Jonathan H. Fox

A small-molecule therapeutic lead for Huntington's disease: Preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse

Huntingtons disease (HD) is a progressive neurodegenerative disease caused by a glutamine expansion within huntingtin protein. The exact pathological mechanisms determining disease onset and progression remain unclear. However, aggregates of insoluble mutant huntingtin (mhtt), a hallmark of HD, are readily detected within neurons in HD brain. Although aggregated polyglutamines may not be inherently toxic, they constitute a biomarker for mutant huntingtin useful for developing therapeutics. We previously reported that the small molecule, C2-8, inhibits polyglutamine aggregation in cell culture and brain slices and rescues degeneration of photoreceptors in a Drosophila model of HD. In this study, we assessed the therapeutic potential of C2-8 in the R6/2 mouse model of HD, which has been used to provide proof-of-concept data in considering whether to advance therapies to human HD. We show that, at nontoxic doses, C2-8 penetrates the blood–brain barrier and is present in brain at a high concentration. C2-8-treated mice showed improved motor performance and reduced neuronal atrophy and had smaller huntingtin aggregates. There have been no prior drug-like, non-toxic, brain-penetrable aggregation inhibitors to arise from cell-based high-throughput screens for reducing huntingtin aggregation that is efficacious in preclinical in vivo models. C2-8 provides an essential tool to help elucidate mechanisms of neurodegeneration in HD and a therapeutic lead for further optimization and development.

Iron accumulates in Huntington's disease neurons: protection by deferoxamine.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.

Cysteine Oxidation within N-terminal Mutant Huntingtin Promotes Oligomerization and Delays Clearance of Soluble Protein

Huntington disease (HD) is a progressive neurodegenerative disorder caused by expression of polyglutamine-expanded mutant huntingtin protein (mhtt). Most evidence indicates that soluble mhtt species, rather than insoluble aggregates, are the important mediators of HD pathogenesis. However, the differential roles of soluble monomeric and oligomeric mhtt species in HD and the mechanisms of oligomer formation are not yet understood. We have shown previously that copper interacts with and oxidizes the polyglutamine-containing N171 fragment of huntingtin. In this study we report that oxidation-dependent oligomers of huntingtin form spontaneously in cell and mouse HD models. Levels of these species are modulated by copper, hydrogen peroxide, and glutathione. Mutagenesis of all cysteine residues within N171 blocks the formation of these oligomers. In cells, levels of oligomerization-blocked mutant N171 were decreased compared with native N171. We further show that a subset of the oligomerization-blocked form of glutamine-expanded N171 huntingtin is rapidly depleted from the soluble pool compared with “native ” mutant N171. Taken together, our data indicate that huntingtin is subject to specific oxidations that are involved in the formation of stable oligomers and that also delay removal from the soluble pool. These findings show that inhibiting formation of oxidation-dependent huntingtin oligomers, or promoting their dissolution, may have protective effects in HD by decreasing the burden of soluble mutant huntingtin.

Altered selenium status in Huntington's disease: neuroprotection by selenite in the N171-82Q mouse model.

Disruption of redox homeostasis is a prominent feature in the pathogenesis of Huntingtons disease (HD). Selenium an essential element nutrient that modulates redox pathways and has been reported to provide protection against both acute neurotoxicity (e.g. methamphetamine) and chronic neurodegeneration (e.g. tauopathy) in mice. The objective of our study was to investigate the effect of sodium selenite, an inorganic form of selenium, on behavioral, brain degeneration and biochemical outcomes in the N171-82Q Huntingtons disease mouse model. HD mice, which were supplemented with sodium selenite from 6 to 14 weeks of age, demonstrated increased motor endurance, decreased loss of brain weight, decreased mutant huntingtin aggregate burden and decreased brain oxidized glutathione levels. Biochemical studies revealed that selenite treatment reverted HD-associated changes in liver selenium and plasma glutathione in N171-82Q mice and had effects on brain selenoprotein transcript expression. Further, we found decreased brain selenium content in human autopsy brain. Taken together, we demonstrate a decreased selenium phenotype in human and mouse HD and additionally show some protective effects of selenite in N171-82Q HD mice. Modification of selenium metabolism results in beneficial effects in mouse HD and thus may represent a therapeutic strategy.

