Despite a narrow therapeutic window (0.6–1.5 mM) and the potential for serious adverse events, lithium has been used as the first-line therapy to reduce manic episodes and suicidality in patients with bipolar disorder owing to lack of better alternatives.32 We have previously shown that FDA-approved lithium carbonate produces very sharp peak plasma and brain lithium concentrations after oral dosing, followed by a rapid decline in rats. In contrast, LISPRO showed steady plasma and brain lithium levels out to 48 h without any sharp peak.21 Based on these findings, we hypothesize that LISPRO may prevent the drastic change of lithium levels in the plasma seen with current lithium drugs, and maintain stable therapeutic doses and, thus, would represent a significant improvement over current lithium medicines for a desired time by slow-release into the peripheral blood. Similar to above findings, our recent data (8 weeks treatment in Tg2576 mice) showed significantly stable plasma lithium levels over the period of time (Figure 2c) as well as higher brain lithium levels compared to Li 2 CO 3 (Figure 2d). More importantly, B6129SF2/J mice treated with LISPRO showed significantly higher brain lithium levels at low (1.125 mM/kg/day) and high (2.25 mM/kg/day) concentrations compared to Li 2 CO 3 (Figures 2a–b). Moreover, our recent study also showed comparable levels of lithium in 3XTg-AD mouse plasma and brain as result of lithium salicylate, Li 2 CO 3 , or LISPRO (28-week treatment). Furthermore, the brain to plasma lithium ratio in the LP-treated group was slightly higher (LP and LS, P=0.98; LP and LC, P=0.84; LS and LC, P=0.85) versus LS- and LC-treated groups (Figures 2e–f). Lithium has been employed in treatment of several neurodegenerative diseases, including AD. It has been reported that lithium prevents the generation of Aβ peptides by inhibiting GSK3α activity, which interferes with APP γ-secretase cleavage.14, 33, 34 In terms of LP, we expected that addition of salicylate, which is the primary metabolite derivative of acetyl-salicylic acid (aspirin), could work together synergistically to improve the safety and modify the pharmacological action of lithium for attenuating AD pathology. Study data suggest that aspirin exerts its effects on the inflammatory cascades, irreversibly inhibiting COX1, and modifying enzyme activity of COX2, suppressing production of prostaglandins and thromboxane. Although lithium has anti-inflammatory properties, several studies indicate that chronic lithium might induce COX2 expression through inhibition of GSK3β activity. Our data also showed that both lithium carbonate and LISPRO inactivate GSK3β, but only lithium carbonate activates COX2 whereas LISPRO suppresses COX2 due to the anti-inflammatory properties of salicylate anion. A recent epidemiological study showed that low-dose aspirin with lithium exert synergistic effects by increasing 17-hydroxy-decosahexanoic acid (17-OH-DHA), an anti-inflammatory brain DHA metabolite, which significantly reduced the risk of disease deterioration in bipolar patients compared to other non-steroidal anti-inflammatory drugs and glucocorticoids, a COX2 inhibitor.35 Together, salicylic acid increased brain 17-OH-DHA,36 and lithium reduced neuroinflammation,37, 38 whereas zwitterionic l-proline significantly reduced the hygroscopic property of parent salicylate salt by influencing the solid phase formation. Assuming the above hypothesis is true, we wanted to investigate the bioactivities of LISPRO in terms of ameliorating AD pathology in cell culture systems and in transgenic (Tg2576 and 3XTg-AD) mouse models. We showed that 8-week LISPRO-treated Tg2576 AD mice had significantly reduced soluble and insoluble Aβ levels as well as Aβ burden compared to Li 2 CO 3 - and control-treated Tg2576 AD mice (Figures 3a–c). To examine LISPRO’s effect on Aβ generation in 5-month old 3XTg-AD mice, we treated them with LISPRO, lithium salicylate, Li 2 CO 3, and control diet for 28 weeks with equal dosages of lithium (2.25 mM/kg/day). We showed that LISPRO treatment significantly reduced extracellular Aβ plaques, as evidenced by IHC staining using 4G8 and 6E10 antibodies (Figures 3d and e). Taken together, these findings demonstrated that LISPRO suppresses generation of both soluble and insoluble Aβ in Tg2576 and 3XTg-AD mouse models.

