The distribution of desmin and titin in normal and dystrophic human muscle

Summary We have used monoclonal antibodies to desmin and titin, and a combination of immunofluoescence and immunogold labelling to study the disposition of these two proteins in normal human muscle fibres and in fibres at various stages of degeneration in dystrophic muscle. The normal pattern of desmin labelling, in particular the subsarcolemmal labelling, became disrupted at an early stage of fibre breakdown. There was a change from a transverse to a longitudinal orientation of the labelled intermediate filaments as the myofibrils sheared relative to one another. Thus, while it is probable that the desmin filaments are able to play a role in the mechanical integration of the myofibrils in healthy muscle, our results suggest that they cannot withstand the excessive forces generated by the hypercontraction and stretching of dystrophic muscle. However, small accumulations of desmin persisted between the damaged myofibrils until necrosis reached an advanced stage. In general, the degradation of titin appeared to occur before the degradation of desmin, and at the ultrastructural level, labelling with antibodies to epitopes from parts of the titin molecule close to the A-I-band junction was lost before labelling with an antibody to an epitope in the A-band. This suggests that different regions of the titin molecule break down at different stages in the breakdown of the fibre. We propose that lysis of titin in the I-band may underlie slippage, an abnormality often seen in dystrophic muscle, in which the A-band slips to one pole of the sarcomere such that it abuts onto the Z-line. Breakdown of the A-band section of titin may facilitate the disassembly of the A-filaments.Supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland     

Fluorescent Arc/Arg3.1 indicator mice: A versatile tool to study brain activity changes in vitro and in vivo

The brain-specific immediate early gene Arc/Arg3.1 is induced in response to a variety of stimuli, including sensory and behavior-linked neural activity. Here we report the generation of transgenic mice, termed TgArc/Arg3.1-d4EGFP, expressing a 4-h half-life form of enhanced green fluorescent protein (d4EGFP) under the control of the Arc/Arg3.1 promoter. We show that d4EGFP-mediated fluorescence faithfully reports Arc/Arg3.1 induction in response to physiological, pathological and pharmacological stimuli, and that this fluorescence permits electrical recording from activated neurons in the live mouse. Moreover, the fluorescent Arc/Arg3.1 indicator revealed activity changes in circumscribed brain areas in distinct modes of stress and in a mouse model of Alzheimer's disease. These findings identify the TgArc/Arg3.1-d4EGFP mouse as a versatile tool to monitor Arc/Arg3.1 induction in neural circuits, both in vitro and in vivo.     

Prion protein genes and prion diseases: studies in transgenic mice

In the past decade, manipulation of PrP genes by transgenesis in mice has provided important insights into mechanisms of prion propagation and the molecular basis of prion strains and species barriers. Despite these advances, our understanding of these unique pathogens is far from complete. This review focuses on PrP gene knockout and gene replacement studies, PrP structure and function, and transgenic models of human and animal prion diseases. Transgenic approaches will doubtless remain the cornerstone of investigations into the prion diseases in the coming years, which will include mechanistic studies of prion pathogenesis and prion transmission barriers. Transgenic models will also be important tools for the evaluation of potential therapeutic agents for prion diseases.     

Desmin and actin associated with cytoplasmic bodies in skeletal muscle fibers: immunocytochemical and fine structural studies,with a note on unusual 18- to 20-nm filaments

Summary In a fine structural and immunocytochemical study, the latter performed on semithin sections of epoxy resin embedded skeletal muscle fibers, three types of cytoplasmic bodies were identified in a case of cytoplasmic body myopathy: (1) The first type, the classical type, showed a central core and a light halo with radiating actin filaments at the periphery. (2) The second type, the spheroid body was characterized by irregularly arranged granular masses associated with intermediate filaments. Desmin immunoreactivity occurred in the central and peripheral parts, where filaments of intermediate size were visualized by electron microscopy. Desmin immunoreactivity was noted also at the Z-bands of striated annulets, within areas of disordered myofibrils, such as sarcoplasmic masses, and in atrophic muscle fibers. (3) The third type of the cytoplasmic body was composed mainly of large masses of uneven granularity and electron density. The center of this type reacted to anti-actin antibody suggesting that the 5- to 6-nm filaments, which ultrastructurally proved to be a major component, were of the actin type. By contrast, neither intermediate filaments nor actin microfilaments were found by electron microscopy in cytoplasmic bodies in a second case where no immunoreaction to desmin or actin occurred. Anti-vimentin antibody stained only the cytoplasm of endomysial cells, but not the inclusion bodies. Some other, unusual inclusions with 18- to 20-nm tubulo-filamentous structures have to be distinguished from the various types of filaments in cytoplasmic bodies. It is concluded, that pleomorphism and heterogeneity of cytoplasmic bodies have to be taken into consideration when classifying cytoplasmic body myopathies.Recipient of a grant from the Deutsche Forschungsgemeinschaft.     

