A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation

We have isolated a dominant mutation, night blindness a (nba), that causes a slow retinal degeneration in zebrafish. Heterozygous nba fish have normal vision through 2–3 months of age but subsequently become night blind. By 9.5 months of age, visual sensitivity of affected fish may be decreased more than two log units, or 100-fold, as measured behaviorally. Electroretinographic (ERG) thresholds of mutant fish are also raised significantly, and the ERG b-wave shows a delayed implicit time. These defects are due primarily to a late-onset photoreceptor cell degeneration involving initially the rods but eventually the cones as well. Homozygous nba fish display an early-onset neuronal degeneration throughout the retina and elsewhere in the central nervous system. As a result, animals develop with small eyes and die by 4–5 days postfertilization (pf). These latter data indicate that the mutation affecting nba fish is not in a photoreceptor cell-specific gene.

Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex

Neurons undergoing targeted photolytic cell death degenerate by apoptosis. Clonal, multipotent neural precursor cells were transplanted into regions of adult mouse neocortex undergoing selective degeneration of layer II/III pyramidal neurons via targeted photolysis. These precursors integrated into the regions of selective neuronal death; 15 ± 7% differentiated into neurons with many characteristics of the degenerated pyramidal neurons. They extended axons and dendrites and established afferent synaptic contacts. In intact and kainic acid-lesioned control adult neocortex, transplanted precursors differentiated exclusively into glia. These results suggest that the microenvironmental alterations produced by this synchronous apoptotic neuronal degeneration in adult neocortex induced multipotent neural precursors to undergo neuronal differentiation which ordinarily occurs only during embryonic corticogenesis. Studying the effects of this defined microenvironmental perturbation on the differentiation of clonal neural precursors may facilitate identification of factors involved in commitment and differentiation during normal development. Because photolytic degeneration simulates some mechanisms underlying apoptotic neurodegenerative diseases, these results also suggest the possibility of neural precursor transplantation as a potential cell replacement or molecular support therapy for some diseases of neocortex, even in the adult.

Pathogenic implications of mutations in the tau gene in pallido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17

Pallido-ponto-nigral degeneration (PPND) is one of the most well characterized familial neurodegenerative disorders linked to chromosome 17q21–22. These hereditary disorders are known collectively as frontotemporal dementia (FTD) and parkinsonism linked to chromosome 17 (FTDP-17). Although the clinical features and associated regional variations in the neuronal loss observed in different FTDP-17 kindreds are diverse, the diagnostic lesions of FTDP-17 brains are tau-rich filaments in the cytoplasm of specific subpopulations of neurons and glial cells. The microtubule associated protein (tau) gene is located on chromosome 17q21–22. For these reasons, we investigated the possibility that PPND and other FTDP-17 syndromes might be caused by mutations in the tau gene. Two missense mutations in exon 10 of the tau gene that segregate with disease, Asn279Lys in the PPND kindred and Pro301Leu in four other FTDP-17 kindreds, were found. A third mutation was found in the intron adjacent to the 3′ splice site of exon 10 in patients from another FTDP-17 family. Transcripts that contain exon 10 encode tau isoforms with four microtubule (MT)-binding repeats (4Rtau) as opposed to tau isoforms with three MT-binding repeats (3Rtau). The insoluble tau aggregates isolated from brains of patients with each mutation were analyzed by immunoblotting using tau-specific antibodies. For each of three mutations, abnormal tau with an apparent Mr of 64 and 69 was observed. The dephosphorylated material comigrated with tau isoforms containing exon 10 having four MT-binding repeats but not with 3Rtau. Thus, the brains of patients with both the missense mutations and the splice junction mutation contain aggregates of insoluble 4Rtau in filamentous inclusions, which may lead to neurodegeneration.

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1−/− mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1−/− mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Degeneration of neurons, synapses, and neuropil and glial activation in a murine Atm knockout model of ataxia–telangiectasia

Neural degeneration is one of the clinical manifestations of ataxia–telangiectasia, a disorder caused by mutations in the Atm protein kinase gene. However, neural degeneration was not detected with general purpose light microscopic methods in previous studies using several different lines of mice with disrupted Atm genes. Here, we show electron microscopic evidence of degeneration of several different types of neurons in the cerebellar cortex of 2-month-old Atm knockout mice, which is accompanied by glial activation, deterioration of neuropil structure, and both pre- and postsynaptic degeneration. These findings are similar to those in patients with ataxia–telangiectasia, indicating that Atm knockout mice are a useful model to elucidate the mechanisms underlying neurodegeneration in this condition and to develop and test strategies to palliate and prevent the disease.

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription–PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration

Inactivation of glycogen synthase kinase-3β (GSK3β) by S9 phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y216, on GSK3β is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y216 phosphorylation on GSK3β in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y216, GSK3β activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3β and adenoviral-mediated transduction of dominant negative GSK3β constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S9 phosphorylation and inactivation of GSK3β but did not affect Y216 phosphorylation, suggesting that S9 phosphorylation is sufficient to override GSK3β activation by Y216 phosphorylation. Under the conditions examined, increased Y216 phosphorylation on GSK3β was not an autophosphorylation response. In resting cells, Y216 phosphorylation was restricted to GSK3β present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y216-phosphorylated GSK3β selectively increased within the nucleus. In rats, Y216 phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y216 phosphorylation of GSK3β represents an important mechanism by which cellular insults can lead to neuronal death.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.

Nontropic actions of neurotrophins: Subcortical nerve growth factor gene delivery reverses age-related degeneration of primate cortical cholinergic innervation

Normal aging is associated with a significant reduction in cognitive function across primate species. However, the structural and molecular basis for this age-related decline in neural function has yet to be defined clearly. Extensive cell loss does not occur as a consequence of normal aging in human and nonhuman primate species. More recent studies have demonstrated significant reductions in functional neuronal markers in subcortical brain regions in primates as a consequence of aging, including dopaminergic and cholinergic systems, although corresponding losses in cortical innervation from these neurons have not been investigated. In the present study, we report that aging is associated with a significant 25% reduction in cortical innervation by cholinergic systems in rhesus monkeys (P < 0.001). Further, these age-related reductions are ameliorated by cellular delivery of human nerve growth factor to cholinergic somata in the basal forebrain, restoring levels of cholinergic innervation in the cortex to those of young monkeys (P = 0.89). Thus, (i) aging is associated with a significant reduction in cortical cholinergic innervation; (ii) this reduction is reversible by growth-factor delivery; and (iii) growth factors can remodel axonal terminal fields at a distance, representing a nontropic action of growth factors in modulating adult neuronal structure and function (i.e., administration of growth factors to cholinergic somata significantly increases axon density in terminal fields). These findings are relevant to potential clinical uses of growth factors to treat neurological disorders.

Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-XL plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-XL homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

Glial cell line-derived neurotrophic factor but not transforming growth factor beta 3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion.

Glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor beta 3 (TGF-beta 3) are members of the TGF-beta superfamily with high neurotrophic activity on cultured nigral dopamine neurons. We investigated the effects of intracerebral administration of GDNF and TGF-beta 3 on the delayed cell death of the dopamine neurons in the rat substantia nigra following 6-hydroxydopamine lesions of dopaminergic terminals in the striatum. Fluorescent retrograde tracer injections and tyrosine hydroxylase immunocytochemistry demonstrated nigral degeneration with an onset 1 week after lesion, leading to extensive death of nigral neurons 4 weeks postlesion. Administration of recombinant human GDNF for 4 weeks over the substantia nigra at a cumulative dose of 140 micrograms, starting on the day of lesion, completely prevented nigral cell death and atrophy, while a single injection of 10 micrograms 1 week postlesion had a partially protective effect. Continuous administration of TGF-beta 3, starting on the day of lesion surgery, did not affect nigral cell death or atrophy. These findings support the notion that GDNF, but not TGF-beta 3, is a potent neurotrophic factor for nigral dopamine neurons in vivo.

Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates.

Although the gene defect responsible for Huntington disease (HD) has recently been identified, the pathogenesis of the disease remains obscure. One potential mechanism is that the gene defect may lead to an impairment of energy metabolism followed by slow excitotoxic neuronal injury. In the present study we examined whether chronic administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, can replicate the neuropathologic and clinical features of HD in nonhuman primates. After 3-6 weeks of 3-NP administration, apomorphine treatment induced a significant increase in motor activity as compared with saline-treated controls. Animals showed both choreiform movements, as well as foot and limb dystonia, which are characteristic of HD. More prolonged 3-NP treatment in two additional primates resulted in spontaneous dystonia and dyskinesia accompanied by lesions in the caudate and putamen seen by magnetic resonance imaging. Histologic evaluation showed that there was a depletion of calbindin neurons, astrogliosis, sparing of NADPH-diaphorase neurons, and growth-related proliferative changes in dendrites of spiny neurons similar to changes in HD. The striosomal organization of the striatum and the nucleus accumbens were spared. These findings show that chronic administration of 3-NP to nonhuman primates can replicate many of the characteristic motor and histologic features of HD, further strengthening the possibility that a subtle impairment of energy metabolism may play a role in its pathogenesis.

Constitutive activation of phototransduction by K296E opsin is not a cause of photoreceptor degeneration.

The missense mutation Lys-296-->Glu (K296E) in the rhodopsin gene produces an opsin with no chromophore binding site and therefore is not activated by light. Nevertheless, the mutant opsin constitutively activates transducin in vitro and causes photoreceptor degeneration in vivo, possibly by continuously activating the phototransduction cascade, analogous to constant exposure to environmental light. We studied the K296E mutation in eight lines of transgenic mice. Each line developed photoreceptor degeneration with the rate of degeneration increasing monotonically as the ratio of mutant:wild-type opsin mRNA increased. At no time in the course of degeneration was there endogenous light adaptation in the retina as measured by the electroretinogram. The mutant opsin was found to be invariably phosphorylated and stably bound to arrestin. Light-independent activation of transducin was demonstrated only after the removal of arrestin and dephosphorylation of K296E opsin. Thus, K296E opsin in vivo does not activate the phototransduction cascade because it is shut off by photoreceptor inactivation mechanisms. Our data show that the K296E mutation does not cause photoreceptor degeneration by continuous activation of phototransduction.