Neuronal excitability in Alzheimer's disease Alzheimers disease (AD), the most common form of dementia, is characterized by progressive neuronal loss, which eventually leads to death. Despite massive efforts over the last few decades, the etiology of AD is not well understood. A major challenge for AD research, and for the development of treatments, is that most AD patients are not diagnosed until neuronal function is irreversibly compromised. Therefore, it is crucial to identify neuronal changes at pre-clinical stages, which could provide a basis for early diagnosis and help to identify novel therapeutic targets. Neuronal hyperexcitability occurs early in the pathogenesis of AD and contributes to network dysfunction in AD patients. Although the Beta-amyloid (Ab) hypothesis suggests AD is caused by extracellular accumulation of insoluble Ab plaques, increasing evidence suggests that synaptic and memory impairments are mediated by soluble Ab. Here, in collaboration with the Roberson lab at the University of Alabama, Ben Throesch, tested the hypothesis that Ab- induced hyperexcitability originates in the dendrites. We found that dendrites, but not somata, of hippocampal neurons were hyperexcitable in mice adult mice overexpressing Aβ. This dendritic hyperexcitability was associated with selective depletion of Kv4.2, a dendritic potassium channel important in regulation of dendritic excitability and synaptic plasticity. In a separate project Eun Young Kim and Jakob Gutzmann investigated synaptic changes in young, 2-month-old transgenic mice that overexpress human amyloid precursor protein (hAPP). At this age, the mice do not exhibit Ab plaque accumulation but show increased soluble Ab levels compared to non-transgenic (NTG) mice. Our findings suggest NMDARs as a possible target for prevention or treatment for memory loss in early stage of AD. We are currently testing the effect of NMDAR subunit antagonists on the progression of AD at both the cellular and behavioral level. The role of DPP6 domain in its localization and function Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K+ channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Lin Lin found that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER. We showed recently (Lin et at., 2013) that DPP6 regulates the formation and stability of dendritic filopodia during early neuronal development, which is independent of Kv4.2. In order to identify additional DPP6 binding proteins, TAP purification approach was employed by Jiahhua Hu to isolate DPP6 protein complex in hippocampal neurons. Mass spectrometry analysis identified known proteins such as Kv4 family members and numerous novel synaptic proteins which Jiahua Hu and Jung Park are currently examining. Dendritic trafficking of voltage-gated calcium channels We are currently investigating the expression and trafficking of the voltage gated calcium channel Cav2.3. Cav2.3 is highly expressed in the dendrites of hippocampal and cortical neurons, where it is capable of generating large calcium spikes in response to both back-propagating action potentials and synaptic activity. Thus, alterations in Cav2.3 mRNA localization and translation could have a dramatic impact on cellular excitability and calcium signaling. Recent evidence suggests that Cav2.3 mRNA can be targeted by the Fragile-X mental retardation protein (FMRP), an mRNA binding protein that regulates translation in dendritic spines. Loss of FMRP results in Fragile X Syndrome, the most common form of inherited intellectual disability in humans. Thus, we are investigating the possibility that FMRP can regulate translation of Cav2.3, and will determine if this regulation may underlie aspects of Fragile X Syndrome. Toward this goal, Ying Liu performed real time PCR on mRNA isolated from the hippocampi or cortex of wild-type and FMRP-KO male mice, and examined the mRNA levels of several dendritic proteins. When comparing hippocampi from FMRP-KO and wild-type mice at 3- or 8-weeks of age, she found no significant difference in the mRNA levels of Cav2.3, Kv4.2, PSD-95, and HCN-1. She will further characterize this regulation by identifying FMRP binding sites on Cav2.3. In conjunction with these experiments, Ivan Trang is determining how FMRP affects Cav2.3 protein expression. From synaptoneurosomes isolated from mouse cortex, Ivan has found that Cav2.3 protein levels are reduced in FMRP-KO mice compared to wild-type mice at 3-weeks of age. In addition, primary neurons cultured from FMRP-KO mice show reduced surface Cav2.3 when compared to levels in wild-type mice. Thus, the loss of FMRP leads to a reduction in both synaptic and surface Cav2.3 protein. To determine how this might affect neuronal physiology, Erin Gray will record Cav2.3-mediated calcium currents as well as basic firing properties from wild-type and FMRP-KO neurons. While FMRP has a clear role in regulating mRNA stability, recent evidence suggests that FMRP may directly regulate the internalization and degradation of voltage gated calcium channels. Little is known about the pathways that underlie Cav2.3 degradation, thus Erin Gray and Joshua Lee have begun experiments aimed at better understanding this process. In heterologous cells overexpressing Cav2.3, Erin and Joshua have shown that Cav2.3 undergoes activity-dependent ubiquitination and degradation by the proteasome. Erin has begun to further investigate a possible role for ubiquitin-mediated alterations in surface levels of Cav2.3, and plans to perform a variety of electrophysiological recordings to determine the physiological consequences of this regulation. Co-regulation of HCN1 and Kv4.2 In CA1 pyramidal neuron dendrites, HCN channels, responsible for Ih, and Kv4.2 channels, responsible for IA, are critically important in signal processing and dendritic integration of synaptic inputs. Both channels show a similar pattern of distribution with an increased density from the soma to the apical dendrite. Using hippocampal primary cultured neurons, Emilie Campanac studied the potential co-regulation of HCN1 and Kv4.2. Results so far indicate reciprocal regulation with overexpression of Kv4.2 being associated with an increase in Ih current density without any change in sodium and calcium current while overexpression of HCN1 leads to an increased in IA current density. Pharmacological blockade of Ih with Cesium (2mM) induced a reduction in both current densities. Our data strongly suggest a homeostatic regulation between IA and Ih currents. We are currently investigating the molecular mechanism underlying this co-regulation.
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