Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Dog training alleviates PTSD symptomatology by emotional and attentional regulation.

Background: Post-Traumatic Stress Disorder (PTSD) symptoms include re-experiencing, avoidance, hyperarousal, and cognitive deficits, reflecting both emotional and cognitive dysregulation. In recent years, non-pharmacological approaches and specifically animal-assisted therapy have been shown to be beneficial for a variety of disorders such as Attention-Deficit/Hyperactivity Disorder, Autism Spectrum Disorder, and PTSD. However, little is mentioned in the literature about the reciprocal effects of the animal-human interaction. Objective: To evaluate the effects of a one-year dog training programme on PTSD symptomatology in youngsters with PTSD and on dogs' behaviour. Methods: Fifty-three adolescents, previously exposed to interpersonal trauma, were clinically diagnosed with PTSD and assigned to a dog-training programme group (n = 30) and a control group (n = 23) that engaged in other training programmes (e.g. cooking, hairstyling, etc.). Both groups were evaluated at baseline and following 12-months by The Clinician-Administered PTSD Scale for DSM-5 in Children and Adolescents (CAPS-CA-5) and Beck-Depression Inventory (BDI). Additionally, we physiologically measured both emotional and attention dysregulation. Results: Post-12-months training, a significant alleviation of PTSD symptomatology accompanied by lower depression severity was observed in the dog-training group, compared with a insignificant recovery in the control group. Furthermore, improved emotional and attentional regulation was observed in the dog-training group. Measuring the dogs' behaviour revealed increased anxiety and decreased selective attention performance, which was inversely correlated with the beneficial effects observed in the dog-training programme group. Conclusions: Our findings emphasize the role of emotional and attentional regulations on the dog-handler interface, as evidence-based support for the beneficial effects of the dog-training programme, as either a non-pharmacological intervention or as complementary to anti-depressants treatment of PTSD. Though pharmacological treatments increase the patients' well-being by treating certain PTSD symptoms, our suggested dog-training programme seems to influence the PTSD diagnostic status, thus may be implemented in civilians and veterans with PTSD.

Antecedentes: Los síntomas de trastorno de estrés postraumático (TEPT) incluyen re-experimentación, evitación, hiperalerta y déficits cognitivos, reflejando desregulación tanto emocional como cognitiva. En los últimos años, se demostró que los enfoques no farmacológicos y específicamente la terapia asistida por animales son beneficiosos para una variedad de trastornos como el Trastorno por Déficit Atencional e Hiperactividad, el Trastorno del Espectro Autista y el TEPT. Sin embargo, poco se menciona en la literatura acerca de los efectos recíprocos de la interacción animal-humano.Objetivo: Evaluar los efectos de un programa de adiestramiento canino de un año en la sintomatología de TEPT en los jóvenes con TEPT y en el comportamiento de los perros.Métodos: Cincuenta y tres adolescentes, previamente expuestos a trauma interpersonal, fueron diagnosticados clínicamente con TEPT y asignados a un grupo de programa de adiestramiento canino (n = 30) y a un grupo control (n = 23) que participaron en otros programas de adiestramiento (ej., cocinar, peluquería, etc). Ambos grupos fueron evaluados al inicio y después de 12 meses mediante la Escala de TEPT administrada por el Clínico del DSM-5 en niños y adolescentes (CAPS-CA-5 por sus siglas en inglés) y el Inventario de Depresión de Beck (BDI). Adicionalmente, medimos fisiológicamente la desregulación emocional y de la atención.Resultados: Después del entrenamiento de 12 meses, se observó un alivio significativo de la sintomatología de TEPT junto con una disminución de la severidad de la depresión en el grupo de adiestramiento canino, comparado con una recuperación insignificante en el grupo control. Además, se observó una mejoría en la regulación emocional y de la atención en el grupo de adiestramiento canino. La medición del comportamiento de los perros reveló un aumento de la ansiedad y disminución del rendimiento de la atención selectiva, que se correlacionó inversamente con los efectos beneficiosos observados en el grupo del programa de adiestramiento canino.Conclusiones: Nuestros hallazgos enfatizan el rol de la regulación emocional y de atención en la interfaz del entrenador de perros, como soporte basado en la evidencia para los efectos beneficiosos del programa de adiestramiento canino, tanto como tratamiento no farmacológico como complementario al tratamiento antidepresivo del TEPT. Aunque los tratamientos farmacológicos fomentan el bienestar de los pacientes al tratar ciertos síntomas del TEPT, nuestro programa de adiestramiento canino sugerido parece influir en el estado diagnóstico de TEPT, por lo que puede implementarse en civiles y veteranos con TEPT.

