Sleep and diurnal alternative polyadenylation sites associated with human APA-linked brain disorders.

Disruption of sleep and circadian rhythms are a comorbid feature of many pathologies, and can negatively influence many health conditions, including neurodegenerative disease, metabolic illness, cancer, and various neurological disorders. Genetic association studies linking sleep and circadian disturbances with disease susceptibility have mainly focused on changes in gene expression due to mutations, such as single-nucleotide polymorphisms. The interaction between sleep and/or circadian rhythms with the use of Alternative Polyadenylation (APA) has been largely undescribed, particularly in the context of other disorders. APA is a process that generates various transcript isoforms of the same gene affecting its mRNA translation, stability, localization, and subsequent function. Here we identified unique APAs expressed in rat brain over time-of-day, immediately following sleep deprivation, and the subsequent recovery period. From these data, we performed a secondary analysis of these sleep- or time-of-day associated PASs with recently described APA-linked human brain disorder susceptibility genes.

Identification of sleep and circadian alternative polyadenylation sites associated with APA-linked human brain disorders.

Sleep and circadian rhythm disruptions are comorbid features of many pathologies and can negatively influence numerous health conditions, including degenerative diseases, metabolic illnesses, cancer, and various neurological disorders. Genetic association studies linking sleep and circadian disturbances with disease susceptibility have mainly focused on changes in gene expression due to mutations, such as single-nucleotide polymorphisms. Thus, associations between sleep and/or circadian rhythm and alternative polyadenylation (APA), particularly in the context of other health challenges, are largely undescribed. APA is a process that generates various transcript isoforms from the same gene, resulting in effects on mRNA translation, stability, localization, and subsequent function. Here, we have identified unique APAs in rat brain that exhibit time-of-day-dependent oscillations in expression as well as APAs that are altered by sleep deprivation and the subsequent recovery period. Genes affected by APA usage include Mapt/Tau, Ntrk2, Homer1A, Sin3band Sorl. Sorl1 has two APAs which cycle with a 24 h period, one additional APA cycles with a 12 h period and one more that is reduced during recovery sleep. Finally, we compared sleep- or circadian-associated APAs with recently described APA-linked brain disorder susceptibility genes and found 46 genes in common.

Humanization of the mouse Tert gene reset telomeres to human length.

Telomeres undergo shortening with each cell division, serving as biomarkers of human aging, which is characterized by short telomeres and restricted telomerase expression in adult tissues. Contrarily, mice, featuring their longer telomeres and widespread telomerase activity, present limitations as models for understanding telomere-related human biology and diseases. To bridge this gap, we engineered a mouse strain with a humanized mTert gene, hmTert, wherein specific non-coding sequences were replaced with their human counterparts. The hmTert gene, encoding the wildtype mTert protein, was repressed in adult tissues beyond the gonads and thymus, closely resembling the regulatory pattern of the human TERT gene. Remarkably, the hmTert gene rescued telomere dysfunction in late generations of mTert-knockout mice. Through successive intercrosses of Terth/- mice, telomere length progressively declined, stabilizing below 10-kb. Terth/h mice achieved a human-like average telomere length of 10-12 kb, contrasting with the 50-kb length in wildtype C57BL/6J mice. Despite shortened telomeres, Terth/h mice maintained normal body weight and cell homeostasis in highly proliferative tissues. Notably, colonocyte proliferation decreased significantly in Terth/h mice during dextran sodium sulfate-induced ulcerative colitis-like pathology, suggesting limitations on cellular renewal due to short telomeres. Our findings underscore the genetic determination of telomere homeostasis in mice by the Tert gene. These mice, exhibiting humanized telomere homeostasis, serve as a valuable model for exploring fundamental questions related to human aging and cancer.

P2X7 receptors in body temperature, locomotor activity, and brain mRNA and lncRNA responses to sleep deprivation.

The ionotropic purine type 2X7 receptor (P2X7R) is a nonspecific cation channel implicated in sleep regulation and brain cytokine release. Many endogenous rhythms covary with sleep, including locomotor activity and core body temperature. Furthermore, brain-hypothalamic cytokines and purines play a role in the regulation of these physiological parameters as well as sleep. We hypothesized that these parameters are also affected by the absence of the P2X7 receptor. Herein, we determine spontaneous expression of body temperature and locomotor activity in wild-type (WT) and P2X7R knockout (KO) mice and how they are affected by sleep deprivation (SD). We also compare hypothalamic, hippocampal, and cortical cytokine- and purine-related receptor and enzyme mRNA expressions before and after SD in WT and P2X7RKO mice. Next, in a hypothesis-generating survey of hypothalamic long noncoding (lnc) RNAs, we compare lncRNA expression levels between strains and after SD. During baseline conditions, P2X7RKO mice had attenuated temperature rhythms compared with WT mice, although locomotor activity patterns were similar in both strains. After 6 h of SD, body temperature and locomotion were enhanced to a greater extent in P2X7RKO mice than in WT mice during the initial 2-3 h after SD. Baseline mRNA levels of cortical TNF-α and P2X4R were higher in the KO mice than WT mice. In response to SD, the KO mice failed to increase hypothalamic adenosine deaminase and P2X4R mRNAs. Further, hypothalamic lncRNA expressions varied by strain, and with SD. Current data are consistent with a role for the P2X7R in thermoregulation and lncRNA involvement in purinergic signaling.

Tumor necrosis factor enhances the sleep-like state and electrical stimulation induces a wake-like state in co-cultures of neurons and glia.

We characterise sleep-like states in cultured neurons and glia during development in vitro as well as after electrical stimulation, the addition of tumor necrosis factor alpha (TNF), and the combination of TNF plus electrical stimulation. We also characterise optogenetic stimulation-induced ATP release and neuronal interleukin-1 and TNF expression in vitro demonstrating the activity dependence of these putative sleep-regulatory substances. Action potential (AP) burstiness, expressed as the burstiness index (BI), synchronization of slow electrical potentials between recording electrodes (SYN), and slow wave (SW) power (0.25-3.75 Hz) determined using fast Fourier analyses emerged as network properties, maturing after 2 weeks in culture. Homologous in vivo measures are used to characterise sleep. Electrical stimulation reduced the BI, SYN and SW power values during and/or after the stimulus period. One day later, homeostasis was evident from rebounds of SYN and SW power values to above baseline levels; the magnitude of the rebound was stimulus pattern-dependent. The addition of TNF enhanced BI, SYN and SW power values, suggesting the induction of a deeper sleep-like state. Electrical stimulation reversed these TNF effects, suggesting the network state was more wake-like. The day after TNF plus electrical stimulation, the changes in SYN and SW power values were dependent upon the stimulus patterns the cells received the day before. We conclude that sleep and wake states in cultured in vitro networks can be controlled and they share molecular regulatory mechanisms with local in vivo networks. Further, sleep is an activity-dependent emergent local network property.

Biochemical regulation of sleep and sleep biomarkers.

Symptoms commonly associated with sleep loss and chronic inflammation include sleepiness, fatigue, poor cognition, enhanced sensitivity to pain and kindling stimuli, excess sleep and increases in circulating levels of tumor necrosis factor α (TNF) in humans and brain levels of interleukin-1 ß (IL1) and TNF in animals. Cytokines including IL1 and TNF partake in non-rapid eye movement sleep (NREMS) regulation under physiological and inflammatory conditions. Administration of exogenous IL1 or TNF mimics the accumulation of these cytokines occurring during sleep loss to the extent that it induces the aforementioned symptoms. Extracellular ATP associated with neuro- and glio-transmission, acting via purine type 2 receptors, e.g., the P2X7 receptor, has a role in glia release of IL1 and TNF. These substances in turn act on neurons to change their intrinsic membrane properties and sensitivities to neurotransmitters and neuromodulators such as adenosine, glutamate and GABA. These actions change the network input-output properties, i.e., a state shift for the network. State oscillations occur locally within cortical columns and are defined using evoked response potentials. One such state, so defined, shares properties with whole animal sleep in that it is dependent on prior cellular activity--it shows homeostasis. The cortical column sleep-like state is induced by TNF and is associated with experimental performance detriments. ATP released extracellularly as a consequence of cellular activity is posited to initiate a mechanism by which the brain tracks its prior sleep-state history to induce/prohibit sleep. Thus, sleep is an emergent property of populations of local neural networks undergoing state transitions. Specific neuronal groups participating in sleep depend upon prior network use driving local network state changes via the ATP-cytokine-adenosine mechanism. Such considerations add complexity to finding biochemical markers for sleepiness.

Involvement of cytokines in slow wave sleep.

Cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1ß) play a role in sleep regulation in health and disease. TNFα or IL1ß injection enhances non-rapid eye movement sleep. Inhibition of TNFα or IL1ß reduces spontaneous sleep. Mice lacking TNFα or IL1ß receptors sleep less. In normal humans and in multiple disease states, plasma levels of TNFα covary with EEG slow wave activity (SWA) and sleep propensity. Many of the symptoms induced by sleep loss, for example, sleepiness, fatigue, poor cognition, enhanced sensitivity to pain, are elicited by injection of exogenous TNFα or IL1ß. IL1ß or TNFα applied unilaterally to the surface of the cortex induces state-dependent enhancement of EEG SWA ipsilaterally, suggesting greater regional sleep intensity. Interventions such as unilateral somatosensory stimulation enhance localized sleep EEG SWA, blood flow, and somatosensory cortical expression of IL1ß and TNFα. State oscillations occur within cortical columns. One such state shares properties with whole animal sleep in that it is dependent on prior cellular activity, shows homeostasis, and is induced by TNFα. Extracellular ATP released during neuro- and gliotransmission enhances cytokine release via purine type 2 receptors. An ATP agonist enhances sleep, while ATP antagonists inhibit sleep. Mice lacking the P2X7 receptor have attenuated sleep rebound responses after sleep loss. TNFα and IL1ß alter neuron sensitivity by changing neuromodulator/neurotransmitter receptor expression, allowing the neuron to scale its activity to the presynaptic neurons. TNFα's role in synaptic scaling is well characterized. Because the sensitivity of the postsynaptic neuron is changed, the same input will result in a different network output signal and this is a state change. The top-down paradigm of sleep regulation requires intentional action from sleep/wake regulatory brain circuits to initiate whole-organism sleep. This raises unresolved questions as to how such purposeful action might itself be initiated. In the new paradigm, sleep is initiated within networks and local sleep is a direct consequence of prior local cell activity. Whole-organism sleep is a bottom-up, self-organizing, and emergent property of the collective states of networks throughout the brain.

ATP and the purine type 2 X7 receptor affect sleep.

Sleep is dependent upon prior brain activities, e.g., after prolonged wakefulness sleep rebound occurs. These effects are mediated, in part, by humoral sleep regulatory substances such as cytokines. However, the property of wakefulness activity that initiates production and release of such substances and thereby provides a signal for indexing prior waking activity is unknown. We propose that extracellular ATP, released during neuro- and gliotransmission and acting via purine type 2 (P2) receptors, is such a signal. ATP induces cytokine release from glia. Cytokines in turn affect sleep. We show here that a P2 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), increased non-rapid eye movement sleep (NREMS) and electroencephalographic (EEG) delta power while two different P2 receptor antagonists, acting by different inhibitory mechanisms, reduced spontaneous NREMS in rats. Rat P2X7 receptor protein varied in the somatosensory cortex with time of day, and P2X7 mRNA was altered by interleukin-1 treatment, by sleep deprivation, and with time of day in the hypothalamus and somatosensory cortex. Mice lacking functional P2X7 receptors had attenuated NREMS and EEG delta power responses to sleep deprivation but not to interleukin-1 treatment compared with wild-type mice. Data are consistent with the hypothesis that extracellular ATP, released as a consequence of cell activity and acting via P2 receptors to release cytokines and other sleep regulatory substances, provides a mechanism by which the brain could monitor prior activity and translate it into sleep.

TNFalpha siRNA reduces brain TNF and EEG delta wave activity in rats.

Tumor necrosis factor alpha (TNFalpha) is a pleiotropic cytokine with several CNS physiological and pathophysiological actions including sleep, memory, thermal and appetite regulation. Short interfering RNAs (siRNA) targeting TNFalpha were incubated with cortical cell cultures and microinjected into the primary somatosensory cortex (SSctx) of rats. The TNFalpha siRNA treatment specifically reduced TNFalpha mRNA by 45% in vitro without affecting interleukin-6 or gluR1-4 mRNA levels. In vivo the TNFalpha siRNAalpha reduced TNFalpha mRNA, interleukin-6 mRNA and gluR1 mRNA levels compared to treatment with a scrambled control siRNA. After in vivo microinjection, the density of TNFalpha-immunoreactive cells in layer V of the SSctx was also reduced. Electroencephalogram (EEG) delta wave power was decreased on days 2 and 3 on the side of the brain that received the TNFalpha siRNA microinjection relative to the side receiving the control siRNA. These findings support the hypothesis that TNFalpha siRNA attenuates TNFalpha mRNA and TNFalpha protein in the rat cortex and that those reductions reduce cortical EEG delta power. Results also are consistent with the notion that TNFalpha is involved in CNS physiology including sleep regulation.

Synthetic ansamycins prepared by a ring-expanding Claisen rearrangement. Synthesis and biological evaluation of ring and conformational analogues of the Hsp90 molecular chaperone inhibitor geldanamycin.

A series of ansa-quinones has been prepared by chemical synthesis, and evaluated by biological techniques. Thus, 19-membered ansa-lactams, simplified analogues of the naturally occurring Hsp90 molecular chaperone inhibitor geldanamycin, were obtained by concise routes, the key steps being the combination of a ring-closing metathesis to give a 17-membered ring followed by Claisen rearrangement to effect ring expansion. The methodology was also used to prepare an "unnatural" 18-membered ring analogue. In ATPase enzyme assays, the synthetic ansa-quinones were weak inhibitors of Hsp90.