Abstract
A Role for Insulin-Like Growth Factor 1 in the Generation of Epileptic Spasms in a Murine Model Ballester-Rosado CJ, Le JT, Lam TT, Mohila CA, Lam S, Anderson AE, Frost JD Jr, Swann JW. Ann Neurol. 2022;92(1):45-60. doi:10.1002/ana.26383
Objective: Infantile spasms are associated with a wide variety of clinical conditions, including perinatal brain injuries. We have created a model in which prolonged infusion of tetrodotoxin (TTX) into the neocortex, beginning in infancy, produces a localized lesion and reproduces the behavioral spasms, electroencephalogram (EEG) abnormalities, and drug responsiveness seen clinically. Here, we undertook experiments to explore the possibility that the growth factor IGF-1 plays a role in generating epileptic spasms. Methods: We combined long-term video EEG recordings with quantitative immunohistochemical and biochemical analyses to unravel IGF-1’s role in spasm generation. Immunohistochemistry was undertaken in surgically resected tissue from infantile spasms patients. We used viral injections in neonatal conditional IGF-1R knock-out mice to show that an IGF-1-derived tripeptide (1-3)IGF-1 acts through the IGF-1 receptor to abolish spasms. Results: Immunohistochemical methods revealed widespread loss of IGF-1 from cortical neurons, but an increase in IGF-1 in the reactive astrocytes in the TTX-induced lesion. Very similar changes were observed in the neocortex from patients with spasms. In animals, we observed reduced signaling through the IGF-1 growth pathways in areas remote from the lesion. To show the reduction in IGF-1 expression plays a role in spasm generation, epileptic rats were treated with (1-3)IGF-1. We provide 3 lines of evidence that (1-3)IGF-1 activates the IGF-1 signaling pathway by acting through the receptor for IGF-1. Treatment with (1-3)IGF-1 abolished spasms and hypsarrhythmia-like activity in the majority of animals. Interpretation: Results implicate IGF-1 in the pathogenesis of infantile spasms and IGF-1 analogues as potential novel therapies for this neurodevelopmental disorder.
Commentary
Infantile spasms (IS) typically manifest with frequent, brief seizures with extension or flexion of the trunk and extremities, accompanied by a high amplitude, chaotic EEG background with multifocal spikes, termed hypsarrhythmia. Long-term neurological impairment often results with IS, and treatment of IS with the first-line treatments, adrenocorticotropic hormone (ACTH), vigabatrin, and prednisolone, are often unsuccessful. The causes of IS are varied, and while many genetic causes have been identified, the molecular underpinnings behind acquired etiologies related to perinatal brain injury are not well delineated. A prior study noted that children with symptomatic (but not idiopathic) IS have lower levels of insulin-like growth factor-1 (IGF-1) in their cerebrospinal fluid compared to controls. 1 Ballester-Rosado and colleagues asked whether IGF-1 has a role in the pathophysiology of IS by using a rat model of acquired IS. 2 They found that IGF-1 expression and downstream signaling are decreased and that treatment with IGF-1 activation resulted in resolution of spasms and hypsarrhythmia in a majority of rats.
While many animal models of IS are not known to have hypsarrhythmia, the investigators used a rat model of acquired IS and hypsarrhythmia, with unilateral infusion of the sodium channel blocker, tetrodotoxin (TTX), into the right motor/sensory cortical areas starting from postnatal day 11 to 12 and lasting 4 weeks. Almost all rats treated with TTX developed spontaneous epileptic spasms associated with electrodecrement and superimposed fast activity about 1 week after the start of the infusion, and they also developed an interictal EEG pattern consistent with hypsarrhythmia, thereby recapitulating major features of IS. While IGF-1 was normally expressed in neurons throughout the rat cortex, the lesioned area in the TTX-treated rats had a loss of neurons from apoptosis and an increase in IGF-1 expression in reactive astrocytes. Remarkably, all other areas of the rat brain that were examined (ipsilateral cortex adjacent and remote to the lesion, and contralateral areas that were homotopic to the lesion, adjacent, and remote regions) had a decrease in IGF-1 compared to control (vehicle-infused) animals. Expression of IGF-1 receptor (IGF-1R) and downstream markers of IGF-1R activation (phosphorylated AKT, S6, and ERK) were all decreased in the cortex contralateral to the TTX infusion compared to that of controls. Furthermore, in surgically resected brain tissue from children with symptomatic IS, there was also an increase in IGF-1 in the lesion and a decrease in IGF-1 adjacent to the lesion, compared to controls (cortex adjacent to brain tumors from children who had seizures and tumor resection).
