Homotaurine limits the spreading of T cell autoreactivity within the CNS and ameliorates disease in a model of multiple sclerosis

Most multiple sclerosis (MS) patients given currently available disease-modifying drugs (DMDs) experience progressive disability. Accordingly, there is a need for new treatments that can limit the generation of new waves T cell autoreactivity that drive disease progression. Notably, immune cells express GABAA-receptors (GABAA-Rs) whose activation has anti-inflammatory effects such that GABA administration can ameliorate disease in models of type 1 diabetes, rheumatoid arthritis, and COVID-19. Here, we show that oral GABA, which cannot cross the blood–brain barrier (BBB), does not affect the course of murine experimental autoimmune encephalomyelitis (EAE). In contrast, oral administration of the BBB-permeable GABAA-R-specific agonist homotaurine ameliorates monophasic EAE, as well as advanced-stage relapsing–remitting EAE (RR-EAE). Homotaurine treatment beginning after the first peak of paralysis reduced the spreading of Th17 and Th1 responses from the priming immunogen to a new myelin T cell epitope within the CNS. Antigen-presenting cells (APC) isolated from homotaurine-treated mice displayed an attenuated ability to promote autoantigen-specific T cell proliferation. The ability of homotaurine treatment to limit epitope spreading within the CNS, along with its safety record, makes it an excellent candidate to help treat MS and other inflammatory disorders of the CNS.

there are ongoing clinical trials that administer GABA to individuals newly diagnosed with T1D (NCT02002130 and NCT03635437).
In regards to treating EAE, Steinman and colleagues showed that anti-seizure medications that increase GABAergic tone in the CNS (topiramate and vigabatrin) can inhibit EAE 7 . These drugs however primarily target other neurotransmitter systems (e.g., sodium and calcium channels) and enzymes 19,20 and have adverse effects. Benzodiazepines and barbiturates are BBB-permeable GABA A -R positive allosteric modulators that can potentiate the opening of GABA A -R Clchannels, but only after a GABA A -R agonist opens the channel, and these drugs can be addictive. Accordingly, there is a need for safe BBB-permeable GABA A -R-specific agonists that could provide a new class of drugs for limiting the spreading of T cell autoreactivities in the CNS.
Homotaurine is an amino acid found in seaweed and it was identified as a compound that could interfere with the ability of amyloid peptide to form fibrils in vitro 21,22 . Subsequent studies found that oral homotaurine can pass through the BBB and limit amyloid plaque deposition in the brain of transgenic mice that over-expressed human amyloid protein 21,22 . Based on these observations, homotaurine (also known as Tramiprosate or Alzhemed™) was tested in a large double-blind phase III clinical trial for its ability to slow cognitive loss over 1.5 years in patients with Alzheimer's disease [23][24][25] . While homotaurine treatment did not slow cognitive decline, it had an excellent safety profile. Recently, it has become appreciated that homotaurine can also act as a GABA A -R-specific agonist. Homotaurine has better pharmacokinetics than GABA (as discussed in 26,27 ). We previously showed that oral homotaurine treatment inhibited both monophasic and RR-EAE (in the C57BL/6 MOG 35-55 and SJL PLP 139-151 mouse models, respectively) when treatment was initiated just after the appearance of clinical symptoms 26 . Histological analysis of their brains and spinal cords revealed reduced mononuclear cell infiltration and areas of myelin loss in the cerebellum and spinal cords of homotaurine-treated mice. Mechanistically, we observed that homotaurine significantly reduces the frequency of splenic PLP 139-151-reactive IL-17A + Th17 cells, as well as the frequency of splenic IFNγ (Th1) responses to PLP 139-151 , but increased IL-10-secreting responses, to PLP 139-151 and the frequency of CD4 + and CD8 + Tregs 26 .
