The Hsp60-(p.V98I) mutation associated with hereditary spastic paraplegia SPG13 compromises chaperonin function both in vitro and in vivo

We have previously reported the association of a mutation (c.292G > A/p.V98I) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin with a dominantly inherited form of hereditary spastic paraplegia. Here, we show that the purified Hsp60-(p.V98I) chaperonin displays decreased ATPase activity and exhibits a strongly reduced capacity to promote folding of denatured malate dehydrogenase in vitro. To test its in vivo functions, we engineered a bacterial model system that lacks the endogenous chaperonin genes and harbors two plasmids carrying differentially inducible operons with human Hsp10 and wild-type Hsp60 or Hsp10 and Hsp60-(p.V98I), respectively. Ten hours after shutdown of the wild-type chaperonin operon and induction of the Hsp60-(p.V98I)/Hsp10 mutant operon, bacterial cell growth was strongly inhibited. No globally increased protein aggregation was observed, and microarray analyses showed that a number of genes involved in metabolic pathways, some of which are essential for robust aerobic growth, were strongly up-regulated in Hsp60-(p.V98I)-expressing bacteria, suggesting that the growth arrest was caused by defective folding of some essential proteins. Co-expression of Hsp60-(p.V98I) and wild-type Hsp60 exerted a dominant negative effect only when the chaperonin genes were expressed at relatively low levels. Based on our in vivo and in vitro data, we propose that the major effect of heterozygosity for the Hsp60-(p.V98I) mutation is a moderately decreased activity of chaperonin complexes composed of mixed wild-type and Hsp60-(p.V98I) mutant subunits.     

HSP60/10 chaperonin systems are inhibited by a variety of approved drugs,natural products,and known bioactive molecules

All living organisms contain a unique class of molecular chaperones called 60?kDa heat shock proteins (HSP60 – also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus – MRSA). Intriguingly, during our studies we found that three known antibiotics – suramin, closantel, and rafoxanide – were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.     

Protrudin Regulates Endoplasmic Reticulum Morphology and Function Associated with the Pathogenesis of Hereditary Spastic Paraplegia

Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.     

Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control individuals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with morpholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP.     

cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock

Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric proteins encoded by both nuclear and mitochondrial genes. We have isolated and sequenced full-length complementary DNAs coding for this mitochondrial chaperonin in Arabidopsis thaliana and Zea mays. Southern-blot analysis indicates the presence of a single hsp60 gene in the genome of A. thaliana. There is a high degree of homology at the predicted amino acid levels (43 to 60%) between plant HSP60s and their homologues in prokaryotes and other eukaryotes which indicates that these proteins must have similar evolutionarily conserved functions in all organisms. Northern- and western-blot analyses indicate that the expression of the hsp60 gene is developmentally regulated during seed germination. It is also heat-inducible. Developmental regulation of the (-subunit) of F₁-ATPase, an enzyme complex that is involved in the cyanide-sensitive mitochondrial electron transport system, indicates that imbibed embryos undergo rapid mitochondrial biogenesis through the early stages of germination. Based on the functional role of HSP60 in macromolecular assembly, these data collectively suggest that the presence of higher levels of HSP60 is necessary during active mitochondrial biogenesis, when the need for this protein is greatest in assisting the rapid assembly of the oligomeric protein structures.     

Expression of human 60-kD heat shock protein (HSP60 or P1) in Escherichia coli and the development and characterization of corresponding monoclonal antibodies.

Human P1 protein, which is the homolog of the 60- to 65-kD heat shock "common" antigenic protein of numerous pathogenic organisms (synonyms: HSP60, GroEL homolog, or chaperonin), has been expressed to high level in Escherichia coli cells. A large number of well-characterized deletions of this protein spanning the entire sequence have been constructed and expressed. Methods to purify recombinant human HSP60 protein and its deletions from E. coli have been worked out. In addition, monoclonal antibodies to the human HSP60 protein have been raised and partially characterized. The availability of these materials should greatly aid in understanding the role of this highly conserved and immunologically important protein in autoimmune diseases and in cell structure and function.     

Heat shock protein 60 modified with O-linked N-acetylglucosamine is involved in pancreatic beta-cell death under hyperglycemic conditions

The objective of this study was to identify proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) in pancreatic beta-cells and to understand their roles in cell death under hyperglycemic conditions. Here we report that heat shock protein 60 (HSP60) is modified with O-GlcNAc. Levels of O-GlcNAcylated HSP60 increased twofold in response to hyperglycemic conditions. HSP60 is a chaperonin known to bind to Bax in the cytoplasm under normoglycemic conditions. Under hyperglycemic conditions, Bax detached from O-GlcNAcylated HSP60 and translocated to mitochondria. Hyperglycemic conditions were also associated with cytochrome c release, caspase-3 activation, and cell death, suggesting that elevated O-GlcNAcylation of HSP60 interferes with HSP60-Bax interactions, leading to pancreatic beta-cell death.     

