Dendrimer nanotherapy targeting of glial dysfunction improves inflammation and neurobehavioral phenotype in adult female Mecp2-heterozygous mouse model of Rett syndrome.

Rett syndrome is an X-linked neurodevelopmental disorder caused by mutation of Mecp2 gene and primarily affects females. Glial cell dysfunction has been implicated in in Rett syndrome (RTT) both in patients and in mouse models of this disorder and can affect synaptogenesis, glial metabolism and inflammation. Here we assessed whether treatment of adult (5-6 months old) symptomatic Mecp2-heterozygous female mice with N-acetyl cysteine conjugated to dendrimer (D-NAC), which is known to target glia and modulate inflammation and oxidative injury, results in improved behavioral phenotype, sleep and glial inflammatory profile. We show that unbiased global metabolomic analysis of the hippocampus and striatum in adult Mecp2-heterozygous mice demonstrates significant differences in lipid metabolism associated with neuroinflammation, providing the rationale for targeting glial inflammation in this model. Our results demonstrate that treatment with D-NAC (10 mg/kg NAC) once weekly is more efficacious than equivalently dosed free NAC in improving the gross neurobehavioral phenotype in symptomatic Mecp2-heterozygous female mice. We also show that D-NAC therapy is significantly better than saline in ameliorating several aspects of the abnormal phenotype including paw clench, mobility, fear memory, REM sleep and epileptiform activity burden. Systemic D-NAC significantly improves microglial proinflammatory cytokine production and is associated with improvements in several aspects of the phenotype including paw clench, mobility, fear memory, and REM sleep, and epileptiform activity burden in comparison to saline-treated Mecp2-hetereozygous mice. Systemic glial-targeted delivery of D-NAC after symptom onset in an older clinically relevant Rett syndrome model shows promise in improving neurobehavioral impairments along with sleep pattern and epileptiform activity burden. These findings argue for the translational value of this approach for treatment of patients with Rett Syndrome.

The Role of the Cerebellum in Repetitive Behavior Across Species: Childhood Stereotypies and Deer Mice.

Recent studies suggest that the cerebellum may have a significant role in repetitive behaviors. In primary complex motor stereotypies, typically developing children have repetitive movements usually involving rhythmic flapping/waving arm/hand movements. Similarly, the deer mouse animal model exhibits inherited repetitive behaviors, with increased frequencies of spontaneous jumping and rearing. In this study, data from both children with motor stereotypies and deer mice were used to investigate the role of the cerebellum in repetitive behaviors. The 3.0-T MRI volumetric imaging of the cerebellum was obtained in 20 children with primary complex motor stereotypies and 20 healthy controls. In deer mice, cerebellar volume (n = 7/group) and cell counts (n = 9/group) were compared between high- and low-activity animals. Levels of cerebellar neurotransmitters were also determined via HPLC (n = 10/group). In children with stereotypies, (a) there were a statistically significant reduction (compared to controls) in the white matter volume of the posterior cerebellar lobule VI-VII that negatively correlated with motor control and (b) an 8% increase in the anterior vermis gray matter that positively correlated with motor Stereotypy Severity Scores (SSS). In deer mice, (a) there was a significant increase in the volume of the anterior vermal granular cell layer that was associated with higher activity and (b) dentate nucleus cell counts were higher in high activity animals. Similar increases in volume were observed in anterior vermis in children with stereotypies and a deer mouse model of repetitive behaviors. These preliminary findings support the need for further investigation of the cerebellum in repetitive behaviors.

Inflammatory profile in a canine model of hypothermic circulatory arrest.

BACKGROUND: Hypothermic circulatory arrest (HCA) is a technique used for complex repair of the aorta, but it can be associated with neurologic morbidity. To better understand the molecular changes that underlie ischemic brain injury, we assessed gene expression and cytokine/chemokine polypeptide concentration in brain tissue and cerebrospinal fluid (CSF) of canines that underwent two hours of HCA. MATERIALS AND METHODS: Adult male canines were cannulated peripherally for cardiopulmonary bypass, cooled to 18°C, and arrested for two hours. Animals were euthanized two, eight, or 24 hours post-HCA (n = 8 per group), and their brains were compared to brains from eight normal canines, using gene expression microarray analysis, cytokine assay, and histopathology. RESULTS: Two to eight hours after HCA, pro-inflammatory cytokine mRNAs increased markedly, and gene expression was enriched within signaling pathways related to neuroinflammation or ischemic injury. Concentrations of pro-inflammatory cytokine polypeptides IL-6, IL-8, IL-1ß, and CCL2 were very low in normal canine brain, whereas anti-inflammatory IL-10 and TGF-ß1 were expressed at moderate levels. Pro-inflammatory cytokine concentrations rose robustly in cerebral tissue and CSF after HCA. IL-6 and IL-8 peaked at eight hours and declined at 24 hours, while IL-1ß and CCL2 remained elevated. Concentrations of anti-inflammatory IL-10 and TGF-ß1 were maintained after HCA, with a significant increase in TGF-ß1 at 24 hours. CONCLUSIONS: These cytokines represent potential diagnostic markers for ischemic neurologic injury that could be used to assess neurologic injury in patients undergoing HCA. The cellular mechanisms underlying this pro-inflammatory, ischemic-induced injury represent potential targets for neuroprotection in the future.