To further assess the therapeutic potential of targeting GABA A -Rs as a new avenue to help treat MS, we compared the therapeutic efficacy of GABA and homotaurine treatment on murine EAE, determined whether homotaurine treatment was efficacious when administered at an advanced stage of RR-EAE, assessed whether homotaurine administration could limit the spreading of T cell autoreactivity from the immunogen PLP 139-151 to PLP 178-191 within the CNS which is essential for disease progression 28,29 , and tested whether homotaurine treatment modulated the antigen-presenting activity of APCs. The results suggest that homotaurine is a promising candidate to help treat MS.

Results
Homotaurine, but not GABA, limits the development of EAE. The spreading of T cell autoreactivity to new myelin epitopes within the CNS is thought to be a major factor driving relapses in EAE and MS 28,[30][31][32] . The inability of oral GABA to enter the CNS may make it ill-suited to limit the spreading of autoreactivities within the CNS. However, administered GABA may modulate cells of CNS-draining lymph nodes (which are outside of the BBB) as well as circulating activated/memory myelin-reactive T cells, and thereby impact the course of the disease. Hence, we tested the therapeutic efficacy of oral GABA versus homotaurine. C57BL/6 mice received MOG 33-55 (as described in 26 ) and all mice developed clinical signs of EAE between days 11-13 post-immunization. As individual mice reached an EAE score of 1 they were randomized to receive plain water or water containing GABA (6 mgs/ml, an effective dose in past studies of autoimmune disease 4,6,33 ) or homotaurine (0.25 mg/ml) for the subsequent 16-day observation period. The disease course in the mice that received GABA was essentially the same as that in mice that received plain water (Fig. 1). In contrast, in homotaurine-treated mice, the disease severity remained near the initial trial entry score of 1 for about two weeks and then declined. The contrasting outcomes between GABA and homotaurine treatment suggest that homotaurine's ability to pass through the BBB underlies its therapeutic effect.
Homotaurine treatment initiated just before the second attack prevents EAE relapse. We previously reported that PLP 139-151 immunized SJL mice that were given homotaurine treatment at the first clinical signs of the disease (EAE score of 1) exhibited a reduced mean EAE score throughout the observation period compared to those given plain water and eventually displayed almost complete remission 26 . To more stringently test the robustness of this therapeutic approach we tested homotaurine's ability to ameliorate disease when given at an advanced stage of EAE. SJL mice were immunized with PLP 139-151 (as in 26 ) and homotaurine treatment (0.25 mg/ml), was initiated 22 days later, just before the start of the expected relapse. Homotaurine treatment progressively reduced the severity of illness (Fig. 2).
Homotaurine treatment limits the spreading of T cell autoreactivity within the CNS. In the SJL PLP 139-151 disease model that we studied, the spreading of T cell autoimmunity from PLP 139-151 to PLP [178][179][180][181][182][183][184][185][186][187][188][189][190][191] is required for disease progression 28,29 and has been shown to occur within the CNS and not the draining cervical lymph node or spleen 30,32 . T cell reactivity to PLP 178-191 is observed to arise in the CNS during the secondary paralysis about 42 days post-PLP 139-151 immunization 30 . Our observation that homotaurine, but not GABA, could modulate disease in mice with EAE suggests that homotaurine's ability to pass through the BBB is key to its mode of action. To further test this hypothesis, we examined whether beginning homotaurine treatment during the first peak of clinical symptoms could modulate the spreading of T autoreactivity to a new myelin T cell epitope within the CNS.
