Neuroprotective Properties of 4-Aminopyridine

4-Aminopyridine in Neurologic Disease

Aminopyridines are a group of monoamino and diamino derivatives of pyridine, which inhibit voltage-gated potassium (Kv) channels. Especially, the 2 broad-spectrum potassium channel blockers 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) have been used as investigational new substances in various neurologic diseases. Despite the fact that 3,4-DAP is a more potent antagonist of potassium channels, 4-AP crosses the blood-brain barrier more readily¹ and was clinically superior in patients with MS, particularly for improving visual function,² fatigue,³ cognition,⁴ and walking speed.¹ In addition, 4-AP has been reported to facilitate neural conduction in neurologic diseases other than MS.^5,6

In healthy axons, the channels Kv1.1 and Kv1.2 are clustered near the nodes of Ranvier.⁷ These channels become exposed after demyelination and migrate through the demyelinated segment. At the same time, expression of these channels is increased several fold.⁸ This misdirected redistribution of the Kv channels impairs the transmission of action potentials, leading to permanent disability. 4-AP blocks these exposed potassium channels and therefore enhances signal transduction.^9,10 The Kv1.3 channel was discovered in human T-cells,¹¹ was found to be highly expressed on inflammatory infiltrates in the MS brain,¹² and is expressed on macrophages, microglia, and effector memory T cells.¹³ Selective and nonselective Kv1.3 channel blockers might thereby provide immunomodulatory properties by inhibiting cell proliferation and proinflammatory cytokine secretion.¹⁴ Studies before 2009 failed to establish 4-AP as a symptomatic treatment for MS because drug blood levels in patients were unpredictable, with excessive doses being associated with the risk of epileptic seizures and impaired consciousness.^15,–,18 Therefore, fampridine, the prolonged release formulation of 4-AP was developed and has subsequently been approved for the symptomatic treatment of walking disability in MS.^19,–,23 Interestingly, more recently, an increasing body of evidence suggests that besides these widely acknowledged symptomatic effects, 4-AP may have additional protective properties.

Evaluation of 4-AP Using In Vitro Models

In vitro, neuroprotective effects of 4-AP have been observed in numerous models. When motor neurons (MNs) differentiated from induced pluripotent stem cells of patients with amyotrophic lateral sclerosis carrying mutations of the FUS and SOD1 gene were exposed to 4-AP, ion-channel imbalances were redressed, neuronal activity levels raised, endoplasmatic reticulum stress diminished, and caspase activation attenuated. The mutant MNs showed lower sodium currents and Na⁺/K⁺ ratios, which may at least in part be the reason for their hypoexcitability. This was reversed after 4-AP treatment, which led to decreased potassium currents and restored spontaneous activity patterns and synaptic input in the MNs.²⁴ 4-AP treatment reduced the release of proinflammatory mediators from human microglia challenged with amyloid beta and protected cultured rat hippocampal neurons bathed in supernatants from amyloid beta treated microglia.²⁵ A 4-AP derivative reportedly reduced α-synuclein accumulation, oxidation, inflammation, and Rho kinase activation in an in vitro model of Parkinson disease.²⁶ Other in vitro studies reported that 4-AP increased cAMP response element-binding protein phosphorylation provided protection from cellular stress by glutamate, NMDA, and 3-nitropropionic acid exerted on rat neonatal cerebellar granule neurons. Glutamate lead to a decreased viability also in cells preconditioned with 4-AP but with no significant activation of caspase-3. These observations suggested that 4-AP is primarily effective against necrotic excitotoxicity.²⁷ It was also shown to protect primary neuronal cultures from oxygen-glucose deprivation or ouabain/DL-threo-β-benzyloxyaspartic acid toxicity.²⁸

Preclinical In Vivo Studies on 4-AP

Several studies have investigated the protective effects of 4-AP in various disease models (table 1). In nerve crush models of peripheral nerve damage, prophylactic and early 4-AP treatment prompted recovery of nerve conduction velocity, promoted remyelination, and augmented the axonal area. The latter observations were explained by effects similar to those appearing after electrical stimulation, for instance, elevations in neuronal brain-derived neurotrophic factor (BDNF) levels.²⁹ In a model of Alzheimer disease, injection of amyloid-beta into the hippocampus of Sprague Dawley rats induced neuronal damage and heightened microglial activation. Daily administration of 1 mg/kg 4-AP was found to suppress microglial activation and provided neuroprotection. This was attributed to 4-AP's ability to block the noninactivating outwardly rectifying K+ current in activated microglia and reduce cellular production of proinflammatory cytokines.²⁵ Investigations using an in vivo model of kinate-induced hippocampal neurotoxicity revealed robust neuroprotective effects of 4-AP that could be abrogated by the noncompetitive NMDA receptor antagonist MK-801 and the adenosine A1 antagonist 8-cyclopenthyltheophylline. These observations suggest that NMDA receptors are relevant for 4-AP mediated protection in this model.³⁰ In an animal model of autoimmune neuropathy in Lewis rats, 4-AP ameliorated the clinical severity and pathologic electrophysiologic findings. The authors suggested that axonal protection was provided by blocking sodium-mediated inward currents in the early phase because high membrane potentials in the acute phase of inflammation may be neurotoxic. In the chronic phase, nervous conduction was potentially improved by blocking potassium-mediated outward currents.³¹

