Insulinotropic treatments exacerbate metabolic syndrome in mice lacking MeCP2 function.

Rett syndrome (RTT), an X-linked postnatal disorder, results from mutations in Methyl CpG-binding protein 2 (MECP2). Survival and breathing in Mecp2(NULL/Y) animals are improved by an N-terminal tripeptide of insulin-like growth factor I (IGF-I) treatment. We determined that Mecp2(NULL/Y) animals also have a metabolic syndrome and investigated whether IGF-I treatment might improve this phenotype. Mecp2(NULL/Y) mice were treated with a full-length IGF-I modified with the addition of polyethylene glycol (PEG-IGF-I), which improves pharmacological properties. Low-dose PEG-IGF-I treatment slightly improved lifespan and heart rate in Mecp2(NULL/Y) mice; however, high-dose PEG-IGF-I decreased lifespan. To determine whether insulinotropic off-target effects of PEG-IGF-I caused the detrimental effect, we treated Mecp2(NULL/Y) mice with insulin, which also decreased lifespan. Thus, the clinical benefit of IGF-I treatment in RTT may critically depend on the dose used, and caution should be taken when initiating clinical trials with these compounds because the beneficial therapeutic window is narrow.

A small-molecule TrkB ligand improves dendritic spine phenotypes and atypical behaviors in female Rett syndrome mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in MECP2, which encodes methyl-CpG-binding protein 2, a transcriptional regulator of many genes, including brain-derived neurotrophic factor (BDNF). BDNF levels are lower in multiple brain regions of Mecp2-deficient mice, and experimentally increasing BDNF levels improve atypical phenotypes in Mecp2 mutant mice. Due to the low blood-brain barrier permeability of BDNF itself, we tested the effects of LM22A-4, a brain-penetrant, small-molecule ligand of the BDNF receptor TrkB (encoded by Ntrk2), on dendritic spine density and form in hippocampal pyramidal neurons and on behavioral phenotypes in female Mecp2 heterozygous (HET) mice. A 4-week systemic treatment of Mecp2 HET mice with LM22A-4 restored spine volume in MeCP2-expressing neurons to wild-type (WT) levels, whereas spine volume in MeCP2-lacking neurons remained comparable to that in neurons from female WT mice. Female Mecp2 HET mice engaged in aggressive behaviors more than WT mice, the levels of which were reduced to WT levels by the 4-week LM22A-4 treatment. These data provide additional support to the potential usefulness of novel therapies not only for RTT but also to other BDNF-related disorders.

Detection of rarely identified multiple mutations in MECP2 gene do not contribute to enhanced severity in Rett syndrome.

The objective of our study was to characterize the influence of multiple mutations in the MECP2 gene in a cohort of individuals with Rett syndrome. Further analysis demonstrated that nearly all resulted from de novo in cis mutations, where the disease severity was indistinguishable from single mutations. Our methods involved enrolling participants in the RTT Natural History Study (NHS). After providing informed consent through their parents or principal caretakers, additional molecular assessments were performed in the participants and their parents to assess the presence and location of more than one mutation in each. Clinical severity was assessed at each visit in those participants in the NHS. Non-contiguous MECP2 gene variations were detected in 12 participants and contiguous mutations involving a deletion and insertion in three participants. Thirteen of 15 participants had mutations that were in cis; four (of 13) had three MECP2 mutations; two (of 15) had mutations that were both in cis and in trans (i.e., on different alleles). Clinical severity did not appear different from NHS participants with a single similar mutation. Mutations in cis were identified in most participants; two individuals had mutations both in cis and in trans. The presence of multiple mutations was not associated with greater severity. Nevertheless, multiple mutations will require greater thought in the future, if genetic assignment to drug treatment protocols is considered.

