The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNASer(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ~70% in the tRNASer(UCN) level and a decrease of ~45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to ~40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ~7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells

Photoreceptor Degeneration in Two Mouse Models for Congenital Stationary Night Blindness Type 2

Light-dependent conductance changes of voltage-gated Cav1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Cav1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Cav1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca2+ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca2+ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca2+ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants

Photoreceptor degeneration in two mouse models for congenital stationary night blindness type 2.

Light-dependent conductance changes of voltage-gated Cav1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Cav1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Cav1.4 channels, but some mutations alter the channels' gating properties and, presumably, disturb Ca(2+) influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14-17) in a longitudinal study up to eight months of age. In ΔEx14-17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca(2+) responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca(2+) response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14-17 mutants

Effects of Presynaptic Mutations on a Postsynaptic Cacna1s Calcium Channel Colocalized with mGluR6 at Mouse Photoreceptor Ribbon Synapses

PURPOSE: Photoreceptor ribbon synapses translate light-dependent changes of membrane potential into graded transmitter release via L-type voltage-dependent calcium channel (VDCC) activity. Functional abnormalities (e.g., a reduced electroretinogram b-wave), arising from mutations of presynaptic proteins, such as Bassoon and the VDCCα1 subunit Cacna1f, have been shown to altered transmitter release. L-type VDCCα1 subtype expression in wild-type and mutant mice was examined, to investigate the underlying pathologic mechanism. METHODS: Two antisera against Cacna1f, and a Cacna1f mouse mutant (Cacna1fΔEx14-17) were generated. Immunocytochemistry for L-type VDCCα1 subunits and additional synaptic marker proteins was performed in wild-type, BassoonΔEx4-5 and Cacna1fΔEx14-17 mice. RESULTS: Active zone staining at photoreceptor ribbon synapses with a panα1 antibody colocalized with staining for Cacna1f in wild-type mouse retina. Similarly, in the BassoonΔEx4-5 mouse, residual mislocalized staining for panα1 and Cacna1f showed colocalization. Unlike the presynaptic location of Cacna1f and panα1 antibody staining, the skeletal muscle VDCCα1 subunit Cacna1s was present postsynaptically at ONbipolar cell dendrites, where it colocalized with metabotropic glutamate receptor 6 (mGluR6). Surprisingly, Cacna1s labeling was severely downregulated in the BassoonΔEx4-5 and Cacna1fΔEx14-17 mutants. Subsequent analyses revealed severely reduced ON-bipolar cell dendritic expression of the sarcoplasmic reticulum Ca²⁺ ATPase Serca2 in both mouse mutants and of mGluR6 in the Cacna1fΔEx14-17 mutant. CONCLUSIONS: Presynaptic mutations leading to reduced photoreceptor- to-bipolar cell signaling are associated with disturbances in protein expression within postsynaptic dendrites. Moreover, detection of Cacna1s and Serca2 in ON-bipolar cell dendrites in wild-type animals suggests a putative role in regulation of postsynaptic Ca²⁺ flux

Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms' tumour

Genomic imprinting has been implicated in the onset of several embryonal tumours but the mechanism is not well understood. Maternal chromosome 11p15 loss of heterozygosity and paternal chromosome 11 isodisomy suggest that imprinted genes are involved in the onset of Wilms' tumour and the Beckwith-Wiedemann syndrome. The insulin-like growth factor II (IGF2) gene located at 11pl5.5 has been put forward as a candidate gene as it is maternally imprinted (paternally expressed) in the mouse , and is expressed at high levels in Wilms' tumours. We report here that the IGF2 gene is expressed from the paternal allele in human fetal tissue, but that in Wilms' tumour expression can occur biallelically. These results provide, to our knowledge, the first evidence that relaxation of imprinting may play a role in the onset of disease and suggest a new genetic mechanism involved in the development of cancer

Age-dependent loss of CACNA1F immunoreactivity in the I756T mutant mouse.

<p>Immunocytochemical labeling of CACNA1F in wild-type and I756T mutant outer plexiform layer (OPL) at P6, P14, P28, two, and eight months. Scale bar: 10 µm.</p

Photoreceptor degeneration in <i>Cacna1f</i> mutant mice.

<p><b>A:</b> Quantification of the percentage of TUNEL positive cells in the ONL from wild-type, ΔEx14–17, and I756T mutants at P28, 2 and 8 months. Values are means ± SD. (*p<0.05; **p<0.01; ***p<0.001, ANOVA). <b>B:</b> Immunocytochemical staining of GFAP on P28 old wild-type, ΔEx14–17, and I756T mutant retina shows more pronounced Müller cell reactivity in the I756T mutant retina. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 µm.</p

Ca²⁺ imaging of photoreceptor terminals.

**<p>p<0.01;</p>*<p>p<0.05; n.s., not significant. Significance levels were determined by Kruskal-Wallis ANOVA.</p

Comparison of the sprouting phenotype in the wild-type, ΔEx14–17, and I756T mutant retinae.

<p><b>A–C:</b> Immunocytochemical triple staining of Calbindin (red), PKCα (green), and VGluT1 (blue) on P28 old wild-type (A), ΔEx14–17 (B), I756T (C) outer retinae shows sprouting of ON-bipolar cell dendrites as well as horizontal cell processes into the ONL of both <i>Cacna1f</i> mutants. The VGluT1 labeling shows the existence of presynaptic contacts with the sprouting elements. <b>D:</b> Comparison of the severity of horizontal cell sprouting in the wild-type, ΔEx14–17, I756T mutant outer retina at P6, P14, P28, two months, and eight months. Asterisks indicate the onset of noticeable sprouting in the <i>Cacna1f</i> mutants. In the I756T mutant retina, noticeable horizontal cell sprouting started earlier (P14) than in the ΔEx14–17 mutant retina (P28), but declined at eight months, when sprouting still continued in the ΔEx14–17 mutant retina. ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar in A for A–C<b>:</b> 10 µm; in D: 20 µm.</p

Age-dependent ONL thickness in <i>Cacna1f</i> mutant mice.

<p><b>A:</b> Labeling of nuclei with DAPI (blue) and of cone photoreceptor outer segments and terminals (arrowheads) with peanut agglutinin (green) on retinal cryostat sections of wild-type, ΔEx14–17, and I756T mutant mice at 8 months. <b>B:</b> Quantification of the number of cell rows in the outer nuclear layer (ONL) from wild-type, ΔEx14–17, and I756T mutants at P28, 2 and 8 months. Values in are means ± SD. (*p<0.05; **p<0.01; ***p<0.001, ANOVA). POS, photoreceptor outer segments; OPL, outer plexiform layer. Scale bar: 10 µm.</p