HER2/ERBB2 immunoreactivity in human retinoblastoma.

Retinoblastoma (RB) is an ocular malignancy of early childhood. Although mutations in the Rb1 gene and expression of stem cell markers have been identified in RB, additional information on RB-specific alterations in signaling pathways and protein expression would be useful for the design of targeted RB therapies. Here we have evaluated the expression of HER2 (ERBB2) in RB. HER2 is a member of the epidermal growth factor family, which is overexpressed in breast, ovarian, gastric, colorectal, pancreatic, and endometrial cancers in a stratified manner. Overexpression and gene amplification of HER2 is associated with aggressive malignancies, accompanied by chemoresistance and poor outcomes. In this study, we present the first evidence of HER2 immunoreactivity in retinoblastoma, as shown by immunocytochemistry, flow cytometry, and western immunoblot, with validation by reverse transcription PCR (RT-PCR) in both RB cell lines and clinical RB tumors. Our results suggest that the HER2 protein expressed in RB is a truncated version that spares the trastuzumab binding site, while HER2 is not detected in normal ocular tissues. Our discovery of HER2 expression in RB may lead to innovative and targeted drug treatment options designed to spare the eye and preserve vision in RB patients.

Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates.

It is unclear how polyglutamine expansion is associated with the pathogenesis of Huntington disease (HD). Here, we provide evidence that polyglutamine expansion leads to the formation of large intracellular aggregates in vitro and in vivo. In vitro these huntingtin-containing aggregates disrupt normal cellular architecture and increase in frequency with polyglutamine length. Huntingtin truncated at nucleotide 1955, close to the caspase-3 cleavage site, forms perinuclear aggregates more readily than full-length huntingtin and increases the susceptibility of cells to death following apoptotic stimuli. Further truncation of huntingtin to nucleotide 436 results in both intranuclear and perinuclear aggregates. For a given protein size, increasing polyglutamine length is associated with increased cellular toxicity. Asymptomatic transgenic mice expressing full-length huntingtin with 138 polyglutamines form exclusively perinuclear aggregates in neurons. These data support the hypothesis that proteolytic cleavage of mutant huntingtin leads to the development of aggregates which compromise cell viability, and that their localization is influenced by protein length.

The influence of huntingtin protein size on nuclear localization and cellular toxicity.

Huntington disease is an autosomal dominant neurodegenerative disorder caused by the pathological expansion of a polyglutamine tract. In this study we directly assess the influence of protein size on the formation and subcellular localization of huntingtin aggregates. We have created numerous deletion constructs expressing successively smaller fragments of huntingtin and show that these smaller proteins containing 128 glutamines form both intranuclear and perinuclear aggregates. In contrast, larger NH2-terminal fragments of huntingtin proteins with 128 glutamines form exclusively perinuclear aggregates. These aggregates can form in the absence of endogenous huntingtin. Furthermore, expression of mutant huntingtin results in increased susceptibility to apoptotic stress that is greater with decreasing protein length and increasing polyglutamine size. As both intranuclear and perinuclear aggregates are clearly associated with increased cellular toxicity, this supports an important role for toxic polyglutamine-containing fragments forming aggregates and playing a key role in the pathogenesis of Huntington disease.

Wild-type huntingtin protects from apoptosis upstream of caspase-3.

Expansion of a polyglutamine sequence in the N terminus of huntingtin is the gain-of-function event that causes Huntington's disease. This mutation affects primarily the medium-size spiny neurons of the striatum. Huntingtin is expressed in many neuronal and non-neuronal cell types, implying a more general function for the wild-type protein. Here we report that wild-type huntingtin acts by protecting CNS cells from a variety of apoptotic stimuli, including serum withdrawal, death receptors, and pro-apoptotic Bcl-2 homologs. This protection may take place at the level of caspase-9 activation. The full-length protein also modulates the toxicity of the poly-Q expansion. Cells expressing full-length mutant protein are susceptible to fewer death stimuli than cells expressing truncated mutant huntingtin.

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

A 100 amino acid region in the GABA rho 1 subunit confers robust homo-oligomeric expression.

Retinal gamma-aminobutyric acid type C (GABAC) receptors are believed to be composed of rho subunits. Although rho 1 and rho 2 are over 80% similar, the whole-cell currents generated by rho 1 receptors in Xenopus oocytes are significantly greater than those generated by rho 2 receptors. In this study, chimeric subunits containing different portions of human rho 1 and human rho 2 were created to localize sequences facilitating robust rho 1 expression. Our results indicate that these sequences reside in a 100 amino acid domain in the N-terminus of rho 1, and may involve N-linked glycosylation. Since the N-terminus also contains subunit assembly signals, rho 1 receptors may be formed more efficiently than rho 2 receptors. Therefore, this study furthers our understanding of the molecular basis of GABA-mediated inhibition in the retina.

