SHANK gene family, as mutations in SHANK2 and SHANK3 have been detected in several autistic individuals. SHANK genes code for a family of scaffolding proteins located in the postsynaptic density of excitatory synapses. To test the hypothesis that a mutation in SHANK1 contributes to the symptoms of autism, we evaluated Shank1−/− null mutant mice for behavioral phenotypes with relevance to autism, focusing on social communication. Ultrasonic vocalizations and the deposition of scent marks appear to be two major modes of mouse communication. Our findings revealed evidence for low levels of ultrasonic vocalizations and scent marks in Shank1/−− mice as compared to wildtype Shank1+/+ littermate controls. Shank1/−− pups emitted fewer vocalizations than +/+Shank1 pups when isolated from mother and littermates. In adulthood, genotype affected scent marking behavior in the presence of female urinary pheromones. Adult Shank1/−− males deposited fewer scent marks in proximity to female urine than +/+Shank1 males. Call emission in response to female urinary pheromones also differed between genotypes. +/+Shank1 mice changed their calling pattern dependent on previous female interactions, while Shank1/−− mice were unaffected, indicating a failure of Shank1/−− males to learn from a social experience. The reduced levels of ultrasonic vocalizations and scent marking behavior in Shank1/−− mice are consistent with a phenotype relevant to social communication deficits in autism.Autism is a neurodevelopmental disorder with a strong genetic component. Core symptoms are abnormal reciprocal social interactions, qualitative impairments in communication, and repetitive and stereotyped patterns of behavior with restricted interests. Candidate genes for autism include the  SHANK gene family SHANK3 and deletions in the chromosome 22q13.2 region containing SHANK3 have been described in autistic patients SHANK2 were recently reported in several individuals with autism Autism is a neurodevelopmental disorder characterized by abnormal reciprocal social interactions, deficits in social communication, motor stereotypies, repetitive behaviors, and narrow restricted interests SHANK genes code for a family of multidomain scaffolding proteins located in the postsynaptic density (PSD) of glutamatergic synapses Shank1 gene in mice, to investigate its function in vivo. Shank1/−− mutant mice showed altered protein composition of the PSD with reduced levels for Shank, Homer, and GKAP. Dendritic spines and synapses were smaller, which correlated with weakening of excitatory synaptic transmission. Behaviorally, Shank1/−− mice displayed reduced locomotion, impaired rotarod performance, higher anxiety-like behavior, but normal levels of social interactions Shank1/−− mice. As suggested by Hung et al. Hung et al. SHANK1 contributes to the symptoms of autism, we evaluated Shank1−/− null mutant, +/Shank1− heterozygote, and +/+Shank1 wildtype littermate control mice for behavioral phenotypes with relevance to autism. Here, we focus on communication deficits in mice that may incorporate conceptual analogies to the qualitative impairments in communication such as delayed language and poor communication skills, which are fundamental to the diagnosis of autism To test the hypothesis that a mutation in In adult mice, high USV levels are detected in males when courting and copulating with females Shank1/−− mutant mice for developmental milestones and pup isolation-induced USV, and in our assays for adult male scent marking and USV to female urine in an open field. Results are consistent with an interpretation of communication deficits relevant to autism.In addition to USV, adult male mice display scent marking behavior, depositing urinary pheromone traces in close proximity to the location of female urine Shank1/−− null mutant mice with a targeted replacement of exons 14 and 15 encoding almost the entire PDZ domain were compared to +/Shank1− and +/+Shank1 littermate control mice. Mice were obtained from mutant lines originally generated by Hung et al. Shank1 mutation, consistent with the other studies on Shank1 mutants +/Shank1− males and females were bred in a conventional vivarium at the National Institute of Mental Health in Bethesda, MD, USA. Approximately 2 weeks after pairing for breeding, females were individually housed and inspected daily for pregnancy and delivery. The day of birth was considered as postnatal day (pnd) 0. After weaning on pnd 21, mice were socially housed in groups of 2–4 with same-sex partners. All mice were housed in polycarbonate Makrolon IVC cages . Bedding, paper strips, a nestlet square and a cardboard tube were provided in each cage. Standard rodent chow and water were available ad libitum. The colony room was maintained on a 12∶12 light/dark cycle with lights on at 06∶00 h, at approximately 20°C and 55% humidity. Pups were identified by paw tattoo, using non-toxic animal tattoo ink . The ink was inserted subcutaneously through a 30 gauge hypodermic needle tip into the center of the paw. All procedures were conducted in strict compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the National Institute of Mental Health Animal Care and Use Committee.+/+Shank1 littermate control mice , n = 11 +/Shank1− , and n = 16 Shank1/−− mutant mice . Mice of cohort 2 were tested for USV in isolation on pnd 8. After measuring pup USV, body weight and body temperature were determined. At the age of 13.00±0.50 weeks, adult male mice were tested for open field activity and basal scent marking behavior when exposed to a clean open field for 60 min. Immediately thereafter, a drop of female urine was added to the open field. Open field activity, female urine elicited scent marking behavior and USV were scored during the 5 min exposure to the female urine. Approximately 7 days later, adult male mice were exposed to females for 5 min. This was the first inter-sexual contact after weaning. Approximately 7 days after female experience, adult male mice were exposed again first to the clean open field for 60 min and then for 5 min to the female urine. A subgroup of cohort 2 was used to measure body weight at approximately 5 months of age. Mice of cohort 2 were obtained from mothers that gave birth twice, named first litter mice from primiparous females  and second litter mice from multiparous females . Cohort 2 consisted of n = 42 +/+Shank1 littermate control mice , n = 44 +/Shank1− , and n = 54 Shank1/−− mutant mice . A heterozygous breeding protocol was used throughout. About half of the +/Shank1− mutant mice were randomly excluded from the study to obtain similar numbers of mice per genotype.Two independent cohorts of mice were tested to avoid potential confounds from using previously handled animals. Mice of Cohort 1  were tested for developmental milestones from pnd 2–12. Cohort 1 consisted of n = 9 Pups of Cohort 1 were tested according to a modified Fox battery for developmental milestones and somatosensory reflexes Surface righting: The pup is gently held on its back and released. Latency to flip over onto the abdomen with four paws touching the surface is measured with a stopwatch.Negative geotaxis: The pup is gently placed head down on a square of grid (8×11 cm) at an angle of 45°. Latency to turn 180° to either side is measured with a stopwatch.Cliff avoidance: The pup's snout and forepaws are gently pushed over the edge of a table. Latency to withdraw from the edge of a flat surface is measured with a stopwatch.Grasping reflex: The pup's paw is stroked with a toothpick. Grasping the shaft of the toothpick is recorded as present or absent.Level screen holding: The pup is dragged across a square grid (8×11 cm) by the tail. Grasping is recorded as present or absent.Vertical screen holding: The pup is placed on a square grid (8×11 cm) at 90° angle. Length of time the pup is able to stay on the grid is measured with a stopwatch.Bar holding: The pup grasps a small elevated wire bar by its forelimbs while the hindlimbs are not in contact with the surface. Length of time the pup is able to hold onto a bar is measured with a stopwatch.Auditory startle: The pup is exposed to an acoustic stimulus (hand clapping). Startle response is recorded as present or absent.Latencies were measured in seconds for surface righting (maximum: 60 s), negative geotaxis (maximum: 60 s), cliff avoidance (maximum: 60 s), vertical screen holding (maximum: 10 s) and bar holding (maximum: 10 s). Other somatic and behavioral variables were rated semi-quantitatively, 0 =  no response/not present, 1 =  slight response/slightly present, 2 =  strong response/strongly present, 3 =  incomplete response/incompletely present, and 4 =  complete adult-like response/presence. Experimenters were trained until the inter-observer reliability was greater than 95%.Pups of Cohort 2 were isolated from their mother and littermates on pnd 8 for 5 min under room temperature (23–24°C). Pups were removed individually from the nest at random and gently placed into an isolation container  made of glass, containing clean bedding material. The isolation container was surrounded by a sound attenuating box (18×18×18 cm) made of Styrofoam . USV emission was monitored by an UltraSoundGate Condenser Microphone CM 16  placed in the roof of the sound attenuating box, 10 cm above the floor. The microphone was connected via an UltraSoundGate 116 USB audio device (Avisoft Bioacoustics) to a personal computer, where acoustic data were recorded with a sampling rate of 250,000 Hz in 16 bit format by Avisoft RECORDER . The microphone that was used for recording was sensitive to frequencies of 15–180 kHz with a flat frequency response (±6 dB) between 25–140 kHz. After the 5 min isolation period, body weight and body temperature were determined as described above. Isolation occurred between 8.00–16.00 h during the light phase of the 12∶12 h light/dark cycle. Prior to each test, behavioral equipment