Una FitzGerald

Caspase‐12 and ER‐Stress‐Mediated Apoptosis

Abstract: The labyrinth of the endoplasmic reticulum (ER) interweaves the cytosol and connects to the nucleus, mitochondria, and the plasma membrane. In the lumen of the ER, the essential function of lipid synthesis, Ca2+ storage, folding, and maturation of proteins take place. Therefore, the tight regulation and maintenance of ER homeostasis is vital. Disturbance of the Ca2+ homeostasis during hypoxia, or imbalance between the demand and capacity of the protein‐folding apparatus, initiates an adaptive response of the cell, termed the unfolded protein response (UPR, ER stress response). As a result, ER‐localized chaperones are induced, protein synthesis is slowed down, and a protein degrading system is initiated. However, if the ER stress cannot be alleviated, it culminates in apoptosis. This paper reviews the newly outlined signaling pathways of the unfolded protein response and describes the central role of caspase‐12 in the initiation of cell death. The complex role of the ER and its signaling pathways provides a novel angle on apoptosis research and may offer a key to apoptosis‐associated diseases.

Methods for Monitoring Endoplasmic Reticulum Stress and the Unfolded Protein Response

The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

Identification of growth factors that promote long-term proliferation of olfactory ensheathing cells and modulate their antigenic phenotype

Olfactory ensheathing cells can develop into distinct subtypes in culture after incubation in serum‐free medium conditioned by astrocytes, which have Schwann cell–like and astrocyte‐like properties. It has not been possible so far to modulate and grow large numbers of these olfactory ensheathing cell subtypes. In this study, we have shown that astrocyte‐conditioned medium, although promoting differentiation of the two olfactory ensheathing cell types, is growth‐restrictive after 14 days, probably due to the upregulation of p16 and p27. Growth arrest can be overridden and cells maintained for a further 11 weeks, by a mitogen mix of fibroblast growth factor 2, forskolin, and heregulin (olfactory mitogen medium) combined with astrocyte‐conditioned medium. In the absence of astrocyte‐conditioned medium, combinations of the same factors can also override growth arrest but to a lesser extent. Olfactory mitogen medium combined with astrocyte‐conditioned medium upregulates O4 and low‐affinity nerve growth factor receptor expression on olfactory ensheathing cells, leading to a 100% Schwann cell–like phenotype. If cells are maintained in olfactory mitogen medium alone, or if they are treated with forskolin or fibroblast growth factor 2 diluted in serum‐free medium, O4 and low‐affinity nerve growth factor receptor expression remains at 100%, but there is also an increase in expression of E‐NCAM, the astrocyte‐like marker. Medium containing serum also overrides growth arrest, but for only 4 weeks, during which time most differentiation‐specific markers disappear. These studies have allowed us to define conditions to modulate the olfactory ensheathing cell phenotype. GLIA 37:349–364, 2002.

Absence of aquaporin-4 expression in lesions of neuromyelitis optica but increased expression in multiple sclerosis lesions and normal-appearing white matter

Aquaporin-4 (AQP4) has recently been implicated in the pathogenesis of neuromyelitis optica (NMO) where it has been identified as the first defined autoantigen pertinent to an inflammatory demyelinating disorder of the human CNS. Furthermore, a recent case report has shown a lack of AQP4 expression in the spinal cord lesions of NMO. However, the pattern of AQP4 expression in multiple sclerosis (MS) tissues has not been well-defined. In the present investigation we have confirmed a lack of expression of AQP4 in optic and spinal cord lesions in NMO which contrasted sharply with the increased levels of AQP4 expression seen in MS lesions. Furthermore a detailed immunohistochemical and semi-quantitative analysis is used to describe the expression pattern of AQP4 on well-characterized tissue microarray samples of MS and control white matter. Anatomically AQP4 was more highly expressed in all categories of MS tissue compared to normal control tissues with the most abundant expression in active lesions. Within active lesions AQP4 expression was significantly correlated with expression of the pro-inflammatory cytokine osteopontin. At the cellular level dual-labeling immunofluoresence demonstrated that increased expression of AQP4 was most pronounced at the astrocytic endfeet but was also associated with the cell bodies of astrocytes in the tissue parenchyma. The finding of increased AQP4 expression in MS lesions in contrast to the lack of expression in NMO lesions may suggest different mechanisms of initiation and progression between the two disease states.

Increased expression of endoplasmic reticulum stress-related signaling pathway molecules in multiple sclerosis lesions

Activation of endoplasmic reticulum (ER) stress-related cell signals has been reported in several neurologic disorders and may contribute to neurodegeneration. Endoplasmic reticulum stress is also linked to ischemic injury. However, activation of an ER stress response has not been investigated in multiple sclerosis (MS) lesions. We detected increased expression of ER stress-associated C/EBP homologous protein, immunoglobulin heavy chain-binding protein, and X-box-binding protein 1 in multiple cell types, including oligodendrocytes, astrocytes, T cells, and microglia in active MS lesions. Semiquantitative analysis of expression in active, chronic active, and chronic inactive lesions indicated that levels of immunoglobulin heavy chain-binding protein were significantly higher in acute lesions than in non-MS controls or MS normal-appearing white matter, and that ER stress-associated C/EBP homologous protein was upregulated to the greatest extent at the edges of chronic active lesions. Because demyelination may be triggered by a tissue response to ischemia-like conditions, changes in the hypoxia-related antigen D-110 were also investigated, and it was found that increased ER stress-associated C/EBP homologous protein expression can occur in either the presence or absence of D-110. A possible link between a perturbed ER and lesion development in MS suggests a signaling pathway that may represent a new therapeutic target in MS.

