Nutrigenomics moves in on neuroprotection …

Mulberry Down-Regulates
Alzheimer’s Genes

This may represent a tipping point for nutritional research

By Will Block

C ontaining high amounts of many bioactive substances, mulberry has been used as a natural medicine for at least 3,000 years in China, and perhaps as long as 5,000 years as a principal food for silk worms. In the Chinese Materia Medica, mulberry is classified as a blood tonic, meaning that it nourishes the yin, whatever the precise meaning of that is. In modern nutritional research, mulberry’s antioxidant and antiinflammatory properties have increasingly been the subject of intense study. Mulberry is thought to address a wide spectrum of ailments, for which there is an expanding amount of Chinese science.

The ability for mulberry to treat many disorders may be due to the fact that it contains significant amounts of nutrients such as vitamins A, B1, B2, C, protein, and especially the flavonoids known as anthocyanins. Anthocyanin-rich substances have long been used to treat a number of conditions (including high blood pressure, colds, and urinary tract infections) and illnesses such as cancer, diabetes, cardiovascular disease, liver disease, and neurodegeneration.

Mulberry Fruit Study Opens Nutrigenomic Doors

In past studies, mulberry fruit extracts (ME) have demonstrated neuroprotection activity. What these studies haven’t done is to present a detailed explanation of how they do it. A new study1 conducted by Chinese researchers set out to investigate the neuroprotective efficacy of ME against amyloid-β- (specifically Aβ25 – 35-) induced PC12 cell injury. While merely a cell study (thus low on the pyramid of proof), the new report packs muscle and thus adds significantly to our understanding of mulberry’s mechanisms, right down to the gene level. The study opens the door to how nutrigenomics can shed more interesting light in the field of Alzheimer’s intervention.

To investigate the role of mulberry,
genomic techniques were used to
quickly and accurately quantify vast
numbers of potential gene expression
changes after mulberry pretreatment.

Mulberry Enhances Neural Growth Factor Regulation

Last year, Life Enhancement ran an article on ME (see “Mulberry Fruit Enhances Memory” in the January 2014 issue). This was particularly interesting because the research cited investigated the memory-enhancing effect of ME in mice, with the goal of determining its effects on neural growth factor regulation (NGF).1 ME was orally administered in doses of 20, 100, and 500 mg  /  kg per day for 7 days, resulting in dose-dependent increases in NGF release in the mouse hippocampus. NGF is essential for the growth, maintenance, and survival of certain neurons.

Producing an Array of Brain Benefits

ME significantly increased pre- and post-synapse formation, acetylcholine synthesis, neuronal cell differentiation, neurite outgrowth and neuronal cell proliferation in the mouse hippocampus. The hippocampi (there are two) belong to the limbic system and play important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation. Furthermore, ME significantly increased latency time in the passive avoidance task and decreased recognition time of novel objects in the object recognition test, demonstrating learning and memory improvements. The passive avoidance test measures recall of compartment-associated pain; the longer the delay (latency) the stronger the memory. The object recognition test measured the speed with which mice recognized objects they had been exposed to 24 hours earlier. Taken together, these data suggest that ME exhibits a memory-enhancing effect via up-regulation of NGF.

Alzheimer’s Correlations Remain Unchanged

Alzheimer’s disease (AD), accounting for 60% to 70% of all cases of dementia, may be the fastest-growing disease in the U.S. Also, it appears to be approaching the mortality rates from cancer and heart disease (see “Alzheimer’s Deaths Rival Cancer’s” in the May 2014 issue). Since we’ve been writing about AD, little has changed in its characterization as a progressive degeneration of cognition and memory, or its correlation with the appearance of neurofibrillary tangles, senile plaques, and loss of neurons in the brain.

Figure 1. Amyloid protein precursor processing. http://www/
(click on thumbnail for full sized image)

Even papers about the processing of amyloid precursor protein (APP)—a type I transmembrane glycoprotein that plays an important role in the development of AD—have been in the scientific literature since 1988. In the amyloidogenic pathway, cleavage of APP by β-secretase results in the release of a soluble fragment (sAPPβ), and a membrane anchored C-terminal fragment (CTFβ). The CTFβ fragment is subsequently cleaved by β-secretase and generates an Aβ peptide (see Fig. 1). Aβ peptide is the major component of senile plaques and has been suggested to play a causal role in the development and progression of AD. Aβ can trigger a cascade of pathogenic events such as culminating of neuronal apoptosis  /  death, dystrophy of neurites, excitoactivation of glutamate receptors, and induction of oxidation stress. The accumulation of this damage can precipitate AD.

The Amyloid Toxin Used is the Equivalent of Full-Length Aβ

In the new Chinese study, the researchers used a synthetic peptide Aβ25 – 35 that correspond to amino acids 25 – 35 in Aβ1 – 40 and Aβ1 – 42. These possess the same β-sheet structure and exhibit large β-sheet fibrils and retain most physical and biological properties of full length-Aβ, including its toxicity. More importantly, Aβ25 – 35 is a particularly intractable peptide because it aggregates rapidly, unlike full-length Aβ, which requires aging for more than 1 week before it aggregates and becomes toxic. As such, it is often used for in vitro studies.

