Transgenic expression of the protein-L-isoaspartyl methyltransferase (PIMT) gene in the brain rescues mice from the fatal epilepsy of PIMT deficiency

Protein-L-isoaspartyl methyltransfearase (PIMT) plays a physiological role in the repair of damaged proteins containing isoaspartyl residues. In previous studies, we showed that PIMT-deficient mice developed a fatal epileptic seizure associated with the accumulation of damaged proteins in the brain. The mutant mice also showed a neurodegenerative pathology in hippocampi and impaired spatial memory. Still undefined, however, is how the accumulation of isoaspartates leads to the death of PIMT-deficient mice. In the present study, we generated PIMT transgenic (Tg) mice to investigate whether the exogenous expression of PIMT could improve the symptoms associated with PIMT deficiency. Rescue experiments showed that Tg expression of PIMT driven by a prion promoter effectively cured the PIMT-deficient mice. Biochemically, a higher expression level of transgene led to the effective repair of damaged proteins in vivo. Although a lower level of expression caused an accumulation of damaged proteins in a partially rescued line, the mice survived. Interestingly, synapsin I, which was extensively modified posttranslationally in PIMT-deficient mice, was specifically repaired in a partially rescued, but symptom-improved, Tg line. Our results suggest that an overall accumulation of damaged proteins does not necessarily lead to a fatal epileptic seizure, whereas certain modifications, such as changes in synapsin I, may play a pivotal pathological role in epilepsy.     

Recovery from spinal cord injury: a new transection model in the C57Bl/6 mouse

Spinal cord transections in mammalian animal models lead to loss of motor function. In this study, we show that functional recovery from complete transection of the adult mouse spinal cord can in fact occur without any intervention if dural injury along with displacement of the ends of the cut cord and fibroblastic infiltration is minimized. Underlying this function is the expression of GAP-43 in axonal growth cones, axonal extension and bridging of the injury site indicated by biocytin retrograde tracing and neuronal remodeling of both the white matter and the gray matter. Such studies suggest a new murine model for the study of spinal cord regeneration.     

Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice

Catechol-O-methyltransferase (COMT) catalyses the O-methylation of compounds having a catechol structure and its main function involves the elimination of biologically active or toxic catechols and their metabolites. By means of homologous recombination in embryonic stem cells, a strain of mice has been produced in which the gene encoding the COMT enzyme is disrupted. We report here the levels of catecholamines and their metabolites in striatal extracellular fluid in these mice as well as in homogenates from different parts of the brain, under normal conditions and after acute levodopa administration. In immunoblotting studies, COMT-knockout mice had no COMT protein in brain or kidney tissues but the amounts of catecholamine synthesizing and other metabolizing enzyme proteins were normal. Under normal conditions, COMT deficiency does not appear to affect significantly brain dopamine and noradrenaline levels in spite of relevant changes in their metabolites. This finding is consistent with previous pharmacological studies with COMT inhibitors and confirms the pivotal role of synaptic reuptake processes and monoamine oxidase-dependent metabolism in terminating the actions of catecholamines at nerve terminals. In contrast, when COMT-deficient mice are challenged with l-dihydroxyphenylalanine, they show an extensive accumulation of 3,4-dihydroxyphenylacetic acid and dihydroxyphenylglycol and even dopamine, revealing an important role for COMT under such situations. Notably, in some cases these changes appear to be Comt gene dosage-dependent, brain-region specific and sexually dimorphic. Our results may have implications for improving the treatment of Parkinson's disease and for understanding the contribution of the natural variation in COMT activity to psychiatric phenotypes.     

