Susan I.V. Judge

Overexpression of SIRT1 Protein in Neurons Protects against Experimental Autoimmune Encephalomyelitis through Activation of Multiple SIRT1 Targets

Treatment of experimental autoimmune encephalomyelitis (EAE) with resveratrol, an activator of sirtuin 1 (SIRT1), reduces disease severity. This suggested that activators of SIRT1, a highly conserved NAD-dependent protein deacetylase, might have immune-modulating or neuroprotective therapeutic effects in EAE. Previously, we showed that SIRT1 expression increases in EAE, suggesting that it is an adaptive response. In this study, we investigated the potential function of SIRT1 in regulating EAE using SIRT1-overexpressing mice. The current studies examine potential neuroprotective and immunomodulatory effects of SIRT1 overexpression in chronic EAE induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35–55. SIRT1 suppressed EAE clinical symptoms compared with wild-type EAE mice and prevented or altered the phenotype of inflammation in spinal cords; as a result, demyelination and axonal injury were reduced. Significant neuroprotective effects were observed, with fewer apoptotic cells found in the spinal cords of SIRT1-overexpressing EAE mice associated with increased brain-derived neurotrophic factor and NAD levels. Earlier, we showed that brain-derived neurotrophic factor and NAD play crucial neuroprotective roles in EAE. These results suggest that SIRT1 reduces neuronal loss in this chronic demyelinating disease model and that this is associated with a reduction in inflammation.

Potassium channels Kv1.3 and Kv1.5 are expressed on blood-derived dendritic cells in the central nervous system

Potassium (K+) channels on immune cells have gained attention recently as promising targets of therapy for immune‐mediated neurological diseases such as multiple sclerosis (MS). We examined K+ channels on dendritic cells (DCs), which infiltrate the brain in MS and may impact disease course.

Voltage-gated potassium channels in multiple sclerosis: Overview and new implications for treatment of central nervous system inflammation and degeneration.

Inflammatory tissue damage and the presence of reactive immunocompetent T lymphocytes, macrophages, microglia, and dendritic cells (DCs) are characteristic features in the human chronic inflammatory demyelinating disease, multiple sclerosis (MS). Together, these cells orchestrate the inflammation and immunopathogenesis underlying the MS autoimmune disease processes and all up-regulate the same voltage-gated potassium (K(v)) channel, K(v)1.3, when fully activated. Only microglia, which mediate central nervous system (CNS) inflammatory processes (possibly playing a dual role of CNS protection and mediation of neuroinflammation/ neurodegeneration), and DC, which are pivotal to the induction of T cell responses, express the distinct K(v)1.5 prior to K(v)1.3 up-regulation. Although the precise functional roles of first K(v)1.5 and then K(v)1.3 channels are unclear, their differential expression is likely a common mechanism used by both microglia and DC, revealing K(v)1.5 (in addition to K(v)1.3) as a potentially important target for the development of new immunomodulatory therapies in MS.

Potassium Channel Blockers and Openers as CNS Neurologic Therapeutic Agents

Potassium (K+) channels are the most heterogeneous and widely distributed class of ion channels. K(+) channels are dynamic pore-forming transmembrane proteins known to play important roles in all cell types underlying both normal and pathophysiological functions. Essential for such diverse physiological processes as nerve impulse propagation, muscle contraction, cellular activation and the secretion of biologically active molecules, various K(+) channels are recognized as potential therapeutic targets in the treatment of multiple sclerosis, Alzheimers disease, Parkinsons disease, epilepsy, stroke, brain tumors, brain/spinal cord ischemia, pain and schizophrenia, migraine, as well as cardiac arrhythmias, pulmonary hypertension, diabetes, cervical cancer, urological diseases and sepsis. In addition to their importance as therapeutic targets, certain K(+) channels are gaining attention for their beneficial roles in anesthesia, neuroprotection and cardioprotection. The K(+) channel gene families (subdividing into multiple subfamilies) include voltage-gated (K(v): K(v)1-K(v)12 or KCNA-KCND, KCNF-KCNH, KCNQ, KCNS), calcium-activated (K(Ca): K(Ca)1-K(Ca)5 or KCNM-KCNN), inwardly rectifying (K(ir): K(ir)1-K(ir)7 or KCNJ) and background/leak or tandem 2-pore (K(2P): K(2P)1-K(2P)7, K(2P)9-K(2P)10, K(2P)12-K(2P)13, K(2P)15-K(2P)18 or KCNK) K(+) channels. Worldwide, the pharmaceutical industry is actively developing better strategies for targeting ion channels, in general, and K(+) channels, in particular, already generating over

Sustained-release fampridine for multiple sclerosis.

