Targeting cellular prion protein reverses early cognitive deficits and neurophysiological dysfunction in prion-infected mice

Currently, no treatment can prevent the cognitive and motor decline associated with widespread neurodegeneration in prion disease. However, we previously showed that targeting endogenous neuronal prion protein (PrP(C)) (the precursor of its disease-associated isoform, PrP(Sc)) in mice with early prion infection reversed spongiform change and prevented clinical symptoms and neuronal loss. We now show that cognitive and behavioral deficits and impaired neurophysiological function accompany early hippocampal spongiform pathology. Remarkably, these behavioral and synaptic impairments recover when neuronal PrP(C) is depleted, in parallel with reversal of spongiosis. Thus, early functional impairments precede neuronal loss in prion disease and can be rescued. Further, they occur before extensive PrP(Sc) deposits accumulate and recover rapidly after PrP(C) depletion, supporting the concept that they are caused by a transient neurotoxic species, distinct from aggregated PrP(Sc). These data suggest that early intervention in human prion disease may lead to recovery of cognitive and behavioral symptoms.     

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Methamphetamine-induced hyperactivity and behavioral sensitization in PACAP deficient mice

Mice lacking the PACAP gene (PACAP(-/-)) display psychomotor abnormalities such as novelty-induced hyperactivity and jumping behavior, and they show different responses to amphetamine, a typical psychostimulant. The present study examined the possible role of endogenous PACAP in methamphetamine (METH)-induced hyperactivity and behavioral sensitization. The locomotor activity of hyperactive PACAP(-/-) mice was measured using the infrared photocell beam detection system, Acti-Track, after a habituation period. Single administration of METH (1 and 2mg/kg) caused a robust increase in locomotor activity of mice, but this effect did not differ between wild-type and PACAP(-/-) mice. Repeated administration of METH (1mg/kg) for 7 days enhanced METH-induced hyperactivity, and this sensitization was observed even when withdrawn for 7 days. There was no difference in the degree of development and expression of METH-induced behavioral sensitization between wild-type and PACAP(-/-) mice. In addition, there was no difference in METH-induced increases in extracellular serotonin and dopamine levels in the prefrontal cortex of the normal and sensitized mice between the two groups. These results suggest that endogenous PACAP is not involved in the locomotor stimulant activity of acute METH and repeated METH-induced behavioral and neurochemical sensitization.     

Structural abnormalities in the primary somatosensory cortex and a normal behavioral profile in Contactin-5 deficient mice

Contactin-5 (Cntn5) is an immunoglobulin cell adhesion molecule that is exclusively expressed in the central nervous system. In view of its association with neurodevelopmental disorders, particularly autism spectrum disorder (ASD), this study focused on Cntn5-positive areas in the forebrain and aimed to explore the morphological and behavioral phenotypes of the Cntn5 null mutant (Cntn5^?/?) mouse in relation to these areas and ASD symptomatology. A newly generated antibody enabled us to elaborately describe the spatial expression pattern of Cntn5 in P7 wild type (Cntn5^+/+) mice. The Cntn5 expression pattern included strong expression in the cerebral cortex, hippocampus and mammillary bodies in addition to described previously brain nuclei of the auditory pathway and the dorsal thalamus. Thinning of the primary somatosensory (S1) cortex was found in Cntn5^?/? mice and ascribed to a misplacement of Cntn5-ablated cells. This phenotype was accompanied by a reduction in the barrel/septa ratio of the S1 barrel field. The structure and morphology of the hippocampus was intact in Cntn5^?/? mice. A set of behavioral experiments including social, exploratory and repetitive behaviors showed that these were unaffected in Cntn5^?/? mice. Taken together, these data demonstrate a selective role of Cntn5 in development of the cerebral cortex without overt behavioral phenotypes.     

Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP(Sc)), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP(C) exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP(C) is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp(+/+)) mice with PrP-ablated mice (TgPrnp(o/o)) to generate Tg1D4(Prnp(o/o)) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp(+/+)) and Tg1D4(Prnp(o/o)) mice, suggesting that cyPrP and PrP(C) function independently in the disease state. Additionally, Tg1D4(Prnp(o/o)) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP(Sc). We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP(C) and the generation of typical prion disease.     