Acute cholinergic rescue of synaptic plasticity in the neurodegenerating cortex of anti-nerve-growth-factor mice

Deficits in cholinergic systems innervating cerebral cortex are associated with cognitive impairment during senescence and in age-related neurodegenerative pathologies. However, little is known about the role of cholinergic pathways in modulating cortical plasticity. Basal forebrain cholinergic neurons are a major target for nerve-growth factor (NGF). In order to investigate the relationship between cholinergic innervation and cortical synaptic plasticity, we exploited a transgenic mouse model in which the activity of NGF in the adult nervous system is neutralized by the expression of blocking antibodies to NGF itself (anti-NGF mice) [Ruberti, F. et al. (2000). J. Neurosci. 20, 2589-2601]. In 6-month-old anti-NGF mice, we show that the reduction in cholinergic innervation of the cortex is associated with different forms of synaptic plasticity impairment. A local, acute increase in the availability of acetylcholine rescues these synaptic plasticity deficits, thus indicating that a cholinergic system mediates the impairment of cortical plasticity at this early stage of the neurodegenerative process triggered by NGF neutralization. Our results represent an important step in unveiling the pivotal role of cholinergic transmission in modulating adult cortical plasticity.     

Transgenic mice expressing a pH and Cl- sensing yellow-fluorescent protein under the control of a potassium channel promoter

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.     

Kindling-induced overexpression of Homer 1A and its functional implications for epileptogenesis

Despite an extensive research on the molecular basis of epilepsy, the essential players in the epileptogenic process leading to epilepsy are not known. Gene expression analysis is one strategy to enhance our understanding of the genes contributing to the functional neuronal changes underlying epileptogenesis. In the present study, we used the novel MPSS (massively parallel signature sequencing) method for analysis of gene expression in the rat kindling model of temporal lobe epilepsy. Kindling by repeated electrical stimulation of the amygdala resulted in the differential expression of 264 genes in the hippocampus compared to sham controls. The most strongly induced gene was Homer 1A, an immediate early gene involved in the modulation of glutamate receptor function. The overexpression of Homer 1A in the hippocampus of kindled rats was confirmed by RT-PCR. In order to evaluate the functional implications of Homer 1A overexpression for kindling, we used transgenic mice that permanently overexpress Homer 1A. Immunohistochemical characterization of these mice showed a marked Homer 1A overexpression in glutamatergic neurons of the hippocampus. Kindling of Homer 1A overexpressing mice resulted in a retardation of seizure generalization compared to wild-type controls. The data demonstrate that kindling-induced epileptogenesis leads to a striking overexpression of Homer 1A in the hippocampus, which may represent an intrinsic antiepileptogenic and anticonvulsant mechanism in the course of epileptogenesis that counteracts progression of the disease.     

Genetic and pharmacological demonstration of a role for cyclic AMP-dependent protein kinase-mediated suppression of protein phosphatases in gating the expression of late LTP

Protein kinases and phosphatases play antagonistic roles in regulating hippocampal long-term potentiation (LTP), with kinase inhibition and phosphatase activation both impairing LTP. The late phase of LTP (L-LTP) requires activation of cAMP-dependent protein kinase (PKA) for its full expression. One way in which PKA may critically modulate L-LTP is by relieving an inhibitory constraint imposed by protein phosphatases. Using mutant PKA mice [R(AB) transgenic mice] that have genetically reduced hippocampal PKA activity, we show that deficient L-LTP in area CA1 of mutant hippocampal slices is rescued by acute application of two inhibitors of protein phosphatase-1 and protein phosphatase-2A (PP1/2A) (okadaic acid and calyculin A). Furthermore, synaptic facilitation induced by forskolin, an adenylyl cyclase activator, was impaired in R(AB) transgenics and was also rescued by a PP1/2A inhibitor in mutant slices. Inhibition of PP1/2A did not affect early LTP (E-LTP) or basal synaptic transmission in mutant and wildtype slices. Our data show that genetic inhibition of PKA impairs L-LTP by reducing PKA-mediated suppression of PP1/2A.     

