S. M. Wu

SCA7 Knockin Mice Model Human SCA7 and Reveal Gradual Accumulation of Mutant Ataxin-7 in Neurons and Abnormalities in Short-Term Plasticity

We targeted 266 CAG repeats (a number that causes infantile-onset disease) into the mouse Sca7 locus to generate an authentic model of spinocerebellar ataxia type 7 (SCA7). These mice reproduced features of infantile SCA7 (ataxia, visual impairments, and premature death) and showed impaired short-term synaptic potentiation; downregulation of photoreceptor-specific genes, despite apparently normal CRX activity, led to shortening of photoreceptor outer segments. Wild-type ataxin-7 was barely detectable, as was mutant ataxin-7 in young animals; with increasing age, however, ataxin-7 staining became more pronounced. Neurons that appeared most vulnerable had relatively high levels of mutant ataxin-7; it is interesting, however, that marked dysfunction occurred in these neurons weeks prior to the appearance of nuclear inclusions. These data demonstrate that glutamine expansion stabilizes mutant ataxin-7, provide an explanation for selective neuronal vulnerability, and show that mutant ataxin-7 impairs posttetanic potentiation (PTP).

Localization and modulatory actions of zinc in vertebrate retina

Zinc ions are colocalized with glutamatergic synaptic vesicles in vertebrate photoreceptors and may act as a diffusible molecular switch regulating neurotransmitter signaling at two distinct sites in the outer retina. In the dark, extracellular zinc acts presynaptically at rods and cones to minimize the depletion of tonically released glutamate, and selectively reduces GABA-mediated depolarization in horizontal cells, accelerating the response kinetics of the second-order cells. The discovery of zinc ions in photoreceptors provides a mechanism for gain control, kinetics modulation, and the balance of rod vs cone output at the first synapse in the visual system.

GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice

Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca2+ and cGMP. Ca2+ regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC‐activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a‐wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild‐type flash responses in the absence of GCAP2.

The Transcription Factor Bhlhb4 Is Required for Rod Bipolar Cell Maturation

Retinal bipolar cells are essential to the transmission of light information. Although bipolar cell dysfunction can result in blindness, little is known about the factors required for bipolar cell development and functional maturation. The basic helix-loop-helix (bHLH) transcription factor Bhlhb4 was found to be expressed in rod bipolar cells (RB). Electroretinograms (ERGs) in the adult Bhlhb4 knockout (Bhlhb4(-/-)) showed that the loss of Bhlhb4 resulted in disrupted rod signaling and profound retinal dysfunction resembling human congenital stationary night blindness (CSNB), characterized by the loss of the scotopic ERG b-wave. A depletion of inner nuclear layer (INL) cells in the adult Bhlhb4 knockout has been ascribed to the abolishment of the RB cell population during postnatal development. Other retinal cell populations including photoreceptors were unaltered. The timing of RB cell depletion in the Bhlhb4(-/-) mouse suggests that Bhlhb4 is essential for RB cell maturation.

Phenotypic characterization of Bbs4 null mice reveals age-dependent penetrance and variable expressivity.

Bardet-Biedl syndrome (BBS) is a rare oligogenic disorder exhibiting both clinical and genetic heterogeneity. Although the BBS phenotype is variable both between and within families, the syndrome is characterized by the hallmarks of developmental and learning difficulties, post-axial polydactylia, obesity, hypogenitalism, renal abnormalities, retinal dystrophy, and several less frequently observed features. Eleven genes mutated in BBS patients have been identified, and more are expected to exist, since about 20–30% of all families cannot be explained by the known loci. To investigate the etiopathogenesis of BBS, we created a mouse null for one of the murine homologues, Bbs4, to assess the contribution of one gene to the pleiotropic murine Bbs phenotype. Bbs4 null mice, although initially runted compared to their littermates, ultimately become obese in a gender-dependent manner, females earlier and with more severity than males. Blood chemistry tests indicated abnormal lipid profiles, signs of liver dysfunction, and elevated insulin and leptin levels reminiscent of metabolic syndrome. As in patients with BBS, we found age-dependent retinal dystrophy. Behavioral assessment revealed that mutant mice displayed more anxiety-related responses and reduced social dominance. We noted the rare occurrence of birth defects, including neural tube defects and hydrometrocolpos, in the null mice. Evaluations of these null mice have uncovered phenotypic features with age-dependent penetrance and variable expressivity, partially recapitulating the human BBS phenotype.

Impaired photoreceptor protein transport and synaptic transmission in a mouse model of Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an oligogenic syndrome whose manifestations include retinal degeneration, renal abnormalities, obesity and polydactylia. Evidence suggests that the main etiopathophysiology of this syndrome is impaired intraflagellar transport (IFT). In this study, we study the Bbs4-null mouse and investigate photoreceptor structure and function after loss of this gene. We find that Bbs4-null mice have defects in the transport of phototransduction proteins from the inner segments to the outer segments, before signs of cell death. Additionally, we show defects in synaptic transmission from the photoreceptors to secondary neurons of the visual system, demonstrating multiple functions for BBS4 in photoreceptors.