Passive immunization reduces behavioral and neuropathological deficits in an alpha-synuclein transgenic model of Lewy body disease

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.     

Prion protein scrapie and the normal cellular prion protein

Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP^C) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed.     

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.     

Cellular prion protein null mice display normal AMPA receptor mediated long term depression

Cellular prion protein (PrP^C) appears to be involved in numerous physiological processes. We have recently shown a novel modulation of NMDA receptors by PrP^C that results in neuroprotection via silencing of NMDA receptors containing NR2D subunits, whereas no effects on AMPA receptor function could be observed (Khosravani, et al. J Cell Biol 2008; 181:551). Here we show that PrP-null mice show a normal response to long-term depression stimuli requiring AMPA receptor activity, thus further supporting our previous findings of a selective action on NMDA receptors among ionotropic glutamate receptors.Key words: AMPA receptor, NMDA receptor, PrP, long term depression, LTDThe role of prion proteins in the pathophysiology of transmissible spongiform encephalopathies is well documented.¹ Although there is a growing body of literature associating normal cellular prion protein (PrP^C) with functions such as regulation of cell proliferation and survival, cell signalling and immune function,¹ the spectrum of physiological roles attributable to PrP^C remains to be determined. This may in part be due to the fact that mice lacking PrP^C display a relatively mild phenotype, unless subjected to insults such as ischemia or seizures, where increased mortality of the PrP-null mice has been reported.^2–5 Interestingly, the increased neuronal damage in PrP-null mice following excitotoxicity is alleviated upon treatment with the N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) inhibitor MK-801,⁶ suggesting a neuroprotective role of PrP^C via an action on NMDARs, but the mechanism was unclear.We recently described a novel action of PrP^C on NMDAR function.⁷ By examining the neurophysiological properties of hippocampal neurons isolated from PrP-null mice, we were able to show that PrP-null mouse neurons exhibit enhanced and drastically prolonged NMDA evoked currents due to a functional upregulation of NMDARs containing NR2D subunits. Biochemical analyses suggested that NR2D subunits, but not NR2B subunits, co-immunoprecipitated with PrP^C, indicating that PrP^C and NMDARs form physical signaling complexes in neurons. The increased NMDAR function could be phenocopied by RNA interference and were rescued upon overexpression of exogenous PrP^C. The enhanced NMDAR activity resulted in increased neuronal excitability, as well as enhanced glutamatergic-based excitotoxicity in both in vitro and in vivo experiments were neurons were transiently exposed to the selective agonist NMDA. Hence, native PrP^C appears to mediate an important neuroprotective role by virtue of its ability to silence NR2D containing NMDARs. In contrast, minor effects on amplitude and rise and decay-time kinetics were observed for both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and GABA_A (miniature and evoked) currents in synaptically mature hippocampal cultures.AMPA and NMDA receptors have been linked to synaptic plasticity, in particular long term potentiation (LTP) and long term depression (LTD). LTP is believed to mostly reflect a strengthening of the postsynaptic response, caused by a brief period of hyperexcitability that releases significant amounts of glutamate such as during a brief tetanic stimulation. This is thought to result in the opening of AMPA receptors, which depolarize the postsynaptic membrane. This in turn increases the activity of postsynaptic NMDARs, because magnesium ions that normally inhibit NMDAR activity, are dislodged by the postsynaptic depolarization, thus allowing NMDARs to become active. This functional activation of NMDARs results in the influx of calcium ions, which in turn initiate a signaling cascade that results in the membrane insertion of additional AMPA receptors, thus strengthening the synapse. This process is thought to involve NMDA receptor isoforms that predominantly contain the NR2A subunits.⁸A synaptic depotentiation process also can take place that results in the opposite effects of LTP; this process is known as long-term depression or LTD, which also has an NMDAR-dependent component. In contrast to brief tetanic stimulation, as is used in the induction of LTP, establishing LTD requires low frequency stimulation (e.g., 1 Hz for 15 min). Successful and repeatable induction of LTD depends on the parameters used for the conditioning stimulus and more importantly on the age of the animal. In juvenile animals (P12–P21) a low frequency protocol is effective and the mechanism of LTD is believed to depend on the activity of NMDA receptors containing NR2B subunits.⁸ Although a clear distinction of the roles between NR2A and NR2B containing NMDARs, in LTP and LTD respectively, has remained controversial,⁹ it is clear that both NR2A and NR2B are key mediators of alterations in synaptic plasticity. In older animals, the conditioning protocol is reported to require modification to include paired-pulses. This is thought to be due to the involvement of predominantly AMPA (and perhaps kainate) receptors in addition to mGluRs responsible for the synaptic depotentiation (reviewed in ref. ¹⁰).As mentioned earlier, our data obtained from hippocampal cultures indicated only a minor effect of PrP^C knockout on AMPA receptor function. Hence, we hypothesized that AMPA receptor-mediated LTD should be similar in both wild type and PrP-null mice. We therefore examined the effect of PrP on LTD in hippocampal slices obtained from P30–P45 wild type mice and Zurich 1 PrP^C knockout mice. Extracellular potentials were recorded using a patch pipette filled with 150 mM NaCl. First, 10 minutes of baseline evoked (every 30 sec) potentials were recorded to ensure stability of the preparation. LTD was then evoked by application of conditioning paired pulses (Δt = 60 ms) delivered at 1 Hz for 15 min. Thereafter, the field response was sampled every 30 sec for 40 min. As shown in Figure 1, this protocol evoked reliable LTD in wild type mice that partially recovered over the time course of about 20 minutes (Fig. 1). In the PrP^C-null slices, LTD was indistinguishable from that observed in the wild type slices (Fig. 1).Open in a separate windowFigure 1LTD in the CA1 region of hippocampal slices from adult (P30–P45) WT and PrP-null mice. The conditioning pulse (arrow head) was delivered as paired-pulses (Δt = 60 ms) at 1 Hz for 15 min at the Schaffer collaterals. Analysis of field excitatory postsynaptic potential (fEPSP) slope revealed no statistically significant differences (Student''s t-test, p > 0.05) in the extent of induced LTD or the time course of its recovery to baseline. Numbers in parentheses indicate number of slices.The age of the animals, combined with the paired pulse protocol used in our experiments was designed to isolate AMPA receptor mediated LTD.¹⁰ The notion that LTD was unaltered in PrP-null mice is consistent with the observation that AMPA currents were not altered in these mice, and that AMPA receptor-mediated spontaneous synaptic events showed only minute changes compared with wild type animals. These data are also consistent with the notion that PrP-null mice show only mild phenotypes in spatial learning, with no apparent overall short-term memory deficits. Collectively, these data further support a selective action of PrP on NMDA receptors, rather than overall glutamatergic synaptic transmission.     