LacZ andOMP are co-expressed during ontogeny and regeneration in olfactory receptor neurons of omp promoter-lacZ transgenic mice

The ontogeny and cellular specificity of expression of β-galactosidase activity and olfactory marker protein (OMP) are compared in olfactory tissue of the H-OMP-lacZ-3 line of transgenic mice. In this line the expression oflacZ is driven by a 0.3 kb fragment of the rat OMP promoter. During fetal development,lacZ expression is detectable in olfactory receptor neurons (ORNs) shortly after the initial appearance of endogenous OMP. The β-galactosidase marker was observed only in mature olfactory receptor neurons where it co-localized with endogenous OMP. It was absent from immature neurons that express the growth associated phosphoprotein B50/GAP43. Lesion of the peripheral olfactory pathway by intranasal irrigation with Triton X-100 eliminated expression of both OMP andIacZ in the olfactory neuroepithelium. Subsequent regeneration of the full complement of olfactory receptor neurons was associated with co-expression of both OMP and β-galactosidase activity. Neither OMP nor β-galactosidase activity was induced in any other cell type of the regenerating olfactory mucosa. Thus, as little as 0.3 kb of the OMP promoter has the ability to targetlacZ expression to olfactory receptor neurons in a temporally and spatially defined manner. We discuss the potential utility of this transgenic line for future studies of the olfactory system.     

Reducing body myopathy and desmin storage in skeletal muscle: morphological and biochemical findings

We describe clinical, morphological and biochemical findings of a patient with reducing body myopathy (RBM). This 15-year-old patient was affected by severe limb-girdle progressive myopathy with asymmetric distribution. Muscle biopsy showed many fibers with cytoplasmic polymorphic masses, which stained dark purple with modified Gomori's trichrome, associated with proliferation of cytoplasmic bodies. Cytoplasmic polymorphic masses showed marked reducing activity with menadione-nitro blue tetrazolium reaction. Ultrastructurally, there was great amount of highly electron-dense tubular-filamentous structures of 16–17 nm in diameter. Immunohistochemistry showed that many fibers were positive for desmin. Sodium dodecyl sulfate-electrophoresis disclosed an increase in two bands of approximately 53 and 70 kDa, and Western blot demonstrated that the 53-kDa band was desmin. It was not possible to characterize the 70-kDa protein further.Supported by Telethon and institutional funds of the Consiglio Nazionale delle Ricerche     

The lacZ gene under the control of the 7 kb of human dystrophin muscle specific promoter is expressed in cardiac muscle but not in adult skeletal muscle in transgenic mice

In previous transgenic studies, we reported a 0.9 kb fragment from a mouse dystrophin muscle promoter that contains the regulatory elements required for expression of dystrophin only in the right heart. In this study, to further characterize the regulation of muscle type of promoter, we analyzed promoter activity and tissue specificity using a total 14 kb fragment around the human dystrophin muscular-specific exon 1 in vitro and in vivo. In vitro analysis showed that the lacZ construct of the 7 kb promoter and 7 kb intron 1 was expressed 2.5 times as strong as the lacZ construct of only the 7 kb promoter in C2/4 myotubes. In vivo analysis revealed expression of both constructs in the whole heart, skeletal muscle and vascular smooth muscle in embryos. However, in adults, the expression in skeletal muscle disappeared. We conclude that the 7 kb upstream region and the 7 kb intronic region included responsible elements for the expression in the heart, but not in skeletal muscle in vivo. It is possible that a strong enhancer element for skeletal muscle exists in some other region.     

