Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease.

Huntington disease is an autosomal dominant neurodegenerative disease with no effective treatment. Minocycline is a tetracycline derivative with proven safety. After ischemia, minocycline inhibits caspase-1 and inducible nitric oxide synthetase upregulation, and reduces infarction. As caspase-1 and nitric oxide seem to play a role in Huntington disease, we evaluated the therapeutic efficacy of minocycline in the R6/2 mouse model of Huntington disease. We report that minocycline delays disease progression, inhibits caspase-1 and caspase-3 mRNA upregulation, and decreases inducible nitric oxide synthetase activity. In addition, effective pharmacotherapy in R6/2 mice requires caspase-1 and caspase-3 inhibition. This is the first demonstration of caspase-1 and caspase-3 transcriptional regulation in a Huntington disease model.

Repeat instability in the 27-39 CAG range of the HD gene in the Venezuelan kindreds: Counseling implications.

The instability of the CAG repeat size of the HD gene when transmitted intergenerationally has critical implications for genetic counseling practices. In particular, CAG repeats between 27 and 35 have been the subject of debate based on small samples. To address this issue, we analyzed allelic instability in the Venezuelan HD kindreds, the largest and most informative families ascertained for HD. We identified 647 transmissions. Our results indicate that repeats in the 27-35 CAG range are highly stable. Out of 69 transmitted alleles in this range, none expand into any penetrant ranges. Contrastingly, 14% of alleles transmitted from the incompletely penetrant range (36-39 CAGs) expand into the completely penetrant range, characterized by alleles with 40 or more CAG repeats. At least 12 of the 534 transmissions from the completely penetrant range contract into the incompletely penetrant range of 36-39 CAG repeats. In these kindreds, none of the individuals with 27-39 CAGs were symptomatic, even though they ranged in age from 11 to 82 years. We expect these findings to be helpful in updating genetic counseling practices.

A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

Complex spatial and temporally defined myelin and axonal degeneration in Huntington disease.

Although much prior work has focused on the basal ganglia and cortical pathology that defines Huntington's disease (HD), recent studies have also begun to characterize cerebral white matter damage (Rosas et al., 2006; Dumas et al., 2012; Poudel et al., 2014). In this study, we investigated differences in the large fascicular bundles of the cerebral white matter of gene-positive HD carriers, including pre-manifest individuals and early symptomatic patients, using recently developed diffusion tractography procedures. We examined eighteen major fiber bundles in 37 patients with early HD (average age 55.2 ±â€¯11.5, 14 male, 23 female), 31 gene-positive, motor negative pre-symptomatic HD (PHD) (average age 48.1 ±â€¯11.5, 13 male, 18 female), and 38 healthy age-matched controls (average age 55.7 ±â€¯8.6, 14 male, 24 female), using the TRActs Constrained by UnderLying Anatomy (TRACULA) procedure available as part of the FreeSurfer image processing software package. We calculated the mean fractional anisotropy (FA) and the mean radial (RD) and axial diffusivities (AD) for each fiber bundle. We also evaluated the relationships between diffusion measures, cognition and regional cortical thinning. We found that early changes in RD of select tracts in PHD subjects were associated with impaired performance on neuropsychological tests, suggesting that early changes in myelin might underlie early cognitive dysfunction. Finally, we found that increases in AD of select tracts were associated with regionally select cortical thinning of areas known to atrophy in HD, including the sensorimotor, supramarginal and fusiform gyrus, suggesting that AD may be reflecting pyramidal cell degeneration in HD. Together, these results suggest that white matter microstructural changes in HD reflect a complex, clinically relevant and dynamic process.

Thalamocortical synapses of pyramidal cells which project from SmI to MsI cortex in the mouse.