A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition.

A class of bicuculline-insensitive gamma-aminobutyric acid (GABA) receptors, GABAC, has been identified in retina. Several lines of evidence indicate that GABAC receptors are formed partially or wholly of GABA rho subunits. These receptors generate a Cl- current in response to GABA but differ from GABAA receptors in a number of ways. Picrotoxin, widely accepted as a noncompetitive antagonist of GABAA receptors, displays competitive and noncompetitive antagonism of GABAC receptors in perch and bovine retina and GABA rho 1 receptors expressed in Xenopus oocytes. The aim of this study was to identify the molecular basis of the two components of picrotoxin inhibition of GABA rho 1 receptors. By using a domain-swapping and mutagenesis strategy, a difference in picrotoxin sensitivity between rho 1 and rho 2 receptors was localized to a single amino acid in the putative second transmembrane domain. Substitution of this amino acid with residues found in the analogous position in highly picrotoxin-sensitive glycine alpha and GABAA subunits increased the sensitivity of rho 1 mutants 10- to 500-fold. Importantly, the competitive component of picrotoxin inhibition of the rho 1 mutant receptors was almost eliminated. These findings demonstrate that an amino acid in the putative channel domain of GABA rho 1 receptors influences picrotoxin sensitivity and mediates agonist binding by an allosteric mechanism.

In vitro evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease.

A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have lead to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system that compared the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. We first assessed the mode of nuclear transport of N-terminal fragments of huntingtin, and show that the predicted endogenous NLS is not functional, providing data in support of passive nuclear transport. This result suggests that proteolysis is a necessary step for nuclear entry of huntingtin. Additionally, insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. The findings of nuclear and cytoplasmic aggregates in affected brains, together with these in vitro data, support the nucleus and cytosol as subcellular sites for pathogenesis in HD.

A single histidine residue is essential for zinc inhibition of GABA rho 1 receptors.

The GABA rho 1 subunit, cloned from a human retina library, can form homooligomeric receptors with properties similar to GABAc receptors characterized in retinal cells. The divalent cation Zn2+, abundant in the CNS and retina, was found to inhibit GABA rho 1 receptors in a voltage-independent manner. Varying the extracellular pH from 7.4 to 5.6 significantly reduced this inhibitory effect. This pH profile suggested that one or more histidine residues might play a role in the interaction between Zn2+ and the GABA rho 1 receptor. Site-directed mutagenesis revealed that a single histidine residue (His 156) in the putative extracellular domain of rho 1 was critical for Zn2+ sensitivity. Substitution of this amino acid with tyrosine (H156Y) created a functional GABA receptor with agonist and channel properties indistinguishable from wildtype. However, the H156Y mutant was insensitive to Zn2+, even at concentrations as high as 1 mM. Mutation to aspartic acid, an amino acid that can interact with Zn2+ in other proteins, preserved sensitivity to Zn2+ but abolished the pH-dependent effect. This histidine residue is also involved in Ni2+ and Cd2+ interaction since the H156Y mutation completely suppressed the inhibition effects of these two cations. These data demonstrate that an extracellular histidine residue is critical for transition metal cation sensitivity of GABA rho 1 receptors.

The N-terminal domain of human GABA receptor rho1 subunits contains signals for homooligomeric and heterooligomeric interaction.

gamma-Aminobutyric acid type C (GABAC) receptors identified in retina appear to be composed of GABA rho subunits. The purpose of this study was to localize signals for homooligomeric assembly of rho1 subunits and to investigate whether the same region contained signals for heterooligomeric interaction with rho2 subunits. In vitro translated human rho1 was shown to be membrane-associated, and proteinase K susceptibility studies indicated that the N terminus was oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of rho1 in the initial steps of subunit assembly. To test this hypothesis, mutants were created containing only N-terminal sequences (N-rho1) or C-terminal sequences (C-rho1) of rho1. Co-immunoprecipitation studies revealed that N-rho1, but not C-rho1, interacted with rho1 in vitro. When coexpressed in Xenopus oocytes, N-rho1 interfered with rho1 receptor formation. Together, these data suggested that signals for rho1 homooligomeric assembly reside in the N-terminal half of the subunit. Sequential immunoprecipitations were then performed upon cotranslated rho1 and rho2 subunits which demonstrated that rho1 and rho2 interacted in vitro. Co-immunoprecipitation indicated that N-rho1 specifically associated with rho2. Therefore, the N-terminal regions of rho subunits contain the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.

