James A. Hammarback

Autophagy Is Activated for Cell Survival after Endoplasmic Reticulum Stress

ABSTRACT Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3“ dots”), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

Gene localization and developmental expression of light chain 3: a common subunit of microtubule-associated protein 1A(MAP1A) and MAP1B.

Microtubule‐associated proteins 1A (MAP1A) and MAP1B are abundant neuronal MAPs thought to be involved in neurite formation and stabilization. The relative levels of MAP1A and MAP1B change dramatically during development, with MAP1B expression highest in forming neurons, and MAP1A expression highest in mature neurons. We examined the expression of light chain 3 (LC3), a subunit of both MAP1A and MAP1B, to see if its expression paralleled that of either heavy chain. Anti‐LC3 immunohistochemistry reveals that LC3 in rat brain is restricted to neurons that are expressing either the MAP1A or MAP1B heavy chain. Although LC3 is expressed exclusively in cells expressing heavy chains, developmental changes in the total amount of LC3 protein are not proportional to changes in the amount of either the MAP1A or MAP1B heavy chain. LC3 protein expression measured by quantitative immunoblotting is twice as high in postnatal brain as in embryonic and adult brain. The localization of the LC3 gene to human chromosome 20cen‐q13 demonstrates that LC3 is the only MAP1 subunit that is not linked to the heavy‐chain genes. Because LC3 is a component of both the MAP1A and MAP1B microtubule‐binding domains, the heavy‐chain‐independent regulation of LC3 expression might modify MAP1 microtubule‐binding activity during development.

Expression of Microtubule-Associated Protein 2 in Benign and Malignant Melanocytes : Implications for Differentiation and Progression of Cutaneous Melanoma

Cutaneous melanocytic neoplasms are known to acquire variable characteristics of neural crest differentiation. Melanocytic nevus cells in the dermis and desmoplastic melanomas often display characteristics of nerve sheath differentiation. The extent and nature of neuronal differentiation characteristics displayed by primary and metastatic melanoma cells are not well understood. Here, we describe induction of a juvenile isoform of microtubule-associated protein 2 (MAP-2c) in cultured metastatic melanoma cells by the differentiation inducer hexamethylene bisacetamide. Up-regulation of this MAP-2 isoform, a marker for immature neurons, is accompanied by extended dendritic morphology and down-regulation of tyrosinase-related protein 1 (TYRP1/gp75), a melanocyte differentiation marker. In a panel of cell lines that represent melanoma tumor progression, MAP-2c mRNA and the corresponding approximately 70-kd protein could be detected predominantly in primary melanomas. Immunohistochemical analysis of 61 benign and malignant melanocytic lesions showed abundant expression of MAP-2 protein in melanocytic nevi and in the in situ and invasive components of primary melanoma, but only focal heterogeneous expression in a few metastatic melanomas. In contrast, MAP-2-positive dermal nevus cells and the invasive cells of primary melanomas were TYRP1-negative. This reciprocal staining pattern in vivo is similar to the in vitro observation that induction of the neuronal marker MAP-2 in metastatic melanoma cells is accompanied by selective extinction of the melanocytic marker TYRP1. Our data show that neoplastic melanocytes, particularly at early stages, retain the plasticity to express the neuron-specific marker MAP-2. These observations are consistent with the premise that both benign and malignant melanocytes in the dermis can express markers of neuronal differentiation.

Diurnal gene expression patterns of T-type calcium channels and their modulation by ethanol

The transient (T-type) calcium channel participates in the generation of normal brain rhythms as well as abnormal rhythms associated with a range of neurological disorders. There are three different isoforms of T-type channels and all are particularly enriched in the thalamus, which is involved in generating many of these rhythms. We report a novel means of T-type channel regulation in the thalamus that involves diurnal regulation of gene expression. Using real time polymerase chain reaction we detected a diurnal pattern of gene expression for all T-type channel transcripts. The peak of gene expression for the CaV3.1 transcript occurred close to the transition from active to inactive (sleep) states, while expression for both CaV3.2 and CaV3.3 peaked near the transition of inactive to active phase. We assessed the effect of chronic consumption of ethanol on these gene expression patterns by examining thalamic tissues of ethanol-consuming cohorts that were housed with the controls, but which received ethanol in the form of a liquid diet. Ethanol consumption resulted in a significant shift of peak gene expression of approximately 5 h for CaV3.2 toward the normally active phase of the mice, as well as increasing the overall gene expression levels by approximately 1.7-fold. Peak gene expression was significantly increased for both CaV3.2 and CaV3.3. Measurements of CaV3.3 protein expression reflected increases in gene expression due to ethanol. Our results illustrate a novel regulatory mechanism for T-type calcium channels that is consistent with their important role in generating thalamocortical sleep rhythms, and suggests that alterations in the pattern of gene expression of these channels could contribute to the disruption of normal sleep by ethanol.

Microtubule-associated protein 1 subunit expression in primary cultures of rat brain

Microtubule-associated protein 1A (MAP1A) and MAP1B are developmentally regulated proteins linked to axon formation. They each consist of a unique heavy chain and three common light chains. We used immunofluorescence microscopy to qualitatively assess the variability in MAP1 subunit expression between individual cells. The ratio of light chain 1 to MAP1 heavy chain varies greatly between cells with some cells expressing MAP1A heavy chain in the apparent absence of light chain 1. The results imply the existence of MAP1 molecules that differ in light chain composition. Transfection experiments indicate that the light chains differ in microtubule binding activity and subcellular targeting activity. This further suggests that the regulation of MAP1 light chain content can control MAP1 function.

Ethanol Inhibition of a T‐Type Ca2+ Channel Through Activity of Protein Kinase C

BACKGROUND T-type calcium channels (T-channels) are widely distributed in the central and peripheral nervous system, where they mediate calcium entry and regulate the intrinsic excitability of neurons. T-channels are dysregulated in response to alcohol administration and withdrawal. We therefore investigated acute ethanol (EtOH) effects and the underlying mechanism of action in human embryonic kidney (HEK) 293 cell lines, as well as effects on native currents recorded from dorsal root ganglion (DRG) neurons cultured from Long-Evans rats. METHODS Whole-cell voltage-clamp recordings were performed at 32 to 34°C in both HEK cell lines and DRG neurons. The recordings were taken after a 10-minute application of EtOH or protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate [PMA]). RESULTS We recorded T-type Ca²⁺ currents (T-currents) from 3 channel isoforms (CaV3.1, CaV3.2, and CaV3.3) before and during administration of EtOH. We found that only 1 isoform, CaV3.2, was significantly affected by EtOH. EtOH reduced current density as well as producing a hyperpolarizing shift in steady-state inactivation of both CaV3.2 currents from HEK 293 cell lines and in native T-currents from DRG neurons that are known to be enriched in CaV3.2. A myristoylated PKC peptide inhibitor (MPI) blocked the major EtOH effects, in both the cell lines and the DRG neurons. However, PMA effects were more complex. Lower concentration PMA (100 nM) replicated the major effects of EtOH, while higher concentration PMA (1 μM) did not, suggesting that the EtOH effects operate through activation of PKC and were mimicked by lower concentration of PMA. CONCLUSIONS EtOH primarily affects the CaV3.2 isoform of T-type Ca²⁺ channels acting through PKC, highlighting a novel target and mechanism for EtOH effects on excitable membranes.