Christopher D. Link

Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a ≈25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitin- and phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies.

A global analysis of Caenorhabditis elegans operons

The nematode worm Caenorhabditis elegans and its relatives are unique among animals in having operons. Operons are regulated multigene transcription units, in which polycistronic pre-messenger RNA (pre-mRNA coding for multiple peptides) is processed to monocistronic mRNAs. This occurs by 3′ end formation and trans-splicing using the specialized SL2 small nuclear ribonucleoprotein particle for downstream mRNAs. Previously, the correlation between downstream location in an operon and SL2 trans-splicing has been strong, but anecdotal. Although only 28 operons have been reported, the complete sequence of the C. elegans genome reveals numerous gene clusters. To determine how many of these clusters represent operons, we probed full-genome microarrays for SL2-containing mRNAs. We found significant enrichment for about 1,200 genes, including most of a group of several hundred genes represented by complementary DNAs that contain SL2 sequence. Analysis of their genomic arrangements indicates that >90% are downstream genes, falling in 790 distinct operons. Our evidence indicates that the genome contains at least 1,000 operons, 2–8 genes long, that contain about 15% of all C. elegans genes. Numerous examples of co-transcription of genes encoding functionally related proteins are evident. Inspection of the operon list should reveal previously unknown functional relationships.

Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid β-peptide (1-42) in a transgenic Caenorhabditis elegans model

Alzheimers disease is a progressive, neurodegenerative disorder characterized by senile plaques and neurofibrillary components. Abeta(1-42) is a principal component of senile plaques and is thought to be central to the pathogenesis of the disease. The Alzheimers disease brain is under significant oxidative stress, and the Abeta(1-42) peptide is known to cause oxidative stress in vitro. One controversy in the amyloid hypothesis is whether or not Abeta plaques are required for toxicity. We have employed a temperature-inducible Abeta expression system in Caenorhabditis elegans to create a strain of worms, CL4176, in which Abeta(1-42) is expressed with a non-permissive temperature of 23 degrees C. The CL4176 strain allows examination of the temporal relationship between Abeta expression, oxidative stress, and Abeta fibril formation. CL4176 were under increased oxidative stress, evidenced by increased protein oxidation indexed by increased carbonyl levels, 24 and 32 h after temperature upshift as compared to the control strain, CL1175. The increased oxidative stress in CL4176 occurred in the absence of Abeta fibril formation, consistent with the notion that the toxic species in Abeta toxicity is pre-fibrillar Abeta and not the Abeta fibril. These results are discussed with reference to Alzheimers disease.

Amyloid-β-Induced Pathological Behaviors Are Suppressed by Ginkgo biloba Extract EGb 761 and Ginkgolides in Transgenic Caenorhabditis elegans

Amyloid-β (Aβ) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimers disease (AD). We previously demonstrated that the standardized Ginkgo biloba extract EGb 761, commonly used to enhance memory and by AD patients for dementia, inhibits Aβ-induced apoptosis in neuroblastoma cells. In this study, we use EGb 761 and its single constituents to associate Aβ species with Aβ-induced pathological behaviors in a model organism, Caenorhabditis elegans. We report that EGb 761 and one of its components, ginkgolide A, alleviates Aβ-induced pathological behaviors, including paralysis, and reduces chemotaxis behavior and 5-HT hypersensitivity in a transgenic C. elegans. We also show that EGb 761 inhibits Aβ oligomerization and Aβ deposits in the worms. Moreover, reducing oxidative stress is not the mechanism by which EGb 761 and ginkgolide A suppress Aβ-induced paralysis because the antioxidant l-ascorbic acid reduced intracellular levels of hydrogen peroxide to the same extent as EGb 761, but was not nearly as effective in suppressing paralysis in the transgenic C. elegans. These findings suggest that (1) EGb 761 suppresses Aβ-related pathological behaviors, (2) the protection against Aβ toxicity by EGb 761 is mediated primarily by modulating Aβ oligomeric species, and (3) ginkgolide A has therapeutic potential for prevention and treatment of AD.

Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer’s disease model

We have engineered transgenic Caenorhabditis elegans animals to inducibly express the human beta amyloid peptide (Abeta). Gene expression changes resulting from Abeta induction have been monitored by cDNA hybridization to glass slide microarrays containing probes for almost all known or predicted C. elegans genes. Using statistical criteria, we have identified 67 up-regulated and 240 down-regulated genes. Subsets of these regulated genes have been tested and confirmed by quantitative RT-PCR. To investigate whether genes identified in this model system also show gene expression changes in Alzheimers disease (AD) brain, we have also used quantitative RT-PCR to examine in post-mortem AD brain tissue transcript levels of alphaB-crystallin (CRYAB) and tumor necrosis factor-induced protein 1 (TNFAIP1), human homologs of genes found to be robustly induced in the transgenic C. elegans model. Both CRYAB and TNFAIP1 show increased transcript levels in AD brains, supporting the validity of this approach.