The Silver syndrome variant of hereditary spastic paraplegia maps to chromosome 11q12-q14, with evidence for genetic heterogeneity within this subtype

The hereditary spastic paraplegias (HSPs) are a complex group of neurodegenerative disorders characterized by lower-limb spasticity and weakness. Silver syndrome (SS) is a particularly disabling dominantly inherited form of HSP, complicated by amyotrophy of the hand muscles. Having excluded the multiple known HSP loci, we undertook a genomewide screen for linkage of SS in one large multigenerational family, which revealed evidence for linkage of the SS locus, which we have designated "SPG17," to chromosome 11q12-q14. Haplotype construction and analysis of recombination events permitted the minimal interval defining SPG17 to be refined to approximately 13 cM, flanked by markers D11S1765 and D11S4136. SS in a second family was not linked to SPG17, demonstrating further genetic heterogeneity in HSP, even within this clinically distinct subtype.     

Gender differences in the expression of heat shock proteins: the effect of estrogen

The heat shock proteins (HSPs) are an important family of endogenous, protective proteins that are found in all tissues. In the heart, HSP72, the inducible form of HSP70, has been the most intensely studied. It is well established that HSP72 is induced with ischemia and is cardioprotective. Overexpression of other HSPs also is protective against cardiac injury. Recently, we observed that 17beta-estradiol increases levels of HSPs in male rat cardiac myocytes. We hypothesized that there were gender differences in HSP72 expression in the heart secondary to estrogen. To test this hypothesis, we examined cardiac levels of HSP72 by ELISA in male and female Sprague-Dawley rats. In addition, three other HSPs were assessed by Western blot (HSP27, HSP60, and HSP90). To determine whether estrogen status affected HSP72 expression in other muscles or tissues, two other muscle tissues, slow twitch muscle (soleus muscle) and fast twitch muscle (gastrocnemius muscle), were studied as well as two other organs, the kidney and liver. Because HSP72 is cardioprotective, and females are known to have less cardiovascular disease premenopause, the effects of ovariectomy were examined. We report that female Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy reduced the level of HSP72 in female hearts, and this could be prevented by estrogen replacement therapy. These data show that the expression of cardiac HSP72 is greater in female rats than in male rats, due to upregulation by estrogen.     

Modeling axonal defects in hereditary spastic paraplegia with human pluripotent stem cells

Background

Cortical motor neurons, also known as upper motor neurons, are large projection neurons whose axons convey signals to lower motor neurons to control the muscle movements. Degeneration of cortical motor neuron axons is implicated in several debilitating disorders including hereditary spastic paraplegia (HSP). Since the discovery of the first HSP gene, SPAST that encodes spastin, over 70 distinct genetic loci associated with HSP have been identified. How the mutations of these functionally diverse genes result in axonal degeneration and why certain axons are affected in HSP remain largely unknown. The development of induced pluripotent stem cell (iPSC) technology has provided researchers an excellent resource to generate patient-specific human neurons to model human neuropathological processes including axonal defects.

Methods

In this article, we will first review the pathology and pathways affected in the common forms of HSP subtypes by searching the PubMed database. We will then summarize the findings and insights gained from studies using iPSC-based models, and discuss challenges and future directions.

Results

HSPs, a heterogeneous group of genetic neurodegenerative disorders, exhibit similar pathological changes that result from retrograde axonal degeneration of cortical motor neurons. Recently, iPSCs have been generated from several common forms of HSP including SPG4, SPG3A, and SPG11 patients. Neurons derived from HSP iPSCs exhibit impaired neurite outgrowth, increased axonal swellings, and reduced axonal transport, recapitulating disease-specific axonal defects.

Conclusions

These patient-derived neurons offer a unique tool to study the pathogenic mechanisms and explore the treatments for rescuing axonal defects in HSP, as well as other diseases involving axonopathy.

     

NIPA1 gene mutations cause autosomal dominant hereditary spastic paraplegia (SPG6)

The hereditary spastic paraplegias (HSPs) are genetically heterogeneous disorders characterized by progressive lower-extremity weakness and spasticity. The molecular pathogenesis is poorly understood. We report discovery of a dominant negative mutation in the NIPA1 gene in a kindred with autosomal dominant HSP (ADHSP), linked to chromosome 15q11-q13 (SPG6 locus); and precisely the same mutation in an unrelated kindred with ADHSP that was too small for meaningful linkage analysis. NIPA1 is highly expressed in neuronal tissues and encodes a putative membrane transporter or receptor. Identification of the NIPA1 function and ligand will aid an understanding of axonal neurodegeneration in HSP and may have important therapeutic implications.     

