Summary: In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC−/− mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC−/−CDKN1A−/− mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.

Ca²⁺ and β-catenin, a 92-kDa negatively charged transcription factor, transduce Wnt signaling via the non-canonical, Wnt/Ca²⁺ and canonical, Wnt/β-catenin pathways independently. The nuclear envelope is a barrier to large protein entry, and this process is regulated by intracellular calcium [Ca²⁺]_i and trans-nuclear potential. How β-catenin traverses the nuclear envelope is not well known. We hypothesized that Wnt/Ca²⁺ and Wnt/β-catenin pathways act in a coordinated manner and that [Ca²⁺]_i release facilitates β-catenin entry into the nucleus in mammalian cells. In a live assay using calcium dyes in PC3 prostate cancer cells, six Wnt peptides (3A, 4, 5A, 7A, 9B, and 10B) mobilized [Ca²⁺]_i but Wnt11 did not. Based upon dwell time (range = 15–30 s) of the calcium waveform, these Wnts could be classified into three classes: short, 3A and 5A; long, 7A and 10B; and very long, 4 and 9B. Wnt-activated [Ca²⁺]_i release was followed by an increase in intranuclear calcium and the depolarization of both the cell and nuclear membranes, determined by using FM4-64. In cells treated with Wnts 5A, 9B, and 10B, paradigm substrates for each Wnt class, increased [Ca²⁺]_i was followed by β-catenin translocation into the nucleus in PC3, MCF7, and 253J, prostate, breast, and bladder cancer cell lines; both the increase in Wnt 5A, 9B, and 10B induced [Ca²⁺]_i release and β-catenin translocation are suppressed by thapsigargin in PC3 cell line. We propose a convergent model of Wnt signaling network where Ca²⁺ and β-catenin pathways may act in a coordinated, interdependent, rather than independent, manner.

Wnt signaling is a critical regulatory pathway in development and disease. Very little is known about the mechanisms of Wnt signaling in prostate cancer, a leading cause of death in men. A quantitative analysis of the expression of Wnt5A protein in human tissue arrays, containing 600 prostate tissue cores, showed >50% increase in malignant compared to benign cores (p<0.0001). In a matched pair of prostate cancer and normal cell line, expression of Wnt5A protein was also increased. Calcium waves were induced in prostate cells in response to Wnt5A with a 3 fold increase in Flou-4 intensity. The activity of Ca²⁺/calmodulin dependent protein kinase (CaMKII), a transducer of the non-canonical Wnt/Ca²⁺ signaling, increased by 8 fold in cancer cells; no change was observed in β-catenin expression, known to activate the canonical Wnt/β-catenin pathway. Mining of publicly available human prostate cancer oligoarray datasets revealed that the expression of numerous genes (e.g., CCND1, CD44) under the control of β-catenin transcription is down-regulated. Confocal and quantitative electron microscopy showed that specific inhibition of CaMKII in cancer cells causes remodeling of the actin cytoskeleton, irregular wound edges and loose intercellular architecture and a 6 and 8 fold increase in the frequency and length of filopodia, respectively. Conversely, untreated normal prostate cells showed an irregular wound edge and loose intercellular architecture; incubation of normal prostate cells with recombinant Wnt5A protein induced actin remodeling with a regular wound edge and increased wound healing capacity. Live cell imaging showed that a functional consequence of CaMKII inhibition was 80% decrease in wound healing capacity and reduced cell motility in cancer cells. We propose that non-canonical Wnt/Ca²⁺ signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.

Summary: We have studied the mechanisms underlying nonpathological age‐related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS‐sensitive dyes, dihydrorhodamine (DHR) and 2‐[6‐(4′‐hydroxy)phenoxy‐3H‐xanthen‐3‐on‐9‐yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12–15 months and 24 months but not in adult or aged AL‐fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox‐cycling drug, menadione. Menadione fails to increase apoptosis in 6‐month neurons. However, in 12–15mAL fed rats, when age‐related cell death begins, menadione induces a 7‐ to 15‐fold increase in the proportion of apoptotic neurons. CR protects age‐matched neurons against ROS‐induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione‐induced cell death, but only in 12–15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age‐related increases in neuronal ROS levels and in ROS‐induced cell death.