Mulberry pretreatment was found to
neutralize the cytotoxicity and
prevent Aβ
25–35-induced cells injury.

The Importance of Anthocyanins

Anthocyanins are a group of naturally occurring phenolic compounds that are responsible for the brilliant colors—blue, red, and purple—of leaves, flowers, and fruits. Because a significant property of anthocyanin is antioxidant activity, the neuroprotective effect of anthocyanin has received a lot of attention in the field of nutrition research, where numerous studies have shown these effects in vitro. In vivo tests have also confirmed that anthocyanins reduce the injury area of cerebral ischemic damage in rats. ME contain high amounts of anthocyanins.

To date, 8 different anthocyanins have been identified by high-performance liquid chromatography in mulberry extract. Cyanidin-3-glucoside and cyanidin-3-rutinoside are the major anthocyanins identified in mulberry. In recent years, many papers have been published on the neuroprotective effects of ME. Animal studies have found that mulberry fruits and their neuroprotective constituent—cyanidin-3-O-β-D-glucopyranoside (C3G)—can alleviate the cerebral ischemic injury and aging-associated neuronal damage in vivo using a mouse-brain-injury.

Among the gene expression changes
analyzed, Apaf1, Bace2, and Plcb4
were enriched in the “Alzheimer’s
disease-reference pathway.”

Other recent research has found that ME can increase the endogenous antioxidant enzymes’ activities (glutathione peroxidase, catalase) and improve learning and memory ability in senescence-accelerated mice. In in vitro experiments, ME anthocyanins can inhibit Aβ25 – 35 (the synthetic peptide used in the current Chinese study) spontaneous aggregation into oligomers and their neurotoxicity in human neuronal cells, and have neuroprotective effects on the PC12 cells exposed to hydrogen peroxide and oxygen glucose deprivation. The PC12 cell line is derived from a tumor of the rat adrenal medulla.

New and Important Light in the Field of AD Intervention

Figure 2. Cytoprotective effects of mulberry extracts in Aβ25 – 35-induced PC12 cells. PC12 cells were pretreated with or without ME (200 Aβg/ml) for 24 h and exposed to Aβ25 – 35 (20 βmol/L) for 24 h. The cytotoxicity was measured by MTT assay. The viability of the untreated cells was set to 100%.
(click on thumbnail for full sized image)

Few studies have used PC12 cells as Aβ25 – 35-induced injury model to investigate cytoprotective and neuroprotective effects of ME in vitro. Still, the mechanisms that ME pretreatment might use to inhibit development of AD have not been elucidated clearly. In the Chinese study, not only did the researchers set out to explore the mechanisms involved, they used Aβ25 – 35 treated PC12 cells as an in vitro model to investigate the role of ME, and used genomic techniques to quickly and accurately quantify vast numbers of potential gene expression changes after ME pretreatment. Thus, this study tenders great nutrigenomics interest and brings new and important light in the field of Alzheimer’s disease intervention.

ME Found to Alter Alzheimer Genes for the Better

Once again, the goal of the Chinese cell study1 was to investigate the neuroprotective efficacy of ME against amyloid-β induced PC12 cells’ injury. PC12 cells stop dividing and undergo terminal differentiation when treated with NGF, making the line a useful model system for nerve cell differentiation.

Figure 3. Hierarchial clustering of PC12 cells analyzed with the microarray chip. PC12 cells were pretreated with or without ME for 24 hours and exposed to Ab25 – 35 (20 μmol / L) for 24 hours. Data are representative of three different experiments. Up- and downregulated genes are represented in red and green colors, respectively. Yellow highlights designate the three principal genes downregulated by mulberry.
(click on thumbnail for full sized image)

Cells preincubated with or without ME (200 μg  /  mL) for 24 hours were treated with Aβ25 – 35 (20 μmol  /  L) for another 24 hours. Cell viability was assessed by the MTT assay, gene expression profiles were examined by cDNA microarrays, and real-time polymerase chain reaction (PCR) to confirm the results of microarray assays (see Fig. 2).

ME pretreatment was found to neutralize the cytotoxicity and prevent Aβ25 – 35-induced cells injury. Analyses of gene expression profile revealed that genes involving cell adhesion, peptidase activity, cytokine activity, ion binding activity, and angiogenesis regulation were significantly altered by ME pretreatment. Among those genes, Apaf1, Bace2, and Plcb4 were enriched in the “Alzheimer’s disease-reference pathway,”* according to the National Center for Biotechnology Information database, and downregulated after ME intervention (see Fig. 3). That is, these genes were found to not only play an important role in the development of AD, but also their genes expression products were involved in apoptosis, Aβ formation, and cell or organelle membrane damage.


