Role for calcium in heat shock-mediated synaptic thermoprotection in Drosophila larvae

Chemical synaptic transmission is the mechanism for fast, excitation-coupled information transfer between neurons. Previous work in larval Drosophila has shown that transmission at synaptic boutons is protected by heat shock exposure from subsequent thermal stress through pre- and postsynaptic modifications. This protective effect has been, at least partially, ascribed to an up-regulation in the inducible heat shock protein, hsp70. Effects of hsp70 are correlated with changes to intracellular calcium handling, and the dynamics of intracellular calcium regulate synaptic transmission. Consistent with such a relationship, synaptic plasticity increases at locust neuromuscular junctions following heat shock, suggesting an effect of heat shock on residual presynaptic calcium. Intracellular recording from single abdominal muscle fibers of Drosophila larvae showed that prior heat shock imparts thermoprotection by increasing the upper temperature limit for synaptic transmission. Heat shock exposure enhances short-term synaptic plasticity and increases its thermosensitivity. Increasing extracellular calcium levels eliminates the physiological differences between control and heat shock preparations; excess calcium itself induces thermoprotection at elevated concentrations. These data support the hypothesis that stress-induced neuroprotection at the nerve terminal acts, at least partially, through an alteration to the physiological effects of residual presynaptic calcium.     

Synaptic thermoprotection in a desert‐dwelling Drosophila species

Synaptic transmission is a critical mechanism for transferring information from the nervous system to the body. Environmental stress, such as extreme temperature, can disrupt synaptic transmission and result in death. Previous work on larval Drosophila has shown that prior heat‐shock exposure protects synaptic transmission against failure during subsequent thermal stress. This induced thermoprotection has been ascribed to an up‐regulation of the inducible heat‐shock protein, Hsp70. However, the mechanisms mediating natural thermoprotection in the wild are unknown. We compared synaptic thermosensitivity between D. melanogaster and a desert species, D. arizonae. Synaptic thermosensitivity and the functional limits of the related locomotor behavior differed significantly between closely related, albeit ecologically distinct species. Locomotory behavior of wandering third instar D. arizonae larvae was less thermosensitive and the upper temperature limit of locomotory function exceeded that of D. melanogaster by 6°C. Behavioral results corresponded with significantly lower synaptic thermosensitivity at the neuromuscular junction in D. arizonae. Prior heat‐shock protected only D. melanogaster by increasing relative excitatory junctional potential (EJP) duration, the time required for EJP failure at 40°C, and the incidence of EJP recovery following heat‐induced failure. Hsp70 induction profiles following heat‐shock demonstrate up‐regulation of inducible Hsp70 in D. melanogaster but not in D. arizonae. However, expression of Hsp70 under control conditions is greater in D. arizonae. These results suggest that the mechanisms of natural thermoprotection involve an increase in baseline Hsp70 expression. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Evolution of the Response to Heat Shock in Genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophilaspecies and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilisgroup exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogasterOregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters ofDrosophila and variation in the numbers of gene copies andhsp70 isoforms in group viriliswere found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genusDrosophilais discussed.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

A role for the cytoskeleton in heat‐shock–mediated thermoprotection of locust neuromuscular junctions

A prior hyperthermic stress (heat shock) can induce thermoprotection of neuromuscular transmission in Locusta migratoria extensor tibiae muscle measured 4 h after the onset of the heat shock. It is not clear what effect an acute hyperthermic stress may have on the nervous system's ability to tolerate thermal stress, that is, before increased expression of heat‐shock proteins. We found that over consecutive thermal stress tests, failure temperature was not altered in either heat‐shock or control animals. This suggests that protective mechanisms are not established in the short term (within one hour). Various members of the heat‐shock protein family interact with elements of the cytoskeleton. We found that preexposure of the preparation to cytoskeletal stabilizing drugs induced thermoprotection, while preexposure to cytoskeletal disrupting drugs disrupted the ability to confer and maintain thermoprotection. We conclude that thermoprotection relies on a stable cytoskeleton and suggest that members of the heat shock protein family are involved. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 453–462, 2004     

Factors affecting plasticity in whole-organism thermal tolerance in common killifish (<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>)

We characterized the degree of plasticity in thermal tolerance (assessed as critical thermal maxima; CTMax) and the relationship between thermal tolerance and underlying physiological and biochemical factors in two subspecies of a teleost fish, Fundulus heteroclitus. CTMax was not affected by repeated daily heat shock, but increased within a few days in response to warm acclimation. Loss of tolerance with acclimation to lowered temperatures occurred more slowly. Exposure to hypoxia decreased CTMax, and hyperoxia had no effect. CTMax showed a daily rhythm in both subspecies. Thermal acclimation changed the value of CTMax but did not affect the amplitude of the rhythm. Exposure to altered photoperiod had complex effects with a summer photoperiod producing a daily rhythm at higher CTMax than a spring photoperiod, and a winter photoperiod removing the rhythm. There was no daily rhythm in routine metabolic rate in either subspecies. There was no relationship between CTMax and the protein levels of the constitutive 70 and 90 kDa heat shock proteins (HSC70, HSP90β) in gill, or with mRNA levels of hsc70 in liver. There was a daily rhythm in the basal levels of the inducible hsp70-2 mRNA. Induction of hsp70-2 mRNA with mild heat shock occurred only in the evening and at night, and not during the day. These results demonstrate that there is substantial plasticity of thermal tolerance in killifish, and that this plasticity does not differ between subspecies. CTMax has a complex relationship with physiological and biochemical mechanisms that have been hypothesized to affect thermal tolerance.     

