Lin W. Hung

The Alzheimer's therapeutic PBT2 promotes amyloid-β degradation and GSK3 phosphorylation via a metal chaperone activity.

J. Neurochem. (2011) 119, 220–230.

The hypoxia imaging agent CuII(atsm) is neuroprotective and improves motor and cognitive functions in multiple animal models of Parkinson’s disease

The PET imaging agent CuII(atsm) improves motor and cognitive function in Parkinson’s disease.

Diacetylbis(N(4)-methylthiosemicarbazonato) Copper(II) (CuII(atsm)) Protects against Peroxynitrite-induced Nitrosative Damage and Prolongs Survival in Amyotrophic Lateral Sclerosis Mouse Model

Background: CuII(atsm) [(diacetylbis(N(4)-methylthiosemicarbazonato) copper(II)] was orally administrated to transgenic SOD1G93A mice. Results: Treatment significantly prolonged lifespan with preservation of motor neurons. Reduced protein oxidation, attenuated astrocyte, and microglial activation also resulted from treatment. Conclusion: CuII(atsm) is neuroprotective in this model even when treatment begins after the onset of disease symptoms. Significance: The drug has therapeutic potential for amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.

Oral Treatment with CuII(atsm) Increases Mutant SOD1 In Vivo but Protects Motor Neurons and Improves the Phenotype of a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copperII [CuII(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with CuII(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched 65CuII(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from CuII(atsm) to SOD1, suggesting the improved locomotor function and survival of the CuII(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with CuII(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1.

Development of a Platinum Complex as an anti-Amyloid Agent for the Therapy of Alzheimer’s Disease

Alzheimer s disease (AD) is an age-related neurodegenerative disease. Its pathological indicators include extracellular amyloid plaques, the main constituent of which is the amyloid b-peptide (Ab), and neurofibrillary tangles composed of hyperphosphorylated tau protein. Current evidence suggests that the aggregation of Abs drives the disease process, as various forms of aggregated Ab have been shown to be toxic, resulting in the development of a variety of therapeutic strategies that target Ab. To date, most Ab aggregation inhibitors have been designed to target the hydrophobic central and C-terminal regions of Ab, which are in general conjugated polyaromatic molecules that are very hydrophobic. Herein, we report a different approach to the design of aggregation inhibitors of Ab and demonstrate that this approach can modify Ab in vivo. Ab contains a metalbinding motif with three histidine residues (6, 13, and 14) near the N terminus, and the interaction of this site with zinc and copper modulates the aggregation and toxicity of Ab. We have previously taken advantage of the metal-binding ability of Ab to show that commercially available Pt complexes of 1,10-phenanthroline ligands target this site, thus inhibiting Ab aggregation in vitro. For a variety of reasons, including lack of novelty, cumbersome multi-step synthetic procedures, and poor bioavailability, these complexes are unsuitable for in vivo studies. Therefore we sought to identify new Pt complexes that are suitable for in vivo studies. The 8-(1H-benzoimidazol-2-yl)-quinoline (8-BQ) scaffold (Scheme 1) was identified as suitable for generating the desired platinum complexes. The ligand requires few steps to synthesize and provides the large aromatic surface area required for a Pt complex to target Ab. Surprisingly, although the initial synthesis of 8-BQ was first reported over 100 years ago, the coordination chemistry of this ligand has not been widely explored and Pt complexes of 8-BQ are novel. Additionally, the presence of an NH functionality on the imidazole moiety of 8-BQ allows easy modification of the ligand through a conventional one-step substitution reaction. Attachment of different groups to this position can be used to modulate the solubility and other pharmacokinetic properties of the complexes. We chose the N,N-dimethylaminoethyl group because it improves drug solubility and stability in aqueous media. Herein, we report the synthesis of the 8BQ ligand (Scheme 1) and its coordination to Pt and Pt complexes (Schemes 2 and 3, respectively).

Localized changes to glycogen synthase kinase-3 and collapsin response mediator protein-2 in the Huntington's disease affected brain

All cases of Huntingtons disease (HD) are caused by mutant huntingtin protein (mhtt), yet the molecular mechanisms that link mhtt to disease symptoms are not fully elucidated. Given glycogen synthase kinase-3 (GSK3) is implicated in several neurodegenerative diseases as a molecular mediator of neuronal decline and widely touted as a therapeutic target, we investigated GSK3 in cells expressing mhtt, brains of R6/1 HD mice and post-mortem human brain samples. Consistency in data across the two models and the human brain samples indicate decreased GSK3 signalling contributes to neuronal dysfunction in HD. Inhibitory phosphorylation of GSK3 (pGSK3) was elevated in mhtt cells and this appeared related to an overall energy metabolism deficit as the mhtt cells had less ATP and inhibiting ATP production in control cells expressing non-pathogenic htt with paraquat also increased pGSK3. pGSK3 was increased and ATP levels decreased in the frontal cortex and striatum of R6/1 mice and levels of cortical pGSK3 inversely correlated with cognitive function of the mice. Consistent with decreased GSK3 activity in the R6/1 mouse brain, β-catenin levels were increased and phosphorylation of collapsin response mediator protein-2 (CRMP2) decreased in the frontal cortex where inhibitory phosphorylation of GSK3 was the greatest. pGSK3 was predominantly undetectable in HD and healthy control human brain samples, but levels of total GSK3 were decreased in the HD-affected frontal cortex and this correlated with decreased pCRMP2. Thus, disruptions to cortical GSK3 signalling, possibly due to localized energy metabolism deficits, appear to contribute to the cognitive symptoms of HD.