N-terminally tagged prion protein supports prion propagation in transgenic mice.

The eight amino acid sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, representing the FLAG peptide, was inserted after codons 22 or 88 of the mouse (Mo) prion protein (PrP) gene. Inclusion of the FLAG sequence at these locations interfered neither with the cellular processing of PrPC nor its conversion into PrPSc. Inclusion of the FLAG epitope at residue 22 but not at residue 88 facilitated immunodetection of tagged PrP by anti-FLAG monoclonal antibodies (mAbs). Inoculation of transgenic (Tg) mice expressing N-terminally tagged MoPrP with Mo prions resulted in abbreviated incubation times, indicating that the FLAG sequence was not deleterious to prion propagation. Immunopurification of FLAG-tagged MoPrPC in the brains of Tg mice was achieved using the calcium-dependent anti-FLAG M1 mAb and non-denaturing procedures. Although the function of PrPC remains unknown, our studies demonstrate that some modifications of PrPC do not inhibit the one biological activity that can be measured, i.e., conversion into PrPSc. Tagged PrP molecules may prove useful in the development of improved assays for prions as well as structural studies of the PrP isoforms.     

Cellular prion protein null mice display normal AMPA receptor mediated long term depression

Cellular prion protein (PrPC) appears to be involved in numerous physiological processes. We have recently shown a novel modulation of NMDA receptors by PrPC that results in neuroprotection via silencing of NMDA receptors containing NR2D subunits, whereas no effects on AMPA receptor function could be observed (Khosravani et al. 2008, J Cell Biol. 181, 551). Here we show that PrP-null mice show a normal response to long-term depression stimuli requiring AMPA receptor activity, thus further supporting our previous findings of a selective action on NMDA receptors among ionotropic glutamate receptors.     

