In Goodman and Gilman's, “The Pharmacological Basis of Therapeutics,” we were all taught to search for the one molecule that targets a single pathway, and specifically targets only that one pathway, underlying a disease or condition to optimally develop a therapeutic. However, neurodegenerative diseases result from perturbations in multiple pathways, not just one [1]. For example, evidence suggests that Parkinson’s disease (PD) is triggered by multiple environmental toxins acting at multiple neurological pathways [2]. Therefore, optimal development of a therapeutic must act at those multiple pathways underlying the disease or condition, not just partially correct the underlying mechanisms of the condition by acting at only one pathway.

We argue for a systems therapeutic approach [3], acting at the multiple pathways underlying the disease or condition, that renormalizes the physiology [4] of the nervous system as a safe and efficacious means for preventative and therapeutic strategies in neurogenerative disorders. Ours is a basic approach that is safe using natural and/or non-destructive means to renormalize physiology [5], and can be utilized for numerous tissues other than the nervous system, including the epithelial tissues such as the respiratory tract and skin [6], the immune system [7], including in the development of vaccines [8]. The new cancer therapeutics, called checkpoint inhibitors, use a similar “systems therapeutic for physiological renormalization” strategy where T-cells are returned to their normal physiological state so that they can attack and destroy cancer cells [9] and are more efficacious when combined with polyphenolic compounds and low protein diets [10]. In our framework, neuroprotection methods now come in fast and slow variants. The slow is the long-standing means of using vitamin [11], mineral [12], fiber [13], polyphenols [14], and other nutrient supplements for individuals who are deficient in and need an increased dose of these nutrients, particularly as they age and face the reduced absorption through their gastro-intestinal tract in parallel with the increase in oxidative stress [15] and inflammation and associated DNA methylation [16] that has enhanced the need for such nutrients. This chronic route of neuroprotection is most well documented for the visual system, where several degenerative diseases can be slowed with high doses of vitamin/mineral supplements (typically referred to as a medical food in the US when high doses are used, or as a food for medicinal purposes in Europe under the same conditions). Neural disorders such as glaucoma involve an inflammatory, autoimmune [17] response of T cells [18] and autoantibodies that attack heat shock proteins [19] that can lead to destruction of retinal ganglion cells, and can be significantly controlled through diet [20,21].

Glutamate and neurodegenerative diseases

 A number of studies have suggested that impaired L-Glutamate homeostasis is linked to neurodegenerative diseases, such as Alzheimer’s disease (AD), PD, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington’s disease [22]. Although a glutamate-rich diet is not neurotoxic when the blood–brain barrier is intact, there is strong evidence that when the barrier is compromised, chronic glutamate neurotoxicity can be a trigger for the development of neurodegeneration and subsequent PTSD [23] and depression [24], for example.

Measurement techniques

Ayoub et al. [25] developed an imaging technique for measuring glutamate release from individual photoreceptors and adapted this method to spatially assay glutamate within intact brain slices [26], allowing for the visualization of endogenous extracellular glutamate within given brain regions. Maguire et al. [27] used this technology to visualize endogenous glutamate released at high levels directly surrounding retinal ganglion cells (RGC) in an experimental model of glaucoma. This high level of glutamate constrained to the RGC layer may be why studies of glaucomatous patients who have had their mid-vitreous sampled, do not exhibit elevated levels of vitreal glutamate [28] whereas more complete samples of vitreous from animal models of glaucoma with retinal ischemia were elevated [29]. Further, assays of extracellular glutamate are dependent on the time course of the disease, where glutamate transporters may be upregulated or downregulated depending on the transporter type and timing of the assay during the disease sequalae [30]. In other words, would the high level of glutamate over the RGC layer in the experimental model be reflected in measurements of the total vitreous? The glutamate transporters are responsible for clearing glutamate from the retinal space, and GLAST1a is localized preferentially to the endfeet and inner stem processes of Muller cells that radiate beyond the inner retina to the distal margins beyond the RGCs towards and abutting the vitreous. This suggests a selective regulation of GLAST function in different compartments of the Muller cells [31]. Further, GLAST1 function has been found to be altered during ischemic conditions of the CNS [32]. Whether GLAST1a in the endfeet of Muller Cells would prevent glutamate spillover into the vitreous during glaucomatous events is not known. Here again, as pointed out previously, we must pay close attention to the spatial-functional compartments of the cells in the CNS [33].