Modulating metals as a therapeutic strategy for Alzheimer’s disease

In 1906, Alois Alzheimer first characterized the disease that bears his name. Despite intensive research, which has led to a better understanding of the pathology, there is no effective treatment for this disease. Of the drugs approved by the US FDA, none are disease modifying, only symptomatic. Unfortunately, there have been a number of failed clinical trials in the past 10 years where studies show either no cognitive improvement or, worse, serious side effects associated with treatment. Hence, there is a need for the field to look at alternative approaches to therapy. In this review, we will discuss how metal dyshomeostasis occurs in aging and Alzheimers disease. Concomitantly, we will discuss how targeting this dyshomeostasis offers an effective and novel therapeutic approach. Thus far, compounds that mediate these effects have shown great potential in both preclinical animal studies as well as in early-stage clinical trials.

Rhenium and technetium complexes that bind to amyloid-β plaques

Alzheimers disease is associated with the presence of insoluble protein deposits in the brain called amyloid plaques. The major constituent of these deposits is aggregated amyloid-β peptide. Technetium-99m complexes that bind to amyloid-β plaques could provide important diagnostic information on amyloid-β plaque burden using Single Photon Emission Computed Tomography (SPECT). Tridentate ligands with a stilbene functional group were used to form complexes with the fac-[M(I)(CO)3](+) (M = Re or (99m)Tc) core. The rhenium carbonyl complexes with tridentate co-ligands that included a stilbene functional group and a dimethylamino substituent bound to amyloid-β present in human frontal cortex brain tissue from subjects with Alzheimers disease. This chemistry was extended to make the analogous [(99m)Tc(I)(CO)3](+) complexes and the complexes were sufficiently stable in human serum. Whilst the lipophilicity (log D7.4) of the technetium complexes appeared ideally suited for penetration of the blood-brain barrier, preliminary biodistribution studies in an AD mouse model (APP/PS1) revealed relatively low brain uptake (0.24% ID g(-1) at 2 min post injection).

Gene dysregulation is restored in the Parkinson's disease MPTP neurotoxic mice model upon treatment of the therapeutic drug Cu(II)(atsm).

The administration of MPTP selectively targets the dopaminergic system resulting in Parkinsonism-like symptoms and is commonly used as a mice model of Parkinson’s disease. We previously demonstrated that the neuroprotective compound CuII(atsm) rescues nigral cell loss and improves dopamine metabolism in the MPTP model. The mechanism of action of CuII(atsm) needs to be further defined to understand how the compound promotes neuronal survival. Whole genome transcriptomic profiling has become a popular method to examine the relationship between gene expression and function. Substantia nigra samples from MPTP-lesioned mice were evaluated using whole transcriptome sequencing to investigate the genes altered upon CuII(atsm) treatment. We identified 143 genes affected by MPTP lesioning that are associated with biological processes related to brain and cognitive development, dopamine synthesis and perturbed synaptic neurotransmission. Upon CuII(atsm) treatment, the expression of 40 genes involved in promoting dopamine synthesis, calcium signaling and synaptic plasticity were restored which were validated by qRT-PCR. The study provides the first detailed whole transcriptomic analysis of pathways involved in MPTP-induced Parkinsonism. In addition, we identify key therapeutic pathways targeted by a potentially new class of neuroprotective agents which may provide therapeutic benefits for other neurodegenerative disorders.

Ablation of tau causes an olfactory deficit in a murine model of Parkinson’s disease

Parkinson’s disease is diagnosed upon the presentation of motor symptoms, resulting from substantial degeneration of dopaminergic neurons in the midbrain. Prior to diagnosis, there is a lengthy prodromal stage in which non-motor symptoms, including olfactory deficits (hyposmia), develop. There is limited information about non-motor impairments and there is a need for directed research into these early pathogenic cellular pathways that precede extensive dopaminergic death in the midbrain. The protein tau has been identified as a genetic risk factor in the development of sporadic PD. Tau knockout mice have been reported as an age-dependent model of PD, and this study has demonstrated that they develop motor deficits at 15-months-old. We have shown that at 7-month-old tau knockout mice present with an overt hyposmic phenotype. This olfactory deficit correlates with an accumulation of α-synuclein, as well as autophagic impairment, in the olfactory bulb. This pathological feature becomes apparent in the striatum and substantia nigra of 15-month-old tau knockout mice, suggesting the potential for a spread of disease. Initial primary cell culture experiments have demonstrated that ablation of tau results in the release of α-synuclein enriched exosomes, providing a potential mechanism for disease spread. These alterations in α-synuclein level as well as a marked autophagy impairment in the tau knockout primary cells recapitulate results seen in the animal model. These data implicate a pathological role for tau in early Parkinson’s disease.