Transgenic mice lacking NMDAR-dependent LTD exhibit deficits in behavioral flexibility

While most studies have focused on the role of long-term potentiation in behavior, far less is known about the role of long-term depression (LTD). To examine the potential involvement of LTD in learning and memory, we generated transgenic mice that express a fragment of the SV40 small t antigen known to inhibit protein phosphatase 2A (PP2A). Small t antigen expression blocked both stimulus-induced and chemically induced NMDAR-dependent LTD at Schaffer collateral synapses but did not affect potentiation, depotentiation, or mGluR-dependent LTD. This physiological phenotype was associated with deficits in behavioral flexibility in both the Morris water maze and a delayed nonmatch to place T-maze task, suggesting that NMDAR-dependent LTD is required for behavioral flexibility and may act by weakening previously encoded memory traces when new information is learned.     

Breaking immune tolerance to the prion protein using prion protein peptides plus oligodeoxynucleotide-CpG in mice

The absence of a detectable immune response during transmissible spongiform encephalopathies is likely due to the fact that the essential component of infectious agents, the prion protein (PrP), is a self Ag expressed on the surface of many cells of the host. To overcome self-tolerance to PrP, we used 30-mer PrP peptides previously shown to be immunogenic in Prnp(-/-) mice, together with CFA or CpG-oligodeoxynucleotides (CpG) in IFA. Generation of anti-PrP T and B cell responses was analyzed in the spleen, lymph nodes, and serum of immunized C57BL/6 wild-type mice. Immunization with PrP peptides emulsified in CFA did not trigger an immune response to PrP. When CpG were used, vaccination with peptides P143-172 and P158-187 generated IFN-gamma-secreting splenic T cells, and only P158-187 significantly stimulated IL-4-secreting T cells. Both peptides induced few Ab-producing B cells, and low and variable serum Ab titers. In contrast, immunization with peptide P98-127 did not induce significant levels of T cell responses but elicited specific peptide Abs. T cell epitope mapping, performed using 15-mer peptides covering PrP segment 142-182, revealed that an immunogenic motif lies between positions 156 and 172. These results demonstrate that T and B cell repertoires against PrP can be stimulated in C57BL/6 when adjuvant of the innate immunity such as CpG, but not CFA, is added to PrP peptides, and that the pattern of immune responses varies according to the epitope.     

Processing of retinal signals in normal and HCN deficient mice

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1^−/− mice or during a pharmacological blockade of I_h show that, contrary to previous reports, I_kx alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I_h and I_kx to the rods'' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.     

Methylphenidate improves the behavioral and cognitive deficits of neurogranin knockout mice

Neurogranin (Ng), a brain‐specific calmodulin‐binding protein, is expressed highly in hippocampus, and is important for cognitive function. Deletion of the Ng gene from mice caused attenuation of signal reaction cascade in hippocampus, impairments in learning and memory and high frequency stimulation‐induced long‐term potentiation (LTP). Environmental enrichment alone failed to improve cognitive function. In this study, behavioral testing revealed that Ng knockout (NgKO) mice were both hyperactive and socially withdrawn. Methylphenidate (MPH) was given to mice while they were also kept under an enrichment condition. MPH treatment reduced the hyperactivity of NgKO mice tested in both the open field and forced swim chamber. MPH improved their social abilities such that mice recognized and interacted better with novel subjects. The cognitive memories of MPH‐treated mutants were improved in both water maze and contextual fear conditioning tests. High frequency stimulation‐induced LTP of NgKO mice was also improved by MPH. The present treatment regimen, however, did not fully reverse the deficits of the mutant mice. In contrast, MPH exerted only a minimal effect on the wild type mice. At the cellular level, MPH increased the number of glial fibrillary acidic protein‐positive cells in hippocampus, particularly within the dentate gyrus of NgKO mice. Therefore it will be of interest to determine the nature of MPH‐mediated astrocyte activation and how it may modulate behavior in future studies. Taken together these NgKO mice may be useful for the development of better drug treatment to improve cognitive and behavioral impairments.     