was cleaned using a 70% ethanol solution, followed by water, and dried with paper towels.For acoustical analysis, recordings were transferred to Avisoft SASLab Pro  and a fast Fourier transform was conducted . Correspondingly, the spectrograms were produced at 488 Hz of frequency resolution and 0.512 ms of time resolution. Call detection was provided by an automatic threshold-based algorithm (amplitude threshold:−40 dB) and a hold-time mechanism (hold time: 10 ms). Since no USV were detected below 30 kHz, a high-pass filter of 30 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. The accuracy of call detection by the software was verified manually by an experienced user. When necessary, missed calls were marked by hand to be included in the automatic parameter analysis. Total number of USV was calculated for the entire session and in 60 s time bins, to visualize the time course of the USV response. Additional parameters, based on previous studies of isolation-induced calling Scent marking behavior and USV in adult male mice of Cohort 2 were recorded during a 5 min session of exposure to a drop of fresh female urine in the center of an open field (40×49×30 cm) as previously described Adult male mice were exposed to fresh urine obtained from randomly-selected C57BL/6J females. Urine was collected from adult females in estrus by using a method adopted from Nyby et al. 2; 1145T; Tecniplast) containing clean bedding, approximately 7 days after the first and approximately 7 days before the second test session. Multiple females were used in order to minimize the number of male exposures and hence aggressive behavior against males.In order to provide a standardized prior history of social experience, each adult male mouse was individually placed with a randomly-selected adult C57BL/6J female for 5 min in a clean polycarbonate Makrolon cage  with fresh bedding. The home cage bedding and any feces deposited during the habituation session were removed from the open field. Scent marks deposited on the paper during the habituation session were visualized under ultraviolet (UV) light using a UV lamp . Visualized scent marks were outlined using a pencil. 15 µl of female urine was then pipetted onto the center of the Strathmore paper, and the mice were placed back into the open field for 5 min. The second set of scent marks deposited on the paper during the 5 min exposure to the female urine was visualized under the UV lamp and outlined with a blue colored pen. Prior to each session with a new subject mouse, the open field was cleaned with a 70% ethanol solution, followed by water, and dried with paper towels. The entire habituation and testing procedure was performed twice. Test 1 was conducted before males had female experience. Approximately 7 days later, males were exposed to females for five minutes, as described above. Test 2 was conducted approximately 7 days after the 5 minute interaction with a female. Open field activity, scent marking behavior and USV were scored during both test 1 and test 2. Habituation and testing was performed under red light.Adult male mice were individually habituated for 60 min to the clean open field lined with a sheet of specialized paper  that effectively absorbed drops of mouse urine, and containing some of their own home cage bedding in one corner of the arena to reduce the stress of the novel open field. At the end of the habituation period, the subject mouse was placed back in a clean polycarbonate Makrolon cage  during the 60 min habituation session to the clean open field without female urine and during the 5 min test session with exposure to female urine. Time spent within an area of 10 cm2) divided into squares, 1 cm2 per square, was placed on the top of the sheet of Strathmore paper. The total number of scent marks and the number of scent marks within an area of 10 cm2 around the female urine spot were counted. Scent marks deposited during the 60 min habituation session and the 5 min test session with exposure to female urine were differentiated based on their visualization with pencil or blue colored pen, respectively.Urinary scent marks were scored as previously described USV emission was recorded with a sampling rate of 300,000 Hz in 16 bit format by Avisoft RECORDER  as previously described Shank1/−− null mutant, +/Shank1−heterozygote and +/+Shank1 littermate control mice with ANOVAs for Repeated Measurements. Between-subject factors were genotype and sex. The within-subject factor was age. For analysis of pup USV emitted in isolation, ANOVAs with between-subjects factors of genotype, sex, and parity, i.e. first versus second litter, were calculated. In order to test whether differences in pup USV emitted in isolation emerged over time during testing, ANOVAs for Repeated Measurements with the same between-subject factors and the within-subject factor test duration were calculated. Three outliers were removed from the data set as their values for all parameters determined deviated by >2 standard deviations from the group mean. For analysis of male open field activity, scent marking behavior, and USV in response to female urine, ANOVAs for Repeated Measurements with the between-subject factor genotype and the within-subject factor female experience were calculated. In order to test whether differences in USV emitted by males in response to female urine emerged across testing, ANOVAs for Repeated Measurements with the between-subject factor genotype and the within-subject factors female experience and test duration were calculated. ANOVAs were followed by Bonferroni or Tukey's post hoc analysis when appropriate. A p-value of <0.050 was considered statistically significant.Developmental milestones were compared between All developmental milestones and somatosensory reflexes varied with age  and incisor eruption  as both were delayed in Shank1/−− null mutant pups. Among somatosensory reflexes, a genotype effect was found for surface righting . When gently held on its back and released, it took Shank1/−− null mutant pups longer to flip over onto the abdomen with four paws touching the surface than +/Shank1− (p = 0.006) and +/+Shank1 littermate control pups (p = 0.030), while the latter did not differ (p = 0.998).No genotype differences were detected on body weight, length, and temperature . Emergence of physical developmental milestones, however, was affected by genotype. An interaction between genotype and age was found for pinnae detachment . Finally, there was an interaction between genotype, sex, and age  as body weight gain was delayed in Shank1/−− null mutant female, but not male mice.Males and female pups did not differ on developmental milestones , with the exception of bar holding, where an interaction between sex and age was found as males displayed an accelerated development of their bar holding capabilities than females . Total calling time was also affected by genotype . As there was no genotype difference in the latency to start calling  and duration of calls . Peak amplitude of calls, i.e. loudness, was not affected  and +/+Shank1 littermate control pups (p = 0.022), while the latter did not differ from each other (p = 0.997).A genotype difference was detected in the number of USV emitted , peak frequency of calls , and peak amplitude of calls . For all three call parameters, this interaction is due to the fact that genotype differences were evident only in females and not in males. In females, genotype effects were detected for number of USV emitted , resulting in a lower total calling time in the former . Furthermore, female Shank1/−− mutant pups emitted USV that had higher peak frequencies than USV emitted by female +/Shank1− (p = 0.021) and +/+Shank1 littermate control pups (p<0.001), while the latter did not differ (p>0.050). Finally, female Shank1/−− mutant pups emitted USV that had lower peak amplitudes than USV emitted by female +/+Shank1 littermate control pups . In males, no genotype effects were detected . However, there were interactions between sex and genotype for total calling time , peak amplitude of calls , and call frequency modulation . First litter mice started to emit USV earlier and their calls were lower in amplitude, but more frequency modulated as compared to second litter mice. No interactions between parity and genotype or sex were detected .An effect of parity, i.e. first versus second litter, was detected in the latency to start calling . While there was an increase over time during testing in number of calls emitted in +/+Shank1 littermate control pups (min 1 vs. min 5: p = 0.013), no such increase was detected in Shank1/−− mutant pups (min 1 vs. min 5: p>0.999), resulting in genotype differences in min 4 (p = 0.023) and min 5 . Similarly, the time spent vocalizing increased across testing . While there was an increase over time during testing in the time spent calling in +/+Shank1 littermate control pups (min 1 vs. min 5: p<0.001), no such increase was detected in Shank1/−− mutant pups (min 1 vs. min 5: p = 0.407), resulting in a genotype difference in min 5 . Call duration increased across testing . While call duration increased in both genotypes, the increase was more pronounced in +/+Shank1 (min 1 vs. min 5: p<0.001) than in Shank1/−− pups (min 1 vs. min 5: p<0.001), but no genotype differences were detected . Peak frequency of calls decreased across the one minute time bins in the five minute isolation test . Peak amplitude of calls did not change across testing and no effect of genotype was found thereon . While there was an increase across testing in call frequency modulation in +/+Shank1 littermate control pups (min 1 vs. min 5: p = 0.003), no such increase was detected in Shank1/−− mutant pups (min 1 vs. min 5: p = 0.284), resulting in a genotype difference in min 4 (p = 0.042) and min 5 . Notably, changes in the USV emission over time during testing were not dependent on sex or parity .Across the