Parkinson-like phenotype in insulin-resistant PED/PEA-15 transgenic mice

Neurological abnormalities, such as Parkinson-like disorders (PlD), are often co-morbidities of Type 2 Diabetic (T2D) patients, although the epidemiological link between these two disorders remains controversial. The PED/PEA-15 protein represents a possible candidate linking T2D and PD, because it is increased in subjects with T2D and is highly expressed in the brain. To test this hypothesis, we have analyzed the neurological and neurochemical phenotype of transgenic mice overexpressing PED/PEA-15 (tgPED). These mice develop impaired glucose tolerance and insulin resistance, accompanied by neurological features resembling PlD: feet clasping, slow and delayed locomotor movements in different behavioral tests in absence of clear cognitive deficits, ataxia or anxiety. Morphological analysis of the brains showed selective modifications of metabolic activity in the striatal region. In the same region, we have observed 26% decrease of dopamine fibers, confirmed by immunohistochemistry and Western Blot for tyrosine hydroxylase. Moreover, they also showed 48% reduction of dopamine levels in the striatum. Thus the tgPED mice may represent a genetic animal model of neurological disease linked to T2D.

Burrowing test. The burrowing test has been adapted from a previously published apparatus (Deacon, R. Assessing Burrowing, Nest Construction, and Hoarding in Mice. J. Vis. Exp.(59), e2607, doi:10.3791/2607(2012). Briefly, an opaque plastic burrow (diameter: 7cm), filled with 66g of food pellet normally supplied as diet wasplaced overnight in the home cage of individually house mice. The amount of pellet displaced was measured by weighing the burrow before and after the test.
Beam walking. Each animal was placed on a 10x10 cm starting platform brightly illuminated, connected to the home cage through a horizontal wooden beam (45cm long, 1.6cm wide). The task was repeated three times and the number of falls (errors) through the beam was counted.  Behav Brain Res. 2008 187:449-54), with minor modifications. Briefly, mice were allowed to explore the square corridor resulting between two squared boxes placed one inside the other (the total length of the corridor was 80 cm, all the walls were made of transparent plexiglas, the floor consisted of a removable white PVC sheet). Mice were allowed to explore the maze for 5 minutes, the behavior was videotaped and the traveled distance and number of rearings were scored off-line.

Delay-dependent one-trial object recognition (ORT).
To test episodic memory, mice have been tested with a one-trial object recognition task. To this aim, after exposure to the arena, mice have been exposed in the same environment to a pair of identical objects (sample trial). After a delay of 24 hours animals have been released again into the open-field, now containing two objects, a familiar one, known from the sample trial, and a novel object. For each mouse, the time spent exploring the objects (in seconds) during the test trial has been scored. Data are presented as recognition index, that is the time spent investigating the novel object relative to the total object investigation.
Barnes circular maze task. To test spatial memory the Barnes' maze has been used, which is a dry version of the Morris water test. This test has several advantages such as to minimize thigmotactic behaviour (no walls are present) and to allow animals to use their normal locomotion (walking, whereas in Morris' maze they are required to swim). Mice have been placed in the middle of a circular platform (diam=1.22m.; elevation from the floor= 40 cm), with 36 equally spaced holes (each 5cm diameter) around the periphery (5 cm from the perimeter). Only one hole led to a dark escape box (5 cm x 5 cm x 11 cm) fixed in relation to the distal environmental cues. The platform surface was brightly illuminated from above as motivation to escape in the dark box. The test lasted 5 min and was repeated for 4 days once a day. The amount of time that the mice took to enter the escape hole (escape latency) and the escape path was recorded.
Spontaneous alternation. Spontaneous alternation is the innate tendency of rodents to alternate free choices in a T-maze over a series of successive runs. The T-maze was made of Plexiglas with a main stem (70cm long x 10 cm wide x 20 cm high) and two arms (30 cm long x 10 cm wide x 20 cm high) positioned at 90° angle relative to the main stem. A start box (15 cm long x 10 cm wide) was separated from the main stem by a sliding door. Cues placed above the arms served as spatial reference points. Mice were free to explore the maze for 9 consecutive trials. At the beginning of a trial, the mouse was placed in the start box for 30sec followed by the opening of the door to the stem. When the mouse entered one of the arms, the door of that arm was then closed. The chosen arm and the elapsed time between the opening of the start box and choice of an arm (choice latency) were recorded. After 30sec confinement in the chosen arm, the mouse was removed and returned to the start box for the next trial. The percentage of alternation over the nine trials was determined for each mouse and used as an index of working memory/attention performance. This percentage was defined as entry in a different arm of the T-maze over successive trials (i.e. left-right-left-right-etc.) Supplementary figures: Figure S1: Explicit memory testing in the Barnes' maze. Animals (n=7 per group) have been tested on the Barnes' circular maze for four days as indicated in Supplementary methods, and the time to reach the target hole recorded. Data represent mean ±SEM.