Michael M. Monaghan

KChIPs and Kv4 α Subunits as Integral Components of A-Type Potassium Channels in Mammalian Brain

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family α subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family α subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 α subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

Generation and Characterization of Subtype-specific Monoclonal Antibodies to K+ Channel α- and β-subunit Polypeptides

Abstract Molecular characterization of mammalian voltage-sensitive K + channel genes and their expression became possible with the cloning of the Shaker locus of Drosophila . However, analysis of the expression patterns and subunit composition of native K + channel protein complexes requires immunological probes specific for the individual K + channel gene products expressed in excitable tissue. Here, we describe the generation and characterization of monoclonal antibodies (mAbs) against eight distinct mammalian K + channel polypeptides; the Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 Shaker -related α-subunits, the Kv2.1 Shab -related α-subunit, and the kvβ1 and Kvβ2 β-subunits. We characterized the subtype-specificity of these mAbs against native K + channels in mammalian brain and against recombinant K + channels expressed in transfected mammalian cells. In addition, we used these mAbs to investigate the cellular and subcellular distribution of the corresponding polypeptides in rat cerebral cortex, as well as their expression levels across brain regions. Copyright

Amiloride is neuroprotective in an MPTP model of Parkinson's disease.

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drugs inhibition of central acid-sensing ion channels (ASICs). Given that Parkinsons disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinsons disease.

Inhibition of c-Jun kinase provides neuroprotection in a model of Alzheimer's disease.

The c-Jun N-terminal kinase (JNK) pathway potentially links together the three major pathological hallmarks of Alzheimers disease (AD): development of amyloid plaques, neurofibrillary tangles, and brain atrophy. As activation of the JNK pathway has been observed in amyloid models of AD in association with peri-plaque regions and neuritic dystrophy, as we confirm here for Tg2576/PS(M146L) transgenic mice, we directly tested whether JNK inhibition could provide neuroprotection in a novel brain slice model for amyloid precursor protein (APP)-induced neurodegeneration. We found that APP/amyloid beta (Abeta)-induced neurodegeneration is blocked by both small molecule and peptide inhibitors of JNK, and provide evidence that this neuroprotection occurs downstream of APP/Abeta production and processing. Our findings demonstrate that Abeta can induce neurodegeneration, at least in part, through the JNK pathway and suggest that inhibition of JNK may be of therapeutic utility in the treatment of AD.

Altered expression and localization of hippocampal A-type potassium channel subunits in the pilocarpine-induced model of temporal lobe epilepsy.

Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of 2-3 weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timms stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE.

Sensitive and selective liquid chromatography/tandem mass spectrometry methods for quantitative analysis of 1-methyl-4-phenyl pyridinium (MPP+) in mouse striatal tissue

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinsons disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP+), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP+ in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP+ in brain extracts with 0.4M perchloric acid (HClO4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP+ using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP+ established the limits of detection (LOD) (S/N=3) at 0.34pg/mg tissue and 0.007pg/mg tissue (5microl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D.<6%), and sensitive over a range of 0.001-1ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP+ concentrations in vivo. Two reference agents, R-(-) Deprenyl and MK-801, known to alter the concentration of MPP+ in MPTP treated mice were evaluated.

Social odor recognition: a novel behavioral model for cognitive dysfunction in Parkinson's disease.

Background: Parkinson’s disease (PD) is a progressive neurodegenerative condition characterized by an increasing loss of dopaminergic neurons resulting in motor dysfunction. However, cognitive impairments in PD patients are a common clinical feature that has gained increased attention. Objective: The purpose of the current study was to evaluate the effects of an MPTP-induced dopaminergic lesion in mice on social odor recognition (SOR) memory. Methods: Mice were acutely treated with MPTP and evaluated for memory impairments in the SOR assay and characterized using biochemical and immunohistochemical methods approximately 2 weeks later. Results: Here we demonstrate that SOR memory is sensitive to MPTP treatment and that it correlates with multiple measures of nigrostriatal integrity. MPTP treatment of C57BL/6N mice produced a profound decrease in dopamine levels, dopamine transporter binding and tyrosine hydroxylase immunoreactivity in the striatum. These impairments in stratial dopaminergic function were blocked by pretreatment with the MAO-B inhibitor deprenyl. Changes in the dopaminergic system parallel those observed in SOR with MPTP treatment impairing recognition memory in the absence of a deficit in odor discrimination during learning. Deprenyl pretreatment blocked the MPTP-induced impairment of SOR memory. Conclusion: The use of the SOR memory model may provide a preclinical method for evaluating cognitive therapies for PD.

P1-055: Impaired induction of neuronal immediate-early gene expression correlates with contextual memory deficits in APP transgenic mice

Background: Transgenic mouse models with neuronal expression of human amyloid precursor protein (APP) develop a range of Alzheimer’s disease (AD)-like alterations, including deposition of A and age-dependent deficits in learning and memory. The most popular hypothesis proposes that soluble, oligomeric forms of A mediate the memory deficits observed in APP transgenic animals prior to the formation of plaques. However, very little is known about the underlying molecular mechanisms leading to A mediated memory dysfunction. Methods: It has been known for some time that normal memory processes are associated with altered gene expression. This makes unraveling the molecular basis of memory dysfunction in AD ideally suited to transcriptional profiling. To that end, we have begun to characterize the molecular changes underlying early memory impairments in the Tg2576 mouse model of AD by analyzing the gene changes associated with memory formation in the brains of both wild-type and cognitively impaired Tg2576 mice. Results: In wild-type mice the formation of a strong contextual fear-related memory was associated with a robust induction (2-4 fold) of known immediate early genes (IEGs): c-fos, Jun-b, Erg1 and Nurr77, as well as the effector gene, Arc/Arg3.1 in both the amygdala and hippocampus. A number of genes not previously associated with memory formation were also significantly upregulated. None of these genes were induced in the Tg2576 implying that A antagonizes memory formation upstream of IEG induction. Conclusions: These data, together with recent reports by others, suggest a scenario where soluble A species impair neuronal signaling in key mechanisms related to memory formation. Many of the genes identified to be differentially regulated are downstream of NMDA receptor signaling, indicating the importance of these pathways in A induced memory impairments. We are currently examining the effect of A -directed therapeutics on reversing the memory and IEG deficits in the Tg2576 mice.