Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest

BACKGROUND: UV-induced damage can induce apoptosis or trigger DNA repair mechanisms. Minor DNA damage is thought to halt the cell cycle to allow effective repair, while more severe damage can induce an apoptotic program. Of the two major types of UV-induced DNA lesions, it has been reported that repair of CPD, but not 6-4PP, abrogates mutation. To address whether the two major forms of UV-induced DNA damage, can induce differential biological effects, NER-deficient cells containing either CPD photolyase or 6-4 PP photolyase were exposed to UV and examined for alterations in cell cycle and apoptosis. In addition, pTpT, a molecular mimic of CPD was tested in vitro and in vivo for the ability to induce cell death and cell cycle alterations. METHODS: NER-deficient XPA cells were stably transfected with CPD-photolyase or 6-4PP photolyase to specifically repair only CPD or only 6-4PP. After 300 J/m(2 )UVB exposure photoreactivation light (PR, UVA 60 kJ/m(2)) was provided for photolyase activation and DNA repair. Apoptosis was monitored 24 hours later by flow cytometric analysis of DNA content, using sub-G1 staining to indicate apoptotic cells. To confirm the effects observed with CPD lesions, the molecular mimic of CPD, pTpT, was also tested in vitro and in vivo for its effect on cell cycle and apoptosis. RESULTS: The specific repair of 6-4PP lesions after UVB exposure resulted in a dramatic reduction in apoptosis. These findings suggested that 6-4PP lesions may be the primary inducer of UVB-induced apoptosis. Repair of CPD lesions (despite their relative abundance in the UV-damaged cell) had little effect on the induction of apoptosis. Supporting these findings, the molecular mimic of CPD, (dinucleotide pTpT) could mimic the effects of UVB on cell cycle arrest, but were ineffective to induce apoptosis. CONCLUSION: The primary response of the cell to UV-induced 6-4PP lesions is to trigger an apoptotic program whereas the response of the cell to CPD lesions appears to principally involve cell cycle arrest. These findings suggest that CPD and 6-4 PP may induce differential biological effects in the UV-damaged cell

Matrix metalloproteinase-7 facilitates immune access to the CNS in experimental autoimmune encephalomyelitis

<p>Abstract</p> <p>Background</p> <p>Metalloproteinase inhibitors can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Matrix metalloproteinase-9 (MMP-9) has been implicated, but it is not clear if other MMPs are also involved, including matrilysin/MMP-7 – an enzyme capable of cleaving proteins that are essential for blood brain barrier integrity and immune suppression.</p> <p>Results</p> <p>Here we report that MMP-7-deficient (<it>mmp7</it>^-/-) mice on the C57Bl/6 background are resistant to EAE induced by myelin oligodendrocyte glycoprotein (MOG). Brain sections from MOG-primed <it>mmp7</it>^-/-mice did not show signs of immune cell infiltration of the CNS, but MOG-primed wild-type mice showed extensive vascular cuffing and mononuclear cell infiltration 15 days after vaccination. At the peak of EAE wild-type mice had MMP-7 immuno-reactive cells in vascular cuffs that also expressed the macrophage markers Iba-1 and Gr-1, as well as tomato lectin. MOG-specific proliferation of splenocytes, lymphocytes, CD4⁺and CD8⁺cells were reduced in cells isolated from MOG-primed <it>mmp7</it>^-/-mice, compared with MOG-primed wild-type mice. However, the adoptive transfer of splenocytes and lymphocytes from MOG-primed <it>mmp7</it>^-/-mice induced EAE in naïve wild-type recipients, but not naïve <it>mmp7</it>^-/-recipients. Finally, we found that recombinant MMP-7 increased permeability between endothelial cells in an <it>in vitro </it>blood-brain barrier model.</p> <p>Conclusion</p> <p>Our findings suggest that MMP-7 may facilitate immune cell access or re-stimulation in perivascular areas, which are critical events in EAE and multiple sclerosis, and provide a new therapeutic target to treat this disorder.</p

