A novel cyclic peptide (Naturido) modulates glia-neuron interactions in vitro and reverses ageing-related deficits in senescence-accelerated mice

Abstract

The use of agents that target both glia and neurons may represent a new strategy for the treatment of ageing disorders. Here, we confirmed the presence of the novel cyclic peptide Naturido that originates from a medicinal fungus (Isaria japonica) grown on domestic silkworm (Bombyx mori). We found that Naturido significantly enhanced astrocyte proliferation and activated the single copy gene encoding the neuropeptide VGF and the neuron-derived NGF gene. The addition of the peptide to the culture medium of primary hippocampal neurons increased dendrite length, dendrite number and axon length. Furthermore, the addition of the peptide to primary microglial cultures shifted CGA-activated microglia towards anti-inflammatory and neuroprotective phenotypes. These findings of in vitro glia-neuron interactions led us to evaluate the effects of oral administration of the peptide on brain function and hair ageing in senescence-accelerated mice (SAMP8). In vivo analyses revealed that spatial learning ability and hair quality were improved in Naturido-treated mice compared with untreated mice, to the same level observed in the normal ageing control (SAMR1). These data suggest that Naturido may be a promising glia-neuron modulator for the treatment of not only senescence, but also Alzheimer's disease and other neurodegenerative diseases.

Fig 1. Isaria japonica, the novel cyclic peptide (Naturido) extracted from it, and the effect of Naturido on astrocyte proliferation.

(A) Photographs of the fungus Isaria japonica collected in the field (left) and grown on silkworm pupae (Bombyx mori) in the laboratory at 25°C for 65 days (right). (B) The chemical structure of Naturido purified from the I. japonica extract and analysed by NMR/MS (see Methods). (C) Histochemical observations (left panel) of primary cultured astrocytes not treated with Naturido (control, upper) and primary cultured astrocytes treated with 25 μM Naturido (lower) were carried out using DAPI for nuclear staining, anti-EAAT2 for astrocyte-specific staining, and anti-GFAP for reactive-type cell staining. The GFAP-positive cell ratio (right panel) of astrocytes (shown as a percentage of all astrocytes) was not significantly changed 24 h after Naturido treatment. (D) The effect of Naturido concentrations of 0.1 μM to 50 μM on the proliferative activity of primary cultured astrocytes, as measured with a BrdU assay. All values are expressed as means ± SEMs. ***P < 0.001, **** P < 0.0001 (C, vs before and D, vs 0 μM, Dunnett test using JMP 10.0.0).

Fig 2

Relationship among Naturido and cell proliferation (A), therapeutic agents (B), acetylcholinesterase inhibitory activity (C), and the gene expression (D). (A) Normal human dermal fibroblasts (NHDFs), human hepatocellular carcinoma (HepG2) cells, human myeloid leukaemia (K562) cells and C6 glial tumour cells were used for proliferation assays with WST-1 (%) after treatment with Naturido at concentrations of 2.5 and 25 μM. (B) Each therapeutic agent (zonisamide, donepezil, eserine, and galantamine) was used at the same concentration as Naturido (25 μM), and proliferation was measured with a BrdU assay (%). (C) Comparison of the inhibitory effects of Naturido and donepezil on acetylcholinesterase (AChE) activity, shown as AChE inhibition (%). (D) The mRNA expression levels of representative neurotrophic factors (nerve growth factor = NGF, brain-derived neurotrophic factor = BDNF, vascular endothelial growth factor = VEGF-A, glial cell line-derived neurotrophic factor = GDNF, and non-acronymic neuropeptide = VGF) in astrocyte cultures were investigated to determine whether Naturido treatment enhances the expression of these genes. The Naturido concentration was 25 μM. All values are expressed as means ± SEMs. *P < 0.05, **P< 0.01, ***P < 0.001, **** P < 0.0001 vs each group (A-C Tukey-Kramer test using JMP 10.0.0) and vs the value at 24 h (D, t-test using JMP 10.0.0).

Fig 3. Effects of Naturido on dendrite and axon development in hippocampal neurons.

(A) Representative photographs (left, non-treated; right, treated with 0.1 μM Naturido; scale = 50 μm); (B) Lengths of dendrites/neurons (μm, left), numbers of dendrites per neuron (middle), and lengths of axons/neurons (μm, right), as evaluated from 30 neurons after 3 days of the addition of Naturido at concentrations from 0.03 μM to 1 μM. (C) Comparison of the effects of 1 ng/ml (= 0.0017 μM) Naturido and 1 ng/ml NGF protein on dendrite and axon development. Lengths of dendrites/neurons (μm, left), numbers of dendrites per neuron (middle), and lengths of axons/neurons (μm, right) and, as evaluated in 30 neurons. (D) Comparison of the effects of 0.1 μM Naturido and 0.1 μM VGF protein on dendrite and axon development. The observations and evaluations were conducted in the same way as in c. All values are expressed as means ± SEMs. *P < 0.05, **P< 0.01, ***P < 0.001, **** P < 0.0001 vs the control group (b, Dunnett test using JMP 10.0.0) or between groups (c and d, expressed as ratio of each control, Turkey-Kramer test using JMP 10.0.0).

Fig 4. Effects of Naturido on the expression of transforming growth factor (TGF)-β and interleukin (IL)-1β.

(A) and (B), Relationship between incubation time (a, 24 h; b, 72 h) and cell viability. (C) and (D), Effects of Naturido (1 μM), chromogranin A (CGA), or combined Naturido and CGA treatment for 24 h or 72 h on TGF-β expression. (E) and (F), Effects of Naturido (1 μM), chromogranin A (CGA), or combined Naturido and CGA treatment for 24 h or 72 h on IL-1β expression. All values are expressed as means ± SEMs. */^#P < 0.05, **^/## P < 0.01, ***^/###P < 0.001 vs the control (*) or CGA (^#) group (Turkey-Kramer test using the GraphPad Prism software package).