Gene expression profile of oxidant stress and neurodegeneration in transgenic mice deficient in alpha-tocopherol transfer protein

Alpha-tocopherol transfer protein (TTP) regulates the retention and secretion of alpha-tocopherol (alpha-T) by the liver. Deletion of the TTP gene (Ttpa) in mice results in systemic deficiency of alpha-T and neurological dysfunctions described in patients with mutated Ttpa. We have explored genome-wide changes in mRNAs from brain cortex and liver of Ttpa-deficient (Ttpa(-/-)) mice and wild-type (Ttpa(+/+)) mice. Selective inductions of genes regulated by antioxidant response elements were detected in Ttpa(-/-) livers compared to Ttpa(+/+) livers, suggesting increased oxidant stress in Ttpa(-/-) livers. The activation of cell proliferation pathways in Ttpa(-/-) livers was indicated by the induction of genes that encode growth factor-binding proteins, mitogen-activated protein kinase kinase 3, and apoptosis inhibitor 6. The induction of synuclein-alpha and repression of synuclein-beta genes was detected in Ttpa(-/-) cortex. This may predispose Ttpa(-/-) cortex to increased formation of synuclein-alpha aggregates and Lewy body, often associated with oxidant stress. Cortex of Ttpa(-/-) mice revealed repression of genes encoding synaptic proteins, protein kinase C family members, and myelin proteins. A 13-fold decrease in the expression of retinoic acid receptor-related orphan receptor-alpha mRNA predicts staggerer-like phenotype (ataxia and deficits of motor coordination) of Ttpa(-/-) mice. The repression of specific genes that determine synaptic plasticity and neuronal development may account for suppressed electrophysiological activities of cortex and impaired behavior in Ttpa(-/-) mice.     

Tau protein phosphorylation in diverse brain areas of normal and CRH deficient mice: up-regulation by stress

Tau protein misfolding is a pathological mechanism, which plays a critical role in the etiopathogenesis of neurodegeneration. However, it is not entirely known what kind of stimuli can induce the misfolding. It is believed that physical and emotional stresses belong to such risk factors. Although the influence of stress on the onset and progression of Alzheimer's disease (AD) has already been proposed, the molecular links between stresses and AD are still unknown. We have therefore focused our attention on determination of the influence of acute immobilization stress (IMO) in normal mice and mice deficient in corticotropin-releasing hormone (CRH). Specifically, we have analyzed levels of hyperphosphorylated tau proteins, bearing the AD-specific phospho-epitopes (AT-8, pT181, and PHF-1), which are implicated in the pathogenesis of AD. We found that IMO induces transient hyperphosphorylation of tau proteins regardless of continuation of the stimulus. Concerning tau modifications, detailed analysis of the mouse brain revealed that neurons in different brain regions including frontal cortex, temporal cortex, hippocampal C1 and CA3 regions, dentate gyrus as well as nucleus basalis Meynert, and several brainstem nuclei such as locus coeruleus but also raphe nucleus and substantia nigra respond similarly to IMO. The strongest tau protein phosphorylation was observed after 30?min of IMO stress. Stress lasting for 120?min led either to the disappearance of tau hyperphosphorylation or to the induction of a second wave of hyperphosphorylation. Noteworthy is the magnitude of pathological phosphorylation of tau protein in CRH and glucocorticoids deficient mice, being much lower in comparison to that observed in wild-type animals, which suggests a critical role of CRH in the pathogenesis of AD. Thus, our results indicate that hyperphosphorylation of tau protein induced by stress may represent the pathogenic event upstream of tau protein misfolding, which leads to progression or eventually initiation of neurodegeneration. The data show that CRH plays an important role in stress induced hyperphosphorylation of tau protein, which might be either a direct effect of CRH innervations in the brain or an effect mediated via the hypothalamo-pituitary-adrenal axis.     

Developmental expression of prion protein gene in brain

Synthesis of the cellular isoform of the prion protein (PrPC) was found to be regulated during development of the hamster brain. PrP poly A(+) RNA was readily detectable 10 days postpartum; after 20 days of age, no change in its level could be detected through 13 months of age. Low levels of PrP poly A(+) RNA were detectable 1 day after birth. By contrast, myelin basic protein poly A(+) RNA was found at high levels in brain at 30 days of age and thereafter declined steadily. Using monospecific PrP antisera, immunoprecipitable cell-free translation products were detected at low levels 2 days after birth and increased progressively through 10 days of age. How the levels of PrP mRNA participate in brain development and function remains to be established.