to PBS sham-treated group in PS19 mice (normalized CD68 mRNA expression level: PBS sham-treated PS19 mice 2.22 ± 0.32, JM4-treated PS19 mice 1.12 ± 0.18, wild-type mice 1.00 ± 0.20, n = 5–6) (Fig. 5g).
It has been shown that levels of proinflammatory cytokines including IL1β, IL6, and TNFα were elevated in PS19 mouse brains [9, 45]. Therefore, we additionally assess the expression levels of IL1β, IL6, and TNFα mRNAs using quantitative RT-PCR of the PS19 mouse brain lysate. We also found that IL1β and TNFα mRNAs levels were elevated in PS19 mouse brains and they were reduced by chronic JM4 treatment. There was a trend toward increase of IL6 mRNA level in PS19 mouse and reduction by chronic JM4 treatment, but it did not reach the statistical significance (normalized IL1β mRNA expression level: PBS sham-treated PS19 mice 7.38 ± 1.79, JM4-treated PS19 mice 3.19 ± 0.61, wild-type mice 1.00 ± 0.23, n = 5–6; normalized IL6 mRNA expression level: PBS sham-treated PS19 mice 2.18 ± 0.55, JM4-treated PS19 mice 1.11 ± 0.32, wild-type mice 1.00 ± 0.33, n = 5–6; normalized TNFα mRNA expression level: PBS sham-treated PS19 mice 4.48 ± 1.12, JM4-treated PS19 mice 1.79 ± 0.50, wild-type mice 1.00 ± 0.41, n = 5–6) (Fig. 5g). Taken together, these results show that chronic JM4 treatment leads to a reduction of activated microglia and downstream proinflammatory cytokines and complement components in PS19 mouse brains.