NMDA Receptor Antagonism for Neuroprotection in a Canine Model of Hypothermic Circulatory Arrest.

BACKGROUND: Hypothermic circulatory arrest (HCA) is associated with neurologic morbidity, in part mediated by activation of the N-methyl-D-aspartate glutamate receptor causing excitotoxicity and neuronal apoptosis. Using a canine model, we hypothesized that the N-methyl-D-aspartate receptor antagonist MK801 would provide neuroprotection and that MK801 conjugation to dendrimer nanoparticles would improve efficacy. MATERIALS AND METHODS: Male hound dogs were placed on cardiopulmonary bypass, cooled to 18°C, and underwent 90 min of HCA. Dendrimer conjugates (d-MK801) were prepared by covalently linking dendrimer surface OH groups to MK801. Six experimental groups received either saline (control), medium- (0.15 mg/kg) or high-dose (1.56 mg/kg) MK801, or low- (0.05 mg/kg), medium-, or high-dose d-MK801. At 24, 48, and 72 h after HCA, animals were scored by a standardized neurobehavioral paradigm (higher scores indicate increasing deficits). Cerebrospinal fluid was obtained at baseline, eight, 24, 48, and 72 h after HCA. At 72 h, brains were examined for histopathologic injury in a blinded manner (higher scores indicate more injury). RESULTS: Neurobehavioral deficit scores were reduced by low-dose d-MK801 on postoperative day two (P < 0.05) and by medium-dose d-MK801 on postoperative day 3 (P = 0.05) compared with saline controls, but free drug had no effect. In contrast, high-dose free MK801 significantly improved histopathology scores compared with saline (P < 0.05) and altered biomarkers of injury in cerebrospinal fluid, with a significant reduction in phosphorylated neurofilament-H for high-dose MK801 versus saline (P < 0.05). CONCLUSIONS: Treatment with MK-801 demonstrated significant improvement in neurobehavioral and histopathology scores after HCA, although not consistently across doses and conjugates.

Altered trajectories of neurodevelopment and behavior in mouse models of Rett syndrome.

Rett Syndrome (RTT) is a genetic disorder that is caused by mutations in the x-linked gene coding for methyl-CpG-biding-protein 2 (MECP2) and that mainly affects females. Male and female transgenic mouse models of RTT have been studied extensively, and we have learned a great deal regarding RTT neuropathology and how MeCP2 deficiency may be influencing brain function and maturation. In this manuscript we review what is known concerning structural and coinciding functional and behavioral deficits in RTT and in mouse models of MeCP2 deficiency. We also introduce our own corroborating data regarding behavioral phenotype and morphological alterations in volume of the cortex and striatum and the density of neurons, aberrations in experience-dependent plasticity within the barrel cortex and the impact of MeCP2 loss on glial structure. We conclude that regional structural changes in genetic models of RTT show great similarity to the alterations in brain structure of patients with RTT. These region-specific modifications often coincide with phenotype onset and contribute to larger issues of circuit connectivity, progression, and severity. Although the alterations seen in mouse models of RTT appear to be primarily due to cell-autonomous effects, there are also non-cell autonomous mechanisms including those caused by MeCP2-deficient glia that negatively impact healthy neuronal function. Collectively, this body of work has provided a solid foundation on which to continue to build our understanding of the role of MeCP2 on neuronal and glial structure and function, its greater impact on neural development, and potential new therapeutic avenues.

Rett Syndrome and CDKL5 Deficiency Disorder: From Bench to Clinic.

