Franck Kalume

Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy

Voltage-gated sodium channels (NaV) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in NaV1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a−/− mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/− mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of NaV1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/− and Scn1a−/− mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of NaV1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/− heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.

Autoimmunity due to molecular mimicry as a cause of neurological disease

One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

NaV1.1 channels and epilepsy

Voltage‐gated sodium channels initiate action potentials in brain neurons, and sodium channel blockers are used in therapy of epilepsy. Mutations in sodium channels are responsible for genetic epilepsy syndromes with a wide range of severity, and the NaV1.1 channel encoded by the SCN1A gene is the most frequent target of mutations. Complete loss‐of‐function mutations in NaV1.1 cause severe myoclonic epilepsy of infancy (SMEI or Dravets Syndrome), which includes severe, intractable epilepsy and comorbidities of ataxia and cognitive impairment. Mice with loss‐of‐function mutations in NaV1.1 channels have severely impaired sodium currents and action potential firing in hippocampal GABAergic inhibitory neurons without detectable effect on the excitatory pyramidal neurons, which would cause hyperexcitability and contribute to seizures in SMEI. Similarly, the sodium currents and action potential firing are also impaired in the GABAergic Purkinje neurons of the cerebellum, which is likely to contribute to ataxia. The imbalance between excitatory and inhibitory transmission in these mice can be partially corrected by compensatory loss‐of‐function mutations of NaV1.6 channels, and thermally induced seizures in these mice can be prevented by drug combinations that enhance GABAergic neurotransmission. Generalized epilepsy with febrile seizures plus (GEFS+) is caused by missense mutations in NaV1.1 channels, which have variable biophysical effects on sodium channels expressed in non‐neuronal cells, but may primarily cause loss of function when expressed in mice. Familial febrile seizures is caused by mild loss‐of‐function mutations in NaV1.1 channels; mutations in these channels are implicated in febrile seizures associated with vaccination; and impaired alternative splicing of the mRNA encoding these channels may also predispose some children to febrile seizures. We propose a unified loss‐of‐function hypothesis for the spectrum of epilepsy syndromes caused by genetic changes in NaV1.1 channels, in which mild impairment predisposes to febrile seizures, intermediate impairment leads to GEFS+ epilepsy, and severe or complete loss of function leads to the intractable seizures and comorbidities of SMEI.

Reduced Sodium Current in Purkinje Neurons from NaV1.1 Mutant Mice: Implications for Ataxia in Severe Myoclonic Epilepsy in Infancy

Loss-of-function mutations of NaV1.1 channels cause severe myoclonic epilepsy in infancy (SMEI), which is accompanied by severe ataxia that contributes substantially to functional impairment and premature deaths. Mutant mice lacking NaV1.1 channels provide a genetic model for SMEI, exhibiting severe seizures and premature death on postnatal day 15. Behavioral assessment indicated severe motor deficits in mutant mice, including irregularity of stride length during locomotion, impaired motor reflexes in grasping, and mild tremor in limbs when immobile, consistent with cerebellar dysfunction. Immunohistochemical studies showed that NaV1.1 and NaV1.6 channels are the primary sodium channel isoforms expressed in cerebellar Purkinje neurons. The amplitudes of whole-cell peak, persistent, and resurgent sodium currents in Purkinje neurons were reduced by 58–69%, without detectable changes in the kinetics or voltage dependence of channel activation or inactivation. Nonlinear loss of sodium current in Purkinje neurons from heterozygous and homozygous mutant animals suggested partial compensatory upregulation of NaV1.6 channel activity. Current-clamp recordings revealed that the firing rates of Purkinje neurons from mutant mice were substantially reduced, with no effect on threshold for action potential generation. Our results show that NaV1.1 channels play a crucial role in the excitability of cerebellar Purkinje neurons, with major contributions to peak, persistent, and resurgent forms of sodium current and to sustained action potential firing. Loss of these channels in Purkinje neurons of mutant mice and SMEI patients may be sufficient to cause their ataxia and related functional deficits.

Temperature- and age-dependent seizures in a mouse model of severe myoclonic epilepsy in infancy

Heterozygous loss-of-function mutations in the α subunit of the type I voltage-gated sodium channel NaV1.1 cause severe myoclonic epilepsy in infancy (SMEI), an infantile-onset epileptic encephalopathy characterized by normal development followed by treatment-refractory febrile and afebrile seizures and psychomotor decline. Mice with SMEI (mSMEI), created by heterozygous deletion of NaV1.1 channels, develop seizures and ataxia. Here we investigated the temperature and age dependence of seizures and interictal epileptiform spike-and-wave activity in mSMEI. Combined video-EEG monitoring demonstrated that mSMEI had seizures induced by elevated body core temperature but wild-type mice were unaffected. In the 3 age groups tested, no postnatal day (P)17–18 mSMEI had temperature-induced seizures, but nearly all P20–22 and P30–46 mSMEI had myoclonic seizures followed by generalized seizures caused by elevated core body temperature. Spontaneous seizures were only observed in mice older than P32, suggesting that mSMEI become susceptible to temperature-induced seizures before spontaneous seizures. Interictal spike activity was seen at normal body temperature in most P30–46 mSMEI but not in P20–22 or P17–18 mSMEI, indicating that interictal epileptic activity correlates with seizure susceptibility. Most P20–22 mSMEI had interictal spike activity with elevated body temperature. Our results define a critical developmental transition for susceptibility to seizures in SMEI, demonstrate that body temperature elevation alone is sufficient to induce seizures, and reveal a close correspondence between human and mouse SMEI in the striking temperature and age dependence of seizure frequency and severity and in the temperature dependence and frequency of interictal epileptiform spike activity.