A small-molecule TrkB ligand improves dendritic spine phenotypes and atypical behaviors in female Rett syndrome mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein-2 (MECP2), encoding a transcriptional regulator of many genes, including brain-derived neurotrophic factor (Bdnf). BDNF mRNA and protein levels are lower in RTT autopsy brains and in multiple brain regions of Mecp2-deficient mice, and experimentally increasing BDNF levels improve atypical phenotypes in Mecp2 mutant mice. Due to the low blood-brain barrier permeability of BDNF itself, we tested the effects of a brain penetrant, small molecule ligand of its TrkB receptors. Applied in vitro, LM22A-4 increased dendritic spine density in pyramidal neurons in cultured hippocampal slices from postnatal day (P) 7 male Mecp2 knockout (KO) mice as much as recombinant BDNF, and both effects were prevented by the TrkB receptor inhibitors K-252a and ANA-12. Consistent with its partial agonist activity, LM22A-4 did not affect spine density in CA1 pyramidal neurons in slice cultures from male wildtype (WT) mice, where typical BDNF levels outcompete its binding to TrkB. To identify neurons of known genotypes in the "mosaic" brain of female Mecp2 heterozygous (HET) mice, we treated 4-6-month-old female MeCP2-GFP WT and HET mice with peripheral injections of LM22A-4 for 4 weeks. Surprisingly, mutant neurons lacking MeCP2-GFP showed dendritic spine volumes comparable to that in WT controls, while MeCP2-GFP-expressing neurons showed larger spines, similar to the phenotype we described in symptomatic male Mecp2 KO mice where all neurons lack MeCP2. Consistent with this non-cell-autonomous mechanism, a 4-week systemic treatment with LM22A-4 had an effect only in MeCP2-GFP-expressing neurons in female Mecp2 HET mice, bringing dendritic spine volumes down to WT control levels, and without affecting spines of MeCP2-GFP-lacking neurons. At the behavioral level, we found that female Mecp2 HET mice engaged in aggressive behaviors significantly more than WT controls, which were reduced to WT levels by a 4-week systemic treatment with LM22A-4. Altogether, these data revealed differences in dendritic spine size and altered behaviors in Mecp2 HET mice, while providing support to the potential usefulness of BDNF-related therapeutic approaches such as the partial TrkB agonist LM22A-4.

HDAC activity is required for BDNF to increase quantal neurotransmitter release and dendritic spine density in CA1 pyramidal neurons.

Molecular mechanisms involved in the strengthening and formation of synapses include the activation and repression of specific genes or subsets of genes by epigenetic modifications that do not alter the genetic code itself. Chromatin modifications mediated by histone acetylation have been shown to be critical for synaptic plasticity at hippocampal excitatory synapses and hippocampal-dependent memory formation. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity and behavioral adaptations, it is not surprising that regulation of this gene is subject to histone acetylation changes during synaptic plasticity and hippocampal-dependent memory formation. Whether the effects of BDNF on dendritic spines and quantal transmitter release require histone modifications remains less known. By using two different inhibitors of histone deacetylases (HDACs), we describe here that their activity is required for BDNF to increase dendritic spine density and excitatory quantal transmitter release onto CA1 pyramidal neurons in hippocampal slice cultures. These results suggest that histone acetylation/deacetylation is a critical step in the modulation of hippocampal synapses by BDNF. Thus, mechanisms of epigenetic modulation of synapse formation and function are novel targets to consider for the amelioration of symptoms of intellectual disabilities and neurodegenerative disorders associated with cognitive and memory deficits.

Divergent roles of p75NTR and Trk receptors in BDNF's effects on dendritic spine density and morphology.

Activation of TrkB receptors by brain-derived neurotrophic factor (BDNF) followed by MAPK/ERK signaling increases dendritic spine density and the proportion of mature spines in hippocampal CA1 pyramidal neurons. Considering the opposing actions of p75(NTR) and Trk receptors in several BDNF actions on CNS neurons, we tested whether these receptors also have divergent actions on dendritic spine density and morphology. A function-blocking anti-p75(NTR) antibody (REX) did not affect spine density by itself but it prevented BDNF's effect on spine density. Intriguingly, REX by itself increased the proportion of immature spines and prevented BDNF's effect on spine morphology. In contrast, the Trk receptor inhibitor k-252a increased spine density by itself, and prevented BDNF from further increasing spine density. However, most of the spines in k-252a-treated slices were of the immature type. These effects of k-252a on spine density and morphology required neuronal activity because they were prevented by TTX. These divergent BDNF actions on spine density and morphology are reminiscent of opposing functional signaling by p75(NTR) and Trk receptors and reveal an unexpected level of complexity in the consequences of BDNF signaling on dendritic morphology.