Attention Dysregulation in Breast Cancer Patients Following a Complementary Alternative Treatment Routine: A Double-Blind Randomized Trial.

INTRODUCTION: Breast cancer patients and survivors frequently report fatigue, emotional, and cognitive disturbances, which reduce performance at all levels of occupation and make life quality issues a considerable clinical concern. The aim of this study is to evaluate attention and emotion regulation across radiotherapy period and the possible effects of complementary alternative medicine (CAM). METHODS: Fifty-seven patients with unilateral breast cancer underwent surgery and systemic chemotherapy before participating in this double-blind randomized study. Two thirds were given CAM (n = 38) while the rest received placebo (carrier only, n = 19). Patients' attention and anxiety were physiologically tested at baseline, 2 and 4 weeks during the radiation period as well as 1-month after the end of radiation session. RESULTS: Both groups showed similar levels of anxiety with no significant differences at baseline nor post-radiotherapy. Long-term significant recovery of attention performance was observed in the CAM patients, accompanied by a similar tendency in anxiety level, measured by the eye-blink probability. CONCLUSIONS: This study physiologically validates the attention impairment reported among breast cancer survivors; also, it depicted a beneficial late-effect of a routine CAM on attention dysregulation. The suggested non-invasive physiological measures can physiologically monitor patients' psychological and cognitive well-being as well as evaluate the beneficial effect of CAM in breast cancer patients by assessing their coping ability to support the treatment plan. Thus, the results have potential clinical implications on patients' and survivors' quality of life. TRIAL REGISTRATION: NIH, NCT02890316. Registered July 2016, http://www.ClinicalTrials.gov.

Developmental effects of environmental enrichment on selective and auditory sustained attention.

Environmental enrichment (EE) has been used as a positive manipulation in different disease models. However, there is conflicting evidence reported in the literature about the effects of EE. Additionally, the time period that would be most beneficial in implementing environmental enrichment as an intervention is not clear. Our study aimed to systematically compare the prenatal, juvenile, mid-adolescence, and adulthood developmental trajectory to further the understanding of enriched environment's effects on selective and auditory sustained attention, corresponding to behavioral (conceived) and physiological-reflexive (non-conceived) measures. Rats were exposed for 21 days to enriched environment during various developmental periods and compared to age-matched controls. All groups were tested for long-term effects (at postnatal day 120 and onward) on selective and sustained attention. We found that the exposure to enriched environment during mid-adolescence has yielded the most significant and long-term pattern of effects, including selective and auditory sustained attention performance, increased foraging-like behavior and a significant decrease in corticosterone level. Similarly, the exposure to EE at juvenile period improved selective attention, increased foraging-like behavior, and reduced anxiety levels as reflected in the open field as well as in low corticosterone levels. These results specify a crucial period along the developmental trajectory for applying environmental enrichment. Mid-adolescence is suggested, in future basic and translational studies, as the sensitive time period that induces the most beneficial and long-term effects of EE on attention. The current findings suggest that the exposure to EE during mid-adolescence should be further considered and studied as behavioral alternative intervention, or as adjuvant behavioral therapy, aimed to decrease the probability to develop ADHD in post-adolescence period. This suggestion is highly relevant due to the debate regarding the pros and cons of screens usage (e.g. Facebook, online games, etc.) during early life that decreases environmental enrichment, especially, direct social interaction.

A probabilistic model of startle response reveals opposite effects of acute versus chronic Methylphenidate treatment.