Effects of subacute administration of methamphetamine and nicotine on locomotor activity in transgenic mice expressing the human tyrosine hydroxylase gene

Summary We produced transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene. To investigate differences in the dopaminergic (DAergic) neuronal activity between the Tg and nTg mice, we examined changes in the locomotor activity induced by methamphetamine (MAP) and nicotine (NIC), which enhances DA release and induces TH enzyme activation, respectively. Surprisingly, however, the intensity of MAP (2.5 mg/kg, once a day for 14 days)-induced hyperlocomotion in the nTg mice was greater than that in the Tg mice, and, furthermore, the Tg mice were less sensitive to subacute administration of NIC (0.5 mg/kg, once a day for 14 days) than the nTg mice. These results suggest that DAergic neuronal function is suppressed in Tg mice to compensate for the overexpression of TH.     

Proteoglycan synthesis by clonal skeletal muscle cells during in vitro myogenesis: Differences detected in the types and patterns from primary cultures

Proteoglycan synthesis by two clonal murine skeletal muscle cell lines, G8-1 and C2, was examined. Cultures of skeletal muscle cells at both the myoblast and myotube stages were radiolabeled using [³⁵S]sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. The cell layer proteoglycans eluted from Sepharose CL-20 as a single peak with a K_av of 0.66 and contained glycosaminoglycan chains with an average molecular weight of 20,000. The glycosaminoglycan chains were composed of nearly equal mixtures of chondroitin sulfate and heparan sulfate with the exception that C2 myoblast cultures contained larger amounts of heparan sulfate. Of interest, this line differentiates more rapidly in our laboratory than G8-1. The medium proteoglycans also eluted from Sepharose CL-2B as a single peak with a K_av of 0.66 but contained glycosaminoglycan chains with an average molecular weight of 32,000. Based upon enzymatic and chemical analysis, the medium glycosaminoglycan chains were composed of a mixture of chondroitin sulfate (71–80%) and heparin sulfate (19–22%). Following chondroitinase ABC digestion, the predominant disaccharide released from all glycosaminoglycan fractions was chondroitin-4-sulfate. When the extracted cell layer proteoglycans were chromatographed on Sepharose CL-28 in the absence of detergent, a small but consistent proportion (14–18%) eluted in the void volume, suggesting the association of at least a portion of this proteoglycan with cellular lipid. These differences distinguish proteoglycan metabolism in fusing clonal lines from primary muscle cell cultures suggesting their utility in evaluating the contribution of these macromolecules in myogenesis.     

Behavioral and neurochemical effects of wild-type and mutated human alpha-synuclein in transgenic mice

Human alpha-synuclein (halpha-SYN) is implicated in the Parkinson's disease phenotype (PDP) based on a variety of studies in man, animal models, and in vitro studies. The normal function of halpha-SYN and the mechanism by which it contributes to the PDP remains unclear. We created transgenic mice expressing either wild-type (hwalpha-SYN) or a doubly mutated (hm2alpha-SYN) form of halpha-SYN under control of the 9-kb rat tyrosine hydroxylase promoter. These mice expressed halpha-SYN in cell bodies, axons, and terminals of the nigrostriatal system. The expression of halpha-SYN in nigrostriatal terminals produced effects in both constructs resulting in increased density of the dopamine transporter and enhanced toxicity to the neurotoxin MPTP. Expression of hm2alpha-SYN reduced locomotor responses to repeated doses of amphetamine and blocked the development of sensitization. Adult hwalpha-SYN-5 transgenic mice had unremarkable dopaminergic axons and terminals, normal age-related measures on two motor coordination screens, and normal age-related measures of dopamine (DA) and its metabolites. Adult hm2alpha-SYN-39 transgenic mice had abnormal axons and terminals, age-related impairments in motor coordination, and age-related reductions in DA and its metabolites. Expression of hm2alpha-SYN adversely affects the integrity of dopaminergic terminals and leads to age-related declines in motor coordination and dopaminergic markers.     