Mulberry Reverses Neuronal Damage in PC12 Cells

The Chinese researchers had previously found that pretreatment of PC12 cells with 200 μg / mL ME could almost completely reverse Aβ25 – 35-induced neuronal injury, counteract ROS formation, and inhibit apoptosis.2 Their new study is a continuation, investigating the possible mechanisms involved.

Gene Chip Exploration of 24,358 Genes

Figure 4. The results of gene ontological classification in ME pretreatment groups.
(click on thumbnail for full sized image)

This led them to the current study design, which use gene chip to explore the molecular mechanisms of neuroprotective effect of ME by analyzing the transcription of 24,358 genes. Combined with bioinformatics analysis, the gene expression profiles in samples were significantly affected.

Further analysis shows that in ME pretreatment group, the downregulated genes were mainly related to cell adhesion, cytokine activity, and angiogenesis regulation, and upregulated genes were mostly related to ion binding activity and multicellular organism reproduction (see Fig. 4).

Apoptotic Protease-Activating Factor 1

Apoptotic protease-activating factor 1 (Apaf1), a tumor suppressor gene, is essential for regulation of mammalian development and induction of cell apoptosis. This factor is at the core of the mitochondria-dependent pathway operating as a “apoptosome.” So the abnormal expression and function of Apaf1 is associated with the development of many human diseases, including neurogenesis. The researchers’ previous result found that ME-preincubated cells can effectively reduce the rate of cell apoptosis.

The Amyloid Toxin Increased Expression of Apaf1

The gene chip results found that Aβ25 – 35 toxin increased expression of Apaf1, and ME pretreatment reversed the upregulation of Apaf1 expression induced by Aβ25 – 35. ME could lessen the apoptosis by the way of suppressing gene expression of Apaf1 in PC12 cells. Apaf1 is essential for the normal development of the brain and transcription level changes which may be associated with the development of AD.

β-Secretase β-site APP Cleaving Enzyme

β-Secretase (β-site APP cleaving enzyme, BACE) is the rate limiting enzymatic activity in the production of the amyloid-β peptide (Aβ) and is thought to be involved in Alzheimer’s disease (AD). Though Bace2, the homologue of Bace1, is also expressed in the brain, its potential role in AD has not been clarified completely.

Figure 5. Bace2 inhibits the production of amyloid-beta (Aβ).
(click on thumbnail for full sized image)

However, Bace2 also exhibits β-secretase-like activity (see Fig. 5). Expression of Bace2 in APP-expressing cells results in an increase in the levels of β-secretase derived cleavage products, sAPPβ and CTFβ. Thus, Bace2 maybe play a critical role in the development of AD, because accumulation and deposition of Aβ fragments in the brain induce Aβ-generated cascade process, a critical step of AD pathologic processes. The researchers speculated that BACE2, not BACE1 may become a promising way to treat AD.

The Apoptotic Effect of PLC-b4

The PLCB4 gene specifies for PLC-β 4, one of the isozymes of phospholipase C-β (PLC-β). Phospholipase C (PLC) as an important enzyme is widespread in various cells, having species and cell specificity in the basic biochemical characteristics, function, and subcellular distribution. PLC participates in the apoptosis (cell suicide) signal transduction in various cells, such as neurons. Other studies have found that PLC is involved in the regulation of oxidative stress caused by oxidative glutamate toxicity induced neuronal cell death in immature cortical neurons and hippocampal neurons.

The Chinese researchers’ previous experimental results (along with others in the literature) found that ME pretreatment alleviated membrane structural damage of cells or organelles, and preserved mitochondrial membrane integrity and membrane potential. Plcb4 mRNA in PC12 cells were obviously more overexpressed in the Aβ25 – 35 group than in the control group, which was inhibited by ME preincubation. The results indicated that ME could inhibit apoptosis by way of suppressing gene expression of Plcb4 in PC12 cells.

Nutrigenomics Pivotal Study—a Tipping Point

This is perhaps the first clear nutrigenomics study to date, bringing illumination to the field of AD intervention. The new Chinese nutrigenomic research may be a tipping point, the beginning of an abundance of studies that enable nutritional supplement researchers to make great leaps forward. It may make it possible to reduce the amount of supplementary nutrients taken by creating more precision in dosing.


  1. Song N, Yang H, Pang W, Qie Z, Lu H, Tan L, Li H, Sun S, Lian F, Qin C, Jiang Y. Mulberry extracts alleviate aβ 25-35-induced injury and change the gene expression profile in PC12 cells. Evid Based Complement Alternat Med. 2014;2014:150617. doi: 10.1155 / 2014 / 150617.
  2. Song N, Pang W, Yang H, Tan L, Fu J, Li H, Jiang Y. [Primary study on protective effect of mulberry extracts on Abeta25-35-induced PC12 cells injury]. Wei Sheng Yan Jiu. 2012 Nov;41(6):925 – 9.

Will Block is the publisher and editorial director of Life Enhancement magazine.

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