Neuronal Ceroid-Lipofuscinosis in a Labrador Retriever

An 8-year-old Labrador Retriever with an 11-month history of progressive partial seizures and necropsy examination findings characteristic of the lamellar form of canine neuronal ceroid-lipofuscinosis (NCL) is presented. The clinical, light microscopic, and ultrastructural features of this case most closely resemble human adult-onset NCL (Kufs disease). This is the first report of NCL occurring in the Labrador Retriever breed.

Characterization of Striatal Neuronal Loss and Atrophy in the R6/2 Mouse Model of Huntington's Disease

Striatal neuronal degeneration and loss is an important feature of human Huntington’s disease (HD). R6/2 HD mice recapitulate many features of human HD including striatal atrophy. While striatal neuronal atrophy and loss is reported in R6/2 HD mice the degree of neuronal loss and the characteristics of cell body atrophy are unclear. We used stereological approaches to estimate whole striatal neuronal numbers and characterize changes in striatal neuronal size distribution. R6/2 HD mice had ~126000 fewer neurons per striatum (~12% decline) at 12 weeks of age than wild-type litter-mates; differences were not present at 5 weeks. Analysis of striatal neuronal numbers per cell body size category revealed declines in neuron numbers in the size ranges 550-1050 µm3 suggesting that larger striatal neurons are more susceptible to atrophy or loss in late stages of disease. R6/2 HD mice have a striatal neuronal loss phenotype. As striatal neuronal loss in human HD is dramatic, neuronal loss in R6/2 striatum provides an important late-stage outcome measure for study of disease modifying interventions

Neonatal iron supplementation potentiates oxidative stress, energetic dysfunction and neurodegeneration in the R6/2 mouse model of Huntington's disease

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in huntingtin (htt) protein. Dysregulation of brain iron homeostasis, oxidative stress and neurodegeneration are consistent features of the HD phenotype. Therefore, environmental factors that exacerbate oxidative stress and iron dysregulation may potentiate HD. Iron supplementation in the human population is common during infant and adult-life stages. In this study, iron supplementation in neonatal HD mice resulted in deterioration of spontaneous motor running activity, elevated levels of brain lactate and oxidized glutathione consistent with increased energetic dysfunction and oxidative stress, and increased striatal and motor cortical neuronal atrophy, collectively demonstrating potentiation of the disease phenotype. Oxidative stress, energetic, and anatomic markers of degeneration were not affected in wild-type littermate iron-supplemented mice. Further, there was no effect of elevated iron intake on disease outcomes in adult HD mice. We have demonstrated an interaction between the mutant huntingtin gene and iron supplementation in neonatal HD mice. Findings indicate that elevated neonatal iron intake potentiates mouse HD and promotes oxidative stress and energetic dysfunction in brain. Neonatal-infant dietary iron intake level may be an environmental modifier of human HD.

Filaroides osleri (Oslerus osleri): Two case reports and a review of canid infections in North America

Infections of domesticated dogs by a worldwide parasitic nematode Filaroides osleri (Oslerus osleri) lead to verminous tracheobronchitis that are often misdiagnosed clinically as kennel cough, due to infection with the bacterium Bordetella bronchiseptica. Diagnosis of two canine cases in Wyoming, USA prompted a search of the literature of canid infections in North America. Infections of domestic dogs are reported in nine US states and four Canadian provinces. Dogs of multiple breeds and both sexes were infected. Most were two years old or younger at diagnosis. Anthelmintic treatments were effective in relieving clinical symptoms, as well as causing resolution of tracheobronchial nodules. Other canid species, including coyotes (Canis latrans) and wolves (Canis lupus), have been infected across North America with a prevalence of 23% and 4%, respectively. Infection with F. osleri should be included in the differential diagnosis of infectious tracheobronchitis of dogs. It can be confirmed most readily by endoscopic detection of distinctive submucosal parasite-filled nodules, combined with histological examination of endoscopic biopsies.

Mast cell tumor in an eastern kingsnake (Lampropeltis getulus getulus).

A 16-year-old captive-bred male eastern kingsnake (Lampropeltis getulus getulus) was presented to the Veterinary Medical Teaching Hospital, University of Florida for evaluation of a bleeding mass which was first noted 2 months prior to presentation. The grayish mass was 2 3 2 3 5 cm, firm, ovoid, raised above the ventral scales, and located ventral to the liver. The snake was anesthetized with isoflurane in 100% oxygen, and the mass was found firmly attached to the fascia of the skin and the underlying skeletal musculature. Because of the invasiveness of the mass into the surrounding tissue, complete excision was not possible. Tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (HE), and sections were examined by light microscopy. Microscopically, the mass was composed of sheets and interwoven bundles of contiguous neoplastic cells ranging from round to polygonal to fusiform. Neoplastic cells had scant to abundant, eosinophilic, foamy to fibrillar cytoplasm and variably distinct borders. Nuclei were moderately anisokaryotic, round to elongate, and euchromatic to vesicular and frequently contained single or multiple nucleoli of various sizes. Mitotic figures were rare, and vascular invasion by tumor cells was not detected. Neoplastic tissue invaded adjoining skeletal muscle and extended to the surgical margins (Fig. 1). No bacterial or fungal organisms were detected by Goodpasture, Fites, or Gomori methenamine silver stains. An anatomic diagnosis of undifferentiated sarcoma was made. Given the invasive nature and the incomplete excision, recurrence was considered likely. After 6 months, the mass had regrown at the original site and was approximately the same size as the previous mass. To stage this neoplastic disease, a diagnostic evaluation was performed. Whole body radiographs were within normal limits. An ultrasonogram revealed a heteroechoic mass extending from the skin into the coelomic cavity, displacing the lung. The liver was uniform in echogenicity and texture. Hematologic parameters revealed a leukocytosis (11,300 cells/ml), lymphocytosis (5,760 cells/ml), and anemia (packed cell volume [PCV] 5 17%). Plasma biochemical parameters were within normal limits. When the mass was reexcised, it was found to be firmly attached to the surrounding skeletal musculature and invading the coelomic cavity. Portions of the mass were processed as described above for histopathologic evaluation. Findings were similar to

Neonatal Iron Supplementation Induces Striatal Atrophy in Female YAC128 Huntington’s Disease Mice

Background: Dysregulation of iron homeostasis is implicated in the pathogenesis of Huntington’s disease. We have previously shown that increased iron intake in R6/2 HD neonatal mice, but not adult R6/2 HD mice potentiates disease outcomes at 12-weeks of age corresponding to advanced HD [Redox Biol. 2015;4 : 363–74]. However, whether these findings extend to other HD models is unknown. In particular, it is unclear if increased neonatal iron intake can promote neurodegeneration in mouse HD models where disease onset is delayed to mid-adult life. Objective: To determine if increased dietary iron intake in neonatal and adult life-stages potentiates HD in the slowly progressive YAC128 HD mouse model. Methods: Female neonatal mice were supplemented daily from days 10–17 with 120μg/g body weight of carbonyl iron. Adult mice were provided diets containing low (50 ppm), medium (150 ppm) and high (500 ppm) iron concentrations from 2-months of age. HD progression was determined using behavioral, brain morphometric and biochemical approaches. Results: Neonatal-iron supplemented YAC128 HD mice had significantly lower striatal volumes and striatal neuronal cell body volumes as compared to control HD mice at 1-year of age. Neonatal-iron supplementation of HD mice had no effect on rota-rod motor endurance and brain iron or glutathione status. Adult iron intake level had no effect on HD progression. YAC128 HD mice had altered peripheral responses to iron intake compared to iron-matched wild-type controls. Conclusions: Female YAC128 HD mice supplemented with nutritionally-relevant levels of iron as neonates demonstrate increased striatal degeneration 1-year later.