BACKGROUND: The startle response is considered as the major physio-behavioral indication of anxiety in health and disease conditions. However, due to different protocols of stimulation and measurement, the magnitude as well as the appearance of the startle response is inconsistent. NEW METHOD: We postulate that the startle probability and not merely the amplitude may bare information that will form a consistent physiological measure of anxiety. RESULTS: To examine the proof-of-concept of our suggested probability model, we evaluated the effects of acute (single) versus chronic (14 days) MPH administration on both startle amplitude and probability. We found that both acute and chronic MPH administration has yielded similar effects on startle amplitude. However, acute MPH increased the startle's probability while chronic MPH decreased it. Next, we evaluated the effects of acute versus chronic stress on the startle's parameters and found a complementary effect. Explicitly, acute stress increased the startle's probability while chronic stress increased the startle amplitude. In contrast, enriched environment had no significant effects. Finally, to further validate the probability measure, we show that Midazolam had significant anxiolytic effects. In the second part, we investigated the acoustic startle response parameters (e.g. background noise and pulse duration), to better understand the interplay between these parameters and the startle amplitude versus probability. CONCLUSIONS: We show that the probabilistic element of the startle response does not only point to deeper physiologic relationships but may also serve as "hidden variables" congruent but not entirely identical to the commonly researched amplitude of the startle response.

Evidence for social cooperation in rodents by automated maze.

Social cooperation is defined as a joint action for mutual benefit that depends on the individual and the counterparts' behaviors. To gain valid evidence for social cooperation behavior we conducted a series of experiments in our suggested fully automated non-conditioned maze and depicted three major findings: (i) During 18 days of training the rats showed a progressive social learning curve as well as latent social learning; (ii) Examining the perceptual communication between the cooperating partners, we found a correlation between the available perceptual modalities and the social cooperation performance; and (iii) Investigating contextual learning as a competing process to the social cooperation, we found that additional contextual cues impaired the social cooperation performance. In conclusion, our suggested automated cooperation maze is designed to further our understanding of social cooperation under normal conditions, such as decision-making, and to examine the neural basis of social cooperation. A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social cognition, including depression, autism spectrum disorders, obsessive-compulsive disorder, and schizophrenia. Thus, on the pathological end, our maze for social cooperation evaluation can contribute significantly to the investigation of a wide range of social cooperation impairments in a rodent model.

l-Carnitine improves cognitive and renal functions in a rat model of chronic kidney disease.

Over the past decade, the prevalence of chronic kidney disease (CKD) has reached epidemic proportions. The search for novel pharmacological treatment for CKD has become an area of intensive clinical research. l-Carnitine, considered as the "gatekeeper" responsible for admitting long chain fatty acids into cell mitochondria. l-Carnitine synthesis and turnover are regulated mainly by the kidney and its levels inversely correlate with serum creatinine of normal subjects and CKD patients. Previous studies showed that l-carnitine administration to elderly people is improving and preserving cognitive function. As yet, there are no clinical intervention studies that investigated the effect of l-carnitine administration on cognitive impairment evidenced in CKD patients. Thus, we aimed to investigate the effects of l-carnitine treatment on renal function and on the cognitive performance in a rat model of progressive CKD. To assess the role of l-carnitine on CKD condition, we estimated the renal function and cognitive abilities in a CKD rat model. We found that all CKD animals exhibited renal function deterioration, as indicated by elevated serum creatinine, BUN, and ample histopathological abnormalities. l-Carnitine treatment of CKD rats significantly reduced serum creatinine and BUN, attenuated renal hypertrophy and decreased renal tissue damage. In addition, in the two way shuttle avoidance learning, CKD animals showed cognitive impairment which recovered by the administration of l-carnitine. We conclude that in a rat model of CKD, l-carnitine administration significantly improved cognitive and renal functions.

Methylphenidate and environmental enrichment ameliorate the deleterious effects of prenatal stress on attention functioning.