Contrasting evolutionary rates in the duplicate chaperonin genes of Mycobacterium tuberculosis and M. leprae

A phylogenetic analysis of chaperonin (heat shock protein 60) sequences from prokaryotes and eukaryotes indicated that a single gene duplication event in the common ancestor of Mycobacterium tuberculosis, M. leprae, and Streptomyces albus gave rise to the duplicate chaperonin genes found in these species (designated HSP65 and GroEL in the mycobacterial species). Comparison of rates of synonymous and nonsynonymous nucleotide substitution in different gene regions suggested that the 5' end of the HSP65 gene was homogenized by an ancient recombination event between M. tuberculosis and M. leprae. In S. albus, the two duplicated chaperonin genes have evolved at essentially the same rate. In both M. tuberculosis and M. leprae, however, the GroEL gene has evolved considerably more rapidly at nonsynonymous nucleotide sites than has the HSP65 gene. Because this difference is not seen at synonymous sites, it must be due to a difference in selective constraint on the proteins encoded by the two genes, rather than to a difference in mutation rate. The difference between GroEL and HSP65 is striking in regions containing epitopes recognized by T cells of the vertebrate host; in certain cross-reactive epitopes conserved across all organisms, nonsynonymous sites in GroEL have evolved twice as fast as those in HSP65. It is suggested that these differences are correlated with differences in the way in which the duplicate chaperonins of M. tuberculosis and M. leprae interact with the host immune system.      

Evidence for hSNM1B/Apollo functioning in the HSP70 mediated DNA damage response

The hSNM1B/Apollo protein is involved in the cellular response to DNA-damage as well as in the maintenance of telomeres during S-phase. TRF2 has been shown to interact physically with hSNM1B. As a core component of shelterin, TRF2 functions in organization and protection of telomeres. However, TRF2 was also shown to have a role in the early DNA-damage response, suggesting that hSNM1B and TRF2 cooperate in this dual function.

Here we have used Tandem-Affinity-Purification in combination with mass spectrometry to identify additional binding partners of hSNM1B. This revealed HSC70, HSP72, HSP60 and β-Tubulin to be hSNM1B-interactors. We have confirmed the interaction of hSNM1B and HSP70 in co-immunoprecipitation assays and found that hSNM1B binds to a C-terminal fragment of HSP72, known to contain the substrate binding domain. Depletion of HSP72 in human fibroblasts resulted in a significant reduction of nuclear hSNM1B foci. We also found the phosphorylation of CHK1 at serine 317 to be attenuated in response to UVC irradiation as a consequence of hSNM1B depletion, a result which extends our previous findings on the DNA-damage response function of hSNM1B.

HSP70 chaperones have been implicated in the maintenance of genome stability and their expression is often aberrant in cancer. Our results presented here, suggest that the role in genome stability might not be specific to HSP70 but rather can be attributed, at least in part, to hSNM1B. This, together with its stimulating effect on ATM and ATR substrate phosphorylation in response to DNA-damage qualify hSNM1B as a putative target in cancer therapy.     

HSP10 selective preference for myeloid and megakaryocytic precursors in normal human bone marrow

Heat shock proteins (HSPs) constitute a heterogeneous family of proteins involved in cell homeostasis. During cell life they are involved in harmful insults, as well as in immune and inflammatory reactions. It is known that they regulate gene expression, and cell proliferation, differentiation and death. HSP60 is a mitochondrial chaperonin, highly preserved during evolution, responsible of protein folding. Its function is strictly dependent on HSP10 in both prokaryotic and eukaryotic elements. We investigated the presence and the expression of HSP60 and HSP10 in a series of 20 normal human bone marrow specimens (NHBM) by the means of immunohistochemistry. NHBM showed no expression of HSP60, probably due to its being below the detectable threshold, as already demonstrated in other normal human tissues. By contrast, HSP10 showed a selective positivity for myeloid and megakaryocytic lineages. The positivity was restricted to precursor cells, while mature elements were constantly negative. We postulate that HSP10 plays a role in bone marrow cell differentiation other than being a mitochondrial co-chaperonin. The present data emphasize the role of HSP10 during cellular homeostasis and encourage further investigations in this field.     

Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase.

