Britta Bartelt-Kirbach

The growing world of small heat shock proteins : From structure to functions

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world’s experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12–15, 2016).

Erratum to: Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons

Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability. We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.

Phosphorylation-dependent subcellular localization of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in cultured hippocampal neurons

The so-called stress response involving upregulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia or oxidative stress. To gain more information about the molecular targets by which HspB1 (Hsp25) and HspB5 (αB-crystallin) might exert their neuroprotective effect we investigated the subcellular localization of unphosphorylated and phosphorylated HspB1 and B5 in neurons by immunocytochemistry and subcellular fractionation. In cultured hippocampal neurons, the unphosphorylated forms of both Hsps were localized in the perikaryon and nucleus, whereas the phosphorylated forms were recruited into neuronal processes. pHspB1-Ser15 and -Ser 86 were found within dendrites with a punctate distribution pattern partially colocalizing with the synaptic marker vGlut-1. pHspB5-Ser19 and -Ser45 localized to axons and dendrites with a filamentous-like staining pattern, whereas pHspB5-Ser59 was found in dendrites, especially along the plasma membrane and in spines. Biochemical analysis, i.e. subcellular fractionation of rat brain with subsequent Western blotting supported these localizations. These data show that in neurons HspB1 and B5 may have various molecular interaction partners at synapses, within dendrites and axons and that this interaction is likely to be regulated by phosphorylation. Stress-induced phosphorylation of HspB1 and B5 may lead to binding of these Hsps to their targets at synapses and neuronal processes which might provide one important mechanism of how they exert their neuroprotective effect.

Sensitive Detection of Deletions of One or More Exons in the Neurofibromatosis Type 2 (NF2) Gene by Multiplexed Gene Dosage Polymerase Chain Reaction

Mutation detection in the neurofibromatosis type 2 (NF2) gene is challenging because when combining mutation detection methods such as single-strand conformational polymorphism and heteroduplex analysis, denaturing gradient gel electrophoresis, and direct sequencing of aberrant polymerase chain reaction (PCR) fragments only 30 to 60% of the constitutional mutations are detected. Because large deletions and complete chromosome rearrangements are also described methods such as microarray-comparative genomic hybridization and fluorescence in situ hybridization are also used. The one type of mutation often missed corresponds to deletions encompassing one or few exons. To detect this type we have developed a swift and reliable method. We perform a gene dosage analysis with two fluorescent multiplex PCR assays that amplify 15 of the 17 NF2 exons. The labeled PCR products are quantified and gene dose is calculated with respect to controls. We tested the reliability of this method with DNA from eight NF2 patients with known heterozygous NF2 deletions, eight controls and four unknown NF2 patients. In all of the patients with known heterozygous deletions we found in several exons a reduction of gene dosage to 50 to 69%. In one NF2 patient with previously unknown mutation and a severe phenotype we found the gene dosage of two exons reduced by 50% indicating a deletion of these two exons on one allele. This finding was validated by reverse transcriptase-PCR on fibroblast and schwannoma cell cultures of this patient and cDNA sequencing. Our gene dosage assay will detect deletions of one or more exons as well as gross deletions of the whole coding region of the gene. It can complement the existing screening methods because it is faster and easier.

Different Regulation of N-Cadherin and Cadherin-11 in Rat Hippocampus

Abstract Cadherin-mediated specific cell adhesion is an important process in brain development as well as in synaptic plasticity in the adult brain. In this study the authors quantified mRNA levels of N-cadherin and cadherin-11 in different brain regions for the first time. In hippocampus N-cadherin mRNA levels were very high at embryonic stages and decreased during further development, whereas cadherin-11 mRNA levels were highest at postnatal stages. However, N-cadherin protein level was not altered during hippocampal development and cadherin-11 protein was low at embryonic but high at postnatal and adult stages. In cultured hippocampal neurons both cadherins became colocalized and recruited to synaptic sites during ongoing differentiation, with especially high accumulation of cadherin-11 at synapses. These data hint at a critical role of N-cadherin at early embryonic stages and early synaptogenesis, whereas cadherin-11 might be more important for further stabilization of synapses in the postnatal period and adulthood.

HspB5/αB-crystallin increases dendritic complexity and protects the dendritic arbor during heat shock in cultured rat hippocampal neurons.

The small heat shock protein ΗspΒ5 (αB-crystallin) exhibits generally cytoprotective functions and possesses powerful neuroprotective capacity in the brain. However, little is known about the mode of action of ΗspΒ5 or other members of the HspB family particularly in neurons. To get clues of the neuronal function of HspBs, we overexpressed several HspBs in cultured rat hippocampal neurons and investigated their effect on neuronal morphology and stress resistance. Whereas axon length and synapse density were not affected by any HspB, dendritic complexity was enhanced by HspB5 and, to a lesser extent, by HspB6. Furthermore, we could show that this process was dependent on phosphorylation, since a non-phosphorylatable mutant of HspB5 did not show this effect. Rarefaction of the dendritic arbor is one hallmark of several neurodegenerative diseases. To investigate if HspB5, which is upregulated at pathophysiological conditions, might be able to protect dendrites during such situations, we exposed HspB5 overexpressing neuronal cultures to heat shock. HspB5 prevented heat shock-induced rarefaction of dendrites. In conclusion, we identified regulation of dendritic complexity as a new function of HspB5 in hippocampal neurons.