Interaction of intracellular β amyloid peptide with chaperone proteins

Expression of the human β amyloid peptide (Aβ) in transgenic Caenorhabditis elegans animals can lead to the formation of intracellular immunoreactive deposits as well as the formation of intracellular amyloid. We have used this model to identify proteins that interact with intracellular Aβ in vivo. Mass spectrometry analysis of proteins that specifically coimmunoprecipitate with Aβ has identified six likely chaperone proteins: two members of the HSP70 family, three αB-crystallin-related small heat shock proteins (HSP-16s), and a putative ortholog of a mammalian small glutamine-rich tetratricopeptide repeat-containing protein proposed to regulate HSP70 function. Quantitative reverse transcription–PCR analysis shows that the small heat shock proteins are also transcriptionally induced by Aβ expression. Immunohistochemistry demonstrates that HSP-16 protein closely colocalizes with intracellular Aβ in this model. Transgenic animals expressing a nonaggregating Aβ variant, a single-chain Aβ dimer, show an altered pattern of coimmunoprecipitating proteins and an altered cellular distribution of HSP-16. Double-stranded RNA inhibition of R05F9.10, the putative C. elegans ortholog of the human small glutamine-rich tetratricopeptide-repeat-containing protein (SGT), results in suppression of toxicity associated with Aβ expression. These results suggest that chaperone function can play a role in modulating intracellular Aβ metabolism and toxicity.

Direct observation of stress response in Caenorhabditis elegans using a reporter transgene.

Transgenic Caenorhabditis elegans expressing jellyfish Green Fluorescent Protein under the control of the promoter for the inducible small heat shock protein gene hsp-16-2 have been constructed. Transgene expression parallels that of the endogenous hsp-16 gene, and, therefore, allows direct visualization, localization, and quantitation of hsp-16 expression in living animals. In addition to the expected upregulation by heat shock, we show that a variety of stresses, including exposure to superoxide-generating redox-cycling quinones and the expression of the human beta amyloid peptide, specifically induce the reporter transgene. The quinone induction is suppressed by coincubation with L-ascorbate. The ability to directly observe the stress response in living animals significantly simplifies the identification of both exogenous treatments and genetic alterations that modulate stress response, and possibly life span, in C. elegans.

Longevity genes in the nematode Caenorhabditis elegans also mediate increased resistance to stress and prevent disease.

More than 40 single-gene mutants in Caenorhabditis elegans have been demonstrated to lead to increased lifespan (a rigorous, operational test for being a gerontogene) of 20% or more; these are referred to collectively as ‘Age’ mutants. Age mutants must change key functions that are rate-limiting determinants of longevity; moreover, important genes can be identified independently of prior hypotheses as to actual mode of gene action in extending longevity and/or ‘slowing’ of ageing. These Age mutants define as many as nine (possibly) distinct pathways and/or modes of action, as defined by primary phenotype. Each of three well-studied mutants (age-1, clk-1, and spe-26) alters age-specific mortality rates in a fashion unique to itself. In age-1 mutants, the decreases in mortality rates are quite dramatic, with an almost tenfold drop in mortality throughout most of life. All Age mutants (so far without exception) increase the ability of the worm to respond to several (but not all) stresses, including heat, UV, and reactive oxidants. We have used directed strategies as well as random mutagenesis to identify novel genes that increase the worms ability to resist stress. Two genes (daf-16 and old-1) are epistatic to the long-life phenotype of most mutants and also yield over-expression strains that are stress-resistant and long-lived. We have also used a variety of approaches to determine what transcriptional alterations are associated with increased longevity (and with ageing itself), including whole-genome expression studies using microarrays and GFP reporter constructs. We suggest that the role of the Age genes in both longevity and stress resistance indicates that a major evolutionary determinant of longevity is the ability to respond to stress. In mammals, both dietary restriction and hormesis are phenomena in which the endogenous level of resistance to stress has been upregulated; both of these interventions extend longevity, suggesting possible evolutionary conservation.

In Vivo Aggregation of β‐Amyloid Peptide Variants

Abstract: Transgenic Caenorhabditis elegans animals have been engineered to express wild‐type and single‐amino acid variants of a long form of human β‐amyloid peptide (Aβ 1–42). These animals express high levels (∼300 ng of Aβ/mg of total protein) of apparently full‐length peptide, as determined by quantitative immunoblot. Expression of wild‐type Aβ in these animals leads to rapid production of amyloid deposits reactive with Congo red and thioflavin S. This model system has been used to examine the effect of Leu17Pro, Leu17Val, Ala30‐Pro, Met35Cys, and Met35Leu substitutions on the in vivo production of amyloid deposits. We find that the Leu17Pro and Met35Cys substitutions completely block the formation of thioflavin S‐reactive deposits, implicating these as key residues for in vivo amyloid formation. We have also constructed transgenic strains expressing a novel Aβ variant, the single‐chain dimer. Animals expressing high levels of this variant also fail to produce thioflavin S‐reactive deposits.

Visualization of fibrillar amyloid deposits in living, transgenic Caenorhabditis elegans animals using the sensitive amyloid dye, X-34

Transgenic Caenorhabditis elegans animals can be engineered to express high levels of the human beta amyloid peptide (Abeta). Histochemistry of fixed tissue from these animals reveals deposits reactive with the amyloid-specific dyes Congo Red and thioflavin S (Fay et al., J. Neurochem 71:1616, 1998). Here we show by immuno-electron microscopy that these animals contain intracellular immunoreactive deposits with classic amyloid fibrillar ultrastructure. These deposits can be visualized in living animals using the newly developed, intensively fluorescent, amyloid-specific dye X-34. This in vivo staining allows monitoring of amyloid deposition in individual animals over time. The specificity of this staining is demonstrated by examining transgenic animals expressing high levels of a non-fibrillar beta peptide variant, the beta single-chain dimer. These animals have deposits immunoreactive with anti-beta antibodies, but do not have X-34 deposits or deposits with a fibrillar ultrastructure. X-34 can also be used in vivo to visualize putative amyloid deposits resulting from accumulation of human transthyretin, another amyloidic protein. In vivo amyloid staining with X-34 may be a useful tool for monitoring anti-amyloidic treatments in real time or screening for genetic alterations that affect amyloid formation.