Omega-3 Hastens and Omega-6 Delays the Progression of Neuropathology in a Murine Model of Familial ALS

1. INTRODUCTION

Amyotrophic Lateral Sclerosis (ALS) is characterized by a progressive loss of motor neurons, with eventual degeneration of muscles themselves, resulting in paralysis and death. There currently is no cure or long-lasting treatment. Mutations in multiple genes underlie ALS, including Cu/Zn superoxide dismutase (SOD1) [1-4]. SOD1 is abundant within motor neurons, where it converts superoxide anions to hydrogen peroxide. More than 90 different mutations have been described that span all exons. SOD1 mutations promote neurodegeneration by a gain of toxic function, which promotes increased reactive oxygen species and widespread oxidative damage to proteins, nucleic acids and lipids. Importantly, however, mutations in SOD-1 account for only 20% of the familial cases, which themselves make up only 5-10% of the total cases. Clearly, one or more additional factors is/are involved in the onset and/or progression. Such factors remain elusive despite decades of study [2, 3]. Several genes have been implicated in ALS to date, such as C9ORF72 and TARDBP [4].

ALS is accompanied by inflammation, mediated in part by astrocyte and microglial activation [5, 6]. Proinflammatory activity of cyclooxygenases COX-1 and 2 is upregulated in ALS; suppressing this activity, and/or activation of glia, to reduce inflammation represents a therapeutic target [5-7]. COX-1 and 2 convert dietary polyunsaturated fats (PUFAs) into a series of thromboxanes and prostaglandins; those derived from Ω-6 are proinflammatory, while those derived from Ω-3 are anti-inflammatory [8, 9]. These collective findings prompted the consideration that Ω-3 would be beneficial during ALS pathology. Accordingly, Yip and colleagues supplemented SOD-1 mice with 1000x Ω-3. This 1000x increase corresponds to common over-the-counter Ω-3 supplements for human consumption. While 1000x Ω-3 suppressed inflammation in SOD-1 mice, it unexpectedly hastened the progression of motor neuron disease [10]. While this seems to contradict the recent clinical study reporting a beneficial effect of consuming Ω-3, we note that participants in that study also consumed similar amounts of Ω-6 [11]. We therefore considered that clinical benefit may have been achieved by supplementation with a balance of Ω-3 and Ω-6 rather than Ω-3 alone. As shown herein, supplementation of SOD-1 mice with Ω-3 along with similar or increased levels of Ω-6 delayed the progression of motor neuron disease.

2. MATERIALS AND METHODS

All procedures were approved by our Institutional Animal Care and Use Committee.

2.4. Analyses of Metabolites

Prostaglandin and thromboxane metabolites derived from Ω-3 and Ω-6 were quantified by liquid chromatographic mass-spectrometry (LC-MS) of lumbar spinal tissue via standard methodologies, with identification by co-migration of purified standards as described [18].

3. RESULTS

Disease Onset as quantified by the standard “wire cling” test was not altered by supplementation (not shown); however, 1000x Ω-3 significantly decreased survival: when the 50% survival point was reached for this diet, 90-100% of mice on the other diets were still alive (Fig. 1A). By contrast, 10xΩ-6 significantly prolonged survival; 100% of mice on this diet were still alive when only 50% of mice on the standard and 4xΩ-3,6 diets survived and 100% mice on the 1000xΩ-3 diet had died (Fig. 1A). The 4xΩ-3,6 diet did not statistically alter survival vs. the standard diet. Death was first observed with the standard diet at 15.8 weeks of age, but not until 18.7 weeks of age with 10xΩ-6 (Fig. 1A), which represents an 18% increase in survival time. Motor neuron function was also preserved for 1-2 weeks longer with 10xΩ-6 (Fig. 1B-D).

Motor neuron numbers, reactive astrocytes and activated microglia were quantified at 5 and 6 weeks of age (corresponding to 1 and 2 weeks of supplementation and prior to the onset of terminal symptoms; e.g., Fig. (1). Motor neuron numbers and reactive astrocytes decline during this pre-symptomatic interval [12, 13]. However, we observed significantly greater numbers of motor neurons, reactive astrocytes and activated microglia in mice receiving 10xΩ-6 vs. 4xΩ-3,6 (Figs. 2, 3).

In a single preliminary experiment, supplementation with 10xΩa-6 induced a >2-fold increases in thromboxane 2 (TXB₂) and prostaglandins D₂ and E₂ (PGD₂/ PGE₂) during terminal stages; 4xΩ-3,6 induced the same increase in PGD₂/PGE₂ but completely prevented the increase in TXB₂ (Fig. 4).