6 billion in sales per annum from drugs designed to block or modulate ion channel function. This review provides an overview of recent patents on emerging K(+) channel blockers and activators (openers) with potential for development as new and improved nervous system therapeutic agents.

Patents related to therapeutic activation of K(ATP) and K(2P) potassium channels for neuroprotection: ischemic/hypoxic/anoxic injury and general anesthetics.

Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system of unknown cause in which neurological impairment and disability result from demyelination and axonal loss. Physiologically, myelin loss leads to changes in axonal ion channels that cause conduction failure. Axonal loss leads to a reduction in signal strength in neuronal pathways. Fampridine (4-aminopyridine) is a potassium channel blocker that can increase action potential duration and amplitude, leading to improved conduction in demyelinated nerve fibers and to increased neurotransmitter release at synaptic endings. Fampridine treatment can improve ambulation in some MS patients, but can also cause seizures and other side effects. Pharmacokinetic studies show that improvement in neurological deficits is primarily related to the total fampridine dose, while seizure induction is related to peak serum levels. To reduce side effects, a slow-release (SR) formulation of fampridine was developed. Two Phase III studies of fampridine SR have now shown that treatment can improve leg strength and walking speed in patients with MS; a new drug application has been filed with the US FDA.

Inactivation gating and 4-AP sensitivity in human brain Kv1.4 potassium channel

Background: Mechanisms of neuroprotection encompass energy deficits in brain arising from insufficient oxygen and glucose levels following respiratory failure; ischemia or stroke, which produce metabolic stresses that lead to unconsciousness and seizures; and the effects of general anesthetics. Foremost among those K+ channels viewed as important for neuroprotection are ATP-sensitive (KATP) channels, which belong to the family of inwardly rectifying K+ channels (Kir) and contain a sulfonylurea subunit (SUR1 or SUR2) combined with either Kir6.1 (KCNJ8) or Kir6.2 (KCNJ11) channel pore-forming α-subunits, and various members of the tandem two-pore or background (K2P) K+ channel family, including K2P1.1 (KCNK1 or TWIK1), K2P2.1 (KCNK2 or TREK/TREK1), K2P3.1 (KCNK3 or TASK), K2P4.1 (KCNK4 or TRAAK), and K2P10.1 (KCNK10 or TREK2). Objectives: This review covers patents and patent applications related to inventions of therapeutics, compound screening methods and diagnostics, including KATP channel openers and blockers, as well as KATP and K2P nucleic/amino acid sequences and proteins, vectors, transformed cells and transgenic animals. Although the focus of this patent review is on brain and neuroprotection, patents covering inventions of KATP channel openers for cardioprotection, diabetes mellitus and obesity, where relevant, are addressed. Results/conclusions: Overall, an important emerging therapeutic mechanism underlying neuroprotection is activation/opening of KATP and K2P channels. To this end substantial progress has been made in identifying and patenting agents that target KATP channels. However, current K2P channels patents encompass compound screening and diagnostics methodo-logies, reflecting an earlier ‘discovery’ stage (target identification/validation) than KATP in the drug development pipeline; this reveals a wide-open field for the discovery and development of K2P-targeting compounds.