Structural features are as important as sequence homologies inDrosophila heat shock gene upstream regions

Summary Pelham has shown that theDrosophila hsp 70 gene is not transcribed under heat shock conditions unless a given upstream region is present. Davidson et al. have recently compiled a list of sequences homologous to this region in otherDrosophila heat shock genes. They proposed that a set of unlinked genes, such as the heat shock genes, could be coordinately induced through an interaction in cis with a common regulatory molecule. That this interaction involves structural elements is suggested by the fact that these upstream regions share inverted repeats as well as areas of Z-DNA potential. Furthermore, using the Calladine-Dickerson rules for local helical parameters, we show that these regions share structural homology. This is significant because the presence of regions homologous to a derived consensus sequence does not necessarily imply structural similarity. Therefore, we suggest that these structural features are at least as important as the sequence homologies in enabling the heat shock response.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

hsp70: Nuclear concentration during environmental stress and cytoplasmic storage during recovery

The intracellular distribution of the major Drosophila heat-shock protein hsp70 was determined by indirect immunofluorescence with monoclonal antibodies. During heat shock the protein concentrates strongly in nuclei while a small quantity remains cytoplasmic. During recovery hsp70 leaves the nuclei and becomes distributed throughout the cytoplasm. With a second heat shock it is rapidly transported back into the nucleus. Nuclear translocation depends not on the temperature per se, but on the physiological state of the cell since it also occurs after exposure to an anoxic atmosphere at normal temperatures. We also provide evidence that hsps protect cells from the toxic effects of anoxia, as well as heat, and conclude that nuclear translocation of hsp70 is related to its function in protecting the organism from both forms of environmental stress.     

Mitochondrial regulation of synaptic plasticity in the hippocampus

Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used an in vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity.     

Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal

We studied various aspects of heat‐shock response with special emphasis on the expression of heat‐shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5‐fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats.     

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos. Dev. Genet. 21:123–133, 1997. © 1997 Wiley-Liss, Inc.     

嗜热四膜虫五个hsp70基因的表达分析

鉴定得到嗜热四膜虫13个含有完整保守结构域的hsp70基因,对其中5个高度相似且无内含子的hsp70基因进行表达分析。在37、39和41℃热激条件下,实时荧光定量PCR结果表明,hsp70-2基因对热激响应最敏感。在四膜虫生长、饥饿和接合生殖这3种生理或发育状态下,Microarray结果显示,hsp70-4基因恒定且高表达;在热激条件下,hsp70-4基因的表达水平随着温度的升高而略微增加,证实hsp70-4基因为热休克相关蛋白hsc70基因;克隆的hsp70-4基因全长2208bp,开放阅读框长1959bp,编码653个氨基酸。Microarray结果提示,hsp70-3可能参与四膜虫饥饿早期(0～12h)的耐受和接合生殖后期(6～10h)的新大小核形成,老大核凋亡等事件;hsp70-5可能参与四膜虫饥饿晚期(12～15h)的耐受和接合生殖早期(0～6h)的小核减数分裂、小核交换和原核(pronuclear)融合事件。Blast2GO分析表明,与hsp70-3和hsp70-5共表达的基因分别参与不同的生物学过程,进一步反映了hsp70-3和hsp70-5这两个基因在功能上是存在差异的。     

Presynaptic effectors contributing to cAMP‐induced synaptic potentiation in Drosophila

cAMP analogs and activation of adenylyl cyclase by forskolin strongly potentiate synaptic transmission at the Drosophila neuromuscular junction. These effects are generally attributed to activation of cAMP‐dependent protein kinase. Recent reports on crustacean and mammalian synapses have implicated other cAMP‐dependent effectors in synaptic potentiation. Drosophila neuromuscular junctions were tested for effects of two known cAMP‐dependent effectors: hyperpolarization‐activated, cyclic nucleotide‐regulated channels (HCNCs) and guanine nucleotide exchange protein activated by cAMP (Epac). Forskolin‐induced enhancement of synaptic transmission was drastically reduced by a blocker of HCNCs, but not completely eliminated. A specific agonist for Epac modestly enhanced synaptic potentials. This agonist also stabilized their amplitudes in the presence of a blocker of HCNCs. The observations implicate HCNCs and Epac in cAMP‐dependent potentiation that does not require cAMP‐dependent protein kinase, indicating that additional previously unexplored factors contribute to synaptic plasticity in Drosophila. Genetic and molecular techniques available for Drosophila can be used to define the underlying molecular basis for cAMP‐dependent synaptic potentiation. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006