one minute time bins in the five minute isolation test, increasing numbers of calls were detected  and +/+Shank1 littermate control pups (p<0.001), while the latter did not differ from each other (p = 0.251). Body temperature was not affected by genotype . Body weight and body temperature were not correlated with the number of USV emitted  or other USV characteristics, with the exception of a very low negative correlation between body weight and total calling time . Sex had no effect on body weight or body temperature . Parity affected body weight , but not body temperature . First litter mice were heavier than second litter mice.In the mouse pups tested for USV in isolation, body weight differed between genotypes  and +/+Shank1 littermate control pups (p<0.001), while the latter did not differ from each other (p = 0.605). In adulthood, a clear effect of sex was found, with males heavier than females , as expected.Lower body weights in 1,72 = 32.817, p<0.001 and F1,72 = 27.297, p<0.001, respectively; When comparing the locomotor activity shown by adult males exposed to the clean open field without female urine before and after female experience, a reduction in number of rearings and distance traveled was found, probably reflecting habituation  and +/+Shank1 littermate control mice (p = 0.001). +/Shank1− and +/+Shank1 mice did not differ from each other (p = 0.724). Distance traveled differed between genotypes  and +/+Shank1 littermate control mice (p = 0.003), while the latter did not differ from each other (p = 0.664). These genotype differences in locomotor activity were still evident after adding the female urine spot to the open field. Again, there was a genotype effect on rearing behavior . There was a trend for a genotype difference in the distance traveled . No such interactions were obtained in males exposed to the open field after adding female urine .There was a genotype effect on rearing behavior when males were initially exposed to the clean open field without female urine . Time spent in proximity to the female urine spot showed similar genotype effects  and +/+Shank1 littermate control mice (p = 0.008), while the latter did not differ from each other (p = 0.738). There was no genotype difference in the number of urine traces deposited in the entire open field in the presence of female urine, nor in the absence of female urine when males were initially exposed to the clean open field  and was not dependent on genotype .The number of USV emitted by males during the entire 5 min exposure to the female urine spot did not differ when subjects were tested before versus after female experience  that was dependent on female experience  as a faster onset of the USV response was seen after female experience. Most importantly, the change in the time course of USV emission that was seen after female experience was dependent on genotype . Specifically, while a slightly inverted U-shaped calling pattern was seen in both genotypes before female experience, +/+Shank1 mice displayed a clearly inverted U-shaped calling pattern characterized by a fast onset response after female experience. In contrast, however, a lack of such an inverted calling pattern was seen in Shank1/−− mice. Indeed, when comparing the time course of female urine-elicited USV in +/+Shank1 mice before and after female experience, a more pronounced inverted U-shaped call pattern with a clear shift towards the beginning of testing was evident after female experience . The calling pattern of Shank1/−− mice did not differ before and after female experience . Accordingly, there was no difference in the calling pattern between Shank1/−− and +/+Shank1 mice before female experience (F29,1276 = 1.090, p = 0.340; 29,1276 = 3.747, p<0.001; +/+Shank1 mice changed their calling pattern dependent on female experience, while Shank1/−− mice did not change their calling pattern as a consequence of prior experience with a female.However, when the time course of USV emission was taken into account, an inverted U-shaped calling pattern became apparent (FSHANK1 have yet been identified in individuals with autism, SHANK1 is a member of the SHANK gene family, in which mutations in SHANK2 and SHANK3 have been detected in several autistic individuals USV emission Shank1. As pups, Shank1/−− mutant mice emitted fewer USV than +/+Shank1 littermate control mice when isolated from mother and littermates. Call characteristics and their changes over time during testing differed between Shank1/−− and +/+Shank1 mice. As adults, both +/+Shank1 and Shank1/−− male mice emitted a similar amount of USV when exposed to female urine. Importantly, however, +/+Shank1 adult males changed their calling pattern dependent on their previous exposure to a female, but Shank1/−− adult males were unaffected by prior female experience. In addition, scent marking behavior in the presence of female urine, but not in the absence of female urine, was affected by genotype. Specifically, Shank1/−− mutant mice deposited fewer urine traces in proximity to the female urine spot than +/+Shank1 littermate control mice. Besides these differences in USV emission and scent marking behavior, reduced levels of locomotor behavior were observed in Shank1/−− mutant mice.Our findings revealed deficits in several elements of social communication and early developmental milestones in mice with a null mutation in Shank1 mutant mice. When tested on an accelerating rotarod and in a wire hang task, the latency to fall was lower in Shank1/−− than in +/+Shank1 mice Shank1/−− as compared to +/+Shank1 mice. Body weight gain and the appearance of some physical landmarks, pinnae detachment and incisor eruption, were delayed in Shank1/−− mice. Our findings further replicated a previously reported open field activity deficit Shank1/−− mutant mice were exposed to a novel or a familiar open field, and whether female urine was present or not, they displayed fewer rearings and their distance traveled was lower than in +/+Shank1 littermate controls.Motor impairments have previously been reported in this line of Shank1/−− mutant pups emitted fewer USV than +/+Shank1 littermate control pups, a finding which could indicate an early communication deficit in Shank1/−− mice. As there was no genotype difference in the latency to start calling, these data represent a lower call repetition rate in Shank1/−− than +/+Shank1 pups. Calls emitted by Shank1/−− were higher in peak frequency and less frequency modulated than the ones emitted by +/+Shank1 pups. Changes in USV emission over testing duration were highly dependent on genotype. While there was an increase in call number, total calling time, duration, and frequency modulation of calls in +/+Shank1 mice, this increase was weaker or absent in Shank1/−− mice. Therefore, all call parameters, with the exception of peak amplitude, i.e. loudness, were either directly affected by genotype, or their temporal pattern was affected by genotype. This is particularly remarkable as the two other factors studied, sex and parity, i.e. litter order, had only minor effects on USV production.When isolated from mother and littermates on postnatal day 8, SHANK2 and SHANK3. As in the present study, a female bias was reported instead Shank1/−− pups persist into adulthood, we currently assess USV emitted during social interactions of adult females as such USV reflect the level of social interest and serve an important communicative function Genotype affected isolation-induced USV primarily in females. This is surprising given the typical 4:1 male:female ratio in autism. It is therefore of particular interest that such a male bias was also not found in human autism studies on mutations in Shank1/−− mice is due to their lower body weight. In light of the small genotype difference in body weight of only 0.75 grams, however, body weight appears unlikely to be the cause of the reduced USV level in Shank1/−− pups. Lack of correlation between body weight and USV emission further argues against an interpretation that genotype differences in call emission were due to differences in body weight.Since a genotype difference was detected for body weight, it is possible that the lower USV level in Shank1/−− null mutant mice displayed higher levels of anxiety-related behavior on some components of the light/dark exploration test as compared to +/+Shank1 mice Shank1/−− mice that display higher levels of anxiety-related behavior.Another potentially confounding factor is a genotype difference in anxiety-related behavior. Shank1/−− males and females. It is tempting to speculate that some of the mouse pups bred by using a homozygous breeding regimen were not nurtured, and hence died before weaning, because the reduced level of call production in Shank1/−− mouse pups was insufficient to elicit maternal care.There is compelling evidence that isolation-induced USV serve a communicative function. Pup calls elicit maternal search and retrieval behavior, as shown in playback experiments [36–31]. A reduced level of calling or an unusual calling pattern has been reported in several mouse models of autism Shank1/−− mouse pups were shorter, higher in peak frequency, but less frequency modulated than the ones emitted by +/+Shank1 pups. This means that all altered parameters of calls emitted by Shank1/−− mouse pups may decrease their signal value and hence elicit less maternal caregiving responses.It appears possible that other call parameters such as call duration, peak frequency, peak amplitude, and frequency modulation, affect the communicative value of USV. Playback studies have shown that lactating mice can distinguish between different call types, and that they prefer certain call types over others if given the choice Shank1. In particular, the time course of the emission was dependent on genotype. Whereas +/+Shank1 emitted high numbers of calls to a spot of urine from a female mouse during the first two minutes