5′-flanking sequences of the human HPRT gene direct neuronal expression in the brain of transgenic mice

Total deficiency of hypoxanthine phosphoribosyl-transferase (HPRT) in humans causes the neurological disorder Lesch-Nyhan syndrome. The HPRT gene is expressed at basal levels in all tissues but at higher levels in the brain, the relevance and mechanism of which is unknown. To determine if cis-acting DNA elements play a role in the tissue-differential pattern of expression, we generated transgenic mice carrying different sequences of the human HPRT (hHPRT) promoter fused to the bacterial lacZ gene. We show that a 1.6 kb fragment of the hHPRT promoter contains essential information to direct β-galactosidase expression preferentially to the basal ganglia, cerebral cortex, hippocampus, and several other areas of the forebrain. At least two elements within the 1.6 kb fragment appear to be required for neuronal expression. A 182 bp element (hHPRT-NE) represents one of those sequences and is involved not only in confering neuronal specificity but also in repressing transgene expression in non-neuronal tissues. These studies provide molecular insight into the mechanism of increased HPRT expression in the brain. © 1994 Wiley-Liss, Inc.     

Progressive abnormalities in skeletal muscle and neuromuscular junctions of transgenic mice expressing the Huntington's disease mutation

Huntington's disease (HD) is a neurodegenerative disorder with complex symptoms dominated by progressive motor dysfunction. Skeletal muscle atrophy is common in HD patients. Because the HD mutation is expressed in skeletal muscle as well as brain, we wondered whether the muscle changes arise from primary pathology. We used R6/2 transgenic mice for our studies. Unlike denervation atrophy, skeletal muscle atrophy in R6/2 mice occurs uniformly. Paradoxically however, skeletal muscles show age-dependent denervation-like abnormalities, including supersensitivity to acetylcholine, decreased sensitivity to mu-conotoxin, and anode-break action potentials. Morphological abnormalities of neuromuscular junctions are also present, particularly in older R6/2 mice. Severely affected R6/2 mice show a progressive increase in the number of motor endplates that fail to respond to nerve stimulation. Surprisingly, there was no constitutive sprouting of motor neurons in R6/2 muscles, even in severely atrophic muscles that showed other denervation-like characteristics. In fact, there was an age-dependent loss of regenerative capacity of motor neurons in R6/2 mice. Because muscle fibers appear to be released from the activity-dependent cues that regulate membrane properties and muscle size, and motor axons and nerve terminals become impaired in their capacity to release neurotransmitter and to respond to stimuli that normally evoke sprouting and adaptive reinnervation, we speculate that in these mice there is a progressive dissociation of trophic signalling between motor neurons and skeletal muscle. However, irrespective of the cause, the abnormalities at neuromuscular junctions we report here are likely to contribute to the pathological phenotype in R6/2 mice, particularly in late stages of the disease.     

Both upstream and intragenic sequences of the human neurofilament light gene direct expression oflacZ in neurons of transgenic mouse embryos

Initial expression of the neurofilament light gene coincides with the appearance of postmitotic neurons. To investigate the molecular mechanisms involved in neuron-specific gene expression during embryogenesis, we generated transgenic mice carrying various regions of the human neurofilament light gene (hNF-L) fused to thelacZ reporter gene. We found that 2.3 or 0.3 kb of the hNF-L promoter region directs expression oflacZ in neurons of transgenic embryos. Addition of 1.8 kb hNF-L intragenic sequences (IS) enlarges the neuronal pattern of transgene expression. The 2.3-kb hNF-L promotelacZ-IS construct contains all regulatory elements essential for both spatial and temporal expression of the hNF-L gene during embryogenesis and in the adult. The use of a heterologous promoter demonstrated that the 1.8-kb hNF-L intragenic sequences are sufficient to direct the expression oflacZ in a NF-L-specific manner both temporally and spatially during development and in the adult. We conclude that these hNF-L intragenic sequences containcis-acting DNA regulatory elements that specify neuronal expression. Taken together, these results show that the neurofilament light gene contains separate upstream and intragenic elements, each of which directslacZ expression in embryonic neurons.     