Electrolytic lesions were made of the nucleus ventralis posterior pars lateralis thalami and of the nucleus posterior thalami in the male CD/1 mice to label thalamocortical axon terminals in mouse SmI cortex. Pyramidal cells in layers II through V of SmI cortex were labeled by the retrograde transport of horseradish peroxidase injected into ipsilateral MsI. Numerous pyramidal cells, particularly in the more superficial layers of SmI cortex, were filled with reaction product so that even their dendritic spines and local axon collaterals were clearly visible. Six well-filled pyramidal cells with somata in layers III and IV were serial thin-sectioned, and portions of their dendrites in layer IV were examined with the electron microscope to determine the distribution of their thalamocortical and other synapses. It was found that, in general, different dendrites of a single pyramidal cell formed similar proportions of thalamocortical synapses and that the six pyramidal cells, as a group, also formed similar proportions of thalamocortical synapses with their dendrites in layer IV. In contrast, when the thalamocortical connectivity of the six cells was considered as a function of their depths within the cortex, a clear trend was seen for the proportion of thalamocortical synapses to increase with increasing proximity of the cell body to layer IV. A hypothesis based on the timing of developmental events is proposed to account for this observation.

A quantitative study of the thalamocortical and other synapses in layer IV of pyramidal cells projecting from mouse SmI cortex to the caudate-putamen nucleus.

The thalamocortical and other synapses of the apical dendrites of corticostriatal projection neurons in mouse primary somatosensory cortex (SmI) were examined by combining anterograde degeneration with the retrograde transport of horseradish peroxidase (HRP). Electrolytic lesions were made in the ventrobasal thalamus, followed 3 days later by injections of 40% HRP into the ipsilateral caudate-putamen nucleus. The next day, the mice were perfused and the SmI cortex ipsilateral to the lesion and injection sites was chopped at 125-micrometer and reacted for HRP using a CoCl2-DAB method. HRP-labeled corticostriatal cells in SmI cortex were medium-sized pyramidal cells, having somata located in the superficial portion of layer V and apical dendrites extending into layer I. Seven corticostriatal cells were serially thin sectioned and the layer IV portions of their apical dendrites were reconstructed. Each apical dendrite formed only one or two thalamocortical synapses (0.3 to 0.9% of their synapses in layer IV) indicating that corticostriatal neurons may be minimally responsive to direct synaptic input from the specific thalamic nuclei. Each apical dendrite formed about 12.6 asymmetrical synapses for every symmetrical synapse, suggesting that the relative numbers of excitatory and inhibitory synapses impinging on apical dendrites belonging to an individual class of neurons may be specified.

Thalamocortical synapses involving identified neurons in mouse primary somatosensory cortex: a terminal degeneration and golgi/EM study.

Synapses involving thalamocortical afferents and hitherto unexamined neuron types of the posteromedial barrel subfield (PMBSF) of the mouse have been identified using a combined degeneration and Golgi/EM technique (Peters et al., '77). Degeneration of thalamocortical axon terminals was produced with electrolytic lesions of the nucleus ventralis posterior, pars lateralis thalami, and the nucleus posterior thalami. Four days after receiving lesions, the animals were perfused, and blocks of cortex containing the PMBSF were processed by the Golgi method. The blocks were tissue-chopped at 125 microns and examined with the light microscope. Sections containing neurons of interest were gold-toned and deimpregnated in preparation for electron microscopy (Fairén et al., '77). Portions of selected neurons contained in layers III-IV were serially thin-sectioned and examined with an electron microscope to determine if they were involved in synapses with degenerating thalamocortical axon terminals. Results showed thalamocortical synapses on the apical dendrites of five different sized pyramidal cells whose somata occurred in layers V and VI, and on dendrites of on spiny bitufted neuron and one non-spiny multipolar neuron with somata in layer V. A non-spiny bitufted neuron of layer IV which was not impregnated also received thalamocortical synapses. Although every neuron examined formed at least one thalamocortical synapse, some formed very few, whereas others formed many. Of the pyramidal cells, small layer V and VI pyramidal cells and a large deep layer V pyramidal cell were involved in small numbers of thalamocortical synapses, while a medium superficial layer V pyramidal cell and a large layer VI pyramidal cell each formed many. The spiny bitufted neuron formed a small number of thalamocortical synapses. The findings suggest that, whereas any type of neuron with a dendrite in layer IV likely receives some synaptic input from the thalamus, individual neurons were involved in very different quantities of thalamocortical synapses.

Thalamocortical and other synapses involving nonspiny multipolar cells of mouse SmI cortex.