Moreover, several lines of evidence demonstrated that lithium is a direct inhibitor of GSK3β and also increases the inhibitory serine-phosphorylation of the enzyme.11, 39 Thus, we wanted to examine whether LISPRO could reduce tau phosphorylation in cell culture and AD mouse models. Using human HeLa/tau, human neuroblastoma SHSY-5Y, and primary neuronal cell lines, we found that LISPRO treatment inhibits phosphorylation of tau at 5–10 mM concentrations, which is associated with increasing inhibitory phosphorylation of GSK3β (Ser9) (Figures 6a–c). Taken together, these findings indicated that LISPRO inactivates GSK3β activity, and thereby reduces tau phosphorylation. Since lithium is a suitable inhibitor for inhibiting GSK3β in vivo, we also examined whether LISPRO-mediated suppression of GSK3β activity is associated with attenuation of tau phosphorylation in Tg2576 mice. In this model, we showed that an 8-week LISPRO treatment significantly reduces p-tau (Thr231) phosphorylation compared to Li 2 CO 3 and control (Figures 4a and b). These findings were also correlated with increased pGSK3β (Ser9) inhibitory phosphorylation, indicating inactivation of GSK3β activity (Figure 4c). To confirm these data obtained in the Tg2576 AD mouse model, we next investigated whether chronic administration of LISPRO could also reduce tau phosphorylation in 3XTg-AD mice. Thus, we treated 5-month old 3XTg-AD mice with LISPRO, lithium salicylate, Li 2 CO 3 , or control diet for 28 weeks with equal doses of lithium (2.25 mM/kg/day). IHC staining using p-tau (Thr231) and p-tau (Ser396) antibodies as well WB analyses using multiple p-tau (Ser396, Ser404, Thr181, and Thr231) amino-acid residues demonstrated that LISPRO, and in many cases lithium salicylate, significantly attenuates tau phosphorylation compared to Li 2 CO 3 and control (Figures 4d–j).

Inflammatory processes are thought to have an active role in AD formation and progression. Preclinical as well as postmortem analyses of AD patient brains have provided tons of evidence indicating the dysregulation and/or uncontrolled activation of microglial and astrocytic cells, activation of complement cascade, inflammatory enzymes such as COX2, inducible nitrate oxide synthase, reactive oxygen species, and calcium dysregulation pathways in brain, CSF, and blood.40, 41, 42 Although it is inconclusive whether these changes are initiating or secondary consequences, pro-inflammatory such as IL-1β, IL-6, TNFα, NO, and anti-inflammatory cytokines such as IL-4, IL-10, TGFβ elevated in the CSF and blood of AD patients.41, 43, 44 Multiple lines of evidence showed that lithium down-modulates the pro-inflammatory cytokine responses in animal models and is of therapeutic benefits in several neurodegenerative diseases.45, 46 Specifically, Nassar and Azab conclude that lithium has anti-inflammatory properties that may contribute to its therapeutic activity by down-regulation of COX2, inhibition of IL-1β, TNFα, and upregulation of IL-2 and IL-10.47 On the other hand, in contrast to above findings, large bodies of evidence indicated that lithium also induces pro-inflammatory cytokines production such as IL-4 and IL-6 in certain disease conditions.48, 49 Based on these reports, we sought to examine if the efficacy of LISPRO for reducing AD-like pathology in transgenic Tg2576 mice is associated with modulation of pro- and anti-inflammatory cytokine responses. We showed that LISPRO treatment significantly increases the expression of anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β1, whereas it decreases the expression of pro-inflammatory cytokines such as INFγ, IL-12p70, and sCD40L in Tg2576 mouse brains compared with control- and LC-treated Tg2576 mouse brains (Figures 5e–h). Taken together, these findings suggest that LISPRO might reduce Aβ pathology at least in part via upregulated anti-inflammatory and down-regulated pro-inflammatory cytokine responses in Tg2576 mice.

