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Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.

Rod outer segment membrane guanylate cyclase1 (ROS-GC1) is the original member of the membrane guanylate cyclase subfamily whose distinctive feature is that it transduces diverse intracellularly generated Ca(2+) signals in the sensory neurons. In the vertebrate retinal neurons, ROS