Hippocampal CA1 pyramidal neurons of Mecp2 mutant mice show a dendritic spine phenotype only in the presymptomatic stage.

Alterations in dendritic spines have been documented in numerous neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, an X chromosome-linked disorder associated with mutations in MECP2, is the leading cause of intellectual disabilities in women. Neurons in Mecp2-deficient mice show lower dendritic spine density in several brain regions. To better understand the role of MeCP2 on excitatory spine synapses, we analyzed dendritic spines of CA1 pyramidal neurons in the hippocampus of Mecp2(tm1.1Jae) male mutant mice by either confocal microscopy or electron microscopy (EM). At postnatal-day 7 (P7), well before the onset of RTT-like symptoms, CA1 pyramidal neurons from mutant mice showed lower dendritic spine density than those from wildtype littermates. On the other hand, at P15 or later showing characteristic RTT-like symptoms, dendritic spine density did not differ between mutant and wildtype neurons. Consistently, stereological analyses at the EM level revealed similar densities of asymmetric spine synapses in CA1 stratum radiatum of symptomatic mutant and wildtype littermates. These results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic Mecp2-deficient mice. However, they underscore the potential role of MeCP2 in the maintenance of excitatory spine synapses.

Acute Liver Failure in a Healthy Young Female With COVID-19.

Several well-described manifestations of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported. Among them, a transient elevation of liver enzymes is the typical presentation of coronavirus disease 2019 (COVID-19) liver-related injury. The mechanism of liver involvement is likely a combination of viral injury and immune-mediated inflammation. In contrast, acute liver failure in the setting of COVID-19 has rarely been reported. Herein, we report a case of pediatric acute liver failure in a previously healthy female adolescent infected with SARS-CoV-2 with biopsy evidence of replicating virus in hepatocytes, which has not been previously reported.

Bdnf overexpression in hippocampal neurons prevents dendritic atrophy caused by Rett-associated MECP2 mutations.

The expression of the methylated DNA-binding protein MeCP2 increases during neuronal development, which suggests that this epigenetic factor is crucial for neuronal terminal differentiation. We evaluated dendritic and axonal development in embryonic day-18 hippocampal neurons in culture by measuring total length and counting branch point numbers at 4 days in vitro, well before synapse formation. Pyramidal neurons transfected with a plasmid encoding a small hairpin RNA (shRNA) to knockdown endogenous Mecp2 had shorter dendrites than control untransfected neurons, without detectable changes in axonal morphology. On the other hand, overexpression of wildtype (wt) human MECP2 increased dendritic branching, in addition to axonal branching and length. Consistent with reduced neuronal growth and complexity in Rett syndrome (RTT) brains, overexpression of human MECP2 carrying missense mutations common in RTT individuals (R106W or T158M) reduced dendritic and axonal length. One of the targets of MeCP2 transcriptional control is the Bdnf gene. Indeed, endogenous Mecp2 knockdown increased the intracellular levels of BDNF protein compared to untransfected neurons, suggesting that MeCP2 represses Bdnf transcription. Surprisingly, overexpression of wt MECP2 also increased BDNF levels, while overexpression of RTT-associated MECP2 mutants failed to affect BDNF levels. The extracellular BDNF scavenger TrkB-Fc prevented dendritic overgrowth in wt MECP2-overexpressing neurons, while overexpression of the Bdnf gene reverted the dendritic atrophy caused by Mecp2-knockdown. However, this effect was only partial, since Bdnf increased dendritic length only to control levels in mutant MECP2-overexpressing neurons, but not as much as in Bdnf-transfected cells. Our results demonstrate that MeCP2 plays varied roles in dendritic and axonal development during neuronal terminal differentiation, and that some of these effects are mediated by autocrine actions of BDNF.

Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations.