Rett syndrome (RTT) and CDKL5 deficiency disorder (CDD) are two rare X-linked developmental brain disorders with overlapping but distinct phenotypic features. This review examines the impact of loss of methyl-CpG-binding protein 2 (MeCP2) and cyclin-dependent kinase-like 5 (CDKL5) on clinical phenotype, deficits in synaptic- and circuit-homeostatic mechanisms, seizures, and sleep. In particular, we compare the overlapping and contrasting features between RTT and CDD in clinic and in preclinical studies. Finally, we discuss lessons learned from recent clinical trials while reviewing the findings from pre-clinical studies.

Correction to: Dendrimer-mediated delivery of N-acetyl cysteine to microglia in a mouse model of Rett syndrome.

After publication of the article [1], it has been brought to our attention that an author's name has been formatted incorrectly.

Dendrimer-mediated delivery of N-acetyl cysteine to microglia in a mouse model of Rett syndrome.

BACKGROUND: Rett syndrome (RTT) is a pervasive developmental disorder that is progressive and has no effective cure. Immune dysregulation, oxidative stress, and excess glutamate in the brain mediated by glial dysfunction have been implicated in the pathogenesis and worsening of symptoms of RTT. In this study, we investigated a new nanotherapeutic approach to target glia for attenuation of brain inflammation/injury both in vitro and in vivo using a Mecp2-null mouse model of Rett syndrome. METHODS: To determine whether inflammation and immune dysregulation were potential targets for dendrimer-based therapeutics in RTT, we assessed the immune response of primary glial cells from Mecp2-null and wild-type (WT) mice to LPS. Using dendrimers that intrinsically target activated microglia and astrocytes, we studied N-acetyl cysteine (NAC) and dendrimer-conjugated N-acetyl cysteine (D-NAC) effects on inflammatory cytokines by PCR and multiplex assay in WT vs Mecp2-null glia. Since the cysteine-glutamate antiporter (Xc-) is upregulated in Mecp2-null glia when compared to WT, the role of Xc- in the uptake of NAC and L-cysteine into the cell was compared to that of D-NAC using BV2 cells in vitro. We then assessed the ability of D-NAC given systemically twice weekly to Mecp2-null mice to improve behavioral phenotype and lifespan. RESULTS: We demonstrated that the mixed glia derived from Mecp2-null mice have an exaggerated inflammatory and oxidative stress response to LPS stimulation when compared to WT glia. Expression of Xc- was significantly upregulated in the Mecp2-null glia when compared to WT and was further increased in the presence of LPS stimulation. Unlike NAC, D-NAC bypasses the Xc- for cell uptake, increasing intracellular GSH levels while preventing extracellular glutamate release and excitotoxicity. Systemically administered dendrimers were localized in microglia in Mecp2-null mice, but not in age-matched WT littermates. Treatment with D-NAC significantly improved behavioral outcomes in Mecp2-null mice, but not survival. CONCLUSIONS: These results suggest that delivery of drugs using dendrimer nanodevices offers a potential strategy for targeting glia and modulating oxidative stress and immune responses in RTT.

Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice.

Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541-545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624-1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT.

Vitamin D Status in an Italian Pediatric Cohort: Is There a Role for Tobacco Smoking Exposure?

Introduction: Vitamin D deficiency is a common public health issue worldwide. The purpose of this study was to investigate the vitamin D status and its potential determinants in children residing in Sardinia (40°N), Italy. Methods: A total of 182 children (males: 51.7%; median age: 9 years) were enrolled over a 12-month period. Serum 25(OH)D was measured by an immune-chemiluminescence assay. A questionnaire was used to gather information on other variables, including passive smoke exposure. Results: Mean (SD) serum 25(OH)D was 25.2 (8.3) ng/mL for the whole group. The majority (n=123, 67.6%) of children had vitamin D sufficient values >20 ng/mL, while about 1/3 had vitamin D insufficient/deficient values (≤20 ng/mL (n=59, 32.4%). Among the variables investigated, passive smoke exposure was significantly associated with insufficient 25(OH)D levels (p<0.0001). Conclusion: Our results further prove that hypovitaminosis D is common in the Italian children and documented that passive smoke exposure is a significant risk factor for hypovitaminosis D.

Ketamine Mitigates Neurobehavioral Deficits in a Canine Model of Hypothermic Circulatory Arrest.

