Increased cortical synaptic activation of TrkB and downstream signaling markers in a mouse model of Down Syndrome.

Down Syndrome (DS), trisomy 21, is characterized by synaptic abnormalities and cognitive deficits throughout the lifespan and with development of Alzheimer's disease (AD) neuropathology and progressive cognitive decline in adults. Synaptic abnormalities are also present in the Ts65Dn mouse model of DS, but which synapses are affected and the mechanisms underlying synaptic dysfunction are unknown. Here we show marked increases in the levels and activation status of TrkB and associated signaling proteins in cortical synapses in Ts65Dn mice. Proteomic analysis at the single synapse level of resolution using array tomography (AT) uncovered increased colocalization of activated TrkB with signaling endosome related proteins, and demonstrated increased TrkB signaling. The extent of increases in TrkB signaling differed in each of the cortical layers examined and with respect to the type of synapse, with the most marked increases seen in inhibitory synapses. These findings are evidence of markedly abnormal TrkB-mediated signaling in synapses. They raise the possibility that dysregulated TrkB signaling contributes to synaptic dysfunction and cognitive deficits in DS.

Discoveries in Down syndrome: moving basic science to clinical care.

This review describes recent discoveries in neurobiology of Down syndrome (DS) achieved with use of mouse genetic models and provides an overview of experimental approaches aimed at development of pharmacological restoration of cognitive function in people with this developmental disorder. Changes in structure and function of synaptic connections within the hippocampal formation of DS model mice, as well as alterations in innervations of the hippocampus by noradrenergic and cholinergic neuromodulatory systems, provided important clues for potential pharmacological treatments of cognitive disabilities in DS. Possible molecular and cellular mechanisms underlying this genetic disorder have been addressed. We discuss novel mechanisms engaging misprocessing of amyloid precursor protein (App) and other proteins, through their affect on axonal transport and endosomal dysfunction, to "Alzheimer-type" neurodegenerative processes that affect cognition later in life. In conclusion, a number of therapeutic strategies have been defined that may restore cognitive function in mouse models of DS. In the juvenile and young animals, these strategists focus on restoration of synaptic plasticity, rate of adult neurogenesis, and functions of the neuromodulatory subcortical systems. Later in life, the major focus is on recuperation of misprocessed App and related proteins. It is hoped that the identification of an increasing number of potential targets for pharmacotherapy of cognitive deficits in DS will add to the momentum for creating and completing clinical trials.

Intrathecal administration of AAV vectors for the treatment of lysosomal storage in the brains of MPS I mice.

Mucopolysaccharidosis type I (MPS I) is caused by an inherited deficiency of alpha-L-iduronidase (IDUA). The result is a progressive, lysosomal storage disease with central nervous system (CNS) as well as systemic involvement. To target gene therapy to the CNS, recombinant adeno-associated virus (AAV) vectors carrying IDUA sequence were administered to MPS I mice via injection into cerebrospinal fluid. In contrast to intravenous administration, this intrathecal administration was effective in generating widespread IDUA activity in the brain, with the cerebellum and olfactory bulbs having highest activities. In general, IDUA levels correlated with vector dose, although this correlation was obscured in cerebellum by particularly high variability. High doses of vector (4 x 10(10) particles) provided IDUA levels approaching or exceeding normal levels in the brain. Histopathology indicated that the number of cells with storage vacuoles was reduced extensively or was eliminated entirely. Elimination of storage material in Purkinje cells was particularly dramatic. A lower vector dose (2 x 10(9) particles) reduced both the number of storage cells and the extent of storage per cell, but the effect was not complete. Some perivascular cells with storage persisted, and this cell type appeared to be more resistant to treatment than neurons or glial cells. We conclude that intrathecal administration of AAV-IDUA delivers vector to brain cells, and that this route of administration is both minimally invasive and effective.

Penetration, diffusion, and uptake of recombinant human alpha-L-iduronidase after intraventricular injection into the rat brain.

Central nervous system disease can have devastating consequences in the severe or Hurler form of mucopolysaccharisosis I (MPS I). Intravenously administered recombinant human alpha-L-iduronidase (rhIDU) is not expected to reach and treat the brain disease due to the blood-brain barrier. To determine whether administration of rhIDU into the cerebrospinal fluid could successfully treat the brain, we studied intraventricular administration of rhIDU in rats. RhIDU was stereotactically administered directly to the lateral ventricle of the intact rat brain and the brain tissues assessed by enzyme assays, immunofluorescence and confocal microscopy 30 min, 24 h, or 7 days later. Quantitation of activity revealed that rhIDU was widely distributed throughout the brain following injection into the lateral ventricle, with activities increased by a factor of 3.3 higher than control in most samples 30 min-24 h after injection and highest levels on the side of injection. The enzyme crossed the ependymal lining of the ventricle and entered neurons into lysosomal-like vesicles. The enzyme was able to diffuse through brain tissue as demonstrated by a decreasing signal gradient from 0.2 to 4.8 mm from the ventricle surface. The largest amount of rhIDU, as detected by immunostaining, was observed 24 h after injection and decreased approximately 50% during the first 7 days. Although the immunostaining decreased with time, specific vesicular staining was still detectable 28 days after injection. The data suggest that rhIDU given into the ventricle can diffuse, penetrate at least several millimeters of brain tissue and be taken up into neurons and glial cells.

Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I.

Enzyme replacement therapy (ERT) has been developed for several lysosomal storage disorders, including mucopolysaccharidosis I (MPS I), and is effective at reducing lysosomal storage in many tissues and in ameliorating clinical disease. However, intravenous ERT does not adequately treat storage disease in the central nervous system (CNS), presumably due to effects of the blood-brain barrier on enzyme distribution. To circumvent this barrier, we studied whether intrathecal (IT) recombinant human alpha-L-iduronidase (rhIDU) could penetrate and treat the brain and meninges. An initial dose-response study showed that doses of 0.46-4.14 mg of IT rhIDU successfully penetrated the brain of normal dogs and reached tissue levels 5.6 to 18.9-fold normal overall and 2.7 to 5.9-fold normal in deep brain sections lacking CSF contact. To assess the efficacy and safety in treating lysosomal storage disease, four weekly doses of approximately 1 mg of IT rhIDU were administered to MPS I-affected dogs resulting in a mean 23- and 300-fold normal levels of iduronidase in total brain and meninges, respectively. Quantitative glycosaminoglycan (GAG) analysis showed that the IT treatment reduced mean total brain GAG to normal levels and achieved a 57% reduction in meningeal GAG levels accompanied by histologic improvement in lysosomal storage in all cell types. The dogs did develop a dose-dependent immune response against the recombinant human protein and a meningeal lymphocytic/plasmacytic infiltrate. The IT route of ERT administration may be an effective way to treat the CNS disease in MPS I and could be applicable to other lysosomal storage disorders.

[Structural changes in dendritic spines of the pyramidal neurons of layer III of the rat sensory-motor cortex during remote postischemic period]. / Strukturnye izmeneniia dendritnykh shipikov piramidnykh neironov sloia III sensomotornoi kory bol'shogo mozga krys v otdalennom postishemicheskom periode.

The combination of Golgi method with the lipid phosphorilate mark Dil (1,1'-dioctadecyl-3,3,3 cents, 3 cents-tetramethyl-indocarbocyanine perchlorate) and confocal laser scanning microscope for demonstration of the structure of dendritic tree and dendritic spines enables exact determination of spatial organization of small dendrites and their spines at significant distances in norm and especially in postischemic period. The regularities of dendritic spine reorganization in layer III pyramidal neurons of the cerebral sensory-motor cortex during 9 months after short-term (10 min) total brain ischemia were established.

Mind and brain in Rett disorder.

Development and retention of speech is reported in 265 people with Rett syndrome: 30% (80) never gained real words, 55% (145) gained real words and lost them, 15%(40) retained some words and 6% of the total (16/265) continued to use phrases appropriately. Morphological studies of the cytoarchitecture of the speech areas in 14 cases indicate the existence of interhemispheric differences which form part of the infrastructure for speech processing. Ten adults with Rett syndrome and with meaningful speech are compared to age matched adults without speech. The profile of mind and strategies for coping with its problems are described by a family. Although the range in severity is wide the mental profile is remarkably consistent across the severity range with regard to both positive and negative aspects.

Brain-directed autoantibodies levels in the serum of Rett syndrome patients.

Increased titer of brain-directed autoantibodies (AAB) may represent a risk for brain development in children with Rett syndrome (RTT). The aims of this work were to study the levels of brain-directed AAB, mainly nerve growth factor (NGF) and S-100 protein AAB, to analyze morphological features of brain labeling by AAB produced in RTT patients, and to correlate with clinical manifestation. The increased titer of anti-NGF AAB, but not of anti-S100 AAB has been determined in the blood of RTT patients. The blood from five RTT girls was investigated repeatedly (two to four times) within 0.5-3 years. In these RTT patients the level of anti-NGF AAB was stable, not depending on the stage of illness, so individual stability of anti-NGF AAB levels have been detected. However, the negative correlation between the level of these AAB and severity of disease has been found: girls with the milder course of illness (with relative preservation of speech and locomotor functions, later disease onset, and later development of regressive symptoms) were characterized by the higher levels of AAB. The study also revealed immunohistochemical labeling of neuronal population with serum from RTT patients. Serum AAB from RTT cases labeled the cytoplasm and apical dendrites of pyramidal neurons in the neocortex and hippocampus, neurons in basal ganglia and brain stem, but not in the cerebellum of rats. Our results show the presence of brain-directed AAB in blood serum of RTT patients, which suggests an autoimmune component in pathogenesis of RTT.

