A spirulina-enhanced diet provides neuroprotection in an α-synuclein model of Parkinson's disease

Abstract

Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc) of PD models when there has been a decrease in tyrosine hydroxylase (TH) positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9) for α-synuclein was injected in the substantia nigra of rats to model Parkinson's disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1) on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action.

Figure 1. Effect of spirulina on TH immunoreactive cells in the SNpc.

TH positive cells in the SNpc after 1 or 4 months of α-synuclein expression. Cells were counted using unbiased stereological counting techniques. (A) One month after α-synuclein gene transfer, there was a significant decrease in TH positive cells as compared to GFP control (N = 12–18/group). The spirulina diet group lesioned with α-synuclein (N = 12) had greater numbers of TH positive cells compared to the control diet group lesioned with α-synuclein (N = 18). There was a diet by vector group interaction in the two way ANOVA analysis [F = 5.569, p<0.01]. (B) Results at four months were similar. There was a similar loss of TH positive cells and protective effect of spirulina. Two-way ANOVA yielded a main effect of diet (F = 81.3), and a main effect of vector group (F = 45.5; p<0.01), although without a significant interaction. Bonferonni post-hoc tests comparing NIH 31 GFP (N = 10) vs NIH 31 synuclein (N = 8) and NIH31 synuclein vs spirulina synuclein (N = 8) groups were significant). Asterisk denotes significance (*P<0.05; **p<0.01).

Figure 2. Effect of spirulina on NeuN immunoreactive cells in the SNpc.

NeuN positive cells in the SNpc after 1 or 4 months of α-synuclein expression. At 1 month, there was a decrease in NeuN positive cells in the SN, mirroring the loss of TH positive cells in Fig. 1. This confirms cell loss rather than loss of TH expression. The effect was similar at 4 months of expression (B). There was neuroprotection in the groups that received a diet enhanced with spirulina at both intervals. Two-way ANOVA yielded a significant interaction of diet and vector treatment at both time points [1 month F = 6.931, df = 1; 4 months F = 8.899 df = 1]. (*p<0.05; **p<0.01) after Bonferonni's post-hoc.

Figure 3. Effect of spirulina on OX-6 positive staining.

There was a decrease in OX-6 positive staining in the groups that received spirulina at both 1 (A) and 4 (B) months of α-synuclein expression as compared to the control groups. Two-way ANOVA was performed for both 1 and 4 month groups and showed a significant interaction between diet and vector treatment [1 month F = 5.592, df = 1] and [4 months F = 8.899, df = 1]. Bonferonni post hoc analysis for individual comparisons is shown (*p<0.05, and ***p<0.001).

Figure 4. Gene transfer efficiency is not affected by spirulina.

Quantification of GFP positive cells in the SN. Stereological estimates of the number of GFP positive cells in the SN one month after gene transfer. There was no significant effect of the spirulina diet on numbers of GFP transduced cells (Student’s two-talied t-test).

Figure 5. Effect of AAV9-synuclein treatment on the LC.

Quantification of TH immunohistochemistry in the locus coeruleus using unbiased stereological techniques. Graph shows that 4 months following α-synuclein gene transfer there is a significant loss of TH positive cells in the LC (2-way ANOVA did not show a significant interaction, but did reveal main effects of diet and treatment, and bonferonni post-hoc revealed a difference between the α-synuclein control and diet treated groups, p<0.01). Treatment with spirulina was able to prevent the loss of TH positive cells in the locus coeruleus. Asterisk denotes significance (**p<0.01 vs control GFP; *p<0.05 vs control α-synuclein).

Figure 6. Effects of spirulina on CX3CR1.

Spirulina diet increased expression of CX3CR1; inset of Western blot in upper right. When the data are analyzed across groups there is a significant increase in expression of CX3CR1 in the spirulina treatment groups. Asterisk denotes significance (p<0.05; Bonferroni post-hoc). Two-way ANOVA shows a main effect of diet (F = 19.90; df = 1) and no interaction or main effect of vector treatment.