Ultrastructural study of mitochondria in the spinal cord of transgenic mice with a G93A mutant SOD1 gene

The purpose of this study was to examine mitochondrial changes in the spinal cord of transgenic mice of a relatively low transgenic copy number (gene copy 10) expressing a G93A mutant human Cu/Zn superoxide dismutase (SOD1) that were generated in our own laboratories by electron and immunoelectron microscopy from presymptomatic to symptomatic stages. Age-matched non-transgenic mice served as controls at each stage. Ultrastructurally, at the early presymptomatic stage, many mitochondria in large myelinated axons exhibited swelling with an increased number of cristae, and bore small vacuoles in the matrix, cristae or both, in the anterior root exit zone, anterior root, and in the neuropils of the ventral portion of the anterior horn. At the late presymptomatic stage, vacuoles of various sizes (including large ones) were observed in the same regions as in the previous stage. The intermembrane space of mitochondria was also vacuolated. In mitochondria with advanced vacuolation, the vacuolar space was filled with a granular or amorphous substance. At the symptomatic stage, mitochondrial vacuolation seen in the late presymptomatic stage persisted, although to a lesser extent. These vacuolated mitochondria were predominantly seen in the axons, but not in the somata of normal-looking neurons or dendrites at any stage, which differs from that described in other reports. Non-transgenic littermates occasionally exhibited vacuolar changes in the axons of anterior horns. However, they were smaller both in size and number than those in transgenic mice. By immunoelectron microscopy using an immunogold labeling method, at the presymptomatic and symptomatic stages both SOD1 and ubiquitin determinants were localized in vacuolated mitochondria, particularly in the granular or amorphous substance of large vacuoles, but were not detected in most normal-appearing mitochondria. The SOD1-immunoreactive mitochondria were exclusively observed in the axons, and not in proximal dendrites or somata. These findings suggest that the toxicity of mutant SOD1 directly affects mitochondria in the axons and increases with the disease progression. Thus, the mutant SOD1 toxicity might disrupt axonal transport of substrates needed for neuronal viability, leading to motor neuron degeneration. The localization of both ubiquitin and SOD1 in vacuolated mitochondria indicates that protein degradation by ubiquitin-proteasomal system may be also disrupted by several pathomechanisms, such as decreased processing of ubiquitinated proteins due to impairment of mitochondrial function or of proteasomal function, both of which are caused by mutant SOD1. Moreover, giant mitochondrial vacuoles occupying almost the entire axonal caliber could be another contributing factor in motor neuron degeneration, in that they could physically block axonal transport.     

Ultrastructural study of aggregates in the spinal cord of transgenic mice with a G93A mutant SOD1 gene

The ultrastructural features of SOD1-positive aggregates were determined to clarify whether these aggregates are associated with the pathogenesis of SOD1 mutant mice. We examined the spinal cord of transgenic mice expressing a G93A mutant human SOD1 gene with fewer copies (gene copy 10). At the early presymptomatic stage (age 24 weeks), SOD1- and ubiquitin-positive granular, linear, or round deposits were found occasionally in the neuropil of the anterior horns. Ultrastructurally, small filamentous aggregates were observed occasionally in the neuronal processes including the axons in the anterior horns. At the late presymptomatic stage (28 weeks), SOD1- and ubiquitin-positive deposits and Lewy body-like inclusions (LIs) were frequently demonstrated in the neuronal processes including cord-like swollen axons and in some remaining anterior horn neurons. Ultrastructurally, larger filamentous aggregates were frequent, predominating in the neuronal processes of the anterior horns including the proximal axons, but were rare in the somata and dendrites. The aggregates usually consisted of interwoven intermediate filaments (about 10–15 nm in diameter) and frequently contained electron-dense cores in the center resembling LIs. Occasionally the aggregates consisted mainly of granular, amorphous, or vesicular substance, showing fewer filamentous structures. At the symptomatic stages (32 and 35 weeks), LIs were frequently demonstrated within the neuronal processes in the anterior horns, particularly in the cord-like swollen axons. Many more prominent SOD1- and ubiquitin-positive deposits were observed over the whole white matter columns and in the gray matter of the anterior and posterior horns than at the previous stage. Ultrastructurally, aggregates frequently contained electron-dense cores, and were frequently observed in cord-like swollen axons consisting of accumulated neurofilaments. A high level of human SOD1-and ubiquitin-immunogold labeling was present in small to large aggregates even at the presymptomatic stages, and the aggregates increased in size and frequency with time. Compactly packed filaments and electron-dense cores of aggregates showed SOD1-and ubiquitin-immunogold labeling more prominently than in loosely packed filaments. These findings suggest that the accumulation of SOD1-positive aggregates in the neuronal processes, predominantly in the axons, constitutes an important determinant of neurotoxicity and the pathogenesis of this animal model, probably causing impairment of axonal transport by the sequestration of mutant SOD1 protein within aggregates, or in part by physically blocking the axonal transport.     