Rett syndrome (RTT) is an X chromosome-linked neurodevelopmental disorder associated with the characteristic neuropathology of dendritic spines common in diseases presenting with mental retardation (MR). Here, we present the first quantitative analyses of dendritic spine density in postmortem brain tissue from female RTT individuals, which revealed that hippocampal CA1 pyramidal neurons have lower spine density than age-matched non-MR female control individuals. The majority of RTT individuals carry mutations in MECP2, the gene coding for a methylated DNA-binding transcriptional regulator. While altered synaptic transmission and plasticity has been demonstrated in Mecp2-deficient mouse models of RTT, observations regarding dendritic spine density and morphology have produced varied results. We investigated the consequences of MeCP2 dysfunction on dendritic spine structure by overexpressing ( approximately twofold) MeCP2-GFP constructs encoding either the wildtype (WT) protein, or missense mutations commonly found in RTT individuals. Pyramidal neurons within hippocampal slice cultures transfected with either WT or mutant MECP2 (either R106W or T158M) showed a significant reduction in total spine density after 48 h of expression. Interestingly, spine density in neurons expressing WT MECP2 for 96 h was comparable to that in control neurons, while neurons expressing mutant MECP2 continued to have lower spine density than controls after 96 h of expression. Knockdown of endogenous Mecp2 with a specific small hairpin interference RNA (shRNA) also reduced dendritic spine density, but only after 96 h of expression. On the other hand, the consequences of manipulating MeCP2 levels for dendritic complexity in CA3 pyramidal neurons were only minor. Together, these results demonstrate reduced dendritic spine density in hippocampal pyramidal neurons from RTT patients, a distinct dendritic phenotype also found in neurons expressing RTT-associated MECP2 mutations or after shRNA-mediated endogenous Mecp2 knockdown, suggesting that this phenotype represent a cell-autonomous consequence of MeCP2 dysfunction.

Transient receptor potential channels as novel effectors of brain-derived neurotrophic factor signaling: potential implications for Rett syndrome.

In addition to their prominent role as survival signals for neurons in the developing nervous system, neurotrophins have established their significance in the adult brain as well, where their modulation of synaptic transmission and plasticity may participate in associative learning and memory. These crucial activities are primarily the result of neurotrophin regulation of intracellular Ca(2+) homeostasis and, ultimately, changes in gene expression. Outlined in the following review is a synopsis of neurotrophin signaling with a particular focus upon brain-derived neurotrophic factor (BDNF) and its role in hippocampal synaptic plasticity and neuronal Ca(2+) homeostasis. Neurotrophin signaling through tropomyosin-related kinase (Trk) and pan-neurotrophin receptor 75 kD (p75(NTR)) receptors are also discussed, reviewing recent results that indicate signaling through these two receptor modalities leads to opposing cellular outcomes. We also provide an intriguing look into the transient receptor potential channel (TRPC) family of ion channels as distinctive targets of BDNF signaling; these channels are critical for capacitative Ca(2+) entry, which, in due course, mediates changes in neuronal structure including dendritic spine density. Finally, we expand these topics into an exploration of mental retardation (MR), in particular Rett Syndrome (RTT), where dendritic spine abnormalities may underlie cognitive impairments. We propose that understanding the role of neurotrophins in synapse formation, plasticity, and maintenance will make fundamental contributions to the development of therapeutic strategies to improve cognitive function in developmental disorders associated with MR.

The actions of BDNF on dendritic spine density and morphology in organotypic slice cultures depend on the presence of serum in culture media.

We have previously shown that brain-derived neurotrophin factor (BDNF) increases dendritic spine density and the proportion of stubby spines in apical dendrites of CA1 pyramidal neurons of hippocampal slice cultures maintained in serum-free media. We show here that serum withdrawal causes an increase in the proportion of thin spines and a decrease in the fraction of stubby spines, without changing the overall density of dendritic spines. When slices are maintained in serum-containing media, BDNF also increased spine density but had the opposite effect on spine morphology: it increased the proportion of mushroom and thin spines and decreased the proportion of stubby spines. Intriguingly, slices maintained in serum media showed a lower p75NTR-to-TrkB expression level than serum-free slices, even after BDNF exposure. The differential actions of BDNF on spine morphology depending on the presence of serum in culture media, together with the difference in neurotrophin receptor expression are reminiscent of opposing functional signaling by p75NTR and Trk receptors, and reveal a complex modulation of dendritic morphology by BDNF signaling.

