Transport of the alpha subunit of the voltage gated Lâ type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138228/1/tra12502-sup-0001-EditorialProcess.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138228/2/tra12502.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138228/3/tra12502_am.pd

Analysis of the Dstac Gene, a Novel Regulator of Neuronal Function and Behavior in Drosophila Melanogaster

The stac genes encode a family of proteins with conserved CRD (cysteine-rich domain) and SH3 (Src Holomogy 3) domains found throughout the animal kingdom. stac1 and stac2 are expressed by subsets of neurons, and stac3 by skeletal muscles in vertebrates. One process regulated by a Stac protein is excitation-contraction (EC) in vertebrate skeletal muscles. EC coupling is the process that transduces changes in muscle membrane potential to increases in cytosolic Ca2+ that initiate muscle contraction. EC coupling is mediated by the interaction between the L-type voltage gated calcium channel (Cav channel), DHPR, in the transverse tubules (T tubules) and the ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). Studies from the Kuwada lab demonstrated that Stac3 regulates EC coupling by regulating the stability, organization and voltage dependency of L-type Cav channel in vertebrate skeletal muscles. The goal of this dissertation is to understand the function of stac genes expressed by neurons, which was completely unknown. Towards this goal, we identified the stac gene in Drosophila melanogaster (Dstac) in order to take advantage of its unparalleled genetic toolbox and found that Dstac is expressed both by muscles and by specific classes of neurons. We first investigated the muscle function of Dstac. We found that Dmca1D, the sole Drosophila L-type Cav channel, and Dstac were expressed in stripes within muscles. Knocking down Dmca1D or Dstac selectively in larval Drosophila body-wall muscles reduced Ca2+ transients during locomotion. Furthermore, immunolabeling showed decreased Dmca1D levels in Dstac mutant muscles, showing that Dstac regulates L-type Cav channels as does Stac3 in vertebrate skeletal muscles. These results suggest that muscle Dstac regulates Dmca1D, which induces cytosolic Ca2+ increases for proper EC coupling in Drosophila body-wall muscles. In the Drosophila adult brain, we found that a set of clock neurons that releases the neuropeptide, pigment dispersing factor (PDF), to regulate circadian rhythm expresses Dstac as well as Dmca1D. Interestingly, selective knockdown of Dstac in PDF neurons disrupted circadian activity. This was the first neural function identified for a Stac protein. The results suggested the hypothesis that Dstac might regulate the Dmca1D L-type Cav channel and this in turn regulates the release of PDF for normal circadian rhythm. We found that Dstac is also expressed by a subset of neurons including motor neurons in the larval CNS. We examined whether Dstac might regulate Dmca1D and neuropeptide release at the larval neuromuscular junction to take advantage of the accessibility of motor boutons for cellular and physiological analysis. We found that Dstac, Dmca1D and the proctolin neuropeptide are expressed by motor boutons. Previously it was shown that proctolin enhances muscle contractions in various insects including Drosophila. By a combination of immunolabeling, Ca2+ imaging, electrophysiology, live imaging of neuropeptide release and behavioral analysis in genetically manipulated larvae we found that Dstac regulates the voltage response of Dmca1D channels and the release of neuropeptides from motor boutons to regulate locomotion by larvae. Since Dstac is expressed by other neuropeptide containing neurons, including the PDF+ clock neurons, in the CNS our results may be applicable to other neurons in the Drosophila CNS. Furthermore the expression of Stac1 and Stac2 in neurons in the vertebrate nervous system opens the intriguing possibility that these Stac proteins may also regulate the release of neuropeptides in at least some vertebrate neurons.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163291/1/iuenhsu_1.pd

Acrolein adducts and responding autoantibodies correlate with metabolic disturbance in Alzheimer’s disease

