Activation of the stress proteome as a mechanism for small molecule therapeutics.

Various small molecule pharmacologic agents with different known functions produce similar outcomes in diverse Mendelian and complex disorders, suggesting that they may induce common cellular effects. These molecules include histone deacetylase inhibitors, 4-phenylbutyrate (4PBA) and trichostatin A, and two small molecules without direct histone deacetylase inhibitor activity, hydroxyurea (HU) and sulforaphane. In some cases, the therapeutic effects of histone deacetylase inhibitors have been attributed to an increase in expression of genes related to the disease-causing gene. However, here we show that the pharmacological induction of mitochondrial biogenesis was necessary for the potentially therapeutic effects of 4PBA or HU in two distinct disease models, X-linked adrenoleukodystrophy and sickle cell disease. We hypothesized that a common cellular response to these four molecules is induction of mitochondrial biogenesis and peroxisome proliferation and activation of the stress proteome, or adaptive cell survival response. Treatment of human fibroblasts with these four agents induced mitochondrial and peroxisomal biogenesis as monitored by flow cytometry, immunofluorescence and/or western analyses. In treated normal human fibroblasts, all four agents induced the adaptive cell survival response: heat shock, unfolded protein, autophagic and antioxidant responses and the c-jun N-terminal kinase pathway, at the transcriptional and translational levels. Thus, activation of the evolutionarily conserved stress proteome and mitochondrial biogenesis may be a common cellular response to such small molecule therapy and a common basis of therapeutic action in various diseases. Modulation of this novel therapeutic target could broaden the range of treatable diseases without directly targeting the causative genetic abnormalities.

Hydroxyurea Improves Spatial Memory and Cognitive Plasticity in Mice and Has a Mild Effect on These Parameters in a Down Syndrome Mouse Model.

Down syndrome (DS), a genetic disorder caused by partial or complete triplication of chromosome 21, is the most common genetic cause of intellectual disability. DS mouse models and cell lines display defects in cellular adaptive stress responses including autophagy, unfolded protein response, and mitochondrial bioenergetics. We tested the ability of hydroxyurea (HU), an FDA-approved pharmacological agent that activates adaptive cellular stress response pathways, to improve the cognitive function of Ts65Dn mice. The chronic HU treatment started at a stage when early mild cognitive deficits are present in this model (∼3 months of age) and continued until a stage of advanced cognitive deficits in untreated mice (∼5-6 months of age). The HU effects on cognitive performance were analyzed using a battery of water maze tasks designed to detect changes in different types of memory with sensitivity wide enough to detect deficits as well as improvements in spatial memory. The most common characteristic of cognitive deficits observed in trisomic mice at 5-6 months of age was their inability to rapidly acquire new information for long-term storage, a feature akin to episodic-like memory. On the background of severe cognitive impairments in untreated trisomic mice, HU-treatment produced mild but significant benefits in Ts65Dn by improving memory acquisition and short-term retention of spatial information. In control mice, HU treatment facilitated memory retention in constant (reference memory) as well as time-variant conditions (episodic-like memory) implicating a robust nootropic effect. This was the first proof-of-concept study of HU treatment in a DS model, and indicates that further studies are warranted to assess a window to optimize timing and dosage of the treatment in this pre-clinical phase. Findings of this study indicate that HU has potential for improving memory retention and cognitive flexibility that can be harnessed for the amelioration of cognitive deficits in normal aging and in cognitive decline (dementia) related to DS and other neurodegenerative diseases.

Hydroxyurea attenuates oxidative, metabolic, and excitotoxic stress in rat hippocampal neurons and improves spatial memory in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by accumulation of amyloid ß-peptide (Aß) plaques in the brain and decreased cognitive function leading to dementia. We tested if hydroxyurea (HU), a ribonucleotide reductase inhibitor known to activate adaptive cellular stress responses and ameliorate abnormalities associated with several genetic disorders, could protect rat hippocampal neurons against oxidative-, excitatory-, mitochondrial-, and Aß-induced stress and if HU treatment could improve learning and memory in the APP/PS1 mouse model of AD. HU treatment attenuated the loss of cell viability induced by treatment of hippocampal neurons with hydrogen peroxide, glutamate, rotenone, and Aß1-42. HU treatment attenuated reductions of mitochondrial reserve capacity, maximal respiration, and cellular adenosine triphosphate content induced by hydrogen peroxide treatment. In vivo, treatment of APP/PS1 mice with HU (45 mg/kg/d) improved spatial memory performance in the hippocampus-dependent Morris water maze task without reducing Aß levels. HU provides neuroprotection against toxic insults including Aß, improves mitochondrial bioenergetics, and improves spatial memory in an AD mouse model. HU may offer a new therapeutic approach to delay cognitive decline in AD.

SOD2 as a potential modifier of X-linked adrenoleukodystrophy clinical phenotypes.

X-linked adrenoleukodystrophy (XALD), a neurological disorder caused by mutations in the peroxisomal membrane protein gene ABCD1, presents as a rapidly progressing, inflammatory cerebral demyelination (cerebral cases) or a slowly progressing, distal axonopathy (non-cerebral cases). Specific ABCD1 defects do not explain this significant phenotypic variation. Patients have increased plasma and tissue very long chain fatty acid levels and increased cellular oxidative stress and oxidative damage. Superoxide dismutase 2 (SOD2), at candidate modifier locus 6q25.3, detoxifies superoxide radicals protecting against oxidative stress and damage. We tested an SOD2 variant C47T (Ala16Val) associated with reduced enzymatic activity as a potential modifier gene of cerebral demyelinating disease by comparing 117 cerebral XALD cases with 105 non-cerebral XALD cases. The hypoactive valine allele of the variant was associated with cerebral disease under a dominant model in the full data set (p = 0.04; ORT* = 1.90, 95% CI 1.01-3.56) and the non-childhood cerebral disease subset (p = 0.03; ORT* = 2.47, 95% CI 1.08-5.61). Three tag SNPs were genotyped to test for additional SNP or haplotype associations. A common haplotype, GTAC, which included the SOD2 valine allele, was associated with cerebral disease in the full data set (p = 0.03; OR = 1.75, 95% CI 1.11-2.75) and the non-childhood cerebral disease subset (p = 0.008; OR = 2.20, 95% CI 1.27-3.83). There was no association between childhood cerebral XALD and the C47T variant or the GTAC haplotype. Thus, reduced SOD2 activity may contribute to the development of cerebral demyelination in adolescent and adult XALD patients.