Hypothermic circulatory arrest is a protective technique used when complete cessation of circulation is required during cardiac surgery. Prior efforts to decrease neurologic injury with the NMDA receptor antagonist MK801 were limited by unacceptable side effects. We hypothesized that ketamine would provide neuroprotection without dose-limiting side effects. Canines were peripherally cannulated for cardiopulmonary bypass, cooled to 18°C, and underwent 90 minutes of circulatory arrest. Ketamine-treated canines (n = 5; total dose 2.85 mg/kg) were compared to untreated controls (n = 10). A validated neurobehavioral deficit score was obtained at 24, 48, and 72 hours (0 = no deficits/normal exam; higher score represents increasing deficits). Biomarkers of neuronal injury in the cerebrospinal fluid were examined at baseline and at 8, 24, 48, and 72 hours. Brain histopathologic injury was scored at 72 hours (higher score indicates more necrosis and apoptosis). Ketamine-treated canines had significantly improved, lower neurobehavioral deficit scores compared to controls (overall P = 0.003; 24 hours: median 72 vs 112, P = 0.030; 48 hours: 47 vs 90, P = 0.021; 72 hours: 30 vs 89, P = 0.069). Although the histopathologic injury scores of ketamine-treated canines (median 12) were lower than controls (16), there was no statistical difference (P = 0.10). Levels of phosphorylated neurofilament-H and neuron specific enolase, markers of neuronal injury, were significantly lower in ketamine-treated animals (P = 0.010 and = 0.039, respectively). Ketamine significantly reduced neurologic deficits and biomarkers of injury in canines after hypothermic circulatory arrest. Ketamine represents a safe and approved medication that may be useful as a pharmacologic neuroprotectant during cardiac surgery with circulatory arrest.

Bone mass in Rett syndrome: association with clinical parameters and MECP2 mutations.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the MECP2 gene. In 49 female RTT children, aged 1.9-17 y, bone mass was assessed and correlated with clinical parameters and mutations involving the MECP2 gene. We also studied five adult females, aged 20-33 y, and one male child, aged 6 y. Lumbar spine bone mineral content (BMC) and bone mineral density (BMD) were correlated with weight, height, BMI, clinical severity, degree of scoliosis, use of anticonvulsants, and ambulatory status. L1-L4 BMD and BMC showed that 48.9% of them had BMD values >2 SD below age-related norms. BMD values were in the osteoporotic range in the five adult females with RTT. Eleven percent of the children and adults with RTT experienced fractures. Low bone mass was correlated with marginal significance to clinical severity and ambulation but not to scoliosis or anticonvulsant use. Lowest bone mass occurred in patients with T158M or R270X mutations but without statistical significance. Studies in a murine model of RTT confirmed low bone mass as an inherent component of this syndrome. MECP2 mutations and clinical parameters impact bone mass in RTT, but an association with a specific mutation was not demonstrable.

Dendrimer-conjugated glutaminase inhibitor selectively targets microglial glutaminase in a mouse model of Rett syndrome.

Background: Elevated glutamate production and release from glial cells is a common feature of many CNS disorders. Inhibitors of glutaminase (GLS), the enzyme responsible for converting glutamine to glutamate have been developed to target glutamate overproduction. However, many GLS inhibitors have poor aqueous solubility, are unable to cross the blood brain barrier, or demonstrate significant toxicity when given systemically, precluding translation. Enhanced aqueous solubility and systemic therapy targeted to activated glia may address this challenge. Here we examine the impact of microglial-targeted GLS inhibition in a mouse model of Rett syndrome (RTT), a developmental disorder with no viable therapies, manifesting profound central nervous system effects, in which elevated glutamatergic tone, upregulation of microglial GLS, oxidative stress and neuroimmune dysregulation are key features. Methods: To enable this, we conjugated a potent glutaminase inhibitor, N-(5-{2-[2-(5-amino-[1,3,4]thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]thiadiazol-2-yl)-2-phenyl-acetamide (JHU29) to a generation 4 hydroxyl PAMAM dendrimer (D-JHU29). We then examined the effect of D-JHU29 in organotypic slice culture on glutamate release. We also examined GLS activity in microglial and non-microglial cells, and neurobehavioral phenotype after systemic administration of D-JHU29 in a mouse model of RTT. Results: We report successful conjugation of JHU29 to dendrimer resulting in enhanced water solubility compared to free JHU29. D-JHU29 reduced the excessive glutamate release observed in tissue culture slices in a clinically relevant Mecp2-knockout (KO) RTT mouse. Microglia isolated from Mecp2-KO mice demonstrated upregulation of GLS activity that normalized to wild-type levels following systemic treatment with D-JHU29. Neurobehavioral assessments in D-JHU29 treated Mecp2-KO mice revealed selective improvements in mobility. Conclusion: These findings demonstrate that glutaminase inhibitors conjugated to dendrimers are a viable mechanism to selectively inhibit microglial GLS to reduce glutamate production and improve mobility in a mouse model of RTT, with broader implications for selectively targeting this pathway in other neurodegenerative disorders.