Implications for glaucoma

While spillage of glutamate into the vitreous from the retina has not been associated with loss of visual function during retinal detachment [34], loss of glutamate from the retina would be energy inefficient given that glutamate is recycled into glutamine within Muller Cells and used for glutamatergic synaptic function. Loss of glutamate into the vitreous for recycling would therefore be deleterious even in the glaucomatous retina [35]. The high levels of glutamate in the RGC layer correlate with the defining characteristic of glaucoma, RGC destruction, and glutamate’s contribution to RGC neurotoxicity [36]. Based on animal studies, Ohguroet al. [37] suggested that a diet with excess glutamate over a period of several years may increase glutamate concentrations in retina and may cause RGC destruction. Therefore, a compromised blood brain barrier and/or blood retinal barrier and a diet high in glutamate may be a contributor to neurodegeneration in the CNS, including the retina.

Fast acting cannabinoid mechanism in neurodegeneration

The endocannabinoid system is critical for CNS function, and if modulated, can provide therapeutic action on fast and slow time course through various mechanisms, including fast acting, direct activation or modulation of cannabinoid receptors. A fast method of neuroprotection from glutamate toxicity has now been demonstrated by Maguire et al. [38] with their work revealing that even in the case of a sudden excitotoxic injury (injection of a toxic dose of NMDA into a mouse eye), application of a cannabinoid acting at multiple receptors (either along with the excitotoxic agent or subsequent to it) reduces cell death by 50%. This stunning neuroprotective efficacy was most pronounced when they coinjected WIN 55,212-2, or applied an extract prepared from the marijuana plant to the corneal surface (unpublished observations), indicating that cannabinoids may rescue neurons from excitotoxic insult, and that these cannabinoids may cross the cornea/sclera [39] to provide such protection (unpublished observations). These two routes to retinal neuroprotection, the chronic and the acute, raise hopes for reducing neural damage with increased age through improved nutrition and tested supplements, as well as providing a potential route to significantly limit neural damage during sudden injuries and in cases of uncontrolled degenerative diseases.

Slow acting, DNA methylation modulation for epigenetic control of neurodegenerative disorders

A slower, long-lasting action of cannabinoids in neurodegenerative disorders is through epigenetic modulation, especially DNA methylation, to reduce inflammation and enhance stem cell controlled regeneration. In the mammalian nervous system, DNA methylation can alter neural stem cell fate, brain development, neurodevelopmental disorders, and neurodegenerative diseases [40-42]. For example, the pleiotropic effects of the cannabinoids CBD and anandamide include DNA methylation modulation in the brain [43] and control of stem cell differentiation through modulation of DNA methylation [44]. Given Gage [45] has found adult neurogenesis (ANG) in brain, and Terreros-Roncal et al. [46] have found that impairment of ANG is related to aged and diseased humans, possibly including ALS, Huntington’s disease, α-synucleinopathies, and frontotemporal dementia, epigenetic modulation of ANG by cannabinoids should be investigated as a possible therapeutic. Epigenetic memory of inflammation through DNA methylation can last a long time [47], and may be passed to offspring through transgenerational epigenetic inheritance [48]. Chronic inflammation is an early factor in neurodegenerative disease such as glaucoma [18], preceding, for example, protein aggregation in AD [49]. Modulation of DNA methylation and the epigenetic memory of chronic inflammation is therefore an important part of the systems therapeutic strategy to prevent and remediate neurodegenerative disorders.

Other epigenetic modulators show promise too. Falckenhayn et al. [50] have screened a library of natural substances to identify active compounds that inhibit DNMT1 (DNA methyltransferase1, a DNA methylation catalyst) in a biochemical assay. They identified dihydromyricetin (DHM), a flavanol compound from plants that is popular in traditional medicines that is already known for its beneficial anti-cancer, antioxidant, and anti-inflammatory properties. Proof of concept studies found that DHM has regenerative effects in human skin models, indicating considerable potential as a safe DNA methylation modulator for the CNS and other tissues. The end goal of such modulators may include reprogramming of glial cells to neurons as a therapeutic solution [51].

Neurodegenerative diseases affecting the retina, a peripheral extension of the CNS, including neurons and glia cells, encompass glaucoma, macular degeneration, retinopathies, and inherited genetic disorders such as retinitis pigmentosa. These retinal pathologies pose a serious burden of visual impairment and blindness worldwide, and a cost to society in the USA of $134.2 billion in 2017 [52]. Three of the most common degenerative eye diseases afflict an increasing fraction of the population with increased age. Age-related macular degeneration (AMD), glaucoma disease (GD) and diabetic retinopathy (DR) share common features of damaging the retina and increasing prevalence with increased age, as shown in the Table 1 (comprising data from the National Eye Institutes data files [53]). With AMD, vision is lost from the center first, while with GD and DR it is lost from the periphery first. But in each, there is a degeneration of retinal cells that leads to vision loss.

It is well-established that many patients can delay the onset and slow the advancement of these conditions with neuroprotection. For AMD, the Age-Related Eye Disease Study-based AREDS and AREDS2 supplements are high dose vitamins and minerals that benefit people with mid-stage or late-stage AMD. The newer AREDS2 supplement discontinued the use of beta-carotene found in AREDS to decrease the risk of lung cancer in those who currently, or have, smoked during their lives [54].