Failed retrograde transport of NGF in a mouse model of Down's syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion.

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer's disease and Down's syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down's syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling.

Effect of transplantation of embryonic nervous tissue on reorganization of interneuronal relationships after mechanical damage to sensorimotor cortex.

The effect of implanted embryonic nervous tissue on restoration of axonal connections in the cerebral cortex after mechanical injury was studied on albino rats using fluorescent lipophilic probe DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate) and confocal laser scanning microscope. Implantation of embryonic tissue to damaged area promotes the growth of axons through the transplant to adjacent tissue. The damaged area is impenetrable for axons growing without implantation.

Guidelines for reporting clinical features in cases with MECP2 mutations.

An international group recommends that papers relating phenotypes to genotypes involving mutations in the X chromosome gene MECP2 should provide a minimum data set reporting the range of disturbances frequently encountered in Rett Syndrome. A simple scoring system is suggested which will facilitate comparison among the various clinical profiles. Features are described which should prompt screening for MECP2 mutations.

Neuronal and fibre organization in neocortical grafts placed in post-ischaemic adult rat brain: a three-dimensional confocal microscopy study.

The dendritic morphology in neocortical grafts was studied with three-dimensional confocal laser scanning microscopy after microinjection of Lucifer Yellow into individual cells. The grafts had been implanted into infarct cavities in the neocortex of hypertensive rats 46 weeks earlier. The carbocyanine dye method was used to identify afferent (host to transplant) and efferent (transplant to host) connections. Pyramidal, nonpyramidal and glial cells were present in the transplants. Some dendrites had an almost normal appearance, but abnormalities (atypical orientation of apical, basal or oblique apical dendrites) were observed. Some bi-apical pyramidal neurons and pyramidal neurons with obliquely oriented apical dendrites were also observed. Carbocyanine dye-labelled fibres of different diameter formed a dense network in the transplant, enabling the border between transplant and host tissue to be clearly recognized. No labelled fibres were observed to enter the host brain. Fibres with "boutons en passant" and no preferential orientation were noted. It is proposed that Lucifer Yellow microinjection may be a useful method in studies aimed at improving graft morphology. Failure to demonstrate host to transplant connections with the carbocyanine dye method was contrary to earlier studies in which tracers were applied in vivo. A combined use of in-vivo and post-mortem tracer techniques is needed to establish the reason for the discrepancy.

Early loss of dendritic spines in murine scrapie revealed by confocal analysis.

Confocal analysis of dye-filled neurons has revealed a significant early loss of dendritic spines in a murine scrapie model in which neuron loss occurs in the hippocampus. An 18% loss of spines was found at 109 days, > 50 days before neuron loss occurs, and by 126 days a 51% spine loss was found. Spine loss is concurrent with synapse loss, axon terminal degeneration and a decrease in long term potentiation in this model. Preceding these changes is the deposition of disease specific PrP at 70 days, which may initiate the damage to dendritic spines and the subsequent degeneration of synapses. We suggest that these changes underlie the development of clinical disease in the transmissible spongiform encephalopathies.

Dendritic and synaptic alterations of hippocampal pyramidal neurones in scrapie-infected mice.

Neurone damage and eventual loss may underlie the clinical signs of disease in the transmissible spongiform encephalopathies (TSEs). Although neurone death appears to be through apoptosis, the trigger for this form of cell death in the TSEs is not known. Using two different murine scrapie models, hippocampal pyramidal cells were studied through microinjection of fluorescent dye, and synaptic integrity, using p38-immunoreactivity (p38-IR), both visualized using confocal laser scanning microscopy. Intradendritic distensions and dendritic spine loss were found to co-localize to areas of vacuolar and prion protein pathology in the hippocampus of mice infected with ME7 or 87 V scrapie. A significant reduction in p38-IR was found concomitantly in the hippocampus in ME7 scrapie mice. These results indicate that both pre- and post-synaptic sites are altered by scrapie infection; this would disrupt neuronal circuitry and may initiate apoptotic cell death, giving rise to the neurological disturbances manifested in clinical TSE cases.

Early destruction of the extracellular matrix around parvalbumin-immunoreactive interneurons in Creutzfeldt-Jakob disease.

