Malfunctioning DNA Damage Response (DDR) Leads to the Degeneration of Nigro-Striatal Pathway in Mouse Brain

Pronounced neuropathology is a feature of ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS), which are both genomic instability syndromes. The Nbs1 protein, which is defective in NBS, is a component of the Mre11/RAD50/NBS1 (MRN) complex. This complex plays a major role in the early phase of the cellular response to double strand breaks (DSBs) in the DNA. Among others, MRN is required for timely activation of the protein kinase ATM (A-T mutated), which is disrupted in patients with A-T. Earlier reports show that Atm-deficient mice exhibit severe degeneration of tyrosine hydroxylase (TH)-positive dopaminergic nigro-striatal neurons and their terminals in the striatum. This cell loss is accompanied by a large reduction in immunoreactivity for the dopamine transporter protein (DAT) in the striatum. To test whether Nbs1 inactivation also affects the integrity of the nigro-striatal pathway, we examined this pathway in a murine model with conditional inactivation of the Nbs1 gene in central nervous system (Nbs1-CNS-Δ). We report that this model has a reduction in TH-positive cells in the substantia nigra. This phenomenon was seen at very early age, while Atm−/− mice showed a progressive age-dependent reduction. Furthermore, we observed an age-dependent increase in the level of TH in the striatum of Atm−/− and Nbs1-CNS-Δ mice. In addition to the altered expression of TH, we also found a reduction of DAT in the striatum of both Atm−/− and Nbs1-CNS-Δ mice at 60 days of age. Finally, microglial recruitment and alterations in the levels of various neurotrophic factors were also observed. These results indicate that malfunctioning DNA damage response severely affects the integrity of the nigro-striatal pathway and suggest a new neurodegenerative pathway in Parkinsonian syndromes.     

Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.

To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S-nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91-kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.     

Illness causal attributions: an exploratory study of their structure and associations with other illness cognitions and perceptions of control

Two studies were conducted to investigate the cognitive organization and psychological meaning of illness causes. Using a direct similarity judgment method (Study 1), illness causes were found cognitively organized in a hierarchical configuration that could meaningfully be represented as a tree with three main branches—environmental, behavioral, and hidden causes—that further divided into subcategories. This classification of illness causes was associated with other components of the illness schema, namely, the consequences and control/cure dimensions, but not with timeline perceptions (Study 2). Perceptions of control were significantly associated with the cognitive organization of illness causal attributions. Personal relevancy was found as a moderator of illness causal attributions, influencing the relationships between attributions and other illness cognitions.     

A Role for Vascular Deficiency in Retinal Pathology in a Mouse Model of Ataxia-Telangiectasia