Similarly, vision loss in glaucoma is slowed with high dose vitamin/mineral supplements. At least one such supplement combination is effective with both high tension and normal tension glaucoma, showing a neuroprotective effect in each type, as well as recently proving efficacious in cases of DN [55]. The formulation is a prescription-based medical food, Ocufolin® (GHF in US, Aprofol in Europe), which includes the AREDS2 cocktail along with several B vitamins that support endothelial cell function, presumably restoring normal vascular perfusion. This supplement was demonstrated to function in GD and DR by improving retinal perfusion and measurably dropping the excess retinal venous pressure that presages the neurotoxic impact of GD and DR [56].

Clinical and preclinical studies have shown that mesenchymal stem cell-secretome, including the exosome fraction, can improve cognitive decline, plaque deposition, inflammatory response, and neuronal degeneration in AD, enhance dopamine levels, reduce loss and apoptosis of dopaminergic neurons, and improve motor performance in PD, boost motor performance, speech, strength, and sleep, and reduce glial cell activation in ALS, and decrease disease severity and improve motor function outcomes in MS. The myriad molecule types (growth factors, HSP, antioxidants, immune modulators, epigenetic regulators, matrix components) released from adipose mesenchymal stem cells (ADSCs) have been found to prevent neurodegeneration using in vitro [57] and animal models [58]. Moving from stem cell therapies to “stem cell therapies without the cells” [59] has proven to be a useful strategy. While the molecules can be optimized in the lab, dosed using defined schedules and abated in the case of an adverse event, the cells are undefined quantity and quality when administered to the body and continuously present despite an adverse event. That is, once injected or otherwise administered to the body, the functionality of the stem cells is unknown.

Key to the effectiveness of the molecules released from stem cells is their ability to modulate the immune system, shifting immunity from an inflammatory state, an early event in neurodegeneration [49] to one of anti-inflammatory and pro-repair [7]. Further, once neurons have differentiated, they stop making or have reduced heat shock protein (HSP) responses to stress [60], and are dependent on HSPs from other cells, including adult mesenchymal stem cells.

Consistent with our systems therapeutic approach, using both the exosome and soluble fractions, not just one or the other, released from the ADSCs has been found to be most efficacious for regenerative healing in the neuromuscular system [61]. This makes sense given the two fractions contain exclusive sets of molecules. As a therapeutic for ALS, Maguire [62] proposed stem cell released molecules be administered intra-nasally as well as intrathecally to perfuse the CNS and spinal cord, both of which are sites of neurodegeneration.

The extracellular matrix (ECM) in the CNS is a critical non-cellular component of the extracellular space, which accounts for about 20% of brain tissue [63]. Crucial intercellular and intracellular signaling capabilities and biophysical stability are provided by the ECM. The ECM also governs the physiological status of the local microenvironment, thus controlling cellular phenotype, including gene expression [64]. The most abundant proteins in the ECM are the collagens, which are produced in the CNS mostly by astrocytes, neurons, and vascular cells [65]. Collagen and other matrix components, such as glycosaminoglycans (GAGs), are degraded by a number of mechanisms, including stress activated cortisol, MMPs, and reactive oxygen species, contributing to a number of neurological disorders [66]. Neurodegeneration in CNS disease and injury is characterized by early biochemical degradation and remodeling of the ECM [67]. Therapies that rebuild or utilize the matrix have been found to have therapeutic potential to promote neural repair and regeneration in animal models [68]. Integrated analysis of multiomic datasets has identified several dysregulated pathways in ALS patients and animal models, including mitochondrial respiration/oxidative stress, transcriptional regulation/splicing, and protein misfolding/degradation. Previously less prioritized pathways, including the dysregulation of the ECM and BCNSB (blood CNS barrier) or the MAPK pathway, have also been identified in these multiomic studies. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes that degrade various components of ECM and mediate ECM remodeling in both physiological and pathological processes [69]. Dysregulation of MMPs during a chronic inflammatory event may lead to matrix breakdown, resulting in even more inflammation. Thus, a chronic neuroinflammatory state may ensue leading to neurodegeneration. Consider one aspect of poor dietary practices in degrading the ECM and inducing inflammation. Many collagens in the ECM are long-lived proteins, having a half-life of 15 to over 100 years [70], with many long lived proteins found in the brain [71], including the retina [72], and responsible for many functions related to neurodegeneration [65]. These long-lived proteins are subject to oxidative damage and their fragments can induce inflammation and glutamatergic excitotoxicity [62]. To rebuild the matrix a general plan for therapeutic development has been proposed, using a supplement that uses the molecules that stem cells release, along with amino acids and prebiotics, probiotics, and postbiotics that help rebuild the extracellular and intracellular matrix, i.e., the cytoskeleton of the cilia and the villi of the epithelial cells lining the gut and nervous system [73].