GABA-interneurons immunoreactive (IR) for the calcium-binding protein parvalbumin are lost during the early stages of Creutzfeldt-Jakob disease (CJD) and diminution in their number may partially account for the neurological disturbances manifested in patients suffering from this condition. The disease is characterized by a transformation of the prion protein, PrP(c)-a host-coded sialoglycoprotein-to its protease-resistant and putatively pathological form, PrP(CJD). And since this conversion is likely to take place at the cell surface, we were curious to know whether the "perineuronal net"-a characteristic accumulation of extracellular matrix in intimate contact with the surface of parvalbumin-IR neurons-is implicated in the early disappearance of the mantled cells. Using various lectins and antibodies as markers for the perineuronal net in brains of 21 CJD victims, we observed that this meshwork of extracellular matrix molecules is lost before the embraced parvalbumin-IR neurons themselves disappear. Hence, an interaction of PrP(c) and/or PrP(CJD) with components of the extracellular matrix around this subpopulation of nerve cells precipitates a sequence of as yet unknown events which culminates in the replacement of perineuronal nets by deposits of insoluble PrP(CJD). This change in the environment of the GABA-interneurons IR for parvalbumin may ultimately provoke their death.

Morphological study of the entorhinal cortex, hippocampal formation, and basal ganglia in Rett syndrome patients.

Entorhinal cortex (EC), fascia dentata (FD), hippocampus (HP), and basal ganglia (BG) were studied in Rett syndrome (RS) cases and compared with control brains and an autism case. Kluver-Barrera and Golgi methods were used. In RS most of the areas of EC, HP, and FD showed severe cell hypochromia. In the EC all cells of layer II and most in layer III were in a state of total chromatolysis or were "ghost" cells, but the cells of layers V and VI were preserved and moderately hyperchromic. In FD and HP the majority of the granular cells and cells of CA3 and CA4 fields were severely hypochromic, whereas in the CA1 field most cells were normal or slightly hypercaryochromic. In BG mostly mild or moderate aberration from normal cell structure was observed: in striatum, mild hypercaryochromia of small neurons and more expressive hyperchromia of large neurons were found; and in pallidum, mild or moderate hypercaryochromia to severe hyperchromia in pallidum internum was found. Degeneration of thick myelinated fibers was evident in pallidum. Large striatal and pallidal neurons showed signs of constructive changes in Golgi slices. These data allow the determination of the cause of the main symptoms of RS. The motor disorders, including specific stereotyped movements, could be related to the enhanced activity of BG cells due to their deafferentation from the side of the neocortex and to supposed hyperactivity of the EC-striatal pathway; the mental retardation and epileptic seizures could be due to FD-HP involvement.

The cellular distribution of GAP-43 immunoreactivity in human neocortical areas using immunofluorescence and confocal microscopy: post-ischemic influence.

The distribution of growth associated protein-43 (GAP-43) immunoreactive (IR) neurons were studied in human neocortical areas, using immunofluorescence and confocal microscopy. The GAP-43-IR cells were generally localised close to blood vessels, and contained fewer lipofuscin granules than GAP-43 negative cells. Quantification of the relative number of GAP-43-IR cells in control cases showed that the highest number of GAP-43-IR cells were present in layers III and V in the motor and visual cortices, fewer in the temporal cortex, and the lowest number in the frontal cortex. After general ischemia, GAP-43-IR cells were significantly reduced at various survival times, with the counts being lowest in the 1 week surviving case, and higher, but still subcontrol, in the 1 year post-ischemic case.

Quantitative analysis of synaptophysin immunoreactivity in human neocortex after cardiac arrest: confocal laser scanning microscopy study.

Transient global ischaemia caused by cardiac arrest results in lesions that involve all brain structures. The aim of this study was to investigate the condition of synapses in patients surviving, but remaining in a persistent vegetative state, following resuscitation after cardiac arrest. We performed a quantitative analysis of the distribution and density of elements containing a synaptic vesicle protein--synaptophysin (p38)--in human neocortex in cases which survived for 1 week, 2 months, and 1 year after the cardiac arrest. Neurologically healthy cases that died following an accident served as control. Dual-channel confocal laser scanning microscopy (CLSM) was used to image p38-immunoreactivity (IR) and lipofuscin autofluorescence. In control cases no statistically significant differences were found for p38-IR between layers II-III and V-VII. However, the temporal cortex had a higher density of p38-immunoreactive structures than the motor cortex. In postischaemic cases a reduction in the density of p38-IR elements was apparent, mainly in the frontal and motor cortices and less pronounced in the temporal cortex. The least decrease compared with controls was observed in the visual cortex. In the 1 week survival case, a maximal decrease in p38-IR (35% below control) was found. In this case, the number of p38-IR elements per visual field was decreased, and big aggregates of p38-IR structures were observed. In general, the amounts of p38-IR structures were higher in all of the control cases compared with the postischaemic cases.