The spontaneous refolding of chemically denatured dihydrofolate reductase (DHFR) is completely arrested by chaperonin 60 (GroEL). This inhibition presumably results from the formation of a stable complex between chaperonin 60 and one or more intermediates in the folding pathway. While sequestered on chaperonin 60, DHFR is considerably more sensitive to proteolysis, suggesting a nonnative structure. Bound DHFR can be released from chaperonin 60 with ATP, and although chaperonin 10 (GroES) is not obligatory, it does potentiate the maximum effect of ATP. Hydrolysis of ATP is also not required for DHFR release since certain nonhydrolyzable analogues are capable of partial discharge. "Native" DHFR can also form a stable complex with chaperonin 60. However, in this case, complex formation is not instantaneous and can be prevented by the presence of DHFR substrates. This suggests that native DHFR exists in equilibrium with at least one conformer which is recognizable by chaperonin 60. Binding studies with 35S-labeled DHFR support these conclusions and further demonstrate that DHFR competes for a common saturable site with another protein (ribulose-1,5-bisphosphate carboxylase) known to interact with chaperonin 60.     

ER network formation and membrane fusion by atlastin1/SPG3A disease variants

At least 38 distinct missense mutations in the neuronal atlastin1/SPG3A GTPase are implicated in an autosomal dominant form of hereditary spastic paraplegia (HSP), a motor-neurological disorder manifested by lower limb weakness and spasticity and length-dependent axonopathy of corticospinal motor neurons. Because the atlastin GTPase is sufficient to catalyze membrane fusion and required to form the ER network, at least in nonneuronal cells, it is logically assumed that defects in ER membrane morphogenesis due to impaired fusion activity are the primary drivers of SPG3A-associated HSP. Here we analyzed a subset of established atlastin1/SPG3A disease variants using cell-based assays for atlastin-mediated ER network formation and biochemical assays for atlastin-catalyzed GTP hydrolysis, dimer formation, and membrane fusion. As anticipated, some variants exhibited clear deficits. Surprisingly however, at least two disease variants, one of which represents that most frequently identified in SPG3A HSP patients, displayed wild-type levels of activity in all assays. The same variants were also capable of co-redistributing ER-localized REEP1, a recently identified function of atlastins that requires its catalytic activity. Taken together, these findings indicate that a deficit in the membrane fusion activity of atlastin1 may be a key contributor, but is not required, for HSP causation.     

Hereditary spastic paraplegia: LOD-score considerations for confirmation of linkage in a heterogeneous trait.

Hereditary spastic paraplegia (HSP) is a degenerative disorder of the motor system, defined by progressive weakness and spasticity of the lower limbs. HSP may be inherited as an autosomal dominant (AD), autosomal recessive, or an X-linked trait. AD HSP is genetically heterogeneous, and three loci have been identified so far: SPG3 maps to chromosome 14q, SPG4 to 2p, and SPG4a to 15q. We have undertaken linkage analysis with 21 uncomplicated AD families to the three AD HSP loci. We report significant linkage for three of our families to the SPG4 locus and exclude several families by multipoint linkage. We used linkage information from several different research teams to evaluate the statistical probability of linkage to the SPG4 locus for uncomplicated AD HSP families and established the critical LOD-score value necessary for confirmation of linkage to the SPG4 locus from Bayesian statistics. In addition, we calculated the empirical P-values for the LOD scores obtained with all families with computer simulation methods. Power to detect significant linkage, as well as type I error probabilities, were evaluated. This combined analytical approach permitted conclusive linkage analyses on small to medium-size families, under the restrictions of genetic heterogeneity.     

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.     

Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal-recessive spastic paraplegia, including Kjellin syndrome

Hereditary spastic paraplegias (HSPs) are genetically and phenotypically heterogeneous disorders. Both "uncomplicated" and "complicated" forms have been described with various modes of inheritance. Sixteen loci for autosomal-recessive "complicated" HSP have been mapped. The SPG15 locus was first reported to account for a rare form of spastic paraplegia variably associated with mental impairment, pigmented maculopathy, dysarthria, cerebellar signs, and distal amyotrophy, sometimes designated as Kjellin syndrome. Here, we report the refinement of SPG15 to a 2.64 Mb genetic interval on chromosome 14q23.3-q24.2 and the identification of ZFYVE26, which encodes a zinc-finger protein with a FYVE domain that we named spastizin, as the cause of SPG15. Six different truncating mutations were found to segregate with the disease in eight families with a phenotype that included variable clinical features of Kjellin syndrome. ZFYVE26 mRNA was widely distributed in human tissues, as well as in rat embryos, suggesting a possible role of this gene during embryonic development. In the adult rodent brain, its expression profile closely resembled that of SPG11, another gene responsible for complicated HSP. In cultured cells, spastizin colocalized partially with markers of endoplasmic reticulum and endosomes, suggesting a role in intracellular trafficking.