4. DISCUSSION

We report the unexpected result that dietary supplementation with equivalent Ω-3 and Ω-6 hastened motor neuron pathology and death, while 10x Ω-6 with no change in Ω-3 significantly delayed motor neuron pathology, including preservation of minor motor neuron function during the terminal stage of a mouse model of familial ALS. These findings support and extend the studies of Yip et al. [10], in which supplementation with Ω-3 hastened a decline in motor neuron function, by demonstrating a dose-dependent delay of ALS-related neuropathology following moderate increases in Ω-6. Acceleration of disease progression with increasing Ω-3, yet delay of disease progression with increasing Ω-6 supports the hypothesis that a balance of Ω-3 and Ω-6 may be beneficial for ALS. While these results in a mouse model seems to contradict the recent clinical study reporting a beneficial effect of consuming Ω-3, we note that participants in that study also consumed similar amounts of Ω-6 [11]. The possibility therefore remains that clinical benefit was actually achieved by supplementation with a balance of Ω-3 and Ω-6 rather than Ω-3 alone.

We observed the intriguing preliminary result that TXB₂ generation was suppressed by the 4xΩ-3,6 diet. Since TXB₂ is derived exclusively from Ω-6 in the nervous system [8], it is possible that the anti-inflammatory properties of Ω-3 suppressed TXB₂ generation. Moreover, since microglia are the source of TXB₂ in the nervous system [19], attenuation of microglial activation is a likely mechanism by which Ω-3 supplementation prevented the TXB₂ increase Fig. (3). Further studies are required to substantiate these possibilities. For example, it remains to be determined whether further increase in Ω-3 suppresses additional Ω-6 inflammatory compounds (e.g., PGD₂/PGE₂); this may have fostered the deleterious impact of 1000x Ω-3 with no compensatory increase in Ω-6 [10]. The clinical implication of this finding is supported by the unexpected observation of downstream benefits in motor neurons in a murine ALS model following supplementation with prostaglandins D2 and PEGs, which are downstream products generated from Ω-6 by COX-2 [20, 21]. These results encompass the challenge that any and all inflammation, including that resulting from consumption of Ω-6, is de facto deleterious. Inappropriate levels of Ω-6 are indeed associated with increased risk of cardiovascular disease, cancer and inflammatory disorders, while appropriate levels of Ω-3 reduces these risks [14]. However, several studies support establishment of an appropriate ratio of these PUFAs, rather than elimination of Ω-6: e.g., (1) a ratio of 4:1 Ω-6:Ω-3 decreased mortality in secondary prevention of cardiovascular disease by 70%; (2) a ratio of 2.5:1 reduced colorectal and breast cancer proliferation, but 4:1 did not; (3) 2-3:1 suppressed rheumatoid arthritic inflammation; (4) 5:1 was beneficial for asthma, but 10:1 was deleterious. The optimal ratio is disease-specific and likely varies in accord with an individual’s genetic predisposition. In this regard, the potential benefit of astroglial and microglial activation (which accompany inflammation) has been advanced in neurodegenerative conditions, including ALS [5, 22]. Comparison of the impact of our omega-supplemented diets on these parameters in wild-type mice would be of interest.

Our findings hold the promise that a simple, non-invasive approach could be initiated presymptomatically in familial ALS (since individuals are likely aware of the possibility of inheritance and could be tested for presence of the conferring mutation), as well as administered late, even via a feeding tube (which routinely accompanies end-stage ALS). Notably, we initiated dietary supplementation prior to onset of symptoms, and we therefore have no indication as to whether or not supplementation would be beneficial after symptomatic manifestation; this will be examined in future studies. Cyclooxygenases (COX-1 and 2) are upregulated in ALS. These enzymes convert dietary PUFAs into thromboxanes and prostaglandins; those derived from Ω-6 are inflammatory while those from Ω-3 are anti-inflammatory. Since Ω-6 and Ω-3 compete for these enzymes, simple dietary manipulation provides a means of regulation of inflammatory pathways without attempting direct enzymatic inhibition; dietary manipulation is therefore ideal for our proposed exploratory clinical studies. Development of precise temporal and spatial promotion of controlled inflammation by pharmacological methodologies may represent an important facet of therapeutic intervention.

CONCLUSION

ALS has no cure or effective treatment. Our demonstration of temporary preservation of motor function in mice relates directly to the quality of life for affected humans: the progressive decline in human motor function eventually prevents speech, the minor finger movement that could operate a computer (with speech capabilities), swallowing and, ultimately, breathing. The terminal stage, (complete loss of movement but prior to death) leaves the individual in a so-called “locked in” state that can last for a year or more. Any approach that could temporarily preserve minor motor function (e.g., finger movement) during this terminal stage would vastly improve an individual’s quality of life, and that of caregivers, by allowing continued communication. Our findings hold the promise that a critical balance of Ω-3 and Ω-6 could provide this temporary window: mice live for 2.5-3yrs; the 1.5 to 3-week increase in survival demonstrated herein extrapolates to ≥1yr of human life based on an 80yr human life span. Moreover, we observed a window of preserved minor motor function during this period. Equally as important, affected individuals may resort to nutritional supplements as a form of self-medication [23]; based on the above preclinical findings, coupled with the popularity of Ω-3, such an approach could actually be harmful.

REFERENCES