Knockin' Out the Spasms

Commentary

APC Conditional Knock-Out Mouse is a Model of Infantile Spasms With Elevated Neuronal β-Catenin Levels, Neonatal Spasms, and Chronic Seizures.

Pirone A, Alexander J, Lau LA, Hampton D, Zayachkivsky A, Yee A, Yee A, Jacob MH, Dulla CG. Neurobiol Dis 2016;98:149–157.27852007

Infantile spasms (IS) are a catastrophic childhood epilepsy syndrome characterized by flexion-extension spasms during infancy that progress to chronic seizures and cognitive deficits in later life. The molecular causes of IS are poorly defined. Genetic screens of individuals with IS have identified multiple risk genes, several of which are predicted to alter β-catenin pathways. However, evidence linking malfunction of β-catenin pathways and IS is lacking. Here, we show that conditional deletion in mice of the adenomatous polyposis coli gene (APC cKO), the major negative regulator of β-catenin, leads to excessive β-catenin levels and multiple salient features of human IS. Compared with wild-type littermates, neonatal APC cKOmice exhibit flexion-extension motor spasms and abnormal high-amplitude electroencephalographic discharges. Additionally, the frequency of excitatory postsynaptic currents is increased in layer V pyramidal cells, the major output neurons of the cerebral cortex. At adult ages, APC cKOs display spontaneous electroclinical seizures. These data provide the first evidence that malfunctions of APC/β-catenin pathways cause pathophysiological changes consistent with IS. Our findings demonstrate that the APC cKO is a new genetic model of IS, provide novel insights into molecular and functional alterations that can lead to IS, and suggest novel targets for therapeutic intervention.

During the past 10 years, a great many articles have been written on the animal models of infantile spasms. In fact, a PubMed search for “infantile spasms model” with limitation to “other animals” (to avoid human studies) returns 51 references, out of which 40 have been published since 2007. This is good: Not only do infantile spasms represent a hard-to-treat epilepsy syndrome of infancy, but this syndrome is devastating for both the patient and the family (1). Additionally, our knowledge of the disorder's neurobiology is evolving quite slowly.

Currently, there are several rodent models of infantile spasms available reproducing more or less human symptomatology and treatment response. In normal or abnormal brain, these models utilize a specific trigger of spasms with either an acute or a delayed response (occurrence of spasms) or they are based on a genetic condition (2). While this may seem broad, this approach to the infantile spasms model somewhat recapitulates the human condition: There are more than 200 (and counting) different etiologies responsible for symptomatic infantile spasms, such as those with identifiable brain damage (3). There is also a second major group of cryptogenic infantile spasms (without identifiable brain damage) likely due to yet unknown genetic or epigenetic conditions (4). Any new model is appreciated when it brings more understanding into this quite diverse and peculiar syndrome, where various etiologies eventually merge into a common pathway (5) involving the triad of infantile spasms of similar semiology (spastic seizures), electrographic signature (interictal hypsarhythmia), and developmental regression (mental deterioration).

The authors of the current study thoroughly reviewed genes linked to the increased risk of infantile spasms in humans (6–8) and found that 11 out of 15 genes described in the human studies are involved in regulation of β-catenin pathways in the brain, stressing the importance of β-catenin regulation for infantile spasms. Besides the role in cadherin synaptic adhesion, β-catenin also controls Wnt signal transduction. An increase in β-catenin signaling alters brain development and function in favor of more complex dendritic branching and changes of synaptic maturation with increased excitatory synapse density (9). The authors then decided to study the adenomatous polyposis coli (APC) gene linked to five risk genes for infantile spasms. This gene encodes a tumor suppressor protein acting as an antagonist of the Wnt signaling pathway with high concentration in the brain. Moreover, APC serves as an mRNA binding protein with β-catenin as one of its targets. The authors have already created conditional APC knock-out (APC cKO) mice in Ca²⁺/calmodulin-dependent protein kinase II-α positive neurons, resulting in loss of control of β-catenin levels. In addition to elevated β-catenin levels in the brain, mice with APC cKO exhibit learning deficits and autism-like behaviors (10). In this study, the mice were investigated using multiple complementary techniques for occurrence of spasms during infancy, seizures during adulthood, EPSCs in neocortical pyramidal cells, and for the expression of both APC and β-catenin.

