Alexander Alexandrovich

Mechanoenzymatics of titin kinase

Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.

Dynamic changes in the recovery after traumatic brain injury in mice: effect of injury severity on T2-weighted MRI abnormalities, and motor and cognitive functions.

Memory and neurobehavioral dysfunctions are among the sequelae of traumatic brain injury (TBI). The Neurological Severity Score (NSS) includes 10 tasks and was previously designed to assess the functional status of mice after TBI. The object recognition task (ORT) measures specific episodic memory and is expressed by the percent time spent by an animal at a novel, unfamiliar object (Discrimination Index [DI]). It is an ideal tool for evaluating cognitive function after TBI. The present study sought to validate the use of the NSS and ORT in severe and mild focal TBI in mice, and to confirm that the spontaneous recovery and the radiological abnormalities, shown by T2-weighted magnetic resonance imaging (MRI), are dependent upon injury severity. Mice were subjected to severe and mild closed head injury (NSS at 1 h 7.52 +/- 0.34 and 4.62 +/- 0.14, respectively). NSS was evaluated for 25 days and showed a decrease by 3.86 +/- 0.26 and 2.54 +/- 0.35 units in the severely and mildly injured mice, respectively. ORT revealed DI in severely injured group of 51.7 +/- 6.15%, (vs approximately 75-80% in uninjured animal) on day 3 and 66.2 +/- 6.81% on day 21. In contrast, the mildly injured mice did not show cognitive impairment throughout the same period. The damage seen by MRI at 24 h after injury, strongly correlated with NSS(1h) (R = 0.87, p < 0.001). We conclude that NSS is a reliable tool for evaluation of neurological damage in head-injured mice, NSS(1h) predicts the motor dysfunction, cognitive damage, and brain-damage characteristics as depicted by T2-weighted MRI. The combined assessment of neurobehavioral and cognitive function along with MRI is most useful in evaluating recovery from injury, especially when testing effectiveness of novel treatments or genetic manipulations.

Recessive TTN truncating mutations define novel forms of core myopathy with heart disease

Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery-Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.

Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury

Despite efforts aimed at developing novel therapeutics for traumatic brain injury (TBI), no specific pharmacological agent is currently clinically available. Here, we show that the pan‐histone deacety‐ lase (HDAC) inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when adminis‐ tered as late as 24 h postinjury. Using a well‐character‐ ized, clinically relevant mouse model of closed head injury (CHI), we demonstrate that a single dose of ITF2357 administered 24 h postinjury improves neu‐ robehavioral recovery from d 6 up to 14 d postinjury (improved neurological score vs. vehicle; P<0.05), and that this functional benefit is accompanied by de‐ creased neuronal degeneration, reduced lesion volume (22% reduction vs. vehicle; P<0.01), and is preceded by increased acetylated histone H3 levels and attenuation of injury‐induced decreases in cytoprotective heat‐ shock protein 70 kDa and phosphorylated Akt. More‐ over, reduced glial accumulation and activation were observed 3 d postinjury, and total p53 levels at the area of injury and caspase‐3 immunoreactivity within microglia/macrophages at the trauma area were ele‐ vated, suggesting enhanced clearance of these cells via apoptosis following treatment. Hence, our find‐ ings underscore the relevance of HDAC inhibitors for ameliorating trauma‐induced functional deficits and warrant consideration of applying ITF2357 for this indication.—Shein, N. A., Grigoriadis, N., Alex‐ androvich, A. G., Simeonidou, C., Lourbopoulos, A., Polyzoidou, E., Trembovler, V., Mascagni, P., Din‐ arello, C. A., Shohami, E. Histone deacetylase inhib‐ itor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following ex‐ perimental traumatic brain injury. FASEB J. 23, 4266‐4275 (2009). www.fasebj.org

Near Infrared Transcranial Laser Therapy Applied at Various Modes to Mice following Traumatic Brain Injury Significantly Reduces Long-Term Neurological Deficits