Either pre- or post-natal environmental factors seem to play a key role in brain and behavioral development and to exert long-term effects. Increasing evidence suggests that exposure to prenatal stress (PS) leads to motor and learning deficits and elevated anxiety, while enriched environment (EE) shows protective effects. The dopaminergic system is also sensitive to environmental life circumstances and affects attention functioning, which serves as the preliminary gate to cognitive processes. However, the effects of methylphenidate (MPH) on the dopaminergic system and attentional functioning, in the context of these life experiences, remain unclear. Therefore, we aimed to examine the effects of EE or PS on distinct types of attention, along with possible effects of MPH exposure. We found that PS impaired selective attention as well as partial sustained attention, while EE had beneficial effects. Both EE and MPH ameliorated the deleterious effects of PS on attention functioning. Considering the possible psychostimulant effect of MPH, we examined both anxiety-like behavior as well as motor learning. We found that PS had a clear anxiogenic effect, whereas EE had an anxiolytic effect. Nevertheless, the treatment with both MPH and/or EE recovered the deleterious effects of PS. In the motor-learning task, the PS group showed superior performance while MPH led to impaired motor learning. Performance decrements were prevented in both the PS + MPH and EE + MPH groups. This study provides evidence that peripubertal exposure to EE (by providing enhanced sensory, motor, and social opportunities) or MPH treatments might be an optional therapeutic intervention in preventing the PS long-term adverse consequences.

Mapping the developmental trajectory of stress effects: pubescence as the risk window.

The exposure to stress at different developmental time points has long been postulated to have a crucial impact on various brain structures involved in mental disorders. The long-term specific effects seem to emerge as a function of timing and duration of the exposure to stress, as well as the characteristics of the stressor. Previous studies have addressed this issue with an effort to describe a single "hyper-sensitive" time point, and have led to disagreement on a particular sensitive period for stress exposure. The primary aim of our study was to investigate the hypothesis that indeed there is a developmental stress risk window in male Wistar rats. We conducted a systematic mapping of the long-term effects of an acute stress protocol, applied both prenatal (gestational days 14-16) and postnatal (days 9-151), overall at 11 different time-points during development. Stress protocol consists of 3 days of either maternal separation (for rats at postnatal days 9-19) or exposure to the stressors forced swim, elevated plus maze and restraint (for both dams and males at postnatal days 24-151). Consequences in adulthood were measured by investigating the animals' behavior in both the open field and startle box, together with the physiological measure of corticosterone. We found both behaviorally and physiologically that the pubescence time points are the most vulnerable to stress compared to all other tested time points along the developmental trajectory. Carefully considering the comparison between rat and human age, our findings may imply the importance of childhood-to-adulthood transition, as a sensitive time-point which may exacerbate a predisposition for the development of stress-induced psychopathologies.

Prenatal Enriched Environment improves emotional and attentional reactivity to adulthood stress.

Environmental factors seem to play a key role in brain and behavioral development, both in humans and animals. Different environmental manipulations, either pre- or post-natal, have been shown to exert long-term physiological and behavioral effects. While studies in the field of Enriched Environment mainly focus on the post weaning period and provide enrichment as a post adverse-experience manipulation, the preceding effects of prenatal Enriched Environment have rarely been investigated. In this study, we investigated the effects of prenatal Enriched Environment (through the entire pregnancy) followed by adulthood acute stress. In the prenatal Enriched Environment offspring, we found anxiety and depressive-like behaviors with poor attentional performance. Surprisingly, when prenatal Enriched Environment was followed by adulthood stress, we observed a dramatic restoration of these behavioral deficits. Our results suggest that prenatal Enriched Environment may substrate resiliency to adulthood stress.

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9ß1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model.

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9ß1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and ßNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9ß1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of ßNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and ßNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9ß1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9ß1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

Losartan, a therapeutic candidate in congenital muscular dystrophy: studies in the dy(2J) /dy(2J) mouse.

OBJECTIVE: Lamininα2-deficient congenital muscular dystrophy type 1A (MDC1A) is a cureless disease associated with severe disability and shortened lifespan. Previous studies have shown reduced fibrosis and restored skeletal muscle remodeling following treatment with losartan, an angiotensin II type I receptor blocker. We therefore evaluated the effect of losartan treatment in the dy(2J) /dy(2J) mouse model of MDC1A. METHODS: Homozygous dy(2J) /dy(2J) and control mice were treated with losartan or placebo for 12 weeks from 6 weeks of age. Outcome measures included hindlimb and forelimb muscle strength by Grip Strength Meter and quantitative muscle fibrosis parameters. Losartan's effects on transforming growth factor ß (TGF-ß) and mitogen-activated protein kinase (MAPK) signaling pathways were evaluated with Western blotting, immunofluorescence, and cytokine measurements. RESULTS: Losartan treatment was associated with significant impressive improvement in muscle strength and amelioration of fibrosis. Administration of losartan inhibited TGF-ß signaling pathway, resulting in decreased serum TGF-ß1 level and reduced downstream phosphorylated (P) Smad2/3 proteins. Moreover, losartan activated Smad7 protein, a key negative regulator of TGF-ß signaling. In addition, losartan treatment inhibited the MAPK cascade as shown by decreased expression of P-p38 MAPK, P-c-jun-N-terminal kinase, and P-extracellular signal-regulated kinases 1 and 2 in the treated mice. INTERPRETATION: Losartan, a commonly prescribed US Food and Drug Administration-approved medication for hypertension, demonstrated clinical improvement and amelioration of fibrosis in the dy(2J) /dy(2J) mouse model of MDC1A via TGF-ß and MAPK signaling pathways. The results of this study support pursuing a clinical trial of losartan treatment in children with MDC1A.

Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes.

The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

Environmental enrichment preceding early adulthood methylphenidate treatment leads to long term increase of corticosterone and testosterone in the rat.

Attention-deficit/hyperactivity disorder (ADD/ADHD) has been emerging as a world-wide psychiatric disorder. There appears to be an increasing rate of stimulant drug abuse, specifically methylphenidate (MPH) which is the most common treatment for ADHD, among individuals who do not meet the criteria for ADHD and particularly for cognitive enhancement among university students. However, the long term effects of exposure to MPH are unknown. Thus, in light of a developmental approach in humans, we aimed to test the effects of adolescence exposure to enriched environment (EE) followed by MPH administration during early adulthood, on reactions to stress in adulthood. Specifically, at approximate adolescence [post natal days (PND) 30-60] rats were reared in EE and were treated with MPH during early adulthood (PND 60-90). Adult (PND 90-92) rats were exposed to mild stress and starting at PND 110, the behavioral and endocrine effects of the combined drug and environmental conditions were assessed. Following adolescence EE, long term exposure to MPH led to decreased locomotor activity and increased sucrose preference. EE had a beneficial effect on PPI (attentive abilities), which was impaired by long term exposure to MPH. Finally, the interaction between EE and, exposure to MPH led to long-term elevated corticosterone and testosterone levels. In view of the marked increase in MPH consumption over the past decade, vigilance is crucial in order to prevent potential drug abuse and its long term detrimental consequences.

The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy.

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J)/dy(2J) mouse model of merosin deficient congenital muscular dystrophy. The dy(2J)/dy(2J) mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J)/dy(2J) mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J)/dy(2J) mouse model of congenital muscular dystrophy.

Improved muscle strength and mobility in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate.

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.

The regulatory domain of protein kinase C delta positively regulates insulin receptor signaling.

Protein kinase C delta (PKCdelta) is induced by insulin to rapidly associate with insulin receptor (IR) and upregulates insulin signaling. We utilized specific JM and CT receptor domains and chimeras of PKCalpha and PKCdelta regulatory and catalytic domains to elucidate which components of PKCdelta are responsible for positive regulatory effects of PKCdelta on IR signaling. Studies were performed on L6 and L8 skeletal muscle myoblasts and myotubes. PKCdelta was preferentially bound to the JM domain of IR, and insulin stimulation increased this binding. Both PKCdelta/alpha and PKCalpha/delta chimeras (regulatory/catalytic) were bound preferentially to the JM but not to the CT domain of IR. Although IR-PKCdelta binding was higher in cells expressing either the PKCdelta/alpha or PKCalpha/delta chimera than in control cells, upregulation of IR signaling was observed only in PKCdelta/alpha cells. Thus, in response to insulin increases in tyrosine phosphorylation of IR and insulin receptor substrate-1, downstream signaling to protein kinase B and glycogen synthase kinase 3 (GSK3) and glucose uptake were greater in cells overexpressing PKCdelta/alpha and the PKCdelta/delta domains than in cells expressing the PKCalpha/delta domains. Basal binding of Src to PKCdelta was higher in both PKCdelta/alpha- and PKCalpha/delta-expressing cells compared to control. Binding of Src to IR was decreased in PKCalpha/delta cells but remained elevated in the PKCdelta/alpha cells in response to insulin. Finally, insulin increased Src activity in PKCdelta/alpha-expressing cells but decreased it in PKCalpha/delta-expressing cells. Thus, the regulatory domain of PKCdelta via interaction with Src appears to determine the role of PKCdelta as a positive regulator of IR signaling in skeletal muscle.