First, the authors identified penetrance of their cKO genotype (depending on developmentally increasing CamKIIa-Cre recombinase expression mostly in excitatory neurons in the forebrain region) into phenotypic changes. Interestingly, there were no changes in APC or β-catenin protein concentrations in lysates from neocortex of P9 mice; the increase in both proteins first occurred in P14 mice and persisted through (young) adulthood (determination at P60). However, when the authors looked at the expression of β-catenin protein in individual layer 5 pyramidal neurons and compared normalized immunoreactivity within the neurons between wt (154 neurons) and APC conditional knock-outs (100 neurons), they found an increase in β-catenin expression. Unfortunately, the authors compared 3 mice in each group, yet their parametric statistics have been performed on neurons—not on mice as subjects—and, as such, there might have been a false positive readout. This is somewhat of a concern as other phenotypic measurements have provided clear differences between APC cKO and wt mice already at P9, such as an increase in probability of occurrence and amplitude of spontaneous excitatory postsynaptic currents, a clear sign of increased action potential-driven excitability in APC cKO mice. Similarly, there was an increase in probability of miniature excitatory postsynaptic currents (mini-EPSPs) in these cKO mice indicating increased spontaneous excitatory transmitter release.

The APC cKO mice have also exhibited spontaneous spasms already on P9 and involving both extensions or flexions and back arching. Once the authors compared frequency of high-amplitude spasms in both groups, there was clear enhancement of spasm-like behaviors in P9 APC cKO mice as well as in time spent on one side compared to the age-matched wt. EEG telemetry in P9–12 mice showed the occurrence of high amplitude discharges (>3 times baseline), followed by a disruption in normal EEG background. The authors were unable to identify patterns resembling mouse “hypsarrhythmia,” which is only a minor setback. The lissencephalic rodent brain is less prone to develop chaotic EEG patterns especially at very young ages: If any abnormality similar to hypsarhythmia occurs, it is later in development (in rats P18 and older; 11, 12). In younger rats (P12–15), only distinct large-amplitude wave patterns different from EEG background can be observed (13). However, the authors did not look at the correlation (temporal or numerical) between the high-amplitude discharges they observed and the spastic behaviors. This is more concerning, as these discharges might have simply been an associated finding and not a correlate for epileptic spasms. Finally, the authors revisited APC cKO mice during adulthood and found electroclinical seizures with EEG phenotype of high-frequency high-amplitude ictal activity and frequent interictal spikes and behaviors involving head bobbing, freezing, and forelimb clonus. This is consistent with progression of infantile spasms in humans into another type of severe seizures. It is unfortunate that the authors were unable to correlate history (frequency of spasms) of individual mice on P9–12 with number and severity of spontaneous seizure events recorded on >P56. This correlation would significantly add value to their model. Also, it is not clear at which age the spasms diminish and the adult-type seizure events start.

The authors show the presence of spastic seizures, EEG abnormalities, and later-life spontaneous seizures in their APC cKO mouse model. These are essential stepping-stones towards a successful model of infantile spasms. The criteria for the “ideal” model have been presented previously (14); nevertheless, it should be emphasized here that as Norbert Wiener noted in Philosophy of Science, “The best material model of a cat is another, or preferably the same, cat.” In other words, no model is perfect; a model will always be an approximation of a condition. Yet there are additional steps to be considered for this model beyond those already mentioned. The subtle anatomic substrate (lack of changes in APC and β-catenin protein expression) in this model during early development strongly suggests that there may be another, possibly intermediary, protein that is already altered and has significance for the occurrence of the behavioral phenotype. Finally, to determine whether the model belongs to the group of ACTH-responsive spasms (13), the authors should further consider testing ACTH as well as vigabatrin treatments in their neonatal mice, not only to find out immediate response of spasms but also the long-term effect in terms of development of spontaneous seizure events in adulthood.