Near-infrared transcranial laser therapy (TLT) has been found to modulate various biological processes including traumatic brain injury (TBI). Following TBI in mice, in this study we assessed the possibility of various near-infrared TLT modes (pulsed versus continuous) in producing a beneficial effect on the long-term neurobehavioral outcome and brain lesions of these mice. TBI was induced by a weight-drop device, and neurobehavioral function was assessed from 1u2009h to 56 days post-trauma using the Neurological Severity Score (NSS). The extent of recovery is expressed as the difference in NSS (dNSS), the difference between the initial score and that at any other later time point. An 808-nm Ga-Al-As diode laser was employed transcranially 4, 6, or 8u2009h post-trauma to illuminate the entire cortex of the brain. Mice were divided into several groups of 6-8 mice: one control group that received a sham treatment and experimental groups that received either TLT continuous wave (CW) or pulsed wave (PW) mode transcranially. MRI was taken prior to sacrifice at 56 days post-injury. From 5-28 days post-TBI, the NSS of the laser-treated mice were significantly lower (p<0.05) than those of the non-laser-treated control mice. The percentage of surviving mice that demonstrated full recovery at 56 days post-CHI (NSS=0, as in intact mice) was the highest (63%) in the group that had received TLT in the PW mode at 100u2009Hz. In addition, magnetic resonance imaging (MRI) analysis demonstrated significantly smaller infarct lesion volumes in laser-treated mice compared to controls. Our data suggest that non-invasive TLT of mice post-TBI provides a significant long-term functional neurological benefit, and that the pulsed laser mode at 100u2009Hz is the preferred mode for such treatment.

Wnt2 is a direct downstream target of GATA6 during early cardiogenesis.

The GATA4, 5 and 6 subfamily of transcription factors are potent transactivators of transcription expressed within the precardiac mesoderm. However, little is known of the immediate downstream targets of GATA-factor regulation during the earliest stages of cardiogenesis. Using the P19-CL6 embryonal carcinoma (EC) cell line as an in vitro model of cardiogenesis, we show that GATA6 is the most abundantly expressed of the GATA factors in presumptive cardiac cells. Consequently, we performed a microarray screen comparing mRNA from control EC cells, early in the cardiac differentiation pathway, with those in which GATA6 had been overexpressed. These studies identified 103 genes whose expression changed significantly and this was verified in a representative array of these genes by real-time RT-PCR. We show that early cardiac expression of one of these genes, Wnt2, mirrors that of GATA6 in vitro and in vivo. In addition, its upregulation by GATA6 in differentiating EC cells is mediated by the direct binding of GATA-factor(s) to the cognate Wnt2 promoter, suggesting Wnt2 is an immediate downstream target of GATA-factor regulation during early cardiogenesis.

The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of Drosophila

Summary Obscurin (also known as Unc-89 in Drosophila) is a large modular protein in the M-line of Drosophila muscles. Drosophila obscurin is similar to the nematode protein UNC-89. Four isoforms are found in the muscles of adult flies: two in the indirect flight muscle (IFM) and two in other muscles. A fifth isoform is found in the larva. The larger IFM isoform has all the domains that were predicted from the gene sequence. Obscurin is in the M-line throughout development of the embryo, larva and pupa. Using P-element mutant flies and RNAi knockdown flies, we have investigated the effect of decreased obscurin expression on the structure of the sarcomere. Embryos, larvae and pupae developed normally. In the pupa, however, the IFM was affected. Although the Z-disc was normal, the H-zone was misaligned. Adults were unable to fly and the structure of the IFM was irregular: M-lines were missing and H-zones misplaced or absent. Isolated thick filaments were asymmetrical, with bare zones that were shifted away from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depends on the symmetry of adjacent thick filaments; shifted bare zones resulted in abnormally long or short thin filaments. We conclude that obscurin in the IFM is necessary for the development of a symmetrical sarcomere in Drosophila IFM.

The sarcomeric cytoskeleton as a target for pharmacological intervention.