Golgi-impregnated and -deimpregnated neurons having somata in layer IV of mouse posteromedial barrel subfield (PMBSF) cortex were identified with the light microscope and then extensive portions of them were examined with the electron microscope. Dendrites of nine nonspiny multipolar cells and eight of their cell bodies were reconstructed from serial thin sections to determine the numbers and types of symmetrical, asymmetrical, and thalamocortical synapses they formed. Results of this analysis show that cells of the same general morphological class may form widely different patterns of synaptic connections: some nonspiny multipolar cells had dendrites that formed a high proportion of their synapses with thalamocortical axon terminals, whereas dendrites belonging to other cells formed only very small proportions of thalamocortical synapses. A similar diversity characterized the synaptic connections of cell bodies: some formed more symmetrical than asymmetrical synapses, others the reverse. Some formed high proportions of thalamocortical synapses, others much less. Comparisons of thalamocortical synaptic input to cell bodies and dendrites showed that one cell formed about the same proportions of thalamocortical synapses with its cell body as with its dendrites. For two other cells the proportions of thalamocortical synapses formed with their somata was about double that formed with their dendrites. The remaining five cell bodies examined formed far higher proportions of thalamocortical synapses than did their dendrites. That different nonspiny multipolar cells form such contrasting synaptic patterns suggests that included within this morphological classification are cells which are likely to have very different functional roles.

Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat.

The m2 muscarinic acetylcholine receptor gene is expressed at high levels in basal forebrain, but the paucity of information about localization of the encoded receptor protein has limited the understanding of cellular and subcellular mechanisms involved in cholinergic actions in this region. The present study sought to determine the cellular localization of m2 protein, its relationship to cholinergic neurons, and its pre- and postsynaptic distribution in the rat medial septum-diagonal band complex using immunocytochemistry with polyclonal rabbit antibodies and a newly developed rat monoclonal antibody specific to the m2 receptor. Light microscopic colocalization studies demonstrated that m2 was present in a subset of choline acetyltransferase immunoreactive neurons, in choline acetyltransferase-negative neurons, and in more neuropil elements than was choline acetyltransferase. Intraventricular injections of 192 IgG-saporin, an immunotoxin directed to the low-affinity nerve growth factor receptor, resulted in depletion of choline acetyltransferase-immunoreactive neurons in the medial septum-diagonal band complex, whereas m2 immunoreactivity in neurons and in the neuropil was unchanged. By electron microscopy, m2 receptor in medial septum-diagonal band complex was localized to the plasmalemma of a small population of small to medium-sized neurons, and it was also found in dendrites, axons, and axon terminals in the neuropil. Neurons expressing m2 immunoreactivity received synaptic contacts from unlabelled axon terminals. A small distinct subpopulation of large neurons, unlabelled by m2 immunoreactivity, received synaptic contacts from m2-immunoreactive terminals. Thus, m2 receptor is situated to mediate the local effects of acetylcholine on basal forebrain cholinergic and noncholinergic neurons and, also, at both pre- and postsynaptic sites.

Subcellular localization and molecular topology of the dopamine transporter in the striatum and substantia nigra.

Plasma membrane transporters remove neurotransmitters from the extracellular space and have been postulated to terminate synaptic activity. Their specific roles in synaptic and nonsynaptic neurotransmission at a cellular level, however, remain unclear. We have determined the subcellular location of the dopamine transporter (DAT) by immunoperoxidase and immunogold electron microscopy, using monoclonal antibodies to both the N-terminus and the second extracellular loop. The two DAT epitopes were found on opposite faces of cellular and intracellular membranes, providing confirmation of the predicted molecular topology of DAT. In the striatum, DAT was localized in the plasma membrane of axons and terminals. Double immunocytochemistry demonstrated DAT colocalization with two other markers of nigrostriatal terminals, tyrosine hydroxylase and D2 dopamine receptors. The latter was thus demonstrated to be an autoreceptor. Labeled striatal terminals formed symmetrical synapses with spines, dendrites, and perikarya. DAT was not identified within any synaptic active zones, however, even using serial section analysis. These results suggest that striatal dopamine reuptake may occur outside of synaptic specializations once dopamine diffuses from the synaptic cleft. In the substantia nigra, DAT appears to be specifically transported into dendrites, where it can be found in smooth endoplasmic reticulum, plasma membrane, and pre- and postsynaptic active zones. These localizations suggest that DAT modulates the intracellular and extracellular dopamine levels of nigral dendrites. Within the perikarya of pars compacta neurons, DAT was localized primarily to rough and smooth endoplasmic reticulum, Golgi complex, and multivesicular bodies, identifying probable sites of synthesis, modification, transport, and degradation.

