Nanocatalytic activity of clean-surfaced, faceted nanocrystalline gold enhances remyelination in animal models of multiple sclerosis

Development of pharmacotherapies that promote remyelination is a high priority for multiple sclerosis (MS), due to their potential for neuroprotection and restoration of function through repair of demyelinated lesions. A novel preparation of clean-surfaced, faceted gold nanocrystals demonstrated robust remyelinating activity in response to demyelinating agents in both chronic cuprizone and acute lysolecithin rodent animal models. Furthermore, oral delivery of gold nanocrystals improved motor functions of cuprizone-treated mice in both open field and kinematic gait studies. Gold nanocrystal treatment of oligodendrocyte precursor cells in culture resulted in oligodendrocyte maturation and expression of myelin differentiation markers. Additional in vitro data demonstrated that these gold nanocrystals act via a novel energy metabolism pathway involving the enhancement of key indicators of aerobic glycolysis. In response to gold nanocrystals, co-cultured central nervous system cells exhibited elevated levels of the redox coenzyme nicotine adenine dinucleotide (NAD+), elevated total intracellular ATP levels, and elevated extracellular lactate levels, along with upregulation of myelin-synthesis related genes, collectively resulting in functional myelin generation. Based on these preclinical studies, clean-surfaced, faceted gold nanocrystals represent a novel remyelinating therapeutic for multiple sclerosis.

. CNM-Au8 nanocrystal characterization Table S2. Functional improvements in each open field parameter toward Sham controls by CNM-Au8 treated groups compared to vehicle controls. Table S3. RNASeq DE genes identified overrepresenting the pathways shown in Fig. 7c

Cuprizone experiments
Cuprizone experiments described in the Supplemental section were performed as described in the main paper Materials and Methods, with the following changes: For the pilot cuprizone experiment, sixteen mice were divided into four groups with N=4 per group. Group 1 served as a negative control with vehicle provided ad libitum in drinking water and normal chow. Group 2 was fed 0.2% cuprizone chow with vehicle provided ad libitum in drinking water. Group 3 received CNM-Au8 (50 µg/mL) in their drinking water, and normal chow. Group 4 received CNM-Au8 (50µg/mL) in their drinking water, and 0.2% cuprizone chow. After five weeks of treatment, animals were prepared for TEM imaging as described. The average intake of CNM-Au8 in the treatment groups was measured on a daily, per cage basis.
From this data, a daily dose of 10 mg CNM-Au8/kg/day was calculated to be a sufficient dose for detectable effects on myelin.
For the early sacrifice cuprizone experiment, four groups of mice were treated as follows: Group 1 (N=4) was given normal chow and vehicle (10mL/kg) by gavage; Group 2 (N=6) was given 0.2% cuprizone chow and vehicle by gavage; Group 3 (N=6) was given 0.2% cuprizone chow and CNM-Au8 by gavage (10 mg/kg); Group 4 (N=6) was given 0.2% cuprizone chow on Day 0, and CNM-Au8 by gavage (10 mg/kg) starting seven days prior to Day 0 when all other groups were started on both cuprizone and CNM-Au8 or vehicle, and continuing through to Day 14. All animals were sacrificed on Day 14 and coronal brain slices were prepared for immunohistochemical staining and quantitation as described in Materials and Methods.

PLP staining and quantitation
7 µm thick serial coronal brain sections between bregma-0.82 mm and bregma-1.82 mm were prepared and analysed. Using previously described methods 3 , paraffin-embedded sections were de-waxed, rehydrated, while housed in a glass container partially filled with 10mM citrate buffer (pH 6.0), and then microwave-heated in a conventional 1.65 KW microwave until the buffer began to boil. Brain sections were then quenched with 0.3% H2O2, blocked for 1 hr in PBS containing 3% normal horse serum and 0.1% Triton X-100. Sections were then incubated at 4°C overnight with anti-PLP antibodies (AbD Serotec) at a dilution factor of 1:500. After washing with PBS buffer (pH 7.4), coronal brain sections were further incubated with biotinylated antimouse IgG secondary antibody (Vector Laboratories) for 1 hr, followed by exposure to peroxidase-coupled avidin-biotin complex (ABC Kit, Vector Laboratories) for 30min and developed with diamino-3,3'benzidine reagent according to the manufacturer's instructions (Vector Laboratories). To determine the total cross-sectional area of all brain matter present on each slide, sections were also stained with hematoxylin to label all brain tissue.
Specifically, to quantify the amount of immunopositive PLP in the coronal portion of each mouse brain, coronal sections (i.e., between bregma-0.82mm and 1.82mm) were scanned using a specially adapted Cannon Scanner (output resolution of 2400 dpi). Each pixel intensity in the scan was assigned a numeric intensity value by Photoshop, with "255" corresponding to the least intensity and "1" corresponding to the highest. The data were then exported to Excel.
Intensity values were analysed and a quantitative weighted average for intensity (corrected for background by haematoxylin stained area) in each myelin-stained coronal slide was determined. The copper-cuprizone complex has been previously characterized and is known to have an absorbance peak at 595 nm. An Agilent 8453 spectrophotometer was used to monitor the binding of copper to cuprizone. All samples were blanked with Tris buffer. For saturation curve experiments, final molarity of cuprizone was 8.8 x 10 -5 M, and copper concentration was 2.2 x 5 curves were produced in the presence of CNM-Au8 (26 μg/mL). After adding copper reagents, the samples were incubated for 5 minutes to stabilize the Cu-CPZ complex, and the full spectrum for each sample was recorded.

Rat spinal cord culture and immunohistochemistry
A standard laboratory protocol was followed; in brief, postnatal rat spinal cord cells were dissociated and plated on PLL coated coverslips. Cells were allowed to adhere for at least 24 hours in base medium and then switched to vehicle or CNM-Au8 containing medium (final concentration of 0.01 ug/mL or 10 ug/mL), grown for 72 hours, fixed, and labelled with antibodies as follows: anti-Olig2, anti-MBP, anti-GFAP, anti-CC1. Fig. S1: TEM image of a representative 13 nm pentagonal bipyramidal gold nanocrystal