Gait control represents a non-cognitive domain that is affected negatively during late stages of Alzheimer’s Disease (AD) and AD Related Dementias (ADRDs). In some cases, early identification can be predictive of worsened cognitive function. Prompt detection could help define intervention windows, perhaps even years prior to formal diagnosis of dementia. In this study, the locomotor function of 3-month-old male 5xFAD mice was evaluated using several variables (speed, deviance from center, paw precision index, stride length, stride length deviation, and stride time) to characterize the change in ambulatory behavior prior to the onset of cognitive decline. The data presented here show no significant change in measures of gait behaviors between the 5XFAD mice and the controls. Gait behavior measures obtained were selected to parallel those used in the clinic (i.e. GAITRite®). Future studies will evaluate the dysregulation of locomotor function longitudinally in the same mouse model at 6, 9, and 12 months of age

Case, Sami L.

Galopin, Leopoldine B

Lin, Ruei-Lung

Pauss, Kate E.

Sims, Sophiya L

Thibault, Olivier

Wright, Nicholas A

English

University of Kentucky

1   Abstract— Gait control represents a non-cognitive domain that is affected negatively during late stages of Alzheimer’s Disease (AD) and AD-Related Dementias (ADRDs).  In some cases, early identification can be predictive of worsened cognitive function.  Prompt detection could help define intervention windows, perhaps even years prior to formal diagnosis of dementia. In this study, the locomotor function of 3-month-old male 5xFAD mice was evaluated using several variables (speed, deviance from center, paw precision index, stride length, stride length deviation, and stride time) to characterize the change in ambulatory behavior prior to the onset of cognitive decline.  The data presented here show no significant change in measures of gait behaviors between the 5XFAD mice and the controls. Gait behavior measures obtained were selected to parallel those used in the clinic (i.e. GAITRite®). Future studies will evaluate the dysregulation of locomotor function longitudinally in the same mouse model at 6, 9, and 12 months of age.  Keywords—gait, aging, AD, locomotor function  I. INTRODUCTION GAIT control represents a non-cognitive domain that is affected negatively during late stages of Alzheimer’s Disease (AD) and AD-Related Dementias (ADRD).   In recent years, it has become evident that motor dysfunction is a predictor of AD development (Beauchet et al., 2016; Buchman & Bennett, 2011). Mobility impairments and muscle weakness, along with altered gait increase the risk of falls and injuries, which, in turn, can lead to institutionalization, permanent loss of function, and decreased quality of life (Aryee et al., 2017; Beauchet et al., 2013; Kusljic et al., 2018). Addressing this decline is essential for not only predicting risk of dementia, but also protecting older adults from injury.  Motor impairment and alterations in gait have been studied in murine models of amyloidosis and aging.  C57BL/6J mice, a strain commonly employed in aging studies, show a clear decline in ambulatory ability during aging.  For example, aged mice demonstrate an increase in the number of foot faults while performing a Rotor-rod test, tend to travel shorter distances at lower velocities, and show larger deviances in stride time deviation while ambulating (Barreto et al., 2010; Case et al., 2023; Mock et al., 2018; Shoji & Miyakawa, 2019). Using video analysis, a longitudinal study conducted by Broom and colleagues corroborates these results and emphasize sex differences in the timing of motor decline (2021), showing that certain characteristics of gait (e.g. swing time dissociation, stance time, and cadence) decline first in females. This finding is surprising, given that this sex difference is not found in the clinic, where females show maintained gait control in older ages (Aryee et al., 2017; Kusljic et al., 2018).  There are several models of mouse used to study amyloidosis, among which include the amyloid precursor protein (APP)/presenilin-1 (PS-1) double knock-in mouse.  This mouse is a humanized model that displays targeted insertions of both APP and PS-1 genes, which are associated with familiar AD (fAD) mutations (Webster et al., 2013).  Importantly, transgenic APP/PS-1 mice on a senescence-accelerated background have been shown to perform as well as age-matched controls on the Rotor-Rod, open field performance, and beam walks until 9 months of age (Lok et al., 2013).  