Specific deletion of NaV1.1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome

Heterozygous loss-of-function mutations in the brain sodium channel NaV1.1 cause Dravet syndrome (DS), a pharmacoresistant infantile-onset epilepsy syndrome with comorbidities of cognitive impairment and premature death. Previous studies using a mouse model of DS revealed reduced sodium currents and impaired excitability in GABAergic interneurons in the hippocampus, leading to the hypothesis that impaired excitability of GABAergic inhibitory neurons is the cause of epilepsy and premature death in DS. However, other classes of GABAergic interneurons are less impaired, so the direct cause of hyperexcitability, epilepsy, and premature death has remained unresolved. We generated a floxed Scn1a mouse line and used the Cre-Lox method driven by an enhancer from the Dlx1,2 locus for conditional deletion of Scn1a in forebrain GABAergic neurons. Immunocytochemical studies demonstrated selective loss of NaV1.1 channels in GABAergic interneurons in cerebral cortex and hippocampus. Mice with this deletion died prematurely following generalized tonic-clonic seizures, and they were equally susceptible to thermal induction of seizures as mice with global deletion of Scn1a. Evidently, loss of NaV1.1 channels in forebrain GABAergic neurons is both necessary and sufficient to cause epilepsy and premature death in DS.

Sudden unexpected death in a mouse model of Dravet syndrome

Sudden unexpected death in epilepsy (SUDEP) is the most common cause of death in intractable epilepsies, but physiological mechanisms that lead to SUDEP are unknown. Dravet syndrome (DS) is an infantile-onset intractable epilepsy caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes brain type-I voltage-gated sodium channel NaV1.1. We studied the mechanism of premature death in Scn1a heterozygous KO mice and conditional brain- and cardiac-specific KOs. Video monitoring demonstrated that SUDEP occurred immediately following generalized tonic-clonic seizures. A history of multiple seizures was a strong risk factor for SUDEP. Combined video-electroencephalography-electrocardiography revealed suppressed interictal resting heart-rate variability and episodes of ictal bradycardia associated with the tonic phases of generalized tonic-clonic seizures. Prolonged atropine-sensitive ictal bradycardia preceded SUDEP. Similar studies in conditional KO mice demonstrated that brain, but not cardiac, KO of Scn1a produced cardiac and SUDEP phenotypes similar to those found in DS mice. Atropine or N-methyl scopolamine treatment reduced the incidence of ictal bradycardia and SUDEP in DS mice. These findings suggest that SUDEP is caused by apparent parasympathetic hyperactivity immediately following tonic-clonic seizures in DS mice, which leads to lethal bradycardia and electrical dysfunction of the ventricle. These results have important implications for prevention of SUDEP in DS patients.

Insights into pathophysiology and therapy from a mouse model of Dravet syndrome

Mutations in voltage‐gated sodium channels are associated with epilepsy syndromes with a wide range of severity. Complete loss of function in the Nav1.1 channel encoded by the SCN1A gene is associated with severe myoclonic epilepsy in infancy (SMEI), a devastating infantile‐onset epilepsy with ataxia, cognitive dysfunction, and febrile and afebrile seizures resistant to current medications. Genetic mouse models of SMEI have been created that strikingly recapitulate the SMEI phenotype including age and temperature dependence of seizures and ataxia. Loss‐of‐function in Nav1.1 channels results in severely impaired sodium current and action potential firing in hippocampal γ‐aminobutyric acid (GABA)ergic interneurons without detectable changes in excitatory pyramidal neurons. The resulting imbalance between excitation and inhibition likely contributes to hyperexcitability and seizures. Reduced sodium current and action potential firing in cerebellar Purkinje neurons likely contributes to comorbid ataxia. A mechanistic understanding of hyperexcitability, seizures, and comorbidities such as ataxia has led to novel strategies for treatment.

Molecular mimicry: Cross‐reactive antibodies from patients with immune‐mediated neurologic disease inhibit neuronal firing

Recent data indicate that molecular mimicry may play a role in the pathogenesis of human‐T‐lymphotropic virus type‐1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP), an immune‐mediated disease of the central nervous system (CNS). Specifically, HAM/TSP patients developed antibodies that cross‐react with heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an antigen highly expressed in neurons. Antibodies to HTLV‐1‐tax cross‐reacted with hnRNP A1, suggesting molecular mimicry between the two proteins. In support of this hypothesis, HAM/TSP IgG and antibodies to hnRNP A1 and HTLV‐1‐tax inhibited neuronal firing, suggesting that these antibodies can be pathogenic. We extended these observations by carrying out studies on over 20 different neurons. We also tested IgG isolated from six different HAM/TSP patients and two HTLV‐1 seronegative controls and added experiments that control for antibody isotype, antibody target, and neuron viability. In these studies, IgG was infused into the extracellular space during whole‐cell current clamp recordings of neurons. Our results confirm that in contrast to normal IgG, IgG from all HAM/TSP patients completely inhibited neuronal firing. Affinity‐purified antibodies specific for hnRNP A1 and a monoclonal antibody to HTLV‐1‐tax (which reacted with hnRNP A1 and whose epitope overlaps the human immunodominant epitope of tax) also inhibited neuronal firing. Monoclonal antibodies to neurofilament did not change neuronal firing. These data indicate that antibodies to neurons can be pathogenic, that biologic activity can be affected by a cross‐reactive epitope between HTLV‐1‐tax and hnRNP A1, and that molecular mimicry may play a role in the pathogenesis of HAM/TSP.

Protective effect of the ketogenic diet in Scn1a mutant mice

Purpose:  We evaluated the ability of the ketogenic diet (KD) to improve thresholds to flurothyl‐induced seizures in two mouse lines with Scn1a mutations: one that models Dravet syndrome (DS) and another that models genetic (generalized) epilepsy with febrile seizures plus (GEFS+).