Abstract Background Alzheimer’s disease (AD) is caused by many intertwining pathologies involving metabolic aberrations. Patients with metabolic syndrome (MetS) generally show hyperglycemia and dyslipidemia, which can lead to the formation of aldehydic adducts such as acrolein on peptides in the brain and blood. However, the pathogenesis from MetS to AD remains elusive. Methods An AD cell model expressing Swedish and Indiana amyloid precursor protein (APP-Swe/Ind) in neuro-2a cells and a 3xTg-AD mouse model were used. Human serum samples (142 control and 117 AD) and related clinical data were collected. Due to the involvement of MetS in AD, human samples were grouped into healthy control (HC), MetS-like, AD with normal metabolism (AD-N), and AD with metabolic disturbance (AD-M). APP, amyloid-beta (Aß), and acrolein adducts in the samples were analyzed using immunofluorescent microscopy, histochemistry, immunoprecipitation, immunoblotting, and/or ELISA. Synthetic Aß1-16 and Aß17-28 peptides were modified with acrolein in vitro and verified using LC–MS/MS. Native and acrolein-modified Aß peptides were used to measure the levels of specific autoantibodies IgG and IgM in the serum. The correlations and diagnostic power of potential biomarkers were evaluated. Results An increased level of acrolein adducts was detected in the AD model cells. Furthermore, acrolein adducts were observed on APP C-terminal fragments (APP-CTFs) containing Aß in 3xTg-AD mouse serum, brain lysates, and human serum. The level of acrolein adducts was correlated positively with fasting glucose and triglycerides and negatively with high-density lipoprotein-cholesterol, which correspond with MetS conditions. Among the four groups of human samples, the level of acrolein adducts was largely increased only in AD-M compared to all other groups. Notably, anti-acrolein-Aß autoantibodies, especially IgM, were largely reduced in AD-M compared to the MetS group, suggesting that the specific antibodies against acrolein adducts may be depleted during pathogenesis from MetS to AD. Conclusions Metabolic disturbance may induce acrolein adduction, however, neutralized by responding autoantibodies. AD may be developed from MetS when these autoantibodies are depleted. Acrolein adducts and the responding autoantibodies may be potential biomarkers for not only diagnosis but also immunotherapy of AD, especially in complication with MetS

Huntingtin-Associated Protein 1 Interacts with Breakpoint Cluster Region Protein to Regulate Neuronal Differentiation

<div><p>Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from <i>Hap1</i>-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in <i>Hap1</i>-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.</p></div

Co-localization of Bcr and Hap1 in differentiated neuro-2A cells.

<p>(<b>A</b>) Confocal imaging of differentiated neuro-2a cells transfected with GFP-Hap1A or GFP-Hap1B (green) with Bcr-myc (red). Scale bar, 10 μm. (<b>B</b>) Statistical analysis showing the percentage of GFP, GFP-Hap1A and GFP-Hap1B co-localized with Bcr in differentiated cells. Five or more sets of image were analyzed. * p < 0.05; ** p < 0.01; *** p < 0.001.</p

Association of Bcr with Hap1 on microtubules in mouse brains.

<p>(<b>A</b>) The relative amount of a microtubule subunit (β-tubulin), a small GTPase (RhoA) and a GAP/GEF (Bcr) in wild-type (WT) and <i>Hap1</i>-null newborn mouse brains normalized to isotope-labeled counterparts in wild type adult mouse brain. (<b>B</b>) Western blotting showing Hap1 isoforms, Bcr, RhoA and α-Tubulin in microtubule pellets precipitated by taxol-GTP treatment from <i>Hap1</i>-null and WT mouse brains. Input is the supernatant of brain lysates after centrifugation at 18,000 × g for 20 min (S2). The nonspecific protein species reacting to Hap1 antibody in the inputs (arrow) was not precipitated with microtubules.</p

Lack of Hap1 inhibits Bcr signaling in mouse hypothalamus.

<p>(<b>A</b>) Western blotting analysis of Bcr and downstream signaling molecules including p38, ERK1/2, PAK, JNK and their phosphorylated forms from the WT and <i>Hap1</i>-null mouse hypothalamic (left panel) and non-hypothalamic regions (right panel). (<b>B</b>) Quantitative and statistical analysis of the changes of Bcr and its downstream signaling molecules in the hypothalamic and non-hypothalamic regions. The presented value was the ratio of the phosphorylated protein level to the total protein level and normalized with the result in WT mouse hypothalamic or non-hypothalamic region, which was set as 1. Three independent experiments were performed for statistical analysis. * p < 0.05; ** p < 0.01.</p