Homotaurine impairs the antigen presenting function of APCs to induce PLP 139-151 -specific T cell proliferation. We next examined whether homotaurine could modulate the ability of APCs to promote autoantigen-specific T cell mitogenesis. We purified CD4 + T cells and APC from PLP 139-151 immunized SJL mice that were/were not treated with homotaurine for nine days. We co-cultured the CD4 + T cells from each group with APC isolated from plain water, or homotaurine-treated mice. We found that cultures of CD4 + T cells and APCs from the homotaurine-treated animals displayed the least levels of proliferation at all concentrations of PLP 139-151 (Fig. 4). Co-cultures of T cells from the homotaurine-treated mice with APCs from the control mice also displayed reduced proliferation at all concentrations of peptide ("H-T/C-APC" in Fig. 4) consistent with the notion that homotaurine acts directly on T cells to limit their mitogenic potential. Notably, co-cultures of CD4 +

Discussion
Based on the success of GABA treatment to ameliorate T cell-mediated autoimmune diseases, we sought to extend this therapeutic approach to EAE and potentially MS. Our previous study showed that homotaurine treatment ameliorated both monophasic and RR-EAE when initiated just after the onset of symptoms. Here, we expanded those studies and also tested homotaurine treatment at advanced stages of RR-EAE since that is the most clinically relevant situation. There are three major findings from the studies presented here. The first is that despite administering GABA at a level that effectively ameliorates T1D, RA, and coronavirus infection in mouse models, it had no effect on the disease course of EAE. In contrast, homotaurine treatment was an effective therapy. Since both GABA and homotaurine are GABA A -R agonists, we ascribe homotaurine's therapeutic effectiveness to its ability to pass through the BBB. This contention is supported by our finding that homotaurine treatment limited the spreading of T cell autoreactivity from the priming immunogen PLP 139-151 to PLP 178-191 which has been shown to occur within the CNS and to be essential for driving relapses 28,[30][31][32] . Since GABA also activates GABA B -Rs, it is possible that its lack of effect on EAE was due to GABA B -R mediated changes that counteracted the beneficial effects of GABA A-R activation. We do not prefer this scenario because GABA B -R agonists have been shown to (1) inhibit murine dendritic cell (DC) activation and immune cell chemotaxis 34,35 , (2) inhibit DC proinflammatory functions 34 , (3) alleviate collagen-induced arthritis and contact dermatitis in mouse models 34,35 , (4) delay T1D onset in NOD mice 36 , and (5) attenuate TLR4-induced inflammatory signaling in human PBMC 37 .
Interestingly, we also observed that homotaurine treatment significantly attenuated the activity of APCs in inducing antigen-specific T cell proliferation ex vivo. The attenuation of antigen-presenting activity by homotaurine could contribute to its inhibition of the spreading of T cell autoimmunity and disease progression. Although GABA can also inhibit the antigen-presenting activity of APCs, its failure to pass the BBB and inhibit EAE progression suggests that suppression of APC's activity in the CNS may be necessary to inhibit the spreading of T cell autoimmunity and EAE progression in mice. Accordingly, these observations may shed new light on the immunoregulation of T cell autoimmunity within the CNS.
Second, our studies showed that homotaurine treatment beginning after the first wave of paralysis in an RR-EAE model greatly reduced the severity of the disease. This observation has potential clinical relevance since there is a critical need for late-stage therapeutic improvements for MS patients whose response to DMDs has www.nature.com/scientificreports/ diminished. Homotaurine had an excellent safety profile in long-term Alzheimer's disease clinical trials making it an excellent candidate for clinical trials in MS patients and other neurological conditions in which limiting inflammation within the CNS is desirable. Third, we observed that homotaurine treatment after the first wave of paralysis limited the spreading of Th17 and Th1 responses from PLP 139-151 to PLP 178-191 within the CNS. This is the first demonstration that a BBBpermeable GABA A -R agonist can limit the spreading of pathogenic Th17 and Th1 responses in the CNS. This diminution of epitope spreading is likely to be a major contributing factor to the reduced clinical severity of the disease in homotaurine-treated mice.