Likewise, O’Leary et al. (2018) report a decrease in locomotor activity is only seen in older (12-month-old) APP/PS-1mouse mice when compared to age-matched controls, as inferred from Morris water maze data.    The transgenic 5xFAD mouse, another model of amyloidosis, exhibits gait dysregulation and balance impairment that increase with age (Sukoff Rizzo et al., 2018).  12-month-old 5xFAD female mice show marked decreases in motor performance when compared to age-matched controls during open field tests (O'Leary, Robertson, et al., 2018). Using the same methods and model, O’Leary et al. indicate motor deficits as early as 9 months of age in a later report (O'Leary et al., 2020).  However, in another study also using open field tests, only 18-month-old 5xFAD mice of either sex demonstrate deficits in both velocity and distance travelled when compared to aged-matched controls (Forner et al., 2021). In addition, work from the Wirths lab provides evidence that Rotor-Rod data show a marked increase in latency in the 4-month-old 5xFAD mouse, which appears to be more pronounced in females than in males (Jawhar et al., 2012).  It is also reported in this study that 12-month-old 5xFAD females tested with balance beam, string suspension, and clasping tests, show significant motor function dysregulation when compared to the aged-matched wild type mice.  Despite these age-, genotype-, and sex-dependent deficits, open field tests show that aged Characterizing Gait in Young Male 5xFAD Mice Sophiya L. Sims1, Kate E. Pauss1, Sami L. Case1,2, Ruei-Lung Lin1, Leopoldine B. Galopin1, Nicholas A. Wright1, Olivier Thibault1 1Published by UKnowledge, 20232  5xFAD females travel as far the age-matched controls (Jawhar et al., 2012).  Clearly, the lack of consensus regarding the exact timeline of motor dysfunction within the 5xFAD strain needs to be further addressed.  Indirect measures of motor function, such as those derived from Morris water maze, suspension tasks, beam tasks, and open field tests, are intrinsically tied to measures of strength, balance, coordination, and anxiety, which could introduce confounds into studies of gait regulation.  Additionally, the idea of sex as a major variable in gait dysregulation is emerging and needs to be further evaluated.  Compared to the wealth of data regarding gait across several pathological conditions (e.g. traumatic brain injury (Reviewed in Dever et al., 2022), Parkinson's disease and Huntington’s disease (Amende et al., 2005), spinal cord injury (Beare et al., 2009), etc.), there are relatively few studies that evaluate motor function longitudinally in mice with AD-related pathologies despite the availability of simple and cost-effective methods to measure gait variables (Barneoud et al., 1997; Broom et al., 2021; Henry et al., 2020; Palliyaguru et al., 2021).    Here, in this study, we present ambulatory data from male 3-month-old 5xFAD and aged-matched control mice to contribute to the current body of knowledge regarding the characterization of gait behavior in this model of cognitive decline. Three-month-old mice were used because this timepoint is thought to precede the onset of cognitive decline (Jawhar et al., 2012).  Future reports will follow up on the same mice at 6-, 9-, and 12-months of age to evaluate longitudinal motor decline in the same mice with the same measures. The timeline of gait dysregulation is currently unclear and yields an area that requires additional focus, especially considering the rising clinical significance of locomotor behavior in the aging population.  II. METHODS Locomotor stability was evaluated in 3-month-old IR double-floxed 5xFAD (C57BL6J; Insrtm1khn/tm1khn X (C57BL6J;Tg(APPSwFlLon,PSEN1*M146L*L286V)6799 Vas)) male mice (n = 5) and control mice (IR double-floxed (C57BL6J), null for 5xFAD alleles) (n = 5), as previously described (Case et al., 2023; Lin et al., 2022).  Each subject was recorded (HD camera, 1280x720, 50 Hz frame rate) ambulating across a 3-plane walking corridor built in-house (Figure 1). The resulting videos were analyzed in ImageJ (v1.52S) using standard pixels measures along the x‐ and y‐axes.  Animals that stopped midway or did not cross the corridor continuously were returned to the beginning of the corridor and tested again until at least five sequential steps were recorded.  