Chris R. Guthrie

Phosphorylation promotes neurotoxicity in a Caenorhabditis elegans model of TDP-43 proteinopathy.

Neurodegenerative disorders characterized by neuronal and glial lesions containing aggregated pathological TDP-43 protein in the cytoplasm, nucleus, or neurites are collectively referred to as TDP-43 proteinopathies. Lesions containing aggregated TDP-43 protein are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). In addition, mutations in human TDP-43 cause ALS. We have developed a Caenorhabditis elegans model of TDP-43 proteinopathies to study the cellular, molecular, and genetic underpinnings of TDP-43-mediated neurotoxicity. Expression of normal human TDP-43 in all C. elegans neurons causes moderate motor defects, whereas ALS-mutant G290A, A315T, or M337V TDP-43 transgenes cause severe motor dysfunction. The model recapitulates some characteristic features of ALS and FTLD-U including age-induced decline in motor function, decreased life span, and degeneration of motor neurons accompanied by hyperphosphorylation, truncation, and ubiquitination of TDP-43 protein that accumulates in detergent-insoluble protein deposits. In C. elegans, TDP-43 neurotoxicity is independent of activity of the cell death caspase CED-3. Furthermore, phosphorylation of TDP-43 at serine residues 409/410 drives mutant TDP-43 toxicity. This model provides a tractable system for additional dissection of the cellular and molecular mechanisms underlying TDP-43 neuropathology.

Cloning of the mouse 5-HT6 serotonin receptor and mutagenesis studies of the third cytoplasmic loop

We have cloned the mouse 5-HT6 serotonin receptor and examined structure-function relationships in the C-terminal end of the third cytoplasmic (CIII) loop, introducing point mutations by site-directed mutagenesis at positions 264 to 268. We examined the ability of 5-HT6 wild type and receptor mutants to activate a cAMP responsive reporter gene when transiently expressed in JEG-3 or COS-7 cells. The wild type 5-HT6 receptor showed strong constitutive activity even when expressed at very low levels and which increased in proportion to the amount of receptor cDNA transfected. Three of the five mutants investigated (K264I, K267A and A268R) showed reduction in constitutive activity compared to wild type. These data suggest that constitutive activity may be important to 5-HT6 receptor activity in vivo and that, unlike some other G-protein coupled receptors, alteration in the BBXXB CIII-loop motif reduces rather than further activates basal activity of the murine 5-HT6 receptor.

Two Novel Brain-specific Splice Variants of the Murine Cβ Gene of cAMP-dependent Protein Kinase

We have previously characterized two murine cAMP-dependent protein kinase catalytic subunit genes, Cα and Cβ1. Targeted disruption of the Cβ1 promoter revealed two splice variants of the Cβ catalytic subunit gene (designated Cβ2 and Cβ3) that continue to be expressed. These variants arise from unique promoters and are brain-specific. Cβ2 is expressed in several discrete areas in the limbic system. These include the lateral septum, the bed nucleus of the stria terminalis, the ventral medial hypothalamus, and the amygdala. In the neocortex, expression is highest in cortical areas such as the prefrontal and insular cortex that are associated limbic structures. By contrast, Cβ1 is most highly expressed in the cortex and hippocampus and is also present in all non-neuronal tissues examined. Cβ3 is expressed at very low levels with wide distribution throughout the brain. Both the Cβ2 and Cβ3 variants are enzymatically active and induce gene expression in transient transfections with a cAMP response element-reporter construct. This activity is inhibited by protein kinase A regulatory subunits, the protein kinase inhibitor, and the chemical inhibitor H-89. We also demonstrate that Cβ1 is myristoylated at the amino terminus like the Cα isoform, but neither Cβ2 nor Cβ3 is myristoylated. The discrete expression of Cβ variants in the brain suggests specific functional roles in neuronal signaling.