Altered ionic conductances in axons of transgenic mouse expressing the human neurofilament heavy gene: A mouse model of amyotrophic lateral sclerosis

Neurofilaments (NFs; made by copolymerization of three intermediate filament proteins NF-L, NF-M, and NF-H, for light, medium, and heavy) constitute the most abundant cytoskeletal structure in large myelinated axons. The presence of aberrant NF accumulation has been associated with neurodegenerative diseases (such as ALS). The possible causal role of NF in neurodegeneration has been supported by studies on recently available transgenic mice in which expression of human NF-H (hNF-H +/+) leads to overt neuropathy. We have examined electrophysiological properties of myelinated axons in hNF-H +/+ mice using intraaxonal microelectrode recording from isolated sciatic and tibial nerves. Transgenic mice showed several deficits in physiological properties of low threshold myelinated fibers: conduction velocity and resting membrane potential were significantly decreased (20 +/- 1.6 vs 40 +/- 2 m/s; -71.3 +/- 0.9 vs -75.5 +/- 0.5 m/s; mean +/- SE; n = 25; 22 degrees C). While the amplitude of action potentials was of comparable size (82 +/- 5 vs 86 +/- 3 mV) duration of action potential (at half-amplitude, AP/2) in hNF-H +/+ was significantly prolonged (0.82 +/- 0.02 vs 0.65 +/- 0.02 ms). Voltage-current properties of axonal membrane indicate a significant decrease in inward and outward rectification. Occasionally, impaled axons of hNF-H +/+ showed membrane oscillations and repetitive activity (reminiscent of fasciculations) never observed in normal animals. These results are compatible with an imbalance between ion conductances in axons from transgenic animals (an increase in Na(+) and a decrease in K(+) conductances), in agreement with recent suggestion based on clinical studies on ALS patients (H. Bostock et al., 1995, Brain 118, 217-225). One may hypothesize that these changes could contribute to neurodegenerative processes (i.e., via an increase in [Na(+)](i)), as well as clinical symptoms (fasciculations) observed in patients with degenerative motor neuron diseases.     

Cell-specific expression and subcellular localization of neurophysin-CAT-fusion proteins expressed from oxytocin and vasopressin gene promoter-driven constructs in transgenic mice

The cell-specific expression of both the oxytocin (OT) and vasopressin (VP) genes in magnocellular neurons (MCNs) of the hypothalamus has been proposed to be under the control of cis-elements in an intergenic region downstream of the VP gene. We examined this hypothesis using transgenic mice containing mouse genomic DNA-derived constructs linked to chloramphenicol acetyltransferase (CAT) reporters. VP gene expression was studied using constructs containing 3.8 kbp of the 5' flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to a CAT reporter. The two VP-transgene constructs differed by the lengths of their VP gene 3' flanking regions (2.1 versus 3.6 kbp). A similar construct for the oxytocin CAT transgene was used which contained the full-length (3.6 kbp) downstream intergenic region between the mouse genes. All three transgenic constructs produced cell-specific expression of the CAT-reporter in the magnocellular neurons as determined by CAT-immunoreactivity. Oxytocin transgene expression was restricted to OT cells in two founders, and the two VP transgenes to VP cells in five founders. Electron microscopic immunocytochemistry showed that the CAT fusion proteins produced from the OT- and VP-transgenes were efficiently trafficked through the regulated secretory pathways in their respective magnocellular neurons, packaged into large dense core vesicles, and transported to nerve terminals in the posterior pituitary.     