Amyloid pathology is associated with progressive monoaminergic neurodegeneration in a transgenic mouse model of Alzheimer's disease.

beta-Amyloid (Abeta) pathology is an essential pathogenic component in Alzheimer's disease (AD). However, the significance of Abeta pathology, including Abeta deposits/oligomers and glial reactions, to neurodegeneration is unclear. In particular, despite the Abeta neurotoxicity indicated by in vitro studies, mouse models with significant Abeta deposition lack robust and progressive loss of forebrain neurons. Such results have fueled the view that Abeta pathology is insufficient for neurodegeneration in vivo. In this study, because monoaminergic (MAergic) neurons show degenerative changes at early stages of AD, we examined whether the APPswe/PS1DeltaE9 mouse model recapitulates progressive MAergic neurodegeneration occurring in AD cases. We show that the progression forebrain Abeta deposition in the APPswe/PS1DeltaE9 model is associated with progressive losses of the forebrain MAergic afferents. Significantly, axonal degeneration is associated with significant atrophy of cell bodies and eventually leads to robust loss (approximately 50%) of subcortical MAergic neurons. Degeneration of these neurons occurs without obvious local Abeta or tau pathology at the subcortical sites and precedes the onset of anxiety-associated behavior in the mice. Our results show that a transgenic mouse model of Abeta pathology develops progressive MAergic neurodegeneration occurring in AD cases.

Are dopamine receptor and transporter changes in Rett syndrome reflected in Mecp2-deficient mice?

We tested the claim that the dopaminergic dysfunction of Rett Syndrome (RTT) also occurs in Mecp2-deficient mice that serve as a model of the syndrome. We used positron emission tomography (PET) to image dopamine D2 receptors (D2R) and transporters (DAT) in women with RTT and in Mecp2-deficient mice, and D1R and D2R density was measured in postmortem human tissue by autoradiography. Results showed 1) significantly reduced D2R density in the striatum of women with RTT compared to control subjects. 2) PET imaging of mouse striatum similarly demonstrated significant reductions in D2R density of 7-10 week-old hemizygous (Mecp2-null) and heterozygous (HET) mice compared to wild type (WT) mice. With age, the density of D2R declined in WT mice but not HET mice. 3) In contrast, postmortem autoradiography revealed no group differences in the density of D1R and D2R in the caudate and putamen of RTT versus normal control subjects. 4) In humans and in the mouse model, PET revealed only marginal group differences in DAT. The results confirm that dopaminergic dysfunction in RTT is also present in Mecp2-deficient mice and that reductions in D2R more likely explain the impaired ambulation and progressive rigidity observed rather than alterations in DAT.

Modeling early cortical serotonergic deficits in autism.

Autism is a developmental brain disorder characterized by deficits in social interaction, language and behavior. Brain imaging studies demonstrate increased cerebral cortical volumes and micro- and macro-scopic neuroanatomic changes in children with this disorder. Alterations in forebrain serotonergic function may underlie the neuroanatomic and behavioral features of autism. Serotonin is involved in neuronal growth and plasticity and these actions are likely mediated via serotonergic and glutamatergic receptors. Few animal models of autism have been described that replicate both etiology and pathophysiology. We report here on a selective serotonin (5-HT) depletion model of this disorder in neonatal mice that mimics neurochemical and structural changes in cortex and, in addition, displays a behavioral phenotype consistent with autism. Newborn male and female mice were depleted of forebrain 5-HT with injections of the serotonergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), into the bilateral medial forebrain bundle (mfb). Behavioral testing of these animals as adults revealed alterations in social, sensory and stereotypic behaviors. Lesioned mice showed significantly increased cortical width. Serotonin immunocytochemistry showed a dramatic long-lasting depletion of 5-HT containing fibers in cerebral cortex until postnatal day (PND) 60. Autoradiographic binding to high affinity 5-HT transporters was significantly but transiently reduced in cerebral cortex of 5,7-DHT-depleted mice. AMPA glutamate receptor binding was decreased at PND 15. We hypothesize that increased cerebral cortical volume and sensorimotor, cognitive and social deficits observed in both 5-HT-depleted animals and in individuals with autism, may be the result of deficiencies in timely axonal pruning to key cerebral cortical areas.