Muscarinic m1 and m2 receptor proteins in local circuit and projection neurons of the primate striatum: anatomical evidence for cholinergic modulation of glutamatergic prefronto-striatal pathways.

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double-labeling immunocytochemistry revealed m1 receptors in calbindin-D28k--positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age-dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse-forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1-positive spines with lesion-induced degenerating prefronto-striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2-positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons.

Treatment trial of oxiracetam in Alzheimer's disease.

Twenty-four carefully assessed patients with probable Alzheimer's disease were enrolled in a double-blind, placebo-controlled treatment study of oxiracetam, a nootropic agent reported to improve memory performance in patients with dementia. A broad battery of neuropsychological tests failed to reveal any improvement in the treated group or in any treated patient when individual test scores were analyzed. These findings indicate that oxiracetam is ineffective in reducing cognitive impairment due to Alzheimer's disease.

Immunocytochemical localization of D1 and D2 dopamine receptors in the basal ganglia of the rat: light and electron microscopy.

The modulatory actions of dopamine on the flow of cortical information through the basal ganglia are mediated mainly through two subtypes of receptors, the D1 and D2 receptors. In order to examine the precise cellular and subcellular location of these receptors, immunocytochemistry using subtype specific antibodies was performed on sections of rat basal ganglia at both the light and electron microscopic levels. Both peroxidase and pre-embedding immunogold methods were utilized. Immunoreactivity for both D1 and D2 receptors was most abundant in the neostriatum where it was mainly contained within spiny dendrites and in perikarya. Although some of the immunoreactive perikarya had characteristics of interneurons, most were identified as medium-sized spiny neurons. Immunoreactivity for D1 receptor but not D2 receptor was associated with the axons of the striatonigral pathway and axons and terminals in the substantia nigra pars reticulata and the entopeduncular nucleus. In contrast, D2 immunoreactivity but not D1 immunoreactivity was present in the dopaminergic neurons in the substantia nigra pars compacta and ventral pars reticulata. In the globus pallidus, little immunoreactivity for either D1 or D2 receptor was detected. At the subcellular level, D1 and D2 receptor immunoreactivity was found to be mainly associated with the internal surface of cell membranes. In dendrites and spines immunoreactivity was seen in contact with the membranes postsynaptic to terminals forming symmetrical synapses and less commonly, asymmetrical synapses. The morphological features and membrane specializations of the terminals forming symmetrical synapses are similar to those of dopaminergic terminals previously identified by immunocytochemistry for tyrosine hydroxylase. In addition to immunoreactivity associated with synapses, a high proportion of the immunoreactivity was also on membranes at non-synaptic sites. It is concluded that dopamine receptor immunoreactivity is mainly associated with spiny output neurons of the neostriatum and that there is a selective association of D1 receptors with the so-called direct pathway of information flow through the basal ganglia, i.e. the striatoentopeduncular and striatonigral pathways. Although there is an association of receptor immunoreactivity with afferent synaptic inputs a high proportion is located at extrasynaptic sites.

Tissue heterogeneity of the FMR1 mutation in a high-functioning male with fragile X syndrome.

Few studies have been conducted comparing the FMR1 mutation in multiple tissues of individuals affected with fragile X syndrome. We report a postmortem study of the FMR1 mutation in multiple tissues from a high-functioning male with fragile X syndrome. This man was not mentally retarded and had only a few manifestations of the disorder such as learning disabilities and mild attention problems. Southern blot analysis of leukocytes demonstrated an unmethylated mutation with a wide span of sizes extending from the premutation to full mutation range. A similar pattern was seen in most regions of the brain. In contrast, a methylated full mutation of a single size was seen in the parietal lobe and in most non-brain tissues studied. Therefore, there were striking differences in both FMR1 mutation size and methylation status between tissues. Lack of mental retardation in this individual may have been due to sufficient expression of FMR1 protein (FMRP) in most areas of the brain. Immunocytochemistry showed FMRP expression in regions of the brain with the unmethylated mutation (superior temporal cortex, frontal cortex, and hippocampus) and no expression in the region with the methylated full mutation (parietal). Neuroanatomical studies showed no dendritic spine pathology in any regions of the brain analyzed.