Open-label add-on treatment trial of minocycline in fragile X syndrome

<p>Abstract</p> <p>Background</p> <p>Fragile X syndrome (FXS) is a disorder characterized by a variety of disabilities, including cognitive deficits, attention-deficit/hyperactivity disorder, autism, and other socio-emotional problems. It is hypothesized that the absence of the fragile X mental retardation protein (FMRP) leads to higher levels of matrix metallo-proteinase-9 activity (MMP-9) in the brain. Minocycline inhibits MMP-9 activity, and alleviates behavioural and synapse abnormalities in <it>fmr1 </it>knockout mice, an established model for FXS. This open-label add-on pilot trial was conducted to evaluate safety and efficacy of minocycline in treating behavioural abnormalities that occur in humans with FXS.</p> <p>Methods</p> <p>Twenty individuals with FXS, ages 13-32, were randomly assigned to receive 100 mg or 200 mg of minocycline daily. Behavioural evaluations were made prior to treatment (baseline) and again 8 weeks after daily minocycline treatment. The primary outcome measure was the Aberrant Behaviour Checklist-Community Edition (ABC-C) Irritability Subscale, and the secondary outcome measures were the other ABC-C subscales, clinical global improvement scale (CGI), and the visual analog scale for behaviour (VAS). Side effects were assessed using an adverse events checklist, a complete blood count (CBC), hepatic and renal function tests, and antinuclear antibody screen (ANA), done at baseline and at 8 weeks.</p> <p>Results</p> <p>The ABC-C Irritability Subscale scores showed significant improvement (p < 0.001), as did the VAS (p = 0.003) and the CGI (p < 0.001). The only significant treatment-related side effects were minor diarrhea (n = 3) and seroconversion to a positive ANA (n = 2).</p> <p>Conclusions</p> <p>Results from this study demonstrate that minocycline provides significant functional benefits to FXS patients and that it is well-tolerated. These findings are consistent with the <it>fmr1 </it>knockout mouse model results, suggesting that minocycline modifies underlying neural defects that account for behavioural abnormalities. A placebo-controlled trial of minocycline in FXS is warranted.</p> <p>Trial registration</p> <p>ClinicalTrials.gov Open-Label Trial NCT00858689.</p

Amyloid-β and APP Deficiencies Cause Severe Cerebrovascular Defects: Important Work for an Old Villain

<div><p>Alzheimer’s disease (AD) is marked by neuritic plaques that contain insoluble deposits of amyloid-β (Aβ), yet the physiological function of this peptide has remained unclear for more than two decades. Using genetics and pharmacology we have established that Aβ plays an important role in regulating capillary bed density within the brain, a function that is distinct from other cleavage products of amyloid precursor protein (APP). APP-deficient zebrafish had fewer cerebrovascular branches and shorter vessels in the hindbrain than wild-type embryos; this phenotype was rescued by treatment with human Aβ peptide, but not a smaller APP fragment called p3. Similar vascular defects were seen in zebrafish treated with a β-secretase inhibitor (BSI) that blocked endogenous Aβ production. BSI-induced vascular defects were also improved by treatment with human Aβ, but not p3. Our results demonstrate a direct correlation between extracellular levels of Aβ and cerebrovascular density in the developing hindbrain. These findings may be relevant to AD etiology where high levels of Aβ in the brain parenchyma precede the development of neuritic plaques and dense aberrantly-branched blood vessel networks that appear between them. The ability of Aβ to modify blood vessels may coordinate capillary density with local metabolic activity, which could explain the evolutionary conservation of this peptide from lobe-finned fish to man.</p> </div

Schematic of APP processing that produces Aβ and p3 peptides.

<p>APP is initially cleaved by either α-secretase or β-secretases to yield a C87 in the alpha pathway, or C99 in the beta pathway, respectively. These cleavage events also produce an extracellular soluble APP (sAPP) fragment, from the amino terminus, that is slightly longer with α-secretase cleavage. The C87 and C99 fragments are subsequently cleaved within the transmembrane domains (tm) by γ-secretase to produce p3 and Aβ peptides, respectively. Both γ-secretase events produce an Aβ-intracellular-domain (AICD) fragment that is entirely cytosolic. Variability of γ-secretase cleavage on C99 produces Aβ fragments from 39–43 amino acids - only Aβ(1–42) is shown – and similar variability with C87 cleavage.</p