Stride length deviation was calculated using the standard deviation of the stride length between step-to-step placements for all paws. Average stride length was calculated using the average distance a paw traveled between step-to-step placements. Stride time deviation was calculated using the standard deviation of time between step-to-step placement for all paws. The paw precision index was calculated using the average of r2 values from each paw. Lastly, deviance from center was calculated using the sum of each paw’s distance from the corridor center per step. Speed was determined by evaluating how quickly the subject completed an entire cycle of 5 steps.  All animals used in this study follow our Institutional Animal Care and Use Committee protocol.     Fig. 1: Three-plane visualization task built in-house to assess gait behavior.  Bottom view of the corridor is shown with an ambulating mouse (Left panel) where mirrors are used to reflect the side views for improved data extraction on time and position of paw placement (red circles).  The top view of the corridor is shown (Right panel); the blue panels show the back side of the 45° mirrors angled towards the camera below. III. RESULTS Analysis of average stride length measures showed no significant differences between 5xFAD and control animals (Fig. 2a; p = 0.977, student’s t-test).  We measured stride length deviation to determine if there was any change between the two groups; again, no significant differences were found (Fig. 2b; p = 0.665, student’s t-test).  We then evaluated the measures of stride time deviation (Fig. 2c; p = 0.519, student’s t-test), paw precision index (Fig. 2d; p = 0.681, student’s t-test), and speed (Fig. 2e; p = 0.657, student’s t-test) and none show significant differences between two groups.  Finally, we measured deviance from center, and did not identify any differences between the 5xFAD and control mice (Fig. 2f; p = 0.4553, student’s t-test).    2https://uknowledge.uky.edu/jpns/vol1/iss2/13   Figure 2: Gait parameters extracted from ambulating mice.  Average stride length (A), stride length deviance (B), stride time deviance (C), paw precision index (D), average speed (E), and deviance from center (F) were all evaluated using unpaired T-tests and show no significant differences.  Error bars represent standard error of the mean.  At least 5 steps from each recording were used to calculate all measures. IV. DISCUSSION As expected, we did not find significant differences between 5xFAD and control mice during the ambulation task when tested on 3 months old animals. These results help establish baseline conditions for measures of gait dysregulation in an animal model of amyloidosis and will be compared to future investigations at more advanced ages when more severe pathological markers are present (e.g., increased amyloid plaque deposition, neuronal loss, gliosis, synaptic loss, etc.).    We use these results to validate that the outcome measures presented here align with prior work in several animal models of AD showing little if any change in gait parameters at 3-months of age. Indeed, the 3xTG, 2xTG, APPS/PS1, and the 5xFAD models, show no overt ambulatory impairment at this age.  The methods used here report on direct measures of gait, which align with those developed in the clinic and reflect on measures of stride-time and stride-length deviance, speed, balance, and posture (Beauchet et al., 2013; Linortner et al., 2014).    Since early detection and subsequent therapeutic intervention may help rescue symptoms of dysregulated gait, it is essential to unravel the pre-clinical motor changes that accompany AD early on (Buchman & Bennett, 2011). Future data will elucidate between-group and individual longitudinal trends to determine the degree and severity of gait dysregulation across lifespan in the 5xFAD model. Given the greater number of aged male mice exhibiting gait impairments when compared to their female counterparts (Case et al., 2023; Oblak et al., 2021), it is also integral for future studies to include both sexes.  Resolving the chronology of the onset of motor dysregulation in this animal model of amyloidosis will help frame the relationship between two separate modalities that occur across different brain regions, and may highlight potentially shared, underlying mechanisms. ACKNOWLEDGMENT This work was supported by the National Institutes of Health (P01AG078116 to OT) and (R01AG033649-S1). 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Characterizing Gait in Young Male 5xFAD Mice

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Characterizing Gait in Young Male 5xFAD Mice

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