Novel variants in outer protein surface of flavin-containing monooxygenase 3 found in an Argentinian case with impaired capacity for trimethylamine N-oxygenation.

Flavin-containing monooxygenase 3 (FMO3) is a polymorphic drug metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped a white Argentinian 11-year-old girl by medical sensory evaluation. After pedigree analysis with her brother and parents, this proband showed to harbor a new allele p.(P73L; E158K; E308G) FMO3 in trans configuration with the second new one p.(F140S) FMO3. Recombinant FMO3 proteins of the wild-type and the novel two variants underwent kinetic analyses of their trimethylamine N-oxygenation activities. P73L; E158K; E308G and F140S FMO3 proteins exhibited moderately and severely decreased trimethylamine N-oxygenation capacities (~50% and ~10% of wild-type FMO3, respectively). Amino acids P73 and F140 were located on the outer surface region in a crystallographic structure recently reported of a FMO3 analog. Changes in these positions would indirectly impact on key FAD-binding residues. This is the first report and characterization of a patient of fish odor syndrome caused by genetic aberrations leading to impaired FMO3-dependent N-oxygenation of trimethylamine found in the Argentinian population. We found novel structural determinants of FAD-binding domains, expanding the list of known disease-causing mutations of FMO3. Our results suggest that individuals homozygous for any of these new variants would develop a severe form of this disorder.

Inner Hair Cell and Neuron Degeneration Contribute to Hearing Loss in a DFNA2-Like Mouse Model.

DFNA2 is a progressive deafness caused by mutations in the voltage-activated potassium channel KCNQ4. Hearing loss develops with age from a mild increase in the hearing threshold to profound deafness. Studies using transgenic mice for Kcnq4 expressed in a mixed background demonstrated the implication of outer hair cells at the initial phase. However, it could not explain the last phase mechanisms of the disease. Genetic backgrounds are known to influence disease expressivity. To unmask the cause of profound deafness phenotype, we backcrossed the Kcnq4 knock-out allele to the inbred strain C3H/HeJ and investigated inner and outer hair cell and spiral ganglion neuron degeneration across the lifespan. In addition to the already reported outer hair cell death, the C3H/HeJ strain also exhibited inner hair cell and spiral ganglion neuron death. We tracked the spatiotemporal survival of cochlear cells by plotting cytocochleograms and neuronal counts at different ages. Cell loss progressed from basal to apical turns with age. Interestingly, the time-course of cell degeneration was different for each cell-type. While for outer hair cells it was already present by week 3, inner hair cell and neuronal loss started 30 weeks later. We also established that outer hair cell loss kinetics slowed down from basal to apical regions correlating with KCNQ4 expression pattern determined in wild-type mice. Our findings indicate that KCNQ4 plays differential roles in each cochlear cell-type impacting in their survival ability. Inner hair cell and spiral ganglion neuron death generates severe hearing loss that could be associated with the last phase of DFNA2.

Reduction of GFAP induced by long dark rearing is not restricted to visual cortex.

A key component of the astrocyte cytoskeleton is the glial fibrillary acidic protein (GFAP), which plays an essential role in neuron/astrocyte interactions. Environmental conditioning, such as visual experience manipulation, can affect neuronal and/or glial plasticity in specific brain areas. Previous work from our laboratory showed that short light deprivation throughout the period of GFAP maturation does not influence the expression profile of GFAP in mouse visual cortex; however, it was strong enough to affect neuronal phenotype. It was suggested that visual experience controls the maturation of the neuronal circuitry in this brain area. Therefore, to see whether the modifications of neuronal activity induced by light deprivation affect the maintenance of normal astrocytic phenotype, the dark rearing protocol was extended until the adult life. GFAP-immunoreactive cells were dramatically affected, showing an 80% decrease in number. In addition, GFAP protein level exhibited a 50% reduction, while its mRNA remained unaffected. Besides the visual cortex, two other areas of the brain not directly involved in vision, the hippocampus and the motor cortex, were chosen as internal controls. Unexpectedly, also in these areas, astrocytes were affected by light deprivation. The present results show that lack of visual experience for long periods of time deeply affects glial phenotype not only in visual areas but also in brain regions not directly involved in sensory processing.

Developmental modulation of synaptic transmission by acetylcholine in the primary visual cortex.

Despite the evidence that cortical synaptic organization and cognitive functions are influenced by the activity of the cholinergic system during postnatal development, so far no information is available on the effects produced by acetylcholine (ACh) on synaptic transmission. In the present article, we show that the ability of visual cortex slices to respond to ACh depends on postnatal age. In adulthood, ACh exerts mainly a facilitatory action on synaptic transmission, depressing field potential (FP) amplitude only if applied at high concentrations (millimolar range). During early postnatal development, at postnatal day 13 (P13), facilitation by ACh was lacking, with depression of FP observed with concentration of ACh in the micromolar range. The magnitude of ACh facilitatory effects increases with age. The time course of ACh-dependent facilitation overlaps the developmental maturation of acetylcholinesterase (AChE), suggesting a close relationship between ACh action and AChE activity. Thus, age-dependent modification of the cholinergic modulatory action may affect cortical maturation by regulating the magnitude of synaptic transmission.

Alpha7 but not alpha4 AChR subunit expression is regulated by light in developing primary visual cortex.