Sequences in the amino termini of GABA rho and GABA(A) subunits specify their selective interaction in vitro.

Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA(A) receptors are composed of alpha, beta, gamma, delta, and epsilon/chi subunits, whereas GABA(C) receptors appear to contain rho subunits. However, retinal cells exhibiting GABA(C) responses express alpha, beta, and rho subunits, raising the possibility that GABA(C) receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA(A) receptor subunits alpha1, alpha5, and beta1 did not coimmunoprecipitate with full-length rho1, rho2, or the N-terminal domain of rho1 that contains signals for rho-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of beta1 to rho1 created a beta1rho1 chimera that coimmunoprecipitated with the alpha1 subunit but not with the rho2 subunit. Furthermore, exchanging the N terminus of the rho1 subunit with the corresponding region of beta1 produced a rho1beta1 chimera that interfered with rho1 receptor expression in Xenopus oocytes, whereas the full-length beta1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of rho subunits and GABA(A) subunits into GABA(C) and GABA(A) receptors, respectively.

A comparison of GABAC and rho subunit receptors from the white perch retina.

There is increasing evidence that GABAC receptors are composed of GABA rho subunits. In this study, we compared the properties of native GABAC receptors with those of receptors composed of a GABA rho subunit. A homologue of the GABA rho gene was cloned from a white perch (Roccus americana) retinal cDNA library. The clone (perch-s) has an open reading frame of 1422 nucleotide base pairs and encodes a predicted protein of 473 amino acids. It is highly homologous to GABA rho subunits cloned from human and rat retinas. The receptors (perch-s receptor) expressed by this gene in Xenopus oocytes show properties similar to those of the GABAC receptors present on white perch retinal neurons. GABA induced a sustained response that had a reversal potential of -27.1 +/- 3.6 mV. The EC50 for the response was 1.74 +/- 1.25 microM, a value similar to that reported for GABAC receptors. Pharmacologically, the responses were bicuculline insensitive and not modulated by either diazepam or pentobarbital as is the case for GABAC receptors. There were, however, some distinct differences between native GABAC and perch-s receptors. I4AA acts as a partial agonist on perch-s receptors whereas it is strictly an antagonist on native GABAC receptors. Picrotoxin inhibition is noncompetitive on perch-s receptors, but both competitive and noncompetitive on GABAC receptors. We conclude that GABAC receptors are formed by GABA rho subunits but that native GABAC receptors probably consist of a mixture of GABA rho subunits.

Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin.

A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have led to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system by comparing the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. Insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. These studies, together with findings in transgenic mice, suggest two phases for the pathogenesis of HD, with the initial toxicity in the cytoplasm followed by proteolytic processing of huntingtin, nuclear translocation with increased nuclear concentration of N-terminal fragments, seeding of aggregates and resultant apoptotic death. These findings support the nucleus and cytosol as subcellular sites for pathogenesis in HD.

Huntingtin interacting protein 1 induces apoptosis via a novel caspase-dependent death effector domain.

Huntington disease is a devastating neurodegenerative disease caused by the expansion of a polymorphic glutamine tract in huntingtin. The huntingtin interacting protein (HIP-1) was identified by its altered interaction with mutant huntingtin. However, the function of HIP-1 was not known. In this study, we identify HIP-1 as a proapoptotic protein. Overexpression of HIP-1 resulted in rapid caspase 3-dependent cell death. Bioinformatics analyses identified a novel domain in HIP-1 with homology to death effector domains (DEDs) present in proteins involved in apoptosis. Expression of the HIP-1 DED alone resulted in cell death indistinguishable from HIP-1, indicating that the DED is responsible for HIP-1 toxicity. Furthermore, substitution of a conserved hydrophobic phenylalanine residue within the HIP-1 DED at position 398 eliminated HIP-1 toxicity entirely. HIP-1 activity was found to be independent of the DED-containing caspase 8 but was significantly inhibited by the antiapoptotic protein Bcl-x(L), implicating the intrinsic pathway of apoptosis in HIP-1-induced cell death. Co-expression of a normal huntingtin fragment capable of binding HIP-1 significantly reduced cell death. Our data identify HIP-1 as a novel proapoptotic mediator and suggest that HIP-1 may be a molecular accomplice in the pathogenesis of Huntington disease.

Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells.

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.

Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity.

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.

Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity.

X-linked spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease-associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase-3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenerative diseases associated with polyglutamine repeat expansions.

Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract.

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.