Neonatal serotonin depletion alters behavioral responses to spatial change and novelty.

Multiple brain disorders that show serotonergic imbalances have a developmental onset. Experimental models indicate a role for serotonin as a morphogen in brain development. To selectively study the effects of serotonin depletions on cortical structural development and subsequent behavior, we developed a mouse model in which a serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), is injected into the medial forebrain bundle (mfb) on the day of birth. Littermates with saline injections into the mfb and age matched mice served as controls. This study characterized the extent and duration of serotonergic denervation after the selective neonatal lesion and investigated effects on exploratory behavior, spatial learning and anxiety in mice of both sexes. We report significant decreases in the serotonergic (5-HT) innervation to cortex and hippocampus, but not to subcortical forebrain structures in 5,7-DHT-lesioned mice. The depletion of 5-HT fibers in cortical areas was long lasting in lesioned mice but autoradiographic binding to high affinity 5-HT transporters was only transiently reduced. Male but not female lesioned mice reduced their exploration significantly in response to spatial rearrangement and object novelty, suggesting increased anxiety in response to change but normal spatial cognition. Our data show that developmental disruptions in the serotonergic innervation of cortex and hippocampus are sufficient to induce permanent, sex specific, behavioral alterations. These results may have significant implications for understanding brain disorders presenting with cortical morphogenetic abnormalities and altered serotonin neurotransmission, such as autism, schizophrenia and affective disorders.

Zoledronic acid improves bone histomorphometry in a murine model of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder predominately affecting young females, caused by deficiency of the global transcriptional protein methyl CpG binding protein 2 (MeCP2). Osteoblasts express MeCP2 and girls with RTT experience early onset osteoporosis, decreased bone mass and an increased fracture risk. There is no defined treatment for osteoporosis associated with RTT. The present study evaluated the effects of zoledronic acid (ZA), a third generation nitrogen-containing bisphosphonate with primarily anti-osteoclastic activity, in a mouse model of MeCP2 deficiency. Mice received weekly injections of 20µg/kg ZA for six weeks. Due to the shortened lifespan of hemizygous male (Mecp2-null) mice, treatment began at 3weeks of age for this group and corresponding wildtype (WT) male mice. Treatment for heterozygous (HET) and WT female mice began at 8weeks of age. Micro-computed tomography (micro-CT) and dynamic analyses of bone turnover were performed. ZA treatment led to significant increases in bone volume fraction, number, connectivity density and apparent density of trabecular bone in all genotypes of mice. In contrast, cortical bone generally was unaffected by ZA injections. Parameters of bone turnover, including mineral apposition rate, labeled bone surface and bone formation rate decreased after treatment with ZA. Mecp2-null mice had reduced labeled bone surface and bone formation rate compared to WT male mice. The results indicate that ZA treatment significantly improved trabecular bone mass in a murine model of RTT with little effect on cortical bone.

Generation-6 hydroxyl PAMAM dendrimers improve CNS penetration from intravenous administration in a large animal brain injury model.

Hypothermic circulatory arrest (HCA) provides neuroprotection during cardiac surgery but entails an ischemic period that can lead to excitotoxicity, neuroinflammation, and subsequent neurologic injury. Hydroxyl polyamidoamine (PAMAM) dendrimers target activated microglia and damaged neurons in the injured brain, and deliver therapeutics in small and large animal models. We investigated the effect of dendrimer size on brain uptake and explored the pharmacokinetics in a clinically-relevant canine model of HCA-induced brain injury. Generation 6 (G6, ~6.7nm) dendrimers showed extended blood circulation times and increased accumulation in the injured brain compared to generation 4 dendrimers (G4, ~4.3nm), which were undetectable in the brain by 48h after final administration. High levels of G6 dendrimers were found in cerebrospinal fluid (CSF) of injured animals with a CSF/serum ratio of ~20% at peak, a ratio higher than that of many neurologic pharmacotherapies already in clinical use. Brain penetration (measured by drug CSF/serum level) of G6 dendrimers correlated with the severity of neuroinflammation observed. G6 dendrimers also showed decreased renal clearance rate, slightly increased liver and spleen uptake compared to G4 dendrimers. These results, in a large animal model, may offer insights into the potential clinical translation of dendrimers.