Cdk5 Is Essential for Amphetamine to Increase Dendritic Spine Density in Hippocampal Pyramidal Neurons.

Psychostimulant drugs of abuse increase dendritic spine density in reward centers of the brain. However, little is known about their effects in the hippocampus, where activity-dependent changes in the density of dendritic spine are associated with learning and memory. Recent reports suggest that Cdk5 plays an important role in drug addiction, but its role in psychostimulant's effects on dendritic spines in hippocampus remain unknown. We used in vivo and in vitro approaches to demonstrate that amphetamine increases dendritic spine density in pyramidal neurons of the hippocampus. Primary cultures and organotypic slice cultures were used for cellular, molecular, pharmacological and biochemical analyses of the role of Cdk5/p25 in amphetamine-induced dendritic spine formation. Amphetamine (two-injection protocol) increased dendritic spine density in hippocampal neurons of thy1-green fluorescent protein (GFP) mice, as well as in hippocampal cultured neurons and organotypic slice cultures. Either genetic or pharmacological inhibition of Cdk5 activity prevented the amphetamine-induced increase in dendritic spine density. Amphetamine also increased spine density in neurons overexpressing the strong Cdk5 activator p25. Finally, inhibition of calpain, the protease necessary for the conversion of p35 to p25, prevented amphetamine's effect on dendritic spine density. We demonstrate, for the first time, that amphetamine increases the density of dendritic spine in hippocampal pyramidal neurons in vivo and in vitro. Moreover, we show that the Cdk5/p25 signaling and calpain activity are both necessary for the effect of amphetamine on dendritic spine density. The identification of molecular mechanisms underlying psychostimulant effects provides novel and promising therapeutic approaches for the treatment of drug addiction.

The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin.

AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

Is memantine a potential therapeutic for Rett syndrome?

Memantine is a low-affinity, voltage-dependent, non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist. It is classified as a neuroprotective aminoadamantane. It does not cure or reverse Alzheimer's but it does effectively treat symptoms, slows the progression of the disease and allows many patients to perform daily cognitive activities with clear thoughts. Based on it's success in patients with Alzheimer's, memantine has been tested in other neurological disorders with impaired learning and memory. In this review, we will discuss the success and failures of memantine in Downs Syndrome and Fragile X research and from those results, assess the potential benefit of memantine in Rett Syndrome (RTT).

Recent Progress in Rett Syndrome and MeCP2 Dysfunction: Assessment of Potential Treatment Options.

Synaptic communication is highly regulated process of contact between cells allowing information to be stored and modified. Synaptic formation and maturation is the result of interactions between intrinsic genetic/molecular factors and the external environment to establish the communication in the brain. One disorder associated with faulty synapse communication is Rett Syndrome (RTT). RTT is the leading form of severe MR in females, affecting approximately 1:10,000 females worldwide, without predisposition to any particular racial or ethnic group. Mutations in MECP2, the gene encoding methyl-CpG-binding protein-2, have been identified in more than 95% of individuals with RTT. Birth and the milestones of early development appear to be normal in individuals with RTT until approximately 6-18 months when in the subsequent months and years that follows, physical, motor, and social-cognitive development enter a period of regression. The clinical management of these individuals is extremely multifaceted, relying on collaborations of specialists and researchers from many different fields. In this critical literature review, we provide an overview of Rett Syndrome, from patient to pathophysiology with a therapeutic summary of clinical trials in RTT and preclinical studies using mouse and cell models of RTT.

Evaluation of current pharmacological treatment options in the management of Rett syndrome: from the present to future therapeutic alternatives.

