Clinicopathologic Dissociation: Robust Lafora Body Accumulation in Malin KO Mice Without Observable Changes in Home-Cage Behavior

Abstract

Lafora disease (LD) is a syndrome of progressive myoclonic epilepsy and cumulative neurocognitive deterioration caused by recessively inherited genetic lesions of EPM2A (laforin) or NHLRC1 (malin). Neuropsychiatric symptomatology in LD is thought to be directly downstream of neuronal and astrocytic polyglucosan aggregates, termed Lafora bodies (LBs), which faithfully accumulate in an age-dependent manner in all mouse models of LD. In this study, we applied home-cage monitoring to examine the extent of neurobehavioral deterioration in a model of malin-deficient LD as a means to identify robust preclinical endpoints that may guide the selection of novel genetic treatments. At 6 weeks, ∼6-7 months, and ∼12 months of age, malin-deficient mice ("KO") and wild-type (WT) littermates underwent a standardized home-cage behavioral assessment designed to non-obtrusively appraise features of rest/arousal, consumptive behaviors, risk aversion, and voluntary wheel-running. At all timepoints, and over a range of metrics that we report transparently, WT and KO mice were essentially indistinguishable. In contrast, within WT mice compared across the same timepoints, we identified age-related nocturnal hypoactivity, diminished sucrose preference, and reduced wheel-running. Neuropathological examinations in subsets of the same mice revealed expected age-dependent LB accumulation, gliosis, and microglial activation in cortical and subcortical brain regions. At 12 months of age, despite the burden of neocortical LBs, we did not identify spontaneous seizures during an electroencephalographic (EEG) survey, and KO and WT mice exhibited similar spectral EEG features. However, in an in vitro assay of neocortical function, paroxysmal bursts of network activity (UP states) in KO slices were more prolonged at 3 and 6 months of age, but similar to WT at 12 months. KO mice displayed a distinct response to pentylenetetrazole, with a greater incidence of clonic seizures and a more pronounced postictal suppression of movement, feeding, and drinking behavior. Together, these results highlight the clinicopathologic dissociation in a mouse model of LD, where the accrual of LBs may latently modify cortical circuit function and seizure threshold without clinically meaningful changes in home-cage behavior. Our findings allude to a delay between LB accumulation and neurobehavioral decline in LD: one that may provide a window for treatment, and whose precise duration may be difficult to ascertain within the typical lifespan of a laboratory mouse.

Keywords: Lafora body disease; astrogliosis; glycogen storage; home‐cage behavior; malin; polyglucosan.

Figure 1.. Histopathological Changes in Malin KO Mice.

(a) Between 6 weeks and 1 year of age, Malin KO mice display a progressive increase in the burden of Lafora bodies (LB, which accumulate GYS1 or glycogen synthase-1) and astrogliosis (GFAP staining). (b) Microglial activation (assessed through IBA1 staining). (c) At 1 year of age, these changes are prominently observed in the hippocampal dentate gyrus (left) and the cerebellar granule cell layer (right). Top panels show PAS-D staining patterns. Scale bar: 50 μm. Images are representative of 2–6 mice per genotype, per age.

Figure 2.. Behavioral Responses to a Novel Home-cage.

(a) Cartoon schematic of home-cage, featuring two lickometered water spouts (water vs 0.8% sucrose-water), a food hopper and an infrared-lucent shelter. RIGHT: representative aerial view from infrared camera with centerpoint tracking. (b) Schematic of the modular design applied for all home-cage assessments, designed to capture a mouse’s initial response to cage novelty (“Intro”), patterns of unperturbed spontaneous behavior (“baseline” recordings), and responses to various provocative maneuvers (see Figure 4). (c) 1-year-old WT and KO mice were similar in body weight (p=0.2), and featured similar patterns of cage exploration. Heatmaps (left) and trackmaps (right) are shown for a representative WT and KO mouse. TOP: Raster plot of distances traversed every minute of trial for every mouse. BOTTOM: Total measures of horizontal displacement (p=0.2), sheltering (p=0.7), water licks (p=0.9), sucrose licks (p=0.8) and feeding entries (p=0.6) were not significantly different between WT and KO mice. (d) Body weights (p=0.8), distances and sheltering curves for 6-week-old mice. (e) Body weights (p=0.9), distances and sheltering curves for 6-month-old mice. Mean ± s.e.m shown for all.

Figure 3.. Baseline Recordings.

(a) On the second 23h-long baseline recording, 1-year-old WT and KO mice displayed similar patterns of hourly horizontal distances (hour x genotype, F_(22,968) = 0.7, p=0.7), total distances (p=0.3) and ultradian oscillations in activity. Raster plot depicts distances moved every minute of the day. (b) 1-year-old WT and KO mice were indistinguishable in measures of total daily “sleep” (p=0.9) and the number of “sleep” bouts per day (p=0.4), without a change in the distributions of “sleep” bout duration and timing. (c) Averaged time budgets for WT and KO mice. (d) WT and KO mice displayed similar sucrose preference (p=0.3), total water licks (p=0.8) and sucrose licks (p=0.5). The overall macrostructure of licking was preserved, with WT and KO mice displaying an identical distribution of lick durations and interlick bouts. (e) At the same age, hourly changes in feeding entries were comparable (hour x genotype, F_(22,968) = 0.7, p=0.8), as were total daily feeding durations (p=0.5). (f) Raster plots of horizontal activity in WT and KO mice at 6 weeks of age, time budgets and total daily distances (p=0.9), feeding durations (p=0.2), “sleep” (p=0.4), sucrose preference (p=0.4) and total daily licks (p=0.5). (g)) Raster plots of horizontal activity in WT and KO mice at 6 months of age, time budgets and total daily distances (p=0.2), feeding durations (p=0.5), “sleep” (p=0.3), sucrose preference (p=0.4) and total daily licks (p=0.1). Mean ± s.e.m shown for all. See Fig.1A for sample sizes. Room lights are off between 1700 and 0500.