Chronology of behavioral symptoms and neuropathological sequela in R6/2 Huntington's disease transgenic mice

Genetic murine models play an important role in the study of human neurological disorders by providing accurate and experimentally accessible systems to study pathogenesis and to test potential therapeutic treatments. One of the most widely employed models of Huntington's disease (HD) is the R6/2 transgenic mouse. To characterize this model further, we have performed behavioral and neuropathological analyses that provide a foundation for the use of R6/2 mice in preclinical therapeutic trials. Behavioral analyses of the R6/2 mouse reveal age-related impairments in dystonic movements, motor performance, grip strength, and body weight that progressively worsen until death. Significant neuropathological sequela, identified as increasing marked reductions in brain weight, are present from 30 days, whereas decreased brain volume is present from 60 days and decreased neostriatal volume and striatal neuron area, with a concomitant reduction in striatal neuron number, are present at 90 days of age. Huntingtin-positive aggregates are present at postnatal day 1 and increase in number and size with age. Our findings suggest that the R6/2 HD model exhibits a progressive HD-like behavioral and neuropathological phenotype that more closely corresponds to human HD than previously believed, providing further assurance that the R6/2 mouse is an appropriate model for testing potential therapies for HD.     

An in vitro and in vivo study of early deficits in associative learning in transgenic mice that over-express a mutant form of human APP associated with Alzheimer's disease

Transgenic mice over-expressing a mutated form of the human amyloid precursor protein (APP, 695 isoform) bearing a mutation associated with Alzheimer's disease (V642I, so-called London mutation, hereafter APPLd2) and wild-type controls were studied at age periods (3 and 10 months) prior to the overt development of neuritic amyloid plaques. Both 3- and 10-month-old APPLd2 mice had reflex eyelid responses like those of controls, but only younger mice were able to acquire a classical conditioning of eyelid responses in a trace paradigm. In vitro studies on hippocampal slices showed that 10-month-old APPLd2 mice also presented deficits in paired-pulse facilitation and long-term potentiation, but presented a normal synaptic activation of CA1 pyramidal cells by the stimulation of Schaffer collaterals. It is proposed that definite functional changes may appear well in advance of noticeable structural alterations in this animal model of Alzheimer's disease, and that specific learning tasks could have a relevant diagnostic value.     

Hyperphosphorylated tau aggregates in the cortex and hippocampus of transgenic mice with mutant human FTDP-17 Tau and lacking the PARK2 gene

Mutations in the PARK2 gene encoding parkin cause autosomal recessive juvenile parkinsonism, but have also been found in patients diagnosed with certain tauopathies. Conversely, mutations in the MAPT gene encoding tau are present in some types of parkinsonism. In order to investigate the possible relationship between these two proteins, we generated a double mutant mouse that is deficient in PARK2 and that over-expresses the hTauVLW transgene, a mutant form of the tau protein present in FTDP-17. Independent deletion of PARK2 or over-expression of the hTauVLW transgene produces mild phenotypic alterations, while a substantial increase in parkin expression is observed in hTauVLW transgenic mice. However, double mutant mice present memory and exploratory deficits, and accumulation of PHF-1 and AT8 hyperphosphorylated tau epitopes in neurons. These phenomena are coupled with reactive astrocytosis, DNA fragmentation, and variable cerebral atrophy. Here, we show that cortical and hippocampal neurons of double mutant mice develop argyrophilic Gallyas-Braak aggregates of phosphorylated tau from 3 months of age. Their number decreases in old animals. Moreover, numerous phosphorylated tau aggregates were identified with the conformation-dependent Alz-50 antibody and the S-Thioflavin staining. Ventral motor nuclei of the spinal cord also present Alz-50, AT8, and PHF1 hyperphosphorylated tau aggregates when parkin is deleted in mice over-expressing the hTauVLW transgene, begining at early ages. Thus, the combination of PARK2 gene deletion with hTauVLW over-expression in mice produces abnormal hyperphosphorylated tau aggregates, similar to those observed in the brain of patients diagnosed with certain tauopathies. In the light of these changes, these mice may help to understand the molecular processes responsible for these diseases, and they may aid the development of new therapeutic strategies to treat neurodegenerative diseases related to tau and parkin proteins.