In experimental autoimmune encephalomyelitis (EAE), a model of immune-mediated CNS inflammation replicating cardinal features of MS, Kv channel blockade has been reported to inhibit T-cell activation, potentially by blocking of channels of the Kv1.3 subfamily^32,33 and attenuated axonal demyelination and degeneration by acting on the K_v3.1 channel on astroglia, potentially inducing the BDNF signalling.³⁴ In proteolipid protein-induced EAE in SJL mice, 4-AP treatment significantly improved the clinical scores which was confirmed pathologically. Glial fibrillary acidic protein expression was observed to be downregulated in 4-AP–treated mice, and T-cell activation and Th1/17 polarization were mitigated. However, in the chronic, myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE model in C57BL/6 mice, 4-AP did not change the EAE course.³⁵ Another study also investigated the effects of 4-AP in a MOG-EAE model in C57BL/6 mice and reported symptomatic but no disease modifying effects. Neither a prophylactic nor a therapeutic 4-AP treatment attenuated the severity of the clinical EAE course, whereas 4-AP treated animals showed improved mobility assessed by foot print and rotarod analysis. Demyelination of the spinal cord, neuronal damage, and MRI imaging of brain volume changes were unaltered. Proliferation, IL17, or IFN-γ production of CD4⁺ T-cells were also unaffected.³⁶

More recently, we have demonstrated that apart from its symptomatic effects on enhancing neural conduction, 4-AP can prevent retinal neurodegeneration during MOG peptide-induced experimental optic neuritis (EAEON) in C5BL6 mice.³⁷ Using in vivo optical coherence tomography (OCT) imaging, visual function testing, and histologic assessment, we observed a reduction in the extent of degeneration of inner retinal layers in models of EAEON, both for prophylactic and therapeutic 4-AP administration. In this model, 4-AP potentiated the effects of immunomodulatory treatment with the sphingosine-1-phosphate receptor modulator fingolimod, suggesting independent modes of action. It is reasonable to assume that this effect is not limited to fingolimod alone and applicable to 4-AP combined with other MS immunomodulatory drugs. In our study, optic nerve histology revealed that in contrast to fingolimod, 4-AP had no significant influence on microglial activation and/or infiltration of lymphocytes or macrophages, suggesting that the protective effects were not related to an anti-inflammatory mode of action. In line with this, 4-AP treatment did not interfere with EAE induction, validated by a T-cell restimulation assay. Furthermore, we observed significant protection from retinal neurodegeneration under 4-AP treatment also in the noninflammatory optic nerve crush model, whereas, here, fingolimod had no effect. Interestingly, in vitro, 4-AP treatment failed to directly protect retinal ganglion cells. Instead, histology and in vitro experiments indicated 4-AP–mediated stabilization of myelin and oligodendrocyte precursor cells. This effect was associated with increased calcium influx and nuclear translocation of the nuclear factor of activated T-cells (NFAT). It has previously been shown that 4-AP regulates calcium homeostasis by elevating inositol trisphosphate levels and thereby causing calcium release from intracellular calcium stores.³⁸ However, additional studies on 4-AP in demyelinating models, for instance, cuprizone-treatment or transgenic mouse models with inducible oligodendrocyte ablation might be helpful to confirm these conclusions. Existing studies in animal models of demyelination mainly focus on the capacity of 4-AP to restore the action potential but lack further investigations of oligodendroglial cells (figure).³⁹

It is important to mention that the doses needed to obtain these effects in vitro are approximately 100-1,000x higher than the concentration achieved in patients.³⁹ Therefore, additional or other mechanisms might be relevant for the observations made in vivo and in vitro. These include but are not limited to a diminished energy dissipation of demyelinated axons because of blockage of potassium leakage and stronger protective and reparative capacities in the brain indirectly resulting from increased mobility and greater exercise.

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Figure Presumed Mode of Action and Additional Potential Effects of 4-AP in Demyelinating Disease

4-AP blocks potassium channels and therefore enhances signal transduction of the axon.^9,10 Potential immunomodulatory effects may be exerted by K_v1.3 channels expressed by microglia²⁵ and T-cells.³⁵ The Kv3.1 channel on astrocytes might be targeted by 4-AP to suppress demyelination and axonal degenerating.³⁴ In another preclinical study, 4-AP stabilized myelin via the NFAT pathway.³⁷ 4-AP = 4-aminopyridine; NFAT = nuclear factor of activated T-cells.

Moreover, immunomodulatory mechanisms of 4-AP cannot be ruled out especially because reduced T-cell activation and Th1/17 polarization have been demonstrated in PLP-induced EAE in SJL mice.³⁵ In addition, preclinical studies of other disease models found an attenuated activation and reduced release of proinflammatory mediators by microglia.²⁵ These controversial results highlight the diverse pathologic mechanisms of the different animal models, where immune cells are more or less susceptible to treatment strategies. The discrepancies between our results and previous reports of only symptomatic effects in MOG peptide-induced EAE in C57BL6 mice^35,36 may at least in part be explained by (1) differences in dosing because others used doses of 100 µg and 600 µg/mouse/day, whereas we administered 250 µg/mouse/d; (2) treatment duration (40, 60, and 90 days by Göbel et al.,³⁶ Moriguchi et al.,³⁵ and Dietrich et al.,³⁷ respectively); and (3) amount of MOG used for immunization because we used 200 µg MOG per mouse, whereas others used only 100 µg MOG per mouse. Recent research focuses on the role of gut microbiota in influencing induction and severity of EAE by altering the balance of effector and regulatory T and B cells.^40,–,42 The microbiome of the rodents may differ between the animal facilities resulting in differences of EAE severity, course, and possibly even response to therapeutics. Taken together, these factors could account for the heterogeneity in study results.