of the test session, Shank1/−− mutant mice emitted remarkably few calls during the first two minutes of the test session. Intriguingly, +/+Shank1 mice changed their calling pattern dependent on a prior experience interacting with a female, but the calling pattern of Shank1/−− mice was unaffected by female experience. The experience-dependent change in calling pattern by +/+Shank1 mice is a typical phenomenon in mice, which has been repeatedly replicated Shank1/−− mice displayed impairment of contextual fear conditioning, but normal cued fear conditioning, indicating a deficit in hippocampus-dependent memory processes. Another interpretation of the lack of experience-induced changes in USV production could therefore be related to the cognitive phenotypes reported for Shank1/−− null mutants, conceivably in the domain of social learning and memory. Finally, the weaker USV response of Shank1/−− mice in response to female urine could also be a consequence of potential social behavior abnormalities during female exposure.In support of a lifelong communication impairment, USV emission in adulthood was also affected by a lack of Shank mice. In this task, scent marking in proximity to the female urine spot appears to be the most sensitive measure for communication deficits Shank1/−− mutant mice deposited fewer urine traces in close proximity to the female urine spot as compared to +/+Shank1 littermate control mice. Importantly, scent marking differences between genotypes were specifically detected in the presence of female urine but not in its absence, as genotypes did not differ on number of scent marks deposited in a clean open field. Therefore, level of urination in general was not affected by genotype. Instead, it appears that the deposition of urinary scent marks within an area of 10 cm2 of social odors around an aliquot of fresh urine from a female represents a species-typical response to social communicative signals, which differs in magnitude between Shank1/−− and +/+Shank1 mice. No evidence for reduced levels of social interactions in Shank1/−− mutant mice was obtained on tests of juvenile reciprocal social interaction and adult social approach Shank1/−− mice as deficits in USV production and scent marking behavior observed are therefore unlikely to be due to a lack of social motivation, though level of social motivation in Shank1/−− mutant mice needs to be tested formally. The social conditioned place preference test has provided a valid measure of social motivation and has been employed for studies of mice Olfaction is the predominant modality for communication in many rodents including mice SHANK1 has not been associated with autism, mutations in SHANK2 and SHANK3, other members of the SHANK gene family, appear in several individuals with autism Shank isoforms in the brain are overlapping, though differences in some structures such as striatum and thalamus were described SHANK gene family appear to fulfill similar physiological roles and display considerable neuroanatomical co-expression, it is intriguing that Shank3/−− null mutant mice display social deficits and repetitive self-grooming behavior Shank1/−− null mutant mice Shank1/−− phenotype relevant to the second diagnostic symptom of autism, communication deficits, the Shank1/−− null mutant mice do therefore not qualify for a genetic mouse model of autism, covering all three diagnostic symptoms.While


Mouse Atm−/− neurons re-enter a cell cycle and replicate their DNA, but they do not die – even months after initiating the cycle. In the current study, we explore whether systemic inflammation or hypoxia-induced oxidative stress can serve as second stressors that can promote cell death in ATM-deficient neurons. We find that after either immune or hypoxic challenge, the levels of cell cycle proteins – PCNA, cyclin A and cyclin B – are significantly elevated in cerebellar Purkinje cells. Both the number of cells that express cell cycle proteins as well as the intensity of the expression levels in each cell is increased in the stressed animals. The cell cycle-positive neurons also increasingly express cell death markers such as activated caspase-3, γ-H2AX and TUNEL staining. Interestingly, nuclear HDAC4 localization is also enhanced in Atm−/− Purkinje neurons after the immune challenge suggesting that both genetic and epigenetic changes in −/−Atm mice respond to environmental challenges. Our findings support the hypothesis that multiple insults are needed to drive even genetically vulnerable neurons to die a cell cycle-related cell death and point to either inflammation or oxidative stressors as potential contributors to the A−T disease process.In ataxia-telangiectasia (A–T) the death of neurons is associated with the loss of neuronal cell cycle control. In most  Ataxia-telangiectasia (A–T) is an autosomal recessive disorder characterized by immune deficiency, radiation sensitivity, sterility and enhanced risk of cancer. Affected individuals also develop a progressive neurodegeneration that strikes particularly hard at the Purkinje and granule cells of the cerebellar cortex. In advanced stages of the disease, neuronal cell loss continues and there is a substantial loss of motor function Our laboratory focuses on cell cycle control in the adult neuron and the relationship between an abortive cell cycle re-entry and neuronal cell death. We have shown, in both human A−T and two different mouse models of ATM deficiency that the neuronal re-expression of cell cycle proteins is associated with the death of Purkinje cells and striatal neurons −/−Atm Purkinje cells are increased in mice exposed to either acute or chronic LPS injection. Sustained LPS treatment drives Purkinje and granule cells to become positive for cell death markers such as TUNEL, γ-H2AX and activated caspase-3. This correlation between inflammation, defective cell cycle regulation, and the initiation of neuronal death offers fresh insight into the question of why neurons die during the course of A–T.The physical status and activity of the ATM protein are known to be sensitive to oxidation −/−Atm Purkinje cells do not die, we tested the hypothesis that a cell cycle-positive neuron, although weakened, might require a second stress to initiate the final death progress. Thus, we explored the roles of two possible stressors – hypoxia induced oxidative stress and LPS induced inflammation – as triggers that might induce cell death in the ‘cycling’ neurons.Our previous work has shown that cell cycle proteins are elevated in Purkinje cells in both human A–T disease and the ATM-deficient mouse 2) for 30 minutes with a 20-minute recovery period between exposures. Expression of PCNA  exposed to the same regimen  but then waited 7 days before sacrificing the animals. Finally, to test for the immediate effects of a surge of inflammatory cytokines, we adopted a paradigm of daily administration of LPS (1 mg/kg) for 3–4 days followed by sacrifice and analysis after 24 hours.−/−Atm mice at 3 days  mice exposed to the same regimen of acute and chronic LPS treatment, the expression of cell cycle proteins also appeared in the nuclei of Purkinje cells . This involvement of more than twice the number of Purkinje cells showing evidence of a neuronal cell cycle is compelling evidence for the impact of a second stressor on the cell cycle status of the vulnerable neurons. Further, the similarity of the LPS and low-oxygen responses suggests that the effects we observed on cell cycle proteins were not specific to any one treatment. We have therefore focused our subsequent efforts solely on the LPS injection paradigm.Both exposure to low oxygen and chronic LPS administration had qualitatively similar effects on the cerebellar Purkinje cell population – a marked increase in both the number of Purkinje cells expressing cell cycle proteins as well as in the intensity of expression of the immunostaining. Counts of the percentages of cell cycle positive Purkinje cells revealed that in either hypoxia or LPS treated −/−Atm animals using the reactive morphology of the microglia seen after Iba1 staining  mRNA can be found in mutant brain tissue Atm message is 10kb in length; therefore we assayed RT-PCR fragments from 3 different portions along its total length – exons 14–15, 34–41 and 55–61. Generally, the expression of all three regions of the Atm message was significantly lower in −/−Atm cerebellum than in wild type  γ-H2AX immunopositive Purkinje cells remained present even though there was little indication of a reduction in Purkinje cell density in cerebellar cortex (not shown). TUNEL-positive Purkinje cells were also visible in the LPS injected mutant animals  or cyclin B  as marke animals . When th markers . In LPS-−/−Atm  and the appearance of DNA breakage as measured by TUNEL. Each of these is a frequently used measure of cell death and, in the aggregate, there seems little doubt that the Purkinje cells shown in This identifies a second complexity of the findings. It would appear that death is at best a slow response to cell cycle induction. Unlike the hypoxia experiments, an apoptotic-like process did begin in the inflammatory model. Yet, it was only observed in a fraction of the The finding that chronic inflammation can exacerbate the course of A–T is consistent with findings in other neurodegenerative diseases. We used systemic LPS injections in our study, as this is a well-tested model of the inflammatory process. LPS is a cell wall component of gram-negative bacteria, which is known to induce a profound inflammatory response both in the periphery and in the brain Atm−/− mice that LPS injection increases the number and intensity of cell cycle staining and leads to the expression of common markers of cell death.The question of whether brain inflammation is involved in the origins of human A–T is not addressed by our study. Our injections were given to animals 2 months old or older. For reference, puberty