Synaptic sequences in mouse SmI cortex involving pyramidal cells labeled by retrograde filling with horseradish peroxidase.

A method combining anterograde degeneration and the retrograde transport of horseradish peroxidase (HRP) has been used to study synapses involving pyramidal cells in mouse SmI cortex. Neurons labeled with HRP are so well filled that even their finer processes, such as dendritic spines and axon collaterals, are clearly visible with both the light and electron microscopes. Results indicate that pyramidal cells projecting from SmI to ipsilateral MsI cortex receive thalamocortical input and have local axon collaterals which form asymmetrical synapses with spines and with varicose, non-spiny dendrites.

The use of lectin transport in the mouse central nervous system as an anterograde axonal marker for electron microscopy.

Lesion-induced axonal degeneration and autoradiography-electron microscopy have been the only reliable anterograde axonal markers available for electron microscopic examination of neuronal circuitry. However, these methods have their limitations. Recently, Phaseolus vulgaris-leucoagglutinin (PHA-L) has been used as an anterograde axonal marker for light microscopy. This report describes the use of this lectin as an anterograde marker for electron microscopy. PHA-L was injected into mouse SmI cortex or ventrobasal thalamus. Using standard immunohistochemical techniques, the transported lectin was tagged with antibody, which was then visualized with avidin-biotin-horseradish peroxidase binding. Light microscopy demonstrated anterograde transport to predicted cortical regions. With the electron microscope, labeled axon terminals were seen forming asymmetric synapses with spines, dendrites and cell bodies.

Diverse pre- and post-synaptic expression of m1-m4 muscarinic receptor proteins in neurons and afferents in the rat neostriatum.

We have utilized subtype specific antibodies to determine the cellular and subcellular distributions of the muscarinic acetylcholine receptor subtypes that are highly expressed in the rat striatum (m1-m4). Each receptor is expressed in distinct populations of striatal neurons in the relative proportions predicted by their mRNAs. They concentrate at post-synaptic sites and each of the four subtypes are also transported to pre-synaptic sites. m2 appears to be the only presynaptic autoreceptor in the striatum, but it is also localized in non-cholinergic terminals. These distinct pre- and post-synaptic localizations suggest that muscarinic receptor subtype diversity evolved to enable increasingly complex responses to acetylcholine release.

CAG repeat expansion in Huntington disease determines age at onset in a fully dominant fashion.

OBJECTIVE: Age at onset of diagnostic motor manifestations in Huntington disease (HD) is strongly correlated with an expanded CAG trinucleotide repeat. The length of the normal CAG repeat allele has been reported also to influence age at onset, in interaction with the expanded allele. Due to profound implications for disease mechanism and modification, we tested whether the normal allele, interaction between the expanded and normal alleles, or presence of a second expanded allele affects age at onset of HD motor signs. METHODS: We modeled natural log-transformed age at onset as a function of CAG repeat lengths of expanded and normal alleles and their interaction by linear regression. RESULTS: An apparently significant effect of interaction on age at motor onset among 4,068 subjects was dependent on a single outlier data point. A rigorous statistical analysis with a well-behaved dataset that conformed to the fundamental assumptions of linear regression (e.g., constant variance and normally distributed error) revealed significance only for the expanded CAG repeat, with no effect of the normal CAG repeat. Ten subjects with 2 expanded alleles showed an age at motor onset consistent with the length of the larger expanded allele. CONCLUSIONS: Normal allele CAG length, interaction between expanded and normal alleles, and presence of a second expanded allele do not influence age at onset of motor manifestations, indicating that the rate of HD pathogenesis leading to motor diagnosis is determined by a completely dominant action of the longest expanded allele and as yet unidentified genetic or environmental factors.