Effects of elevated intraocular pressure on mouse retinal ganglion cells

We developed and characterized a mouse model of elevated intraocular pressure (IOP) to investigate the underlying cellular and genetic mechanisms of retinal ganglion cell (RGC) death. IOP was unilaterally increased in C57BL/6J mice by photocoagulation of the episcleral and limbal veins. IOP was measured using an indentation tonometer. RGC survival was measured by retrograde labeling using DiI applied to the superior colliculous. The mechanism of RGC death was investigated using TUNEL staining, immunostaining for cleaved caspase-3, and Western blot for Bcl-2 and Bax expression. RT-PCR was used to measure changes in Bcl-2, Bax, Bad, Bak, P53, ICE and Fas. Mean IOP was increased in the treated eyes from 13+/-1.8 to 20.0+/-2.8 mmHg at four weeks and 17+/-2.2 mmHg at eight weeks. RGC loss was 15.6+/-3.4% at two weeks and 27.3+/-4.5% at four weeks after laser photocoagulation. TUNEL staining and caspase-3 positive cells were increased in the ganglion cell layer (GCL) in the treated eyes and seldom found in the control eyes. Bcl-2 expression in control group was higher than in the experimental group, while Bax expression in the control group was less than in experimental group. This mouse model resulted in a consistent, sustained increase in IOP with a reduction in the number of RGCs in the treated eye. The RGCs in eyes with elevated IOP were TUNEL-positive, with increased caspase-3 and decreased Bcl-2, consistent with apoptosis as the mechanism of neuronal cell death.

Guanylate cyclase-activating protein (GCAP) 1 rescues cone recovery kinetics in GCAP1/GCAP2 knockout mice

Mediated by guanylate cyclase-activating proteins (GCAPs), cytoplasmic Ca2+ levels regulate the activity of photoreceptor guanylate cyclase (GC) and the synthesis of cGMP, the internal transmitter of phototransduction. When GCAP1 is expressed in transgenic mice on a GCAP null background, it restores the wild-type flash responses in rod photoreceptors. In this communication, we explored the role of GCAP1 in cone photoreceptors by using electroretinograms (ERGs). Under cone isolation conditions, ERGs recorded from mice lacking both GCAP1 and GCAP2 had normal amplitudes of the saturated a-wave and b-wave. However, recordings from these mice demonstrated a widened b-wave and increased sensitivity of both M- and UV-cone systems. Paired-flash ERGs revealed a delayed recovery of both the cone driven b-wave and a-wave and suggest that the delay originated from the photoreceptors. To test whether GCAP1 could restore normal cone response recovery, mice that expressed only transgenic GCAP1 in the absence of wild-type GCAP expression were tested. Immunohistochemical analysis demonstrated that cones of these mice expressed high levels of GCAP1. Paired-flash ERGs showed that the recovery of the cone-driven a-wave was restored to normal, whereas recovery of the cone-driven b-wave was slightly faster than that observed in wild-type mice. These studies reveal that, similar to rods, deletion of GCAP1 and GCAP2 delays the recovery of light responses in cones and GCAP1 restores the recovery of cone responses in the absence of GCAP2.

Photoreceptor degeneration and rd1 mutation in the grizzled/mocha mouse strain

The mocha mouse is a spontaneous mutant carrying a defective adaptor-like protein complex AP-3delta subunit. We examined retinal function and histology of the mocha mutant. We found that not only mocha homozygotes but also other littermates in the inbred strain are blind due to severe defects in both rod and cone photoreceptors on electroretinogram recordings. The functional deficit was caused by rapid, early postnatal photoreceptor degeneration. Genotyping confirmed the presence of a viral insertion of rd1 gene in the mocha strain. We conclude that rd1 allele contamination is primarily responsible for photoreceptor degeneration, and caution against behavioral tests with visual cues in the present stocks.

A role for bHLH transcription factors in retinal degeneration and dysfunction.

The basic helix loop helix (bHLH) transcription factors collectively mediate cellular differentiation in almost every type of tissue including the retina (Murre et al. 1989; Jan and Jan 1993; Cepko 1999). Class A factors are ubiquitously expressed throughout mammalian tissue, while the expression of class B factors are cell type specific. These factors have both a DNA binding domain and helix loop helix domain (HLH) protein dimerization domain. Class B factors usually heterodimerize with the ubiquitously expressed, bHLH factors, such as E12/E47. Because of their importance during photoreceptor development, bHLH factors are candidate genes for photoreceptor degeneration. We have examined the roles of two bHLH factors, both which are expressed during retinal development, but also share the property of continued expression in the adult retina.