Many diseases of heart and skeletal muscle, from heart failure to muscle atrophy, pose unmet needs for specific and effective treatments. Recent advances suggest that sarcomeres, the smallest contractile units of heart and skeletal muscles, can be viable pharmacological targets. In sarcomeres, the contractile actin and myosin filaments are organised by a network of proteins combining structural and signalling functions, forming the sarcomeric cytoskeleton. This includes the giant proteins titin, obscurin and nebulin, which contain protein-binding sites along with signalling domains such as protein kinase, Rho activator, and Src-homology domains. These signalling domains have recently been implicated in sarcomere assembly, and the regulation of muscle contractile and metabolic adaptation. Although many functions of sarcomeric proteins remain to be discovered, their potential as pharmacological targets is now emerging. Here, we will review recent insight into the physiological and pathological signalling functions of sarcomeric cytoskeletal proteins and discuss new aspects and strategies in skeletal muscle signalling, pathomechanisms and therapy.

CD38 Facilitates Recovery from Traumatic Brain Injury

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. It causes progressive tissue atrophy and consequent neurological dysfunctions. TBI is accompanied by neuroinflammation, a process mediated largely by microglia. CD38 is an ectoenzyme that promotes transmembrane signaling via the synthesis of potent calcium mobilizing agents or via its receptor activity. CD38 is expressed in the brain in various cell types including microglia. In previous studies, we showed that CD38 regulates microglial activation and response to chemokines. In view of the important role of neuroinflammation in TBI and the effects of CD38 on microglial responses, the present study examines the role of CD38 in the recovery of mice from closed head injury (CHI), a model of focal TBI. For this purpose, CD38-deficient and wild-type (WT) mice were subjected to a similar severity of CHI and the effect of the injury on neurobehavioral and cognitive functions was assessed by the Neurological Severity Score (NSS) and the Object Recognition Test, at various time points post-injury. The results show that recovery after CHI (as indicated by the NSS) was significantly lower in CD38-deficient mice than in WT mice and that the object recognition performance after injury was significantly impaired in injured CD38-deficient mice than in WT mice. In addition, we also observed that the amount of activated microglia/macrophages at the injury site was significantly lower in CD38-deficient mice compared with WT mice. Taken together, our findings indicate that CD38 plays a beneficial role in the recovery of mice from CHI and that this effect is mediated, at least in part, via the effect of CD38 on microglia responses.

Neuroprotection after Traumatic Brain Injury in Heat-Acclimated Mice Involves Induced Neurogenesis and Activation of Angiotensin Receptor Type 2 Signaling

Long-term exposure of mice to mild heat (34°C ± 1°C) confers neuroprotection against traumatic brain injury (TBI); however, the underling mechanisms are not fully understood. Heat acclimation (HA) increases hypothalamic angiotensin II receptor type 2 (AT2) expression and hypothalamic neurogenesis. Accumulating data suggest that activation of the brain AT2 receptor confers protection against several types of brain pathologies, including ischemia, a hallmark of the secondary injury occurring following TBI. As AT2 activates the same pro-survival pathways involved in HA-mediated neuroprotection (e.g., Akt phosphorylation, hypoxia-inducible factor 1α (HIF-1α), and brain-derived neurotrophic factor (BDNF)), we examined the role of AT2 in HA-mediated neuroprotection after TBI. Using an AT2-specific antagonist PD123319, we found that the improvements in motor and cognitive recovery as well as reduced lesion volume and neurogenesis seen in HA mice were all diminished by AT2 inhibition, whereas no significant alternations were observed in control mice. We also found that nerve growth factor/tropomyosin-related kinase receptor A (TrkA), BDNF/TrkB, and HIF-1α pathways are upregulated by HA and inhibited on PD123319 administration, suggesting that these pathways play a role in AT2 signaling in HA mice. In conclusion, AT2 is involved in HA-mediated neuroprotection, and AT2 activation may be protective and should be considered a novel drug target in the treatment of TBI patients.