Figure 4.. Home-cage provocative maneuvers.

(a) 1-year-old WT and KO mice displayed comparable locomotor suppression and shelter engagement in response to an hour-long light spot stimulation. (b) In response to a single 60s long monotone stimulus (“beep”), KO mice displayed a blunted response, featuring lower initial peak velocities (startle) and less emphatic shelter engagement. (c) Light spot and beep responses at 6 weeks of age. (d) Light spot and beep responses at 6 months of age. (e) When presented with a running wheel, 1-year-old WT and KO mice displayed patterns of running wheel engagement (hour x genotype, F_(22,950) = 1.02, p=0.4) and overall wheel running (p=0.5). Similar results were seen in 6-week old cohorts (hour x genotype, F_(22,700) = 0.5, p=0.9, total wheel rotations, p = 0.7). 6-month-old KO mice did display fewer wheel rotations overall (hour x genotype, F_(22,960) = 1.8, p<0.05). (f) In all age groups, WT and KO mice responded comparably to a 2h-long daytime cage-swap maneuver. Mean ± s.e.m shown for all, with * depicting p<0.05. Room lights are off between 1700 and 0500.

Figure 5.. Age-dependent changes in home-cage behavior.

(a) On the second baseline day 2, hourly rates of total distances moved (hour x genotype, F_(44,1229) = 2.9, p<0.0001), sheltering (hour x genotype, F_(44,1229) = 2.8, p<0.0001) and feeding duration (hour x genotype, F_(44,1229) = 1.6, p<0.01). Body weights (F_(2,56) = 62.2, p<0.001), sucrose preference (F_(2,56) = 2.9, p<0.05) and feeding durations (F_(2,56) = 10.55, p<0.001) varied by age, while total daily distances (F_(2,56) = 2.5, p=0.08), sheltering (F_(2,56) = 0.4, p=0.6) and feeding entries (F_(2,56) = 0.18, p=0.3) were not significantly altered. (b) Averaged time budgets in WT mice across age groups with sample sizes shown. Total daily “sleep” (F_(2,56) = 0.93, p=0.4) and “sleep” bouts/day (F_(2,56) = 1.12, p=0.3) remained stable with age, without a change in the distributions of “sleep” bout duration and timing. (c) Responses to light spot stimulation across 6 weeks, 6 months and 1-year cohorts. (d) Responses to beep stimulation across 6 weeks, 6 months and 1-year cohorts. (e) Hourly rates of voluntary wheel running varied by age (F_(44,1120) = 4.5, p<0.0001), as did total wheel rotations (F_(2,56)=9.1, p<0.001). *, **, ***, **** refer to post-hoc comparisons and depict p<0.05, <0.01, <0.001 or <0.0001 respectively. Room lights are off between 1700 and 0500.

Figure 6.. EEG and PTZ Responses.

(a) Representative single-channel wireless electrocorticography from 1-year old WT and KO mice. (b) EEG power spectra calculated during wakefulness. (c) Representative EEG responses to a single intraperitoneal injection of PTZ (60mg/kg), demonstrating a prolonged epoch of spike/wave discharges, followed by a discrete epoch of evolving rhythmicity. Red bars annotate epochs of absent EEG signal while the mouse receives the intraperitoneal injection. (d) Distance and sheltering responses to a single subconvulsant PTZ injection (30mg/kg, provided at approximately 12 noon) measured within home-cages, with a manual tally of convulsive events (inset) for both WT (n=14) and KO (n = 18). (e) Home-cage metrics during the post-ictal period, defined as 1600 to 1100 the following day. WT and KO distances measured during the baseline trial are shown in the background. After PTZ injections, WT and KO mice displayed a similar decline in locomotor activity (F_(18,522) = 1.18, p=0.3). Total sheltering times were higher in KO mice, but this effect did not vary with time (F_(18,522) = 0.97, p=0.5). Malin KO mice also displayed fewer feeding entries in the post-ictal period (F_(18,522)= 1.95, p<0.05). Mean ± s.e.m shown for all. * depicts p<0.05.

Figure 7:. UP States in WT vs KO Somatosensory Cortex.

A: Example traces of extracellular recordings in brain slices exhibiting spontaneously occurring activity bursts (from 12-month-old mice). B: The average duration of activity bursts in MKO slices is longer at 3 and 6 months of age, but not at 1 year (Mann-Whitney test, *p< 0.05) C: UP State burst amplitudes and frequencies. D: Relative power over all activity. Mean ± s.e.m shown for all. Sample sizes at 3 months: WT (9 slices, 4 mice), KO (13 slices, 5 mice). 6 months: WT (19 slices, 6 mice), KO (29 slices, 7 mice). 1 year: WT (12 slices, 6 mice), KO (15 slices, 5 mice).