We demonstrated that CD40-CD40L interaction is critical for brain pro-inflammatory responses in aggravating AD-like pathology.50 As LISPRO treatment reduced Aβ production in cell culture and transgenic (Tg2576 and 3XTg-AD) mouse models, we next hypothesized that reduction of Aβ pathology might correlate with decreased microglial CD40 expression and/or increased phagocytosis by microglia. In this regard, we found that decreased expression of microglial CD40 and brain soluble CD40L expression by LISPRO treatment might help attenuate Aβ associated pathology, suggesting that disruption of CD40-CD40L signaling could also be involved in attenuation of Aβ pathology in Tg2576 and 3XTg-AD mouse models. As expected, LISPRO suppresses IFNγ-induced CD40 expression (Figures 5b and c) and enhances microglial phagocytosis of Aβ (Figure 5d) in cultured primary microglial cells. Moreover, multiple lines of evidence demonstrated that lithium enhances autophagy at low doses (10 mM).27, 28 In this regard, we found that LISPRO treatment enhances autophagy markers LC3B in cultured primary microglial cells (Figure 5a). Collectively, our data suggest that LISPRO-mediated attenuation of Aβ pathology is associated with several therapeutic endpoints, including upregulated anti-inflammatory and down-regulated pro-inflammatory cytokines, suppression of CD40 that disrupts CD40-CD40L signaling, increased microglial phagocytosis of Aβ, and upregulated autophagy.

Furthermore, to investigate whether LISPRO treatment could modulate neuronal cell differentiation, cultured mouse neuroblastoma N2a, as well as murine and human stem cells was treated with LISPRO, Li 2 CO 3, and control. Our data from IHC staining and supportive WB analyses using β-tubulin III, phospho-synapsin I (Ser62–67), MAP2, and total tau antibodies demonstrated that LISPRO treatment significantly promotes neuronal cell differentiation compared to Li 2 CO 3 (Figures 7a–g). Cheng and Chuang reported that lithium increases the suppression of p53 and expression Bcl-2 providing neuronal survival.51 In addition, it has been shown that administration of lithium as well as mood-stabilizing agent valproate, increases Bcl-2 levels in the cortical region.52 Based on these findings, we also wanted to examine whether LISPRO could prevent cortical neuronal loss in 5-month-old 3XTg-AD mice treated with LISPRO, lithium salicylate, Li 2 CO 3 , or control diet for 28 weeks. Quantitative analysis of neuronal cell numbers using the neuronal marker anti-NeuN antibody, displayed that LISPRO and lithium salicylate treatments, respectively, yield an increased survival neurons in the neocortex region of 3XTg-AD mice (Figure 7h). We further examined whether LISPRO treatment could modulate the expression of synaptic proteins in 3XTg-AD mice brain, and found that LISPRO and lithium salicylate significantly increase the protein expression of synaptophysin (Pre-synaptic) and PSD95 (Post synaptic) in these transgenic mice (Figure 7i).

Finally, one of the major side-effects of lithium includes renal toxicity secondary to increased expression of COX2 and ensuing inflammation. It has been shown that acute and chronic administration of lithium could enhance COX2 expression by suppressing GSK3β activity in renal cell lines and mouse models.29, 30 We observed the effect of LISPRO on COX2 expression in renal cells from the Tg2576 AD as well as wild-type B6129SF2/J mouse models. We treated HRPT with LISPRO and Li 2 CO 3 . IHC staining and supportive WB data indicated that LISPRO treatment does not enhance COX2 expression in HRPT renal cells (Figures 8a and b). To further test the effect of LISPRO treatment on COX2 expression in vivo, we orally fed B6129SF2/J and Tg2576 mouse lines with LISPRO, lithium salicylate, and Li 2 CO 3 for 2, and 8 weeks, respectively, with low (1.125 mM/kg/day) and high doses (2.25 mM/kg/day). Our IHC and supportive WB findings indicated that LISPRO treatment does not increase COX2 expression (Figures 8c–g).

In sum, our data support our hypothesis that LISPRO is a better alternative formulation of lithium in terms of safety and efficacy in ameliorating AD pathology in cell culture and two different transgenic mouse models. Nevertheless, further translational research is warranted to fully validate LISPRO as a safe and effective disease modifying therapy for AD and other neurodegenerative diseases.