In addition, homotaurine may have modulated macrophages, DCs, microglia, and astrocytes in the CNS which express GABA A -Rs in ways that down-regulated their pro-inflammatory activities and contributed to the lower frequencies of autoreactive Th17 and Th1 cells in the CNS. Macrophages, DCs, microglia, and astrocytes also express GABA A -Rs and GABA treatment reduces their inflammatory activities [6][7][8][9][10][11] . Consistent with those observations, our co-culture studies showed splenic APC isolated from homotaurine-treated mice had reduced capacity to promote the proliferation of PLP 139-151 -reactive T cell cells isolated from mice that did not receive homotaurine treatment. These data confirm and extend previous findings that GABA A -R agonist can modulate the functions of both T cells and APCs. However, further studies are needed to determine whether homotaurine treatment can modulate the activities of macrophages, DCs, microglia, and astrocytes in the CNS. Finally, glutamate excitotoxicity is a key feature of MS and EAE [38][39][40][41] and homotaurine-mediated activation of CNS GABA A -Rs may have helped quell excitotoxicity.
Since GABA is at very low levels in tissues outside the CNS, we believe that the reason that immune cells express GABA-Rs is so that locally produced GABA can limit inflammation in the CNS. In this case, the activation of immune cell GABA-Rs is a natural mechanism to limit inflammation in the CNS, contributing to immune privilege in the CNS. This natural regulatory mechanism is insufficient to control the robust autoimmunity induced by priming with an encephalitic antigen, however, exogenous administration of homotaurine can inhibit the disease process. Similarly, although islet ß-cells secrete GABA, it is insufficient to prevent T1D in NOD mice, but exogenous GABA or homotaurine administration can prevent and/or reverse the disease [2][3][4]27 . Consistent with our observations, the administration of the benzodiazepine diazepam has been shown to inhibit EAE (e.g., 42,43 ). However, many benzodiazepines such as diazepam also bind to the mitochondrial translocator protein (TSPO, previously referred to as a "peripheral benzodiazepine receptor"), making it difficult to discern effects mediated through TSPO versus those through GABA A -Rs. Homotaurine's safety record and its unique mechanisms of action make it an excellent candidate to test as an adjunct therapy with current DMDs for MS to potentially achieve improved therapeutic outcomes.

EAE induction.
All experimental protocols were approved by the UCLA Animal Protection Committee and carried out in compliance with the ARRIVE guidelines and all relevant guidelines and regulations were followed for the experiment. Nine weeks old female C57BL/6 or SJL mice were obtained from the Jackson Laboratory and housed in a specific pathogen-free facility with free access to food and water. C57BL/6 mice were immunized subcutaneously with MOG 35-55 (200 µg, > 95% purity, GenScript)) in 50% IFA containing Mycobacterium tuberculosis H37R (5 mg/ml, Difco) in multiple sites near the base of their tail on day 1 and injected intraperitoneally with pertussis toxin (200 ng/mouse) on day 0 and 2. Individual SJL mice were immunized with PLP 139-151 (100 µg/mouse, > 95% purity, GenScript) using a similar protocol to that described for C57BL/6 mice. The mice were monitored for EAE onset daily: 0, no disease; 1, limp tail; 2, hind limb weakness; 3, complete hind limb paralysis; 4, quadriplegia; and 5, death. Mice that were in between the clear-cut gradations were scored intermediate in increments of 0.5. When the mice developed EAE with a score of 1 at 10-13 days post-immunization, they were randomized to receive plain water or water containing homotaurine (0.25 mg/ml) or GABA (6 mg/ ml).

Proliferation assays.
Female SJL mice at 9 weeks of age were immunized with 100 µg/mouse of PLP 139-151 peptide in 50% CFA in their foot-pads and treated with, or without, homotaurine in their drinking water (0.25 mg/ml). Nine days later, CD4 + T cells were purified from their popliteal lymph nodes (the responding T cells) and T cell-depleted splenic mononuclear cells (APCs) were prepared by negative selection using microbeads and magnetic sorting (BD PharMigen). The responding T cells were mixed with APCs in a ratio of 5:1, and were stimulated in triplicate with the indicated concentrations of PLP 139-151 in 1% fetal calf serum HL-1 medium for 3 days. The mixed cells without peptide stimulation served as the negative control. During the last 16-h incubation, individual wells of cells were treated with 1 µCi 3 H-thymidine and the T cell proliferation was determined by a β-counter.