Phosphorylated MAP1B is induced in central sprouting of primary afferents in response to peripheral injury but not in response to rhizotomy

A peripheral nerve lesion induces sprouting of primary afferents from dorsal root ganglion (DRG) neurons into lamina II of the dorsal horn. Modifications of the environment in consequence to the axotomy provide an extrinsic stimulus. A potential neuron-intrinsic factor that may permit axonal sprouting is microtubule-associated protein 1B (MAP1B) in a specific phosphorylated form (MAP1B-P), restricted to growing or regenerating axons. We show here that both in rat and mouse, a sciatic nerve cut is rapidly followed by the appearance of MAP1B-P expression in lamina II, increasing to a maximum between 8 and 15 days, and diminishing after three months. Evidence is provided that sprouting and induction of MAP1B-P expression after peripheral injury are phenomena concerning essentially myelinated axons. This is in accordance with in situ hybridization data showing especially high MAP1B-mRNA levels in large size DRG neurons that give rise to myelinated fibers. We then employed a second lesion model, multiple rhizotomy with one spared root. In this case, unmyelinated CGRP expressing fibers do indeed sprout, but coexpression of MAP1B-P and CGRP is never observed in lamina II. Finally, because a characteristic of myelinated fibers is their high content in neurofilament protein heavy subunit (NF-H), we used NF-H-LacZ transgenic mice to verify that MAP1B-P induction and central sprouting were not affected by perturbing the axonal organization of neurofilaments. We conclude that MAP1B-P is well suited as a rapidly expressed, axon-intrinsic marker associated with plasticity of myelinated fibers.     

Selective lesions of basal forebrain cholinergic neurons produce anterograde and retrograde deficits in a social transmission of food preference task in rats

We examined the performance of Long-Evans rats with 192 IgG-saporin lesions of the medial septum/vertical limb of the diagonal band (MS/VDB) or nucleus basalis magnocellularis/substantia innominata (NBM/SI), which removed cholinergic projections mainly to hippocampus or neocortex, respectively. We studied the effects of these lesions on anterograde and retrograde memory for a natural form of hippocampal-dependent associative memory, the social transmission of food preference. In a study of anterograde memory, MS/VDB lesions did not affect the immediate, 24-h or 3-week retention of the task. In contrast, NBM/SI lesions severely impaired immediate and 24-h retention. In a study of retrograde memory in which rats acquired the food preference 5 days or 1 day before surgery and they were tested 10-11 days after surgery, MS/VDB-lesioned rats showed striking memory deficits for the preference acquired at a long delay (5 days) before surgery, although all lesioned rats exhibited poorer retention on both retest sessions than on their pretest performance. Subsequent testing of new anterograde learning in these rats revealed no disrupting effects of lesions on a standard two-choice test. When rats were administered a three-choice test, in which the target food was presented along with two more options, NBM/SI-lesioned rats were somewhat impaired on a 24-h retention test. These results provide evidence that NBM/SI and MS/VDB cholinergic neurons are differentially involved in a social memory task that uses olfactory cues, suggesting a role for these neurons in acquisition and consolidation/retrieval of nonspatial declarative memory.     

Overexpression of corticotropin-releasing hormone in transgenic mice and chronic stress-like autonomic and physiological alterations

To gain a greater insight into the relationship between hyperactivity of the corticotropin-releasing hormone (CRH) system and autonomic and physiological changes associated with chronic stress, we developed a transgenic mouse model of central CRH overproduction. The extent of central and peripheral CRH overexpression, and the amount of bioactive CRH in the hypothalamus were determined in two lines of CRH-overexpressing (CRH-OE) mice. Furthermore, 24 h patterns of body temperature, heart rate, and activity were assessed using radiotelemetry, as well as cumulative water and food consumption and body weight gain over a 7-day period. CRH-OE mice showed increased amounts of CRH peptide and mRNA only in the central nervous system. Despite the presence of the same CRH transgene in their genome, only in one of the two established lines of CRH-OE mice (line 2122, but not 2123) was overexpression of CRH associated with increased levels of bioactive CRH in the hypothalamus, increased body temperature and heart rate (predominantly during the light (inactive) phase of the diurnal cycle), decreased heart rate variability during the dark (active) phase, and increased food and water consumption, when compared with littermate wildtype mice. Because line 2122 of the CRH transgenic mice showed chronic stress-like neuroendocrine and autonomic changes, these mice appear to represent a valid animal model for chronic stress and might be valuable in the research on the consequences of CRH excess in situations of chronic stress.