Induction and phosphorylation of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in the rat retina upon optic nerve injury

Several eye diseases are associated with axonal injury in the optic nerve, which normally leads to degeneration of retinal ganglion cells (RGCs) and subsequently to loss of vision. There is experimental evidence that some members of the small heat shock protein family (HspBs) are upregulated upon optic nerve injury (ONI) in the retina and sufficient to promote RGC survival. These data raise the question as to whether other family members may play a similar role in this context. Here, we performed a comprehensive comparative study comprising all HspBs in an experimental model of ONI. We found that five HspBs were expressed in the adult rat retina at control conditions but only HspB1 and HspB5 were upregulated in response to ONI. Furthermore, HspB1 and HspB5 were constitutively phosphorylated in Müller cells at serine 15 and serine 59, respectively. In RGCs, phosphorylation was stimulated by ONI and occurred at serine 86 of HspB1 and at serine 19 and 45 of HspB5. These data suggest that of all small heat shock proteins, only HspB1 and HspB5 might be of protective value for RGCs after ONI and that this process might be regulated by phosphorylation at serine 86 of HspB1 and serine 19 and serine 45 of HspB5. The molecular targets of phosphoHspB1 and phosphoHspB5 remain to be identified.

Water transport through the intestinal epithelial barrier under different osmotic conditions is dependent on LI-cadherin trans-interaction

ABSTRACT In the intestine water has to be reabsorbed from the chymus across the intestinal epithelium. The osmolarity within the lumen is subjected to high variations meaning that water transport often has to take place against osmotic gradients. It has been hypothesized that LI-cadherin is important in this process by keeping the intercellular cleft narrow facilitating the buildup of an osmotic gradient allowing water reabsorption. LI-cadherin is exceptional among the cadherin superfamily with respect to its localization along the lateral plasma membrane of epithelial cells being excluded from adherens junction. Furthermore it has 7 but not 5 extracellular cadherin repeats (EC1-EC7) and a small cytosolic domain. In this study we identified the peptide VAALD as an inhibitor of LI-cadherin trans-interaction by modeling the structure of LI-cadherin and comparison with the known adhesive interfaces of E-cadherin. This inhibitory peptide was used to measure LI-cadherin dependency of water transport through a monolayer of epithelial CACO2 cells under various osmotic conditions. If LI-cadherin trans-interaction was inhibited by use of the peptide, water transport from the luminal to the basolateral side was impaired and even reversed in the case of hypertonic conditions whereas no effect could be observed at isotonic conditions. These data are in line with a recently published model predicting LI-cadherin to keep the width of the lateral intercellular cleft small. In this narrow cleft a high osmolarity can be achieved due to ion pumps yielding a standing osmotic gradient allowing water absorption from the gut even if the faeces is highly hypertonic.

The Impact of Small Heat Shock Proteins (HspBs) in Alzheimer's and Other Neurological Diseases.

BACKGROUND Heat shock proteins are powerful endogenous cytoprotective proteins which help cells to survive recurrent cellular stress events. Identifying the underlying molecular mechanisms and molecular targets is especially interesting since it may help to develop new therapeutic strategies for the treatment of diseases. OBJECTIVE This review will focus on the group of small heat shock proteins, also named HspBs. HspBs play an important role in various neurological diseases. Most neurodegenerative diseases are characterized by a distinct pathology with accumulation and aggregation of misfolded proteins, such as deposits of amyloid plaques or neurofibrillary tangles in Alzheimer`s disease. Such pathological protein aggregates are thought to lead to cellular dysfunction and finally to cell death. HspBs display chaperone-like functions and are able to prevent protein aggregation by which they may slow down progression of these diseases. However, HspBs have multiple additional functions which also may contribute to neuroprotection. RESULTS/CONCLUSIONS In this review we will first give an overview of the HspB protein family, their structure, functions and expression pattern. Then we will highlight their impact in the brain, in neurodegenerative diseases and especially in Alzheimer`s disease and try to unravel their multifactorial effects in several aspects of the disease pathologies.

HspB5/αB-Crystallin in the Brain

Many organs including the brain exhibit powerful endogenous cytoprotective mechanisms to survive recurrent cellular stress events, i.e. the development of stress tolerance. Investigation of the molecular mechanisms underlying this neuroprotective phenomenon is of special interest since it may provide the basis to develop new therapeutic strategies for the treatment of neurological diseases. One important mechanism is the upregulation of heat shock proteins. Here, we will review the neuroprotective potential of HspB5/αB-crystallin. HspB5 is expressed in glia as well as in neurons and upregulated in certain cell types or subset of cells at pathophysiological conditions. HspB5 is found to be associated with the disease-causing pathological protein aggregates, such as amyloid plaques in Alzheimer’s disease or Rosenthal fibers in Alexander disease. One possible function of HspB5 is to counteract the aggregation process leading to increased cell survival. However, HspB5 may act additionally via its non-chaperone functions, such as anti-inflammatory, anti-apoptotic properties or association with cytoskeletal proteins influencing filament assembly. The cytoprotective activity of HspB5 is regulated by phosphorylation. Interestingly, in neurons HspB5 is recruited to axons and dendrites by phosphorylation, however, to this end little is known about the molecular targets of phosphoHspB5 in neurons. Identifying the impact of phosphorylation of HspB5 in glia and neurons and the targets of HspB5 may be useful to develop new therapeutic strategies for neurological diseases.