SUT-2 potentiates tau-induced neurotoxicity in Caenorhabditis elegans

Expression of human tau in Caenorhabditis elegans neurons causes accumulation of aggregated tau leading to neurodegeneration and uncoordinated movement. We used this model of human tauopathy disorders to screen for genes required for tau neurotoxicity. Recessive loss-of-function mutations in the sut-2 locus suppress the Unc phenotype, tau aggregation and neurodegenerative changes caused by human tau. We cloned the sut-2 gene and found it encodes a novel sub-type of CCCH zinc finger protein conserved across animal phyla. SUT-2 shares significant identity with the mammalian SUT-2 (MSUT-2). To identify SUT-2 interacting proteins, we conducted a yeast two hybrid screen and found SUT-2 binds to ZYG-12, the sole C. elegans HOOK protein family member. Likewise, SUT-2 binds ZYG-12 in in vitro protein binding assays. Furthermore, loss of ZYG-12 leads to a marked upregulation of SUT-2 protein supporting the connection between SUT-2 and ZYG-12. The human genome encodes three homologs of ZYG-12: HOOK1, HOOK2 and HOOK3. Of these, the human ortholog of SUT-2 (MSUT-2) binds only to HOOK2 suggesting the interaction between SUT-2 and HOOK family proteins is conserved across animal phyla. The identification of sut-2 as a gene required for tau neurotoxicity in C. elegans may suggest new neuroprotective strategies capable of arresting tau pathogenesis in tauopathy disorders.

CDC7 inhibition blocks pathological TDP‐43 phosphorylation and neurodegeneration

Kinase hyperactivity occurs in both neurodegenerative disease and cancer. Lesions containing hyperphosphorylated aggregated TDP‐43 characterize amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP‐43 inclusions. Dual phosphorylation of TDP‐43 at serines 409/410 (S409/410) drives neurotoxicity in disease models; therefore, TDP‐43–specific kinases are candidate targets for intervention.

Monoubiquitination Promotes Calpain Cleavage of the Protein Phosphatase 2A (PP2A) Regulatory Subunit α4, Altering PP2A Stability and Microtubule-associated Protein Phosphorylation

Background: α4 binds to the PP2A catalytic subunit and the microtubule-associated E3 ligase MID1. Results: MID1-dependent monoubiquitination promotes calpain-mediated cleavage of α4, altering its phosphatase regulatory function. Conclusion: Defects in this regulatory process may underlie the MAP hypophosphorylation and hyperphosphorylation seen in Opitz syndrome and Alzheimer disease. Significance: Pharmacological agents that interfere with α4 monoubiquitination or cleavage are potential therapeutics to treat Alzheimer disease. Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4s activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.

Dopamine D2 Receptor Antagonism Suppresses Tau Aggregation and Neurotoxicity

BACKGROUND Tauopathies, including Alzheimers disease and frontotemporal dementia, are diseases characterized by the formation of pathological tau protein aggregates in the brain and progressive neurodegeneration. Presently no effective disease-modifying treatments exist for tauopathies. METHODS To identify drugs targeting tau neurotoxicity, we have used a Caenorhabditis elegans model of tauopathy to screen a drug library containing 1120 compounds approved for human use for the ability to suppress tau-induced behavioral effects. RESULTS One compound, the typical antipsychotic azaperone, improved the motility of tau transgenic worms, reduced levels of insoluble tau, and was protective against neurodegeneration. We found that azaperone reduces insoluble tau in a human cell culture model of tau aggregation and that other antipsychotic drugs (flupenthixol, perphenazine, and zotepine) also ameliorate the effects of tau expression in both models. CONCLUSIONS Reduction of dopamine signaling through the dopamine D2 receptor with the use of gene knockouts in Caenorhabditis elegans or RNA interference knockdown in human cell culture has similar protective effects against tau toxicity. These results suggest dopamine D2 receptor antagonism holds promise as a potential neuroprotective strategy for targeting tau aggregation and neurotoxicity.

Proteasome inhibition drives HDAC6-dependent recruitment of tau to aggresomes.

Lesions containing aggregated and hyperphosphorylated tau protein are characteristic of neurodegenerative tauopathies. We have developed a cellular model of pathological tau deposition and clearance by overexpressing wild type human tau in HEK293 cells. When proteasome activity is inhibited, HEK293/tau cells accumulate tau protein in structures that bear many of the hallmarks of aggresomes. These include recruitment of tau into large spherical inclusions, accumulation of the retrograde motor protein dynein at the centrosome, formation of an intermediate filament cage around inclusions, and clustering of mitochondria at the aggresome. Tau aggresomes form rapidly and can be cleared upon relief of proteasome inhibition. We observe recruitment of pathological misfolded phospho-tau species to aggresomes. Immunoblotting reveals accumulation of detergent insoluble aggregated tau species. Knockdown of histone deacetylase 6, a protein known to interact with tau, reveals a requirement for HDAC6 activity in tau aggresome formation. Direct observation of the accumulation and clearance of abnormal tau species will allow us to dissect the cellular and molecular mechanisms at work in clearing aggresomal tau and its similarity to disease relevant pathological tau clearance mechanisms.

MSUT2 is a determinant of susceptibility to tau neurotoxicity

Lesions containing abnormal aggregated tau protein are one of the diagnostic hallmarks of Alzheimers disease (AD) and related tauopathy disorders. How aggregated tau leads to dementia remains enigmatic, although neuronal dysfunction and loss clearly contribute. We previously identified sut-2 as a gene required for tau neurotoxicity in a transgenic Caenorhabditis elegans model of tauopathy. Here, we further explore the role of sut-2 and show that overexpression of SUT-2 protein enhances tau-induced neuronal dysfunction, neurotoxicity and accumulation of insoluble tau. We also explore the relationship between sut-2 and its human homolog, mammalian SUT-2 (MSUT2) and find both proteins to be predominantly nuclear and localized to SC35-positive nuclear speckles. Using a cell culture model for the accumulation of pathological tau, we find that high tau levels lead to increased expression of MSUT2 protein. We analyzed MSUT2 protein in age-matched post-mortem brain samples from AD patients and observe a marked decrease in overall MSUT2 levels in the temporal lobe of AD patients. Analysis of post-mortem tissue from AD cases shows a clear reduction in neuronal MSUT2 levels in brain regions affected by tau pathology, but little change in regions lacking tau pathology. RNAi knockdown of MSUT2 in cultured human cells overexpressing tau causes a marked decrease in tau aggregation. Both cell culture and post-mortem tissue studies suggest that MSUT2 levels may influence neuronal vulnerability to tau toxicity and aggregation. Thus, neuroprotective strategies targeting MSUT2 may be of therapeutic interest for tauopathy disorders.

The role of MSUT-2 in tau neurotoxicity: a target for neuroprotection in tauopathy?

We previously developed a transgenic Caenorhabditis elegans model of human tauopathy disorders by expressing human tau in nematode worm neurons to explore genetic pathways contributing to tau-induced neurodegeneration. This animal model recapitulates several hallmarks of human tauopathies, including altered behaviour, accumulation of detergent-insoluble phosphorylated tau protein and neurodegeneration. To identify genes required for tau neurotoxicity, we carried out a forward genetic screen for mutations that suppress tau neurotoxicity. We ultimately cloned the sut-2 (suppressor of tau pathology-2) gene, mutations in which alleviate tau neurotoxicity in C. elegans. SUT-2 encodes a novel subtype of CCCH zinc-finger protein conserved across animal phyla. SUT-2 shares significant identity with the mammalian SUT-2 (MSUT-2). We identified components of the aggresome as binding partners of MSUT-2. Thus we hypothesize that MSUT-2 plays a role in the formation and/or clearance of protein aggregates. We are currently exploring the role of MSUT-2 in tauopathy using mammalian systems. The identification of sut-2 as a gene required for tau neurotoxicity in C. elegans suggests new neuroprotective strategies targeting MSUT-2 that may be effective in modulating tau neurotoxicity in human tauopathy disorders.