InterfERing with endoplasmic reticulum stress

Stress to the endoplasmic reticulum (ER) is a recognized factor in Alzheimers and Parkinsons diseases, diabetes, heart disease, liver disorders and cancer. Thus, drugs that interfere with ER stress have wide therapeutic potential. Here we review the effects of drugs on three arms of ER stress: the protein kinase RNA-activated (PKR)-like ER kinase (PERK) arm, the activated transcription factor 6 (ATF6) arm and the inositol-requiring enzyme 1 (IRE1) arm. Drugs fall into five groups: (i) compounds directly binding to ER stress molecules; (ii) chemical chaperones; (iii) inhibitors of protein degradation; (iv) antioxidants; (v) drugs affecting calcium signaling. Treatments are generally inhibitory and lead to increased viability, except when applied to cancer cells. A focus on interfering with the ATF6 arm is required, and more in vivo testing of these compounds concurrently across all three arms is needed if the full importance of ER stress to human disease is to be realized.

The effects of blood–brain barrier disruption on glial cell function in multiple sclerosis

Dysfunction of the BBB (blood-brain barrier) is a major hallmark of MS (multiple sclerosis). Studies in our laboratories over the last decade have shown that increased BBB permeability is associated with decreased expression of TJ (tight junction) proteins in brain capillary endothelial cells. Results have revealed that TJ abnormalities were most common in active lesions (42% of vessels affected), but were also present in inactive lesions (23%) and in MS normal-appearing white matter (13%). Importantly, TJ abnormality was also positively associated with leakage of the serum protein fibrinogen which has recently been shown to be an activator of microglia. TJ abnormality and the resultant vascular permeability in both lesional and non-lesional white matter may impair tissue homoeostasis, which may have effects on disease progression, repair mechanisms and drug delivery.

Krp1, a novel kelch related protein that is involved in pseudopod elongation in transformed cells.

We have previously shown that the transcription factor AP-1 regulates the expression of genes which allow neoplastically transformed rat fibroblasts to become invasive. Searches for further AP-1 target genes led to the identification of a gene encoding a novel rat kelch family member, named kelch related protein 1 (Krp1). Kelch family members are characterized by a series of repeats at their carboxyl terminus and a BTB/POZ domain near their amino terminus. Rat Krp1 has a primarily cytoplasmic localization, and a small fraction appears to accumulate and co-localize with F-actin at membrane ruffle-like structures in the tips of pseudopodia. Overexpression of Krp1 in transformed rat fibroblasts led to the formation of dramatically elongated pseudopodia, while expression of truncated Krp1 polypeptides resulted in a reduction in the length of pseudopodia. We propose that the transformation-specific expression of Krp1 is required for pseudopod elongation, which are structures that are required for cell motility and invasion.

Expression profiles of endoplasmic reticulum stress-related molecules in demyelinating lesions and multiple sclerosis

Background: Increasing evidence associates the endoplasmic reticulum (ER) stress signalling pathway as a potential treatment target in multiple sclerosis (MS). Objective: To establish the expression profile of markers of ER stress both in demyelinating biopsy specimens and microdissected lesions in human post-mortem MS tissue. Methods: Immunohistochemical detection of C/EBP homologous protein (CHOP), immunoglobulin heavy chain binding protein (BiP), and hypoxia marker antigen D-110 in biopsies from three patients with MS primary or secondary progressive, three patients with clinically isolated syndrome, and one patient with lesional epilepsy was carried out. Laser capture microdissection of normal, perilesion and lesion tissue from post-mortem MS tissue and non-diseased control tissue was performed, followed by real-time PCR to detect ER stress genes. Results: In biopsy specimens, increased expression of the ER and hypoxic stress molecules in a range of cell types in most of the actively demyelinating lesions and perilesions was detected. Real-time PCR analysis demonstrated statistically significant elevated expression of the ER stress genes in normal-appearing white matter relative to control white matter. Moreover, significantly increased expression of CHOP was detected in the perilesion of active plaques (p < 0.01). Conclusions: Our results, showing detection of elevated expression of ER stress molecules in lesional tissue, offer compelling evidence for further investigation of the ER stress signalling pathway as a potential therapeutic target for the treatment of MS.

Increased expression of ER stress- and hypoxia-associated molecules in grey matter lesions in multiple sclerosis.

Background: The endoplasmic reticulum (ER) stress pathway may play a role in the pathogenesis multiple sclerosis (MS), and while ER stress-associated molecules have been demonstrated in white matter (WM) lesions, these have not been analysed in grey matter (GM) demyelination. Objective: The objective was to characterise the type and frequency of GM lesions and establish expression profiles of ER stress- and hypoxia-associated markers. Methods: Sections from 16 MS cases and 12 non-MS controls were stained for ER stress molecules (BiP and CHOP) and hypoxia-associated D110 antigen. Results: Of the GM lesions analysed, 24% were type 1 (continuous between GM and WM), 22% were type 2 (entirely within GM) and the majority (54%) were type 3 (extending from pia mater). Comparison of GM lesions, MS normal-appearing grey matter (NAGM) and non-MS control tissue showed that NAGM, type 1 and type 3 lesions all had significantly increased levels of CHOP compared to controls. According to morphological and dual-labelling criteria, the majority of CHOP-positive cells were microglia. Approximately 50% of GM lesions contained D110-positive cells. Conclusion: These data suggest that ER stress plays an important role in GM lesion development and may be critical in activation of microglia in pre-lesional NAGM. The high number of lesions containing D110-positive cells suggests a role for hypoxic-like insult in GM lesion development.