Cytosolic protein tyrosine phosphatase-epsilon is a negative regulator of insulin signaling in skeletal muscle.

Whereas positive regulatory events triggered by insulin binding to insulin receptor (IR) have been well documented, the mechanism by which the activated IR is returned to the basal status is not completely understood. Recently studies focused on the involvement of protein tyrosine phosphatases (PTPs) and how they might influence IR signaling. In this study, we examined the possibility that cytosolic PTPepsilon (cytPTPepsilon) is involved in IR signaling. Studies were performed on L6 skeletal muscle cells. cytPTPepsilon was overexpressed by using pBABE retroviral expression vectors. In addition, we inhibited cytPTPepsilon by RNA silencing. We found that insulin induced rapid association of cytPTPepsilon with IR. Interestingly, this association appeared to occur in the plasma membrane and on stimulation with insulin the two proteins internalized together. Moreover, it appeared that almost all internalized IR was associated with cytPTPepsilon. We found that knockdown of cytPTPepsilon by RNA silencing increased insulin-induced tyrosine phosphorylation of IR and IR substrate (IRS)-1 as well as phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Moreover, overexpression of wild-type cytPTPepsilon reduced insulin-induced tyrosine phosphorylation of IR, IRS-1, and phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Finally, insulin-induced tyrosine phosphorylation of IR and IRS-1 was greater in skeletal muscle from mice lacking the cytPTPepsilon gene than that from wild-type control animals. We conclude that cytPTPepsilon serves as another major candidate negative regulator of IR signaling in skeletal muscle.

Protein kinase Calpha regulates insulin receptor signaling in skeletal muscle.

Certain PKC isoforms are stimulated by insulin and interact with IR as well as with IRS, but it is still not clear if specific PKC isoforms regulate IR signaling directly or through IRS-1. PKCalpha may regulate IRS activity in response to insulin. We investigated the possibility that PKCalpha may be important in insulin signaling. Studies were conducted on skeletal muscle in adult mice and on L6 skeletal cells. PKCalpha is constitutively associated with IRS-1, and insulin stimulation of PKCalpha causes disassociation of the two proteins within 5 min. Blockade of PKCalpha inhibited insulin-induced disassociation of PKCalpha from IRS1. Selective inhibition of PKCalpha increased the ability of insulin to reduce blood glucose levels. Insulin stimulation activates PKB and increases the association of PKCalpha with PKB. Blockade of PKCalpha increased threonine phosphorylation of PKB. We suggest that PKCalpha regulates insulin signaling in skeletal muscle through its disassociation from IRS-1 and association with PKB.

Src tyrosine kinase regulates insulin-induced activation of protein kinase C (PKC) delta in skeletal muscle.

Insulin stimulation of skeletal muscle results in rapid activation of protein kinase Cdelta (PKCdelta), which is associated with its tyrosine phosphorylation and physical association with insulin receptor (IR). The mechanisms underlying tyrosine phosphorylation of PKCdelta have not been determined. In this study, we investigated the possibility that the Src family of nonreceptor tyrosine kinases may be involved upstream insulin signaling. Studies were done on differentiated rat skeletal myotubes in primary culture. Insulin caused an immediate stimulation of Src and induced its physical association with both IR and PKCdelta. Inhibition of Src by treatment with the Src family inhibitor PP2 reduced insulin-stimulated Src-PKCdelta association, PKCdelta tyrosine phosphorylation and PKCdelta activation. PP2 inhibition of Src also decreased insulin-induced IR tyrosine phosphorylation, IR-PKCdelta association and association of Src with both PKCdelta and IR. Finally, inhibition of Src decreased insulin-induced glucose uptake. We conclude that insulin activates Src tyrosine kinase, which regulates PKCdelta activity. Thus, Src tyrosine kinase may play an important role in insulin-induced tyrosine phosphorylation of both IR and PKCdelta. Moreover, both Src and PKCdelta appear to be involved in IR activation and subsequent downstream signaling.