Neurodevelopmental disorders are a large family of conditions of genetic or environmental origin that are characterized by deficiencies in cognitive and behavioral functions. The therapeutic management of individuals with these disorders is typically complex and is limited to the treatment of specific symptoms that characterize each disorder. The neurodevelopmental disorder Rett syndrome (RTT) is the leading cause of severe intellectual disability in females. Mutations in the gene encoding the transcriptional regulator methyl-CpG-binding protein 2 (MECP2), located on the X chromosome, have been confirmed in more than 95% of individuals meeting diagnostic criteria for classical RTT. RTT is characterized by an uneventful early infancy followed by stagnation and regression of growth, motor, language, and social skills later in development. This review will discuss the genetics, pathology, and symptoms that distinguish RTT from other neurodevelopmental disorders associated with intellectual disability. Because great progress has been made in the basic and clinical science of RTT, the goal of this review is to provide a thorough assessment of current pharmacotherapeutic options to treat the symptoms associated with this disorder. Furthermore, we will highlight recent discoveries made with novel pharmacological interventions in experimental preclinical phases, and which have reversed pathological phenotypes in mouse and cell culture models of RTT and may result in clinical trials.

Desired professional development pathways for clinical pharmacists.

The 2012 American College of Clinical Pharmacy (ACCP) Certification Affairs Committee was charged with developing guidelines for the desired professional development pathways for clinical pharmacists. This document summarizes recommendations for postgraduate education and training for graduates of U.S. schools and colleges of pharmacy and describes the preferred pathways for achieving, demonstrating, and maintaining competence as clinical pharmacists. After initial licensure within the state or jurisdiction in which the pharmacist intends to practice, completion of an accredited PGY1 pharmacy residency is recommended to further develop the knowledge and skills needed to optimize medication therapy outcomes. An accredited PGY2 pharmacy residency should be completed if a pharmacist wishes to seek employment in a specific therapeutic area or practice setting, if such a residency exists. Clinical pharmacists intending to conduct advanced research that is competitive for federal funding are encouraged to complete a fellowship or graduate education. Initial certification by the Board of Pharmacy Specialties (BPS) or other appropriate sponsoring organizations should be completed in the desired primary therapeutic area or practice setting within 2 years after accepting a position within the desired specific therapeutic area or practice setting. Clinical pharmacists subsequently will need to meet the requirements to maintain pharmacist licensure and board certification. Traineeships, practice-based activities, and certificate programs can be used to obtain additional knowledge and skills that support professional growth. Pharmacists are strongly encouraged to adopt a lifelong, systematic process for professional development and work with ACCP and other professional organizations to facilitate the development and implementation of innovative strategies to assess core practice competencies.

The effect of Rho-associated kinase inhibition on the ocular penetration of timolol maleate.

PURPOSE: To assess the effects of Rho-associated kinase (ROCK) inhibition on the intraocular penetration of timolol maleate. METHODS: Ex vivo porcine corneal penetration of timolol maleate, sotalol hydrochloride, or brinzolamide incubated with or without Y-27632 was determined in vertical Franz diffusion cells. The effect of ROCK inhibition on the vasodilation of porcine conjunctival vasculature was assessed by scanning electron microscopy (SEM) and immunohistochemical staining with subsequent laser-scanning confocal microscopy (LSCM). Experiments were conducted in New Zealand White (NZW) rabbits to assess the effect of ROCK inhibition on the intraocular distribution of timolol maleate. RESULTS: ROCK inhibition resulted in minimal alteration of ex vivo porcine corneal drug penetration of timolol, sotalol, or brinzolamide. SEM and LSCM experiments conducted with conjunctiva and sclera tissue in Franz diffusion cells suggested vasodilation in the conjunctival vasculature in the presence of Y-27632. Pretreatment of the eyes of NZW rabbits with Y-27632 resulted in aggregate fold reductions (1 hour, 0.25-fold; 4 hours, 0.45-fold) of timolol maleate drug concentrations in intraocular tissues (aqueous humor, lens, and iris) versus eyes not receiving Y-27632 pretreatment. Pretreatment with a vasoconstrictor, phenylephrine, resulted in a reversal of the effect of Y-27632 on diminished timolol maleate intraocular penetration in NZW rabbits. CONCLUSIONS: ROCK inhibition reduced the intraocular penetration of administered timolol maleate presumably due to increased systemic elimination through the conjunctival vasculature. It is anticipated that care in order and timing of ROCK inhibitor administration will be warranted for those patients who may be on a multiple topical drug regimen for primary open-angle glaucoma.

MeCP2 regulates the synaptic expression of a Dysbindin-BLOC-1 network component in mouse brain and human induced pluripotent stem cell-derived neurons.

Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.