While integrative approaches to neurodegenerative disorders have been considered as using complementary methods along with traditional medicine [74] and treating the whole and not just acute symptoms [75], we also embrace a polypharmacy approach with combinations of traditional drugs and medical foods, for example. Disorders of the nervous system reflect perturbations in many different pathways at many different levels, with genomics a minor contributor. Polygenic risk scores, representing the weighted sum of independent DNA sequence variants present in an individual's genome that are associated with the risk of a particular disease, are poor at predicting disease [76]. Genetic determinism has often led us down the wrong path [77]. Rather, the exposome, the complex sum of all exposures in an individual, accounts for most disorders [78]. As Rappaport and Smith [79] have written, “toxic effects are mediated through chemicals that alter critical molecules, cells, and physiological processes inside the body…under this view, exposures are not restricted to chemicals (toxicants) entering the body from air, water, or food, for example, but also include chemicals produced by inflammation, oxidative stress, lipid peroxidation, infections, gut flora, and other natural processes.” Complex exposure can act at many pathways to perturb physiology. Proteins involved in different pathways can be particularly vulnerable to our exposome [80]. Thus, the perturbation of many pathways needs to be renormalized through a systems therapeutic approach, including, for example, the use of “nutritional pharmacology” [81]. We’ve briefly highlighted the potential for integrated fast and slow systems therapeutic approaches to neurodegenerative disorders. Our highlights reflect the work of the authors and related studies, however, many other potential therapeutic strategies have not been discussed here, such as targeting the newly discovered mechanism of DNA repair, a form of autophagy called nucleophagy [82], that could play a role in the many neurodegenerative disorders involving compromised DNA repair mechanism [83]. Newer therapeutic studies of a number of diseases, both animal models as well as clinical trials, provide evidence that neurodegenerative diseases are best treated and prevented by using combinations of molecule types, a systems therapeutic, in fast and slow regimens, often used at different levels and pathways to renormalize physiological pathways underlying the affliction. The different types of molecules used for therapeutic purposes are often endogenous molecules (stem cell released molecules, for example) and molecules with which we have co-evolved that are ingested (vitamins and polyphenols, for example). Thus, the substrate of neural disorders is complex, involving multiple pathways, and therefore the therapeutic must also be complex, targeting multiple pathways. Recent examples of clinical studies of AD suggest our approach can be powerful. Masitinib, a tyrosine kinase inhibitor, is used in the treatment of mast cell tumors in dogs. Animal models of AD have found that mast cell depletion increases the immunoreactivity of synaptic markers [84], has a protective effect on synapses, and reduces the secretion of specific synaptic toxins [85], resulting in significant improvement in cognitive function [86]. Further, using a polypharmacy approach, Masitinib was administered as add-on therapy to standard care, i.e. memantine or cholinesterase inhibitors, during 24 weeks was associated with slower cognitive decline in AD [86]. In terms of glutamatergic excitotoxicity, new strategies in the clinic are unfolding. Excitotoxicity can be caused by excessive activity of NMDA receptors, leading to cell death and neurodegeneration. Therefore, some pharmacological treatments of ALS patients counteract glutamate excitotoxicity. Riluzole, an inhibitor of glutamate release, was approved by the US Food and Drug Administration (FDA) in 1995. But the drug provided only a short survival benefit of 2–3 months [87]. Considering the exposome, evidence from several open?label, non?randomized, trials have suggested that the greatest benefit of Riluzole occurs at early in the disease sequalae [88]. Riluzole's therapeutic benefit is likely to affect different physiological pathways depending on the disease stage, hence, the effects depend on the person’s exposome [89]. Another therapeutic found to protect against excitotoxicity is the antibiotic ceftriaxone, which up?regulates glutamate transport and decreases glutamate?induced toxicity [90]. A Phase 3 trial of ceftriaxone did not show clinical efficacy when used alone [91], but a combinatorial investigation has not been attempted. Given that ALS, like most other neurodegenerative disorders, is a multifactorial disorder rather than a single disease, there are many perturbed physiological pathways associated with the underlying pathogenesis. Factors include oxidative stress, misfolding and aggregation of proteins, mitochondrial dysfunction, glutamate-induced excitotoxicity, apoptosis, neuroinflammation, axonal degeneration, skeletal muscle deterioration, and viruses [92]. Given the overreliance on reductionist therapeutic strategies, where one or only a few pathways are considered as targets in each trial, ALS treatment has failed and is largely limited to palliative care. A combinatorial, systems therapeutic, approach is warranted in this and other neurodegenerative disorders.