Gavage of D-Ribose induces Aβ-like deposits, Tau hyperphosphorylation as well as memory loss and anxiety-like behavior in mice.

In addition to D-Glucose, D-Ribose is also abnormally elevated in the urine of type 2 diabetic patients, establishing a positive correlation between the concentration of uric D-Ribose and the severity of diabetes. Intraperitoneal injection of D-Ribose causes memory loss and brain inflammation in mice. To simulate a chronic progression of age-related cognitive impairment, we orally administered D-Ribose by gavage at both a low and high dose to 8 week-old male C57BL/6J mice daily for a total of 6 months, followed by behavioral, histological and biochemical analysis. We found that long-term oral administration of D-Ribose impairs spatial learning and memory, accompanied by anxiety-like behavior. Tau was hyperphosphorylated at AT8, S396, S214 and T181 in the brain. Aβ-like deposition was also found in the hippocampus for the high dose group. D-Glucose-gavaged mice did not show significant memory loss and anxiety-like behavior under the same experimental conditions. These results demonstrate that a long-term oral administration of D-Ribose not only induces memory loss with anxiety-like behavior, but also elevates Aβ-like deposition and Tau hyperphosphorylation, presenting D-Ribose-gavaged mouse as a model for age-related cognitive impairment and diabetic encephalopathy.

metabolism [11]. (3) D-Ribose and ribosylated proteins are more cytotoxic than D-Glucose and its glycated products both in vivo and in vitro [12]; (4) Alzheimer's disease is regarded as the 'type 3 diabetes' which is correlated with imbalance of energetic metabolism [13]; (5) oral administration of D-Ribose induces memory loss and anxiety-like behavior, similar to the symptoms of AD patients; (6) D-Ribose-gavaged mice display AD-like symptoms without abnormal phenotypes of altered fasting blood D-Glucose levels, glycation (AGEs), increased body weight, reduced muscle strength, and impaired motor ability and co-ordination; (7) according to the definition of AD which is an age-related acquired disease except for very rare incidence of the genetic frontal-temporal dementia [14], risk factors in vivo and in vitro are important in the progression of AD, for instance an imbalance in energy and substance metabolism [15]; and finally, (8) oral administration of D-Ribose to mice, rats and other mammals is very conveniently performed with a highly reproducible phenotype.

Gavage procedure
The gavage procedure was performed by professional workers in the animal house of Institute of Biophysics, Chinese Academy of Sciences. Grab the skin over the mouse shoulder firmly with the thumb and middle fingers, stretch the head and neck with the index finger to make the esophagus straight. Direct the ball-tip of the feeding needle along the roof of the mouth and toward the right side of the back of the pharynx, then gently pass down into the esophagus and inject the solution. No resistance should be felt. When being sacrificed, mice were first anesthetized with 10% chloral hydrate and then perfused through heart with at least 50 ml saline per one mouse, for eliminating blood from organs. After that, the organs were fetched out. All processes were carried out according to the Animal Welfare and Research Ethics Committee of the Institute of Biophysics, Chinese Academy of Sciences (Permit number: SYXK2013-77).

Gel electrophoresis and Western blotting/dot blotting
Levels of AGEs in hippocampus, cortex, liver, kidney and serum were determined by Western blotting. The same method was used to analyze the expression of phosphorylated T181 For dot blotting, 5 µl of each tissue lysate was spotted to the NC membrane. After the membrane was air-dried for about 10 minutes, it was incubated with 5% skimmed milk, followed by the same procedure as Western blotting.

Immunohistochemicaly analysis
Immunohistochemical staining for pT181, pS214, pS396, AT8 and Aβ 1-42 was performed as described. Mice brains were immersed in 4% paraformaldehyde for 48 h immediately after they were dissected. After fixation, brains were embedded in paraffin blocks. Five-m thick sections were processed for immunohistochemical analyses. Deparaffinized and rehydrated sections were incubated in Target Retrieval Solution at 95C for 30 min for enhancement of immunoreactivity and then permeabilized with 0.3% H 2 O 2 in absolute methanol for 10 min to block endogenous peroxidase, followed by incubation in 10% normal goat serum in PBS at room temperature for 30 min. The specimens were incubated overnight at 4C with anti-pT181, anti-pS214 anti-pS396, AT8 or anti-Aβ1-42 antibody (Abcam, UK) solution diluted in PBS. After washing with PBS, sections were incubated with biotin-labeled secondary antibodies (37C, 1 h).
The immunoreaction was detected using horseradish peroxidase-labeled antibodies (37C, 1 h) and red staining was visualized with an AEC system (Zhongshan Goldenbridge Biotechnology, China). Slides were lightly counterstained with Harris' haematoxylin and mounted with GVA aqueous mounting medium (Zhongshan Goldenbridge Biotechnology, China) for observation by light microscopy (Nikon Optical, Japan).

Detection of brain D-Ribose and D-Glucose in brain by UV-HPLC
D-Ribose and D-Glucose were measured as described previously [16]. The brain was quickly dissected out and 0.1g brain was homogenized in 1 ml lysis buffer immediately. Then, 3 ml acetonitrile was added and vortexed vigorously for 30 s before centrifugation (12, Samples were vortexed vigorously for 30 s before centrifugation (12,000 rpm, 4°C, and 10 min) and then heated in a 70°C water bath for 90 min, followed by additional centrifugation (12,000 rpm, 4°C, and 10 min). The mixture was acidified by adding 150 μl of an aqueous 2 M HCl solution to precipitate the excess MOPBA. The mixture was vigorously vortexed and then centrifuged (12,000 rpm, 4°C, and 10 min), and then filtrated (0.22 μm). 20 μl of the solution was then subjected to high-performance liquid chromatography (HPLC).
The HPLC system (LC-20A, Shimadzu, Japan) was equipped with an ultraviolet detector. The MOPBA-sugar derivative was collected from the C18 column with a binary mobile phase gradient. Mobile phase A was 10 mM of sodium 1-hexanesulfonate; the pH was stabilized at 2.5 by phosphoric acid. Mobile phase B was 50% acetonitrile solution. The elution conditions were 38%-60% B for 15 min, 100% B for 5 min and 38% for 5 min. The flow rate was 1 ml/min and the column temperature was 40°C. The procedure for D-Glucose analysis was the same procedure for detecting D-Ribose, except in the latter procedure, elution conditions were 42%-60% B for 15 min, 100% B for 5 min and 42% for 5 min, and 20 μl of the solution was injected into the analytical column. The reference concentrations of D-Ribose and D-Glucose were determined according to the standard curve.