To determine whether IGF-1 signaling activation can have therapeutic effects for the TTX-treated rats, the investigators used (1-3)IGF-1, an active tripeptide fragment from the N-terminal side of IGF-1. The researchers showed that (1-3)IGF-1 treatment of normal mice resulted in an increase in downstream markers of IGF-1 signaling. To demonstrate that this signaling occurs through IGF-1R, conditional IGF-1R knockout mice received unilateral viral delivery of either Cre recombinase or control into the brain and were treated with (1-3)IGF-1 or vehicle control. A downstream marker of IGF-1 signaling (phosphorylated AKT) did not increase with (1-3)IGF-1 treatment when IGF-1R was knocked out with Cre recombinase. In addition, wild-type mice pretreated with picropodophyllin, a small molecule inhibitor of IGF-1R, also prevented the ability of (1-3)IGF-1 to increase phosphorylated AKT levels. Therefore, treatment with (1-3)IGF-1 activates the IGF-1 signaling pathway in healthy mice, via the IGF-1R. Tetrodotoxin-treated rats were implanted with EEG electrodes shortly after weaning, and then a 5-day baseline recording was performed, and then 5 weeks of recording (postnatal week 6-10) were performed. Administration of (1-3)IGF-1 during the first 3 weeks of recording resulted in a resolution of spasms in 5 (62.5%) of 8 rats, and a resolution of hypsarrhythmia in 4 (67%) of 6 rats at week 5 of recording. Conversely, all (11/11) of the vehicle-treated rats had spasms throughout the recording period, and only 2 (22%) of 9 had resolution of hypsarrhythmia.
The importance of the study is that it uncovers an additional potential pathophysiological mechanism and therapeutic target for IS. The effectiveness of (1-3)IGF-1 was comparable to that of ACTH and vigabatrin treatment in the TTX rat model, as each results in about 60% of rats to be spasm-free. 3,4 The study has many strengths, including validation of finding decreased IGF-1 expression in the brain tissue of individuals with IS and the usage of a rat model that recapitulates many of the clinical features seen in affected infants (including hypsarrhythmia). A limitation of the study is that there were a relatively low number of animals used in the study. As a result, the P value for resolution of hypsarrhythmia with (1-3)IGF-1 treatment was .06 (above the arbitrary .05 threshold for significance), and sexually dimorphic findings could not be fully explored.
Of all the anti-seizure medications, only ACTH and prednisolone are hormonal therapies. They affect the corticotropin-releasing hormone (CRH)/ACTH/cortisol pathway in the hypothalamic/pituitary axis, and both treat IS, a relatively specific type of epilepsy. It is intriguing that IGF-1 is also a hormonal therapy and is a potential treatment for IS but affects the growth hormone releasing hormone (GHRH)/growth hormone (GH)/IGF-1 pathway in the hypothalamic/pituitary axis. Does ACTH/prednisolone therapy affect IGF-1 levels and signaling (or vice versa), and are there overlapping mechanisms between these treatments? There is crosstalk between the CRH/ACTH/cortisol and the GHRH/GH/IGF-1 axes, 5 although it is unclear how their interplay might affect brain regions involved in IS. In addition, the ketogenic diet, which can be effective for IS, is also known to increase IGF-1R expression in the brain, raising the possibility of shared mechanisms between these treatment modalities. 6
Although (1-3)IGF-1 can activate signaling through IGF-1R in healthy mice, it is unclear what brain regions and cell types (1-3)IGF-1 acts upon in TTX-treated rats in order to alleviate spasms and hypsarrhythmia. Since the lesioned area displayed upregulation of IGF-1 in reactive astrocytes, the (1-3)IGF-1 may be acting on the rest of the cortex (which has downregulated IGF-1 expression), but it could also act on the hypothalamus and pituitary to inhibit GHRH and GH, similar to ACTH’s inhibiting the release of CRH, one of the postulated mechanisms of action for ACTH. 7 Furthermore, if the therapeutic action of (1-3)IGF-1 results from its effect upon cortical neurons, it would be interesting to determine whether there is a differential effect on excitatory and inhibitory neurons.
Another limitation of this study is that a single rodent model of acquired spasms was used. In genetic causes of IS, would similar mechanisms (and therapies) be relevant? A decrease in IGF-1 signaling results in a decrease in PI3K-AKT-mTOR signaling, and this is opposite of the several diseases (e.g., tuberous sclerosis complex) in which hyperactivity of the mTOR pathway is a cause of spasms. In addition, another rat model of acquired IS, which uses multiple hits with intracerebral infusion of doxorubicin and lipopolysaccharide and subsequent intraperitoneal injection of a serotonin depleter, was found to have increased perilesional phosphorylated S6, consistent with activated mTOR signaling, and treatment with rapamycin stopped spasms and improved learning. 8 In the multiple hit rat model, upstream IGF-1 signaling was not measured, and in the TTX model, decreased IGF-1 signaling may be exerting its effect through an mTOR-independent mechanism. While findings from these 2 models may not be directly contradictory, it is unlikely that all causes of IS share a common final mechanistic pathway, and further studies will be necessary to determine whether decreased IGF-1 signaling is common to acquired causes of IS.
Ballester-Rosado and colleagues have uncovered a new potential pathophysiological mechanism and treatment for IS. After promising preclinical studies, recombinant human IGF-1 (rhIGF-1) was used in clinical trials for Rett and Phelan-McDermid Syndromes with demonstration of safety and tolerability, but rhIGF-1 did not demonstrate significant improvement with primary end points. 9,10 Important outstanding questions for the path toward clinical translation for (1-3)IGF-1 in IS include what timing of treatment is needed for efficacy, whether long-term neurodevelopmental sequelae of IS would improve with treatment, and whether (1-3)IGF-1 used in combination with ACTH/prednisolone and/or vigabatrin would provide additional (or even synergistic) benefit.
