Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurological disorder that affects premutation carriers with 55-200 CGG-expansion repeats (preCGG) in FMR1, presenting with early alterations in neuronal network formation and function that precede neurodegeneration. Whether intranuclear inclusions containing DNA damage response (DDR) proteins are causally linked to abnormal synaptic function, neuronal growth and survival are unknown. In a mouse that harbors a premutation CGG expansion (preCGG), cortical and hippocampal FMRP expression is moderately reduced from birth through adulthood, with greater FMRP reductions in the soma than in the neurite, despite several-fold elevation of Fmr1 mRNA levels. Resting cytoplasmic calcium concentration ([Ca2+]i) in cultured preCGG hippocampal neurons is chronically elevated, 3-fold compared to Wt; elevated ROS and abnormal glutamatergic responses are detected at 14 DIV. Elevated µ-calpain activity and a higher p25/p35 ratio in the cortex of preCGG young adult mice indicate abnormal Cdk5 regulation. In support, the Cdk5 substrate, ATM, is upregulated by 1.5- to 2-fold at P0 and 6 months in preCGG brain, as is p-Ser1981-ATM. Bax:Bcl-2 is 30% higher in preCGG brain, indicating a greater vulnerability to apoptotic activation. Elevated [Ca2+]i, ROS, and DDR signals are normalized with dantrolene. Chronic [Ca2+]i dysregulation amplifies Cdk5-ATM signaling, possibly linking impaired glutamatergic signaling and DDR to neurodegeneration in preCGG brain.

Nineteen ortho-substituted PCBs are chiral and found enantioselectively enriched in ecosystems. Their differential actions on biological targets are not understood. PCB 95 (2,2′,3,5′,6-pentachlorobiphenyl), a chiral PCB of current environmental relevance, is among the most potent toward modifying ryanodine receptors (RyR) function and Ca2+ signaling. PCB 95 enantiomers are separated and assigned aR- and aS-PCB 95 using three chiral-column HPLC and circular dichroism spectroscopy. Studies of RyR1-enriched microsomes show aR-PCB 95 with >4× greater potency (EC50 = 0.20 ± 0.05 μM), ~ 1.3× higher efficacy (Bmax = 3.74 ± 0.07 μM) in [3H]Ryanodine-binding and >3× greater rates (R = 7.72 ± 0.31 nmol/sec/mg) of Ca2+ efflux compared with aS-PCB 95, whereas racemate has intermediate activity. aR-PCB 95 has modest selectivity for RyR2, and lower potency than racemate toward the RyR isoform mixture in brain membranes. Chronic exposure of hippocampal neuronal networks to nanomolar PCB 95 during a critical developmental period shows divergent influences on synchronous Ca2+ oscillation (SCO): rac-PCB 95 increasing and aR-PCB 95 decreasing SCO frequency at 50 nM, although the latter’s effects are nonmonotonic at higher concentration. aS-PCB95 shows the greatest influence on inhibiting responses to 20 Hz electrical pulse trains. Considering persistence of PCB 95 in the environment, stereoselectivity toward RyRs and developing neuronal networks may clarify health risks associated with enantioisomeric enrichment of PCBs.

Under resting conditions, external Ca2+ is known to enter skeletal muscle cells, whereas Ca2+ stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca2+ entry and in the SR Ca2+ leak is still a matter of debate, but several lines of evidence suggest that these Ca2+ fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca2+ dye Fura2, we first demonstrated that the rate of Ca2+ increase in response to cyclopiazonic acid (CPA)–induced inhibition of SR Ca2+-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca2+ leak. However, removal of external Ca2+ reduced the rate of CPA-induced Ca2+ increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca2+ influx in mdx fibers. Fibers were then loaded with the low-affinity Ca2+ dye Fluo5N-AM to measure intraluminal SR Ca2+ changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca2+ changes. Voltage dependence and magnitude of depolarization-induced SR Ca2+ depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca2+ reuptake activity in mdx fibers. Overall, CPA-induced SR Ca2+ leak at −80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca2+ in control fibers. The elevated passive SR Ca2+ leak may contribute to alteration of Ca2+ homeostasis in mdx muscle.

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