In the present paper we analyzed the expression pattern of the alpha4 and alpha7 nicotinic acetylcholine receptor (nAChR) subunits in the rat visual cortex through postnatal development, to clarify whether their expression is developmentally regulated and whether eventual developmental changes are regulated by visual experience. We found that both alpha4 and alpha7 mRNA levels accumulate from postnatal day 12 (P12) before eye opening, to around P35. The immunohistochemical results indicated that both subunits are expressed throughout all cortical laminae, except layer I. Alpha4 subunit immunohistochemistry revealed significant increments in the number of positive cells in layers V and VI after eye opening. In the case of the alpha7 subunit, the number of immunoreactive cells increased in all cortical layers soon after eye opening, except in layer VI, matching the results found at the transcriptional level. In animals reared in darkness from P9 to P22, the relative amount of the alpha4 mRNA and the number of immunoreactive cells exhibited no changes. 3H-epibatidine binding experiments showed that the number of heteromeric nAChR subunits in dark-reared rats did not change with respect to age-matched controls, thus confirming the immunohistochemical results. The mRNA of the alpha7 subunit remained stable in dark-reared rats, whereas the number and distribution of immunoreactive cells changed. Moreover, the number of 125I alphabungarotoxin-binding nAChRs was significantly increased in dark-reared animals. These results indicate that visual cortex stimulation by visual input is an essential step for alpha7 nAChR normal expression, suggesting a possible role for these receptors in an experience-dependent fashion on the maturation of this cortical area.

Septal grafts restore cognitive abilities and amyloid precursor protein metabolism.

Cortical cholinergic loss and amyloidogenic processing of the beta-amyloid precursor protein (APP), may functionally interact in Alzheimer's disease. However, it is still unknown whether biological restoration of regulatory cholinergic inputs affects APP metabolism in vivo. Rats immunolesioned with 192 IgG-saporin exhibited severe acquisition deficits in place navigation that were paralleled by a dramatic loss of terminal cholinergic innervation and by marked changes in the regional expression of APP-like immunoreactivity. Moreover, in these animals, we observed a drastic reduction of soluble APP (sAPP) and a concomitant increase of the unsoluble, membrane-bound fraction (mAPP). Notably, at about 6 months post-surgery, lesioned animals implanted with reinnervating cholinergic-rich septal tissue grafts exhibited fairly normal spatial navigation abilities, as well as cortical and hippocampal APP levels that were restored up to normal or near-normal values. APP levels correlated significantly with lesion- or graft-induced changes in cholinergic innervation density, and both these measures correlated with performance in the spatial navigation task. Thus, integrity of ascending cholinergic inputs may be required to prevent amyloidogenic processing of APP in vivo and to modulate cognitive performance.

Muscarinic acetylcholine receptor knockout mice show distinct synaptic plasticity impairments in the visual cortex.

In the present report, we focused our attention on the role played by the muscarinic acetylcholine receptors (mAChRs) in different forms of long-term synaptic plasticity. Specifically, we investigated long-term potentiation (LTP) and long-term depression (LTD) expression elicited by theta-burst stimulation (TBS) and low-frequency stimulation (LFS), respectively, in visual cortical slices obtained from different mAChR knockout (KO) mice. A normal LTP was evoked in M(1)/M(3) double KO mice, while LTP was impaired in the M(2)/M(4) double KO animals. On the other hand, LFS induced LTD in M(2)/M(4) double KO mice, but failed to do so in M(1)/M(3) KO mice. Interestingly, LFS produced LTP instead of LTD in M(1)/M(3) KO mice. Analysis of mAChR single KO mice revealed that LTP was affected only by the simultaneous absence of both M(2) and M(4) receptors. A LFS-dependent shift from LTD to LTP was also observed in slices from M(1) KO mice, while LTD was simply abolished in slices from M(3) KO mice. Using pharmacological tools, we showed that LTP in control mice was blocked by pertussis toxin, an inhibitor of G(i/o) proteins, but not by raising intracellular cAMP levels. In addition, the inhibition of phospholipase C by U73122 induced the same shift from LTD to LTP after LFS observed in M(1) single KO and M(1)/M(3) double KO mice. Our results indicate that different mAChR subtypes regulate different forms of long-term synaptic plasticity in the mouse visual cortex, activating specific G proteins and downstream intracellular mechanisms.

Acetylcholine modulates cortical synaptic transmission via different muscarinic receptors, as studied with receptor knockout mice.

The central cholinergic system plays a crucial role in synaptic plasticity and spatial attention; however, the roles of the individual cholinergic receptors involved in these activities are not well understood at present. In the present study, we show that acetylcholine (ACh) can facilitate or depress synaptic transmission in occipital slices of mouse visual cortex. The precise nature of the ACh effects depends on the ACh concentration, and is input specific, as shown by stimulating different synaptic pathways. Pharmacological blockade of muscarinic receptor (mAChR) subtypes and the use of M1-M5 mAChR-deficient mice showed that specific mAChR subtypes, together with the activity of the cholinesterases (ChEs), mediate facilitation or depression of synaptic transmission. The present data suggest that local ACh, acting through mAChRs, regulates the cortical dynamics making cortical circuits respond to specific stimuli.