C5b-9-activated, Kv1.3 channels mediate oligodendrocyte cell cycle activation and dedifferentiation

Voltage-gated K(+) channels vary in sensitivity to block by 4-aminopyridine (4-AP) over a 1000-fold range. Most K(+) channel phenotypes with leucine at the fourth position (L4) in the leucine heptad repeat region, spanning the S4-S5 linker, exhibit low 4-AP sensitivity, while channels with phenylalanine exhibit high sensitivity. Mutational analysis on delayed rectifier type K(+) channels demonstrate increased 4-AP sensitivity upon mutation of the L4 heptad leucine to phenylalanine. This mutation can also influence inactivation gating, which is known to compete with 4-AP in rapidly inactivating A-type K(+) channels. Here, in a rapidly inactivating human brain Kv1.4 channel, we demonstrate a 400-fold increase in 4-AP sensitivity following substitution of L4 with phenylalanine. Accompanying this mutation is a slowing of inactivation, an acceleration of deactivation, and depolarizing shifts in the voltage dependence of activation and steady-state inactivation. To test the relative role of fast inactivation in modulating 4-AP block, N-terminal deletions of the fast inactivation gate were carried out in both channels. These deletions produced no change in 4-AP sensitivity in the mutant channel and approximately a six-fold increase in the wild type channel. These results support the view that changes at L4 which increase 4-AP sensitivity are largely due to 4-AP binding and may, in part, arise from alterations in channel conformation. Primarily, this study demonstrates that the fast inactivation gate is not a critical determinant of 4-AP sensitivity in Kv1.4 channels.

Evaluation of combination gene therapy with PTEN and antisense hTERT for malignant glioma in vitro and xenografts

Voltage-gated potassium (K(v)) channels play an important role in the regulation of growth factor-induced cell proliferation. We have previously shown that cell cycle activation is induced in oligodendrocytes (OLGs) by complement C5b-9, but the role of K(v) channels in these cells had not been investigated. Differentiated OLGs were found to express K(v)1.4 channels, but little K(v)1.3. Exposure of OLGs to C5b-9 modulated K(v)1.3 functional channels and increased protein expression, whereas C5b6 had no effect. Pretreatment with the recombinant scorpion toxin rOsK-1, a highly selective K(v)1.3 inhibitor, blocked the expression of K(v)1.3 induced by C5b-9. rOsK-1 inhibited Akt phosphorylation and activation by C5b-9 but had no effect on ERK1 activation. These data strongly suggest a role for K(v)1.3 in controlling the Akt activation induced by C5b-9. Since Akt plays a major role in C5b-9-induced cell cycle activation, we also investigated the effect of inhibiting K(v)1.3 channels on DNA synthesis. rOsK-1 significantly inhibited the DNA synthesis induced by C5b-9 in OLG, indicating that K(v)1.3 plays an important role in the C5b-9-induced cell cycle. In addition, C5b-9-mediated myelin basic protein and proteolipid protein mRNA decay was completely abrogated by inhibition of K(v)1.3 expression. In the brains of multiple sclerosis patients, C5b-9 co-localized with NG2(+) OLG progenitor cells that expressed K(v)1.3 channels. Taken together, these data suggest that K(v)1.3 channels play an important role in controlling C5b-9-induced cell cycle activation and OLG dedifferentiation, both in vitro and in vivo.

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells

Abstract.Telomerase activation is a critical event in cell immortalization, and an increase in human telomerase reverse transcriptase (hTERT) expression is the key step in activating telomerase. The phosphatase and tensin homolog (PTEN) gene encodes a double-specific phosphatase that induces cell cycle arrest, inhibits cell growth, and causes apoptotic cell death. Here, we evaluated a combined PTEN and antisense hTERT gene therapy for experimental glioma in vitro and in vivo. We demonstrated that infection with antisense-hTERT and wild-type-PTEN adenoviruses significantly inhibited human U251 glioma cell proliferation in vitro and glioma growth in a xenograft mouse model. The efficacy of therapy was obviously higher in the tumor xenografts infected with both PTEN and antisense hTERT than in the gliomas infected with either agent alone at the same total viral dose. Consistent with these results, we showed that telomerase activity and hTERT protein levels were markedly reduced in the glioma cells following adenovirus infection. In contrast, the levels of PTEN protein expression were dramatically increased in these cells. Our data indicate that combination treatment with antisense hTERT and wild-type PTEN effectively suppresses the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach in glioma gene therapy that warrants further investigation.