in the mouse occurs at 6–8 weeks. Our results are, therefore, not likely to have relevance for the onset of human A–T symptoms at 2–3 years of age . There is no reason to assume that inflammation is a necessary step in the human disease, but it is possible that environmental factors such as inflammation may alter the severity or timing of the onset of symptoms. This possibility is suggested by the observation in tm1AwbAtm gene +/−Atm males and +/−Atm females. The mice were maintained on a 129/SvJ genetic background. Genotyping was performed on extracted tail DNA using PCR techniques were described previously A breeding colony of mice with a targeted disruption of the Atm+/+ or −/−Atm genotype. Three treatment groups were as follows: (1) The first group of animals (3 Atm+/+ and 3 −/−Atm) received daily intraperitoneal (i.p.) injections of LPS (1 mg/kg) for 4 days to elicit an acute systemic inflammatory response. A control group (3 Atm+/+ and 3 −/−Atm) were treated on the same schedule, but injected with filtered saline only. During the treatment, mice were monitored carefully. Mice were killed 24 h after the last injection; the brains were dissected and the tissues prepared described below. (2) A second experiment involved groups of 3 animals; each was given a single i.p. injection of LPS, but sacrifice and subsequent analysis did not occur until one week later. This experiment was repeated 3 times. (3) A third experiment involved groups of 3 animals; each was given weekly intraperitoneal injection of LPS once a week for 4 weeks and animals were sacrificed within 24 h after the final injection. This experiment was also repeated 3 times.LPS administration: LPS (Escherichia coli serotype 055:B5) was purchased from Sigma-Aldrich  and dissolved in filtered saline as a stock at concentration of 3 mg/ml. LPS was administrated intraperitoneally to adult mice (2 to 3 month old) of either SuperPlus glass slides.Animals were deeply anesthetized with Avertin (0.02 cc/g body weight) and transcardially perfused with 1.5 ml cold PBS, followed by approximately 30 ml of 4% paraformaldehyde in 0.1M phosphate buffer saline (PBS). After perfusion, the brain was dissected out and was transferred to 30% sucrose solution at 4°C overnight for cryoprotection. After bisecting along the midline, the brain was embedded in OCT; 10 µm cryostat sections were cut and allowed to air dry on pre-coated γ-H2AX antibody against the residues 100–200 of human γ-H2AX (phosphorylated at S139) was purchased from Abcam . Polyclonal rabbit antibody against cleaved caspase-3  and polyclonal rabbit antibody against HDAC4  were used at dilutions of 1∶400 and 1∶200, respectively. To detect whether inflammation had been induced efficiently in the CNS after LPS injection, adjacent sections were stained with Iba1 antibody to detect activated microglial cells  and GFAP antibody to reveal activated astrocytes. Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) was performed with a kit purchased from Millipore  according to the manufacturer’s instructions. Some sections were co-stained with polyclonal chicken antibody against MAP2  to visualize the neurons for cell counting.The proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody  was diluted 1∶2000 in 10% goat serum/PBS blocking buffer before use. The rabbit polyclonal cyclin A antibody  was raised against amino acids 1–432 representing full length cyclin A of human origin. Polyclonal rabbit Flurochrome-conjugated secondary antibodies (Alexa 488 or 594) were raised against either rabbit or mouse immunoglobulin  or chicken immunoglobulin .Single or double fluorescence immunocytochemistry was performed on mouse brain cryosections according to standard methods. Sections were rinsed in PBS, followed by pretreatment at 95–100°C in 0.1M citrate buffer for 8–10 minutes. After the slides had cooled in buffer for 45–60 minutes at room temperature, slides were rinsed in PBS. Sections were incubated in 10% goat serum in PBS for 1 hour at room temperature to block non-specific binding. All primary antibodies were diluted in PBS containing 0.3% Triton X-100 and 10% goat serum and then were applied to sections and incubated overnight at 4°C. After rinsing in PBS, they were incubated for 2 hours at room temperature with secondary antibody conjugated with fluorescent Alexa dyes. Sections were then rinsed in PBS and counter-stained with DAPI for 5 minutes at room temperature. After rinsing, all sections were mounted with anti-fading fluorescence media (Jackson Laboratories) under a glass coverslip. All experiments were conducted in triplicate.Whole cerebellum tissues were homogenized in lysis buffer and processed to RNA extraction using PureLink RNA Mini Kit (Invitrogen) according to manufacturer’s protocol. Subsequently, 500 ng of total RNA was reverse transcribed into total cDNA using ThermoScript RT-PCR System (Invitrogen). The mRNA level of 36B4 was used as an internal control. The following sets of primers were used:ATM exons 14–155′-gattacaagttctgaaaccc-3′ (sense)5′-gcttggtatgtatacacaagc-3′(antisense)ATM exons 34–415′-ggaaggtgtttgggagaaatagg-3′ (sense)5′-ccgtattctagtaagggatgtaacattttc-3′ (antisense)ATM exons 55–615′-gatgtaagatggtgaaggacatgg-3′ (sense)5′-cacagcaccttctgaagaccaattc-3′ (antisense)The PCR program was set to 55°C for 30 min and 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1–2 min with an extension at 68°C for 10 min. The PCR products were analyzed on 1.5% agarose gels stained with ethidium bromide and visualized under ultraviolet light.γ-H2AX, cleaved caspase-3 or HDAC4. The percentage of the positive cells with cell cycle markers or cell death markers were counted out of total Purkinje cells. Percentages of area occupied by Iba-1 or GFAP positive glial cells were calculated using software ImageJ . One-way ANOVA  was used to determine the differences in staining patterns between different groups. p<0.05 was considered statistically significant.Five fields were randomly chosen for quantification using a 20× objective on an Olympus or a Leica fluorescent microscope. In each field the total number of Purkinje cells was determined using either MAP2 or calbindin staining. We then separately determined the numbers of Purkinje cells that were positive for these experimental markers: PCNA, cyclin A,


Activation of the master energy-regulator AMP-activated protein kinase (AMPK) in the heart reduces the severity of ischemia-reperfusion injury (IRI) but the role of AMPK in renal IRI is not known. The aim of this study was to determine whether activation of AMPK by acute renal ischemia influences the severity of renal IRI.AMPK expression and activation and the severity of renal IRI was studied in mice lacking the AMPK β1 subunit and compared to wild type (WT) mice.172, was markedly reduced by 96% in AMPK-β1−/− mice. Acute renal ischaemia caused a 3.2-fold increase in α1-AMPK activity and a 2.5-fold increase in α2-AMPK activity (P<0.001) that was associated with an increase in AMPK phosphorylation of the AMPK-α subunit at Thr172 and Ser485, and increased inhibitory phosphorylation of the AMPK substrate acetyl-CoA carboxylase. After acute renal ischemia AMPK activity was reduced by 66% in AMPK-β1−/− mice compared with WT. There was no difference, however, in the severity of renal IRI at 24-hours between AMPK-β1−/− and WT mice, as measured by serum urea and creatinine and histological injury score. In the heart, macrophage migration inhibitory factor (MIF) released during IRI contributes to AMPK activation and protects from injury. In the kidney, however, no difference in AMPK activation by acute ischemia was observed between MIF−/− and WT mice. Compared with the heart, expression of the MIF receptor CD74 was found to be reduced in the kidney.Basal expression of activated AMPK, phosphorylayed at αThrThe failure of AMPK activation to influence the outcome of IRI in the kidney contrasts with what is reported in the heart. This difference might be due to a lack of effect of MIF on AMPK activation and lower CD74 expression in the kidney. AMPK is a ubiquitously expressed, energy-sensing kinase that is activated during energy stress by an increase in cellular [AMP] Stimulation of AMPK in the ischemic heart by macrophage migration inhibitory factor (MIF) is reported to protect against myocardial ischemia-reperfusion injury (IRI) −/− mice). It also seeks to determine whether MIF contributes to AMPK activation in acute renal ischemia as it does in the heart.The present study aims to determine the functional significance of AMPK activation in acute renal ischemia by determining the outcome of IRI in mice deficient for the AMPK β1 subunit . Experiments using the AMPK knockout strains were performed using littermate WT controls from heterozygous mating. The MIF−/− mice used for study are on a C57Bl/6 background, as previously described AMPK-β1All animal procedures were performed in the animal facilities of the Austin Hospital or St Vincent's Hospital and were approved by the animal ethics committees of these hospitals. All animal work was conducted according to national and international ethical guidelines.Mice were anaesthetized by intraperitoneal injection of ketamine (85 mg/kg) and xylazine (15 mg/kg). The left kidney was accessed by a midline laparotomy and acute renal ischemia was induced by applying a clamp across the renal pedicle for 10 minutes. The kidneys were rapidly removed and snap frozen in liquid nitrogen for subsequent biochemical analysis. Control kidneys (no ischemia) were removed rapidly without being subject to ischemia.After being anesthetized  mice were placed on a heating pad to maintain their core body temperature at 37°C during surgery. A midline abdominal incision was made and the renal pedicles were bluntly dissected. After right nephrectomy, a microvascular clamp  was placed on the left renal pedicle for either 18 or 20 min while the animal was kept at 37°C in an incubator and well hydrated. The clamp was then removed and the kidney observed to confirm complete reperfusion. The surgical wounds were then sutured in two layers with 5–0 silk. Warm normal saline (100 mL/kg) was instilled into the peritoneal cavity during the procedure. Mice were allowed to recover for 2 hr under a heating lamp and then kept on a heating pad. After 24 hr of reperfusion, the mice were euthanized, blood was taken and kidney samples were obtained for histological analysis.Whole blood was collected from an inferior vena cava puncture and sent for biochemical analysis. Serum creatinine level (modified Jaffe rate reaction) and urea level were measured by the Department of Pathology at the St. Vincent's Hospital Melbourne .2, 200 mM sucrose, 1% Triton X-100, 50 mM NaF, and protease inhibitors 2 mM PMSF, 1 µM leupeptin, 0.2 µM aprotonin, 1 mM benzamidine, and 1 mM AEBSF) and centrifugation . The resultant supernatant was further clarified by high-speed centrifugation .Lysates of mouse kidney were prepared by homogenization into 2 ml of homogenization buffer  with 2 mg of kidney lysate at 4°C for 1 hour. Immunocomplexes were precipitated by mixing the sample with Protein A beads (20 µl)  at 4°C for 30 minutes. After immunoprecipitation the beads were collected by centrifugation and washed 3 times in ice cold wash buffer (1% TritonX-100 in PBS) and once in cold PBS. Reducing Laemmli sample buffer (20 µl) was added to the beads, which were then heated to 95°C for 4 minutes prior to analysis by SDS-PAGE and Western blot.Samples were separated by SDS-PAGE and electrically transferred to a PVDF membrane. The membrane was blocked in 5% casein in Tris-buffered saline (TBS) for 1 hour and then incubated in primary antibody overnight. After washing in TBS 0.05% Tween20 (TBS-T) the membrane was then incubated for 30 minutes in secondary antibody at 1/2500 dilution. After further washing, immunoreactive proteins were detected by enhanced chemiluminescence with the SuperSignal Chemiluminescent System (Perkin Elmer). Western blots were quantified by densitometry .2, 5% glycerol, 1 mM DTT, 0.05% Triton X-100, 250 µM (γ-32P)-ATP (500 cpm/pmole), 100 µM ADR1 peptide substrate] AMPK activity was measured as previously described Ten percent formalin-fixed and paraffin-embedded kidney tissue sections (4 µm) were stained with hematoxylin-eosin. Three high-power fields in each of the cortex, corticomedullary junction and medulla were assessed in a blinded fashion (DP). Scores from the 3 high power fields were then averaged. The degree of tubular necrosis was graded and a modified scoring system was used as previously described Tissue was immersion-fixed in 4% paraformaldehyde , processed, and embedded in paraffin. 4 µm thick paraffin sections were incubated overnight with the anti-MIF antibody (1 µg/ml) at 4°C. Mouse IgG was detected using the mouse peroxidase-antiperoxidase (PAP) technique, comprising a goat anti-mouse Ig  diluted at 1/100, followed by mouse PAP (Dako) diluted 1/100. Peroxidase labelling was revealed using the liquid DAB substrate-chromagen system (Dako). Sections were counterstained with hematoxylin.Statistics were performed using GraphPad Prism Version 4.03 . Data are presented as mean ± SD. Unless otherwise stated, data were analyzed by ANOVA; if significant, Bonferroni's test for multiple comparisons was used. P values less than 0.05 were deemed significant.−/− and AMPK-β1−/− mice have been described previously −/− mice, basal α1-AMPK activity was reduced by 70% and no increase in α1-AMPK activity was seen with ischemia  total (α1 plus α2) AMPK activity compared to WT was reduced from 16.1±4.8 pmol/mg/min to 4.9±3.5 pmol/mg/min      . In thes= 0.002) . In addiP<0.001) . Whilst = 0.013) . In contischemia .−/− mice, kidney lysates from WT and β1−/− were analyzed by Western blot for expression of the activated form of AMPK, which is phosphorylated at αThr172 in the activation loop of the catalytic subunit. In kidneys from AMPK-β1−/− mice, phospho-αThr172 expression was profoundly reduced, although not absent . Importantly, however, no differences in serum urea and creatinine level were observed between WT and AMPK-β1−/− mice.Given the significant reductions in α1 and α2 AMPK expression, AMPK activity and activation in the AMPK-β124-hours . After 2−/− mice and WT mice.The severity of renal IRI was also assessed by measurement of the histological injury score 24-hours after reperfusion in cortex, corticomedullary junction (CMJ) and medulla . The mor−/− mice. Immunohistochemical staining confirmed widespread constitutive renal MIF expression, with the predominant site of expression being tubular epithelium  (−/− mice (P<0.001)   and 2.79-fold in MIFP<0.001) . SimilarP<0.001) . There wo groups . Interesischemia , althoug α1-AMPK  and West α1-AMPK . ActivatC-Ser79) , which iC-Ser79) . In the C-Ser79) . CD74 exet al. have reported that pre-activation of AMPK before renal IRI by the diabetes drug metformin reduced both histological injury and the level of the urinary biomarker neutrophil gelatinase associated lipocalin (NGAL) The lack of effect of deletion of AMPK-β1 on renal IRI in the present study contrasts with a previous study that found the AMPK activator AICAR, in combination with N-acetylcysteine, attenuated IRI in a canine model of autologous renal transplantation et al. found that AMPK activation increased hypertonicity-induced apoptosis in medullary interstitial cells et al found in cultured proximal tubular cells that activation of AMPK is protective against apoptosis induced by metabolic stress by an Akt dependant mechanism in vivo. The finding of the present study that AMPK activation appeared to have a neutral effect in renal IRI suggest that any possible effect of AMPK on renal cell apoptosis in response to ischaemia appears to be not having an effect on the overall severity of kidney injury and renal dysfunction.Cell culture studies examining the effect of AMPK activation on apoptosis of renal cells in culture have produced variable results. Han The finding that reduced AMPK activation does not alter the severity of renal IRI contrasts with studies of myocardial ischemia, where activation of AMPK appears protective −/− despite the fact that basal AMPK expression, activity and activation were significantly reduced. This suggests that AMPK is unlikely to have a vital protective role in renal IRI. A limitation of the present study, however, is that, while significantly and substantially reduced, a low level of AMPK activity and activation was still detectable in kidneys from AMPK-β1−/− mice. The AMPK activity data estimates a reduction of renal AMPK activity in the AMPK-β1−/− mice of approximately 70%, whereas the Western blot data suggests a more profound 96% reduction in the expression of activated AMPK. This discrepancy might be explained, at least in part, by the observation that the AMPK activity assay does detect a low but measurable level of non-specific background activity that is not altered by the presence or absence of AMPK expression. Interestingly, while AMPK activity was reduced in AMPK-β1−/− mice, the relative level of AMPK activation with ischemia was similar in WT and AMPK-β1−/− mice. The residual AMPK activity and activation in the AMPK-β1−/− can be explained by expression of the AMPK-β2 subunit that was detectable, at a proportionally low level, in the mouse kidney. Interestingly, in the rat kidney β2 has been observed as the predominant AMPK scaffolding subunit The principle finding of this study is that the severity of IRI was unaltered in AMPK-β1172 and AMPK-αSer485. Whilst the role of AMPK-αThr172 phosphorylation in the activation of AMPK is well-characterized 485 site, which exists in the βγ binding domain of the α subunit 485 might in fact be inhibitory 485 have not been extensively studied, although both insulin and hepatitis C have been reported to inhibit AMPK activation by mediating phosphorylation of this site by Akt/PKB 485 is reported to be phosphorylated by liver purified AMPK-kinase, but whether this was mediated by LKB1, CaMKKβ or autophosphorylation was not defined et al. where AMPK-αSer485 appears inhibitory, phosphorylation of AMPK-αSer485 preceded phosphorylation of AMPK-αThr172. In contrast, in the present study in which AMPK-αSer485 phosphorylation appears to occur either after or simultaneous to AMPK-αThr172 phosphorylation, a marked increase in AMPK activity was seen despite the fact that phosphorylation at AMPK-αSer485 was increased.In the present study we observed that AMPK activation by acute renal ischaemia occurred simultaneously with increased phosphorylation of both AMPK-αThret al. have reported constitutive CD74 expression in the rat kidney, which was localized to glomeruli and tubules and increased with diabetes 2+], acting via the upstream kinase CaMKKβ 2+] 172 by upstream kinase(s) and this is not influenced by the presence or absence of MIF.The finding in the present study that the presence of MIF did not contribute to activation of AMPK in the ischemic kidney contrasts with what has been reported in the ischemic heart and cultured cardiomyocytes 172 and αSer485 and inhibitory phosphorylation of ACC-Ser79. Significant constitutive expression of MIF in tubular epithelium was found, but there was no difference in AMPK activation by acute renal ischemia between WT and MIF−/− mice. Expression of AMPK and activation of AMPK by acute renal ischemia was markedly attenuated in AMPK-β1−/− mice. Despite this there was no difference in the severity of renal IRI, as measured biochemically or histologically, between AMPK-β1−/− and WT mice. The lack of effect of reduced AMPK activation on the outcome of renal IRI suggests that renal protection in situations of severe energy stress is unlikely to be a critical function of AMPK in the kidney. This supports a more general view that the primary role of AMPK in the kidney is likely to be to detect and respond to more minor perturbations of cellular energy status that occur within the context of normal renal physiology.In summary, this study found that AMPK is activated in response to acute renal ischemia and that activation is associated with phosphorylation of AMPK at αThr


We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer. In the past, the nuclear role of Protein Kinase C (PKC) has been dominated by the cytoplasmic signaling role of this kinase family. A complex series of molecular events is initiated upon stimulation of the TCR and CD28 co-receptor. This results in the selective activation of the novel PKC family member, PKC-θ, which activates transcription factors such as NF-κB, ultimately leading to induction of a distinct cohort of immune response genes  and indeed a putative NLS domain has been described for PKC-θ  motifs and corresponding NLS sequences previously described  were examined for these motifs  of three motifs, where the most frequently occurring superfamily member was used and the frequency of three motifs occurring within 300 bp of each other was calculated. MYCMAX_03 (EBOX) and AHRARNT_02 (AHRR) commonly occurred together and this was attributed to the similarity of their matrices. The combination NFKB_SP1F_E2FF occurs in these promoter regions significantly more than expected by chance Figure B, whereaCurrently it is unknown what targets chromatin-remodeling factors and signaling kinases, such as PKC-θ, to specific chromatin regions. This data provides a clue that a novel NF-κB transcription factor module may be involved in tethering PKC-θ to chromatin in T cells.The NF-κB family of transcription factors encompasses a heterogenous set of inducible proteins, including NF-κB1 (p50 and precursor p105), NF-κB2 (p52 and precursor p100), RelA (p65), RelB, and c-Rel  results clearly show that the lack of NF-κB severely reduced assembly of the PKC-θ-containing active transcription complex Figure B. These To examine whether PKC-θ and LSD1 have the potential to directly interact with each other, we modeled the interaction between PKC-θ and LSD1 using the ClusPro server  the delayed kinetics of nuclear c-Rel translocation and activation following T cell stimulation closely correlates with the formation of the active transcription complex; (2) pentoxifylline is a c-Rel specific inhibitor that successfully abolishes the assembly of the PKC-θ complex; (3) our preliminary c-Rel knock-out studies suggest that c-Rel is essential for anchoring PKC-θ to chromatin (data not shown); and (4) both c-Rel and PKC-θ are essential for chromatin accessibility necessary for transcription of immune response genes  and 1 μM of CaI (Sigma) as previously described , 1:200 Protease Inhibitor Cocktail Set III, EDTA-Free , 1:500 Phosphatase Inhibitor Cocktail Set V ] and incubated on ice for 5 min. Samples were centrifuged at 3000 rpm for 3 min at 4°C then the supernatant was removed. The remaining pellet was resuspended in Nuclei Lysis Buffer A− (as in Nuclei Lysis Buffer A but without NP-40) and centrifuged for 3000 rpm for 3 min at 4°C. The supernatant was removed and the nuclei were resuspended in SDS Lysis Buffer .Both whole cell and nuclear extracts were prepared for PKC kinase assays. For whole cell extracts, cells were collected and washed once in 1 X PBS then resuspended in RIPA buffer  and incubated on ice for 15 min. Samples were centrifuged at 13,000 rpm for 15 min at 4°C and the supernatant was collected as whole cell extracts. For nuclear extracts, cells were collected and washed once in 1× PBS then resuspended in Nuclei Lysis Buffer A+ . Inducible gene transcript levels were normalized to the housekeeping gene, GAPDH (Hs99999905). MiR-200c and RNU6B microRNA assays were performed using the TaqMan® MicroRNA Reverse Transcription Kit (ABI 4366596) and probes previously described . Primers for human CD69 were:−241 to −52 relative to the TSS (Fwd: CCCACTTTCCTCCTGCTACA and Rev: GCCGCCTACTTGCTTGACTA) and +137 to +377 relative to the TSS (Fwd: CCGGAGAGTGGACAAGAAAG and Rev: GGGGTTTACCTCTTCCCTGA); and for the human miR200c promoter: (Fwd: CCACTGCCTTAACCCCTTC and Rev: AGGGGTGAGACTAGGCAGGT) previously described in reference (Wiklund et al., ChIPs were performed following the protocol supplied by Upstate Biotechnology, as previously detailed (Sutcliffe et al., To help determine which transcription factors may be aiding the recruitment of PKC to DNA, we used the Genomatix program to examine the promoters of 69 of the top PKC binding genes. Our strategy was to look for transcription factor binding sites as defined by their TRANSFAC position weight matrices. The sequences of the PKC binding regions (±1 kb; Sutcliffe et al., The interaction between PKC-θ and the LSD1-CoREST complex was modeled using the ClusPro server (Kozakov et al., Protein sequences were retrieved from Uniprot for 11 of the PKC members (see Table in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.Utilizing This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors. Colorectal cancer (CRC) is one of the most common malignancies with high incidence rates globally Tumor suppressor genes (TSG) contribute to the induction of apoptosis in response to stress. The failure to induce apoptosis in response to various types of cellular damage has been long recognized as contributing to oncogenesis. One example of TSG loss contributing to cancer formation and progression is TGFβ signaling We have shown that TGFβ signaling in an early stage non-metastatic colon carcinoma model leads to cell death in colon cancer cells in response to stress in association with inactivation of pAKT and inhibition of the expression of the IAP protein survivin 20TGFβ signaling has been shown to activate cAMP- dependent Protein Kinase A (PKA) in vivo. Rescue of specific tumor suppressor aspects of the TGFβ signaling pathway may provide therapeutic benefits without promoting the cell survival and metastatic effects of TGFβ. If these TGFβ tumor suppressor effects can be mimicked in late stage tumors, therapeutic value against metastatic CRC might ultimately be obtained.The molecular mechanisms involved in the TGFβ mediated downregulation of IAP were investigated in order to determine the effects of TGFβ receptor signaling on metastasis in a metastatic orthotopic model of colon carcinoma. We now report the identification of a novel TGFβ/PKA/AKAP mediated transduceome that converges on XIAP function in controlling aberrant cell survival. Additionally, we have shown that TGFβ receptor rescue in highly metastatic cells with epigenetic silencing of TGFβ receptors leads to decreased metastases in a TGFβ/PKA signaling pathway dependent mechanism in a highly metastatic orthotopic colon cancer models A critical report from the Simeone laboratory noted that TGFβ signaling activates PKA in Mv1Lu cells and mediates control of TGFβ growth inhibitory responses 20 in the cytosol, but not in mitochondria 20). Earlier reports indicate that PKA phosphorylates the Rpt6 ATPase, which unfolds and transports substrates into the proteasome and this phosphorylation enhances the chymotrypsin proteasomal activities of the proteasome XIAP and survivin are well characterized IAP family members recently documented as metastatic genes Collectively, these data lead to the hypothesis that the TGFβ/PKA signaling regulates XIAP expression at multiple levels with activation of PKA leading to phosphorylation of key proteasomal components that promote proteasomal degradation of XIAP.There is an abundance of A-kinase anchoring proteins (AKAPs) which compartmentalize the PKA and other enzymes to the vicinity of specific cellular organelles for carrying out enzyme function. We hypothesized that AKAPs are a critical component in TGFβ/PKA mediated XIAP downregulation. It is well documented that the subcellular localization of PKA regulatory subunits (PKARI and/or PKARII) is mediated through interactions with AKAPs Since the AKAP family includes more than 50 members We next questioned whether the effect of AKAP149 was selective in this TGFβ/PKA effect. While AKAP-PKA interaction is a global phenomenon, validation of AKAP149 as selectively regulating TGFβ/PKA signaling would be a novel extension of our understanding of the TGFβ/PKA mediated XIAP downregulation. AKAP220 has been shown to regulate protein phosphatase 1 (PP1) activity by coordinating the location of PKA and PP1 catalytic subunit It has been observed that activation of PKA leads to the inactivation of AKT through dephosphorylation by protein phosphatase 2A (PP2A) To further validate the role of PP2A in TGFβ/PKA signaling, we generated stable shRNA PP2A catalytic subunit knockdown in FET cells designated FET PP2A Cat KD . TGFβ tr87 which leads to increased stabilization of XIAP and decreased apoptosis of ovarian cancer cells in response to cisplatin While inhibition of AKT activation would have potential for many effects on cell survival, one effect that is of particular interest as related to TGFβ/PKA function is in the context that it targets XIAP stability. A previous study reported that XIAP is phosphorylated by AKT on Serin vivo  subunits bound to two (R) subunits (RIα/β and RIIα/β) that renders it inactive An intriguing observation made in our studies which originally showed that TGFβ signaling inhibits survivin expression was that TGFβ signaling also mediated inactivation of AKT 87, thereby stabilizing XIAP and preventing it from auto-ubiquitination We have shown that XIAP is associated with pAKT and this association is disrupted following TGFβ treatment . AKT aloIn association with direct regulation of XIAP by TGFβ/PKA signaling, we also demonstrated that chymotrypsin-like activity of the proteasome is induced by TGFβ in a PKA dependent manner. XIAP can be auto-ubiquitinated and degraded when treated with DNA damaging agents like chemotherapeutic drugs We have made the novel observation that reconstitution of TGFβ receptor TGFβRI in highly metastatic GEO colon cancer cells rescues TGFβ signaling and inhibited metastatic colonization from orthotopic xenografts. This observation raises the therapeutic possibility that TGFβ can suppress metastatic competence after this advanced state of progression is attained in cells with attenuated type I and/or II TGFβ receptor expression (designated TGFβRI or TGFβRII).in vivo even though invasion at the primary tumor site was maintained (We have made the novel observation that rescue of attenuated receptor expression does inhibit metastatic capability in GEORI (reconstituted with RI) transplanted orthotopic models intained . This fiStudies on different cancer types provide extensive support for loss of TGFβ receptor expression as an important contributor to tumor progression in subgroups of several types of cancer In an effort to determine the relationship between the TGFβ/PKA transduceome and the capability of a tumor for metastatic spread, we investigated the molecular aspects of the TGFβ/PKA transduceome signaling in the context of metastatic progression in highly metastatic GEO and CBS cells and their poorly metastatic counterpart GEORI and CBSRII where receptor reconstitution has led to a decrease in metastatic spread. Since metastatic deposits, like primary cancers, are faced with hypoxic stress and must face the additional challenge of growth in a foreign microenvironment, it seems likely that even established metastases must retain their aberrant survival characteristics.Striking difference in TGFβ/PKA signaling activity and downstream signaling leading to XIAP downregulation was observed when highly metastatic  and their poorly metastatic colon cancer counterparts (GEORI and CBSRII) were compared. Therefore, reconstitution of TGFβ receptors in metastatic models with attenuated TGFβ signaling leading to metastasis might also reconstitute TGFβ/PKA signaling in established metastases which could affect their survival. These lines of evidence lead to the consideration that the TGFβ/PKA transduceome is a critical component in steady-state suppression of abnormal survival signaling that prevents formation of metastases by disrupting the XIAP/survivin cell survival pathway.In conclusion, understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases (most prominent cause of death from solid cancers). The role of loss of tumor suppressor activities in the metastatic process is essentially unknown. Presently, loss of TGFβ signaling is largely regarded as a driver for transition from a benign to a malignant state subsequently leading to metastatic spread by other mechanisms or even by usurping aspects of TGFβ signaling for oncogenic mechanisms such as the acquisition of EMT. However, we have now shown that loss of TGFβ tumor suppressor signaling is a driver for metastatic colonization of distant organs from the primary tumor. Importantly, loss of TGFβ signaling does not affect invasion as reflected by retention of a 100% rate of invasion at the primary site of metastatic cells in which TGFβ signaling has been rescued and metastatic potential inhibited. This indicates that while loss of TGFβ signaling enables the transition from a benign to a malignant state its loss does not drive the earliest step of the metastatic process.However, we have shown here that loss of TGFβ signaling is necessary to allow the last step of the metastatic process- colonization of the metastatic site. Moreover, we have identified a novel TGFβ tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. This survival mechanism is apparently not necessary for malignant behavior at the primary site once the cancer cells have made the transition from the benign state since its inhibition by restoration of TGFβ tumor suppressor signaling does not alter malignant behavior at the primary site.Our work indicates the potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by this novel, multifaceted TGFβ induced pathway. Our previous body of work showing that HDACi's act at least in part through the rescue of TGFβ receptor I and II expression and TGFβ tumor suppressor activity in a wide variety of cancer cells indicates the clinical potential of this concept.Since TGFβ signaling responses can be supportive of events that support malignant progression such as EMT and suppression of TGFβ signaling as a therapy for cancer has been supported by experimental evidence, therapeutic strategies for the inhibition of TGFβ signaling in cancer therapy are currently being pursued. However, our work indicates that the relationship between TGFβ signaling and tumor suppressor function may be governed by context even in the later stages of cancer. The concept that the disruption of the balance between TGFβ tumor suppressor activity and the survivin/XIAP cell stress response enables metastatic colonization of distant organs raises the concern that therapies aimed at inhibition of TGFβ signaling may be deleterious to at least a subset of cancer patients. For example, it has been shown in several types of cancer that progressive epigenetic silencing of TGFβ receptor I and/or II correlates with malignant progression. Pharmacological inhibition of TGFβ receptor signaling may promote metastases in these patients.All experiments involving animals were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC #: 07-047-08-FC).The FET Cells were lysed in a Tris-HCl based buffer containing 0.5% NP-40 and appropriate protease and phosphate inhibitors as described previously Smad 3 (sc-38376-SH), PKA Catalytic α (sc-36240-SH) and PP2A Catalytic α (sc-43509-SH) shRNA was obtained from Santa Cruz. These vectors were co-transfected by electroporation as described previously PKA activity was measured using the PepTag non-radioactive protein kinase assay (Promega) using kemptide (LRRASLG) following the manufacturer's protocol. For quantitative determination of cellular cAMP, the non-radioactive Direct Cyclic AMP Enzyme Immunoassay kit (Assay Design) was used.For quantitative determination of cAMP, non-radioactive Direct Cyclic AMP Enzyme Immunoassay kit (Assay Design) was utilized. Manufacturer's protocol was followed for the assay.Protein Phosphatase 2A (PP2A) activity was measured using the non-radioactive PP2A immunoprecipitation assay (Millipore).The fluorometric Proteasome substrate III for chymotrypsin (SUC-LLVY-AMC) and Peptidyl-glutamyl peptide hydrolyzing (PGPH)-caspase-like activity proteasome substrate II (Z-LLG-AMC) and the corresponding inhibitors were obtained from Calbiochem. FET cells were plated at 800,000 cells/100 mm dish in SF media. On day 5, cells were treated with TGFβ (5 ng/ml) with or without H89 (15 µM) pretreatment for 1 h and harvested in proteasomal assay buffer . After quantification of protein concentration, cell lysates were pretreated with 25 uM of the chymotrypsin-like activity inhibitor Benzyloxy carbonyl Leu Leu phenylalaninal Inhibitor (ZLLF-CHO) or the PGPH activity inhibitor Z-Gly-Pro-Phe-Leu-CH (Z-GPFL-CHO) followed by incubation with chymotrypsin or PGPH substrate for 1 h at 37°C as described by Apoptosis was measured by the Cell Death Detection ELISA Plus kit (Roche) as described previously 3 pieces. These pieces were then orthotopically implanted into other BALB/c nude mice. For operative procedures, animals were anesthetized with isofluorane inhalation. A 1 cm laparotomy was performed and both the cecum and ascending colon were exteriorized. Using 7× magnification and microsurgical techniques, the serosa was disrupted by scraping in two locations. The 1 mm3 pieces of xenograft were sub-serosally implanted using an 8-0 nylon suture at the two points of serosal disruption. The bowel was then returned to the peritoneal cavity and the abdomen was closed with 5-0 vicryl suture. Subsequently, animals were anesthetized with a 1∶1 mixture of ketamine (10 mg/ml) and xylazine (1 mg/ml) by intraperitoneal injection (0.01 ml/mg) and weekly GFP fluorescence imaging was performed for up to 5 weeksWeekly GFP fluorescence imaging was performed for up to 5 weeks. Excitation of GFP in the light box facilitated identification of primary and metastatic disease by direct near-real time visualization of fluorescence in live animals. Both GEO and GEORI cells gave rise to an invasive primary tumor in 100% of the animals implanted.All experiments involving animals were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. The orthotopic implantation methodology has been described in details in the following publications from Brattain laboratory Approximately 50 d post-implantation, the animals were euthanized following proper IACUC protocol. The colon (with primary tumor), liver, lungs, and heart were harvested. Following the harvesting, organs were explanted, imaged, and immediately placed in 10% neutral buffered formalin fixative for 24 h. This was followed by tissues processing and embedding in paraffin. Slides were then cut for hematoxylin and eosin (H and E) and immunohistochemical characterizations. Serial sections were cut to complement the H and E sections and were stained with an IgG1 rabbit polyclonal antibody for Ki-67 . Slides from paraffin embedded tissue blocks were stained according to the Apotag  terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay kit. Detailed methodology and quantitative analysis for these individual experiments has been described in details in the Figure S1Activation of pSmad2.(TIF)Click here for additional data file.Figure S2PKA catalytic subunit shRNA knockdown in FET cells.(TIF)Click here for additional data file.Figure S3No change in IκB-α with TGFβ treatment.(TIF)Click here for additional data file.Figure S4Smad3 shRNA knockdown in FET cells.(TIF)Click here for additional data file.Figure S5TGFβ treatment downregulates survivin. Blocking the proteasome with MG132 abrogates the TGFβ mediated survivin loss.(TIF)Click here for additional data file.