Ataxia-telangiectasia is a multifaceted syndrome caused by null mutations in the ATM gene, which encodes the protein kinase ATM, a key participant in the DNA damage response. Retinal neurons are highly susceptible to DNA damage because they are terminally differentiated and have the highest metabolic activity in the central nervous system. In this study, we characterized the retina in young and aged Atm-deficient mice (Atm^−/−). At 2 months of age, angiography revealed faint retinal vasculature in Atm^−/− animals relative to wild-type controls. This finding was accompanied by increased expression of vascular endothelial growth factor protein and mRNA. Fibrinogen, generally absent from wild-type retinal tissue, was evident in Atm^−/− retinas, whereas mRNA of the tight junction protein occludin was significantly decreased. Immunohistochemistry labeling for occludin in 6-month-old mice showed that this decrease persists in advanced stages of the disease. Concurrently, we noticed vascular leakage in Atm^−/− retinas. Labeling for glial fibrillary acidic protein demonstrated morphological alterations in glial cells in Atm^−/− retinas. Electroretinographic examination revealed amplitude aberrations in 2-month-old Atm^−/− mice, which progressed to significant functional deficits in the older mice. These results suggest that impaired vascularization and astrocyte–endothelial cell interactions in the central nervous system play an important role in the etiology of ataxia-telangiectasia and that vascular abnormalities may underlie or aggravate neurodegeneration.Some brain disorders may have a vascular origin,^1,2 and vascular diseases can be directly linked to neuronal and synaptic dysfunction through changes in the blood flow, increase in blood–brain barrier permeability, and in nutrient supply.³ A healthy brain relies on the proper function and communication of all cells comprising the neurovascular unit: neurons, astrocytes, brain endothelium, and vascular smooth muscle cells.⁴Impaired genomic stability interferes with cellular homeostasis and poses a constant threat to cellular viability.⁵ The cell combats this threat by activating the DNA damage response (DDR), a complex signaling network that detects the DNA lesions, promotes their repair, and temporarily modulates cellular metabolism while the damage is being repaired.⁶ The DDR is vigorously activated by DNA double-strand breaks (DSBs), a particularly cytotoxic DNA lesion induced by ionizing radiation, radiomimetic chemicals, and oxygen radicals.^7,8 The DNA damage response is a hierarchical process executed by sensor/mediator proteins that accumulate at DSB sites and by protein kinases that serve as transducers of the DNA damage alarm to numerous downstream effectors.⁶ The primary transducer of the cellular response to DSBs is the protein kinase ATM, which phosphorylates many key players in the various branches of the DDR.⁹Ataxia-telangiectasia (A-T) is an autosomal recessive disorder caused by mutations in the ATM gene that encodes the ATM protein.¹⁰ A-T is characterized by progressive neurodegeneration affecting mainly the cerebellum, which develops into severe neuromotor dysfunction; peripheral neuropathy; immunodeficiency that spans the B- and T-cell systems; thymic and gonadal atrophy; marked predisposition to lymphoreticular malignancies; and chromosomal fragility and acute sensitivity to ionizing radiation. Cultured ATM-deficient cells exhibit severe cellular sensitivity to DSB-inducing agents, with markedly defective DSB response.DSBs are constantly induced in all body cells by metabolic byproducts such as oxygen radicals. Oxidative stress has been consistently associated with various neurodegenerative conditions.^11–14 Indeed, there is substantial evidence for a role of oxidative damage in the progression of neurodegenerative disorders, including Parkinson''s and Alzheimer''s diseases.¹⁵ Similarly, elevated oxidative stress has been identified in several DNA repair deficiencies, including A-T.^16–22 Notably, ATM has recently been shown to be activated by oxidative stress.²³ Ocular tissues, especially the retina, are exposed to extremely high levels of reactive oxygen species. Nevertheless, retinal neurodegeneration has not been reported in A-T patients. The ocular manifestation of the disease reported to date is scleral telangiectasia^24,25 and saccadic abnormalities.²⁶ Moreover, no retinal pathology has been described to date in Atm-deficient mice despite their increased sensitivity to reactive oxygen species–inducing agents.²⁷ We examined the link between retinal vascular pathology and function in young and aging Atm-deficient mice. We present evidence for vascular changes that accompany neuronal deficiencies in the retina.     

Homozygous deletion of RAG1, RAG2 and 5′ region TRAF6 causes severe immune suppression and atypical osteopetrosis

Mutations of several genes have been implicated in autosomal recessive osteopetrosis (OP), a disease caused by impaired function and differentiation of osteoclasts. Severe combined immune deficiencies (SCID) can likewise result from different genetic mutations. We report two siblings with SCID and an atypical phenotype of OP. A biallelic microdeletion encompassing the 5′ region of TRAF6, RAG1 and RAG2 genes was identified. TRAF6, a tumor necrosis factor receptor‐associated family member, plays an important role in T cell signaling and in RANKL‐dependent osteoclast differentiation and activation but its role in human OP has not been previously reported. The RAG proteins are essential for recombination of B and T cell receptors, and for the survival and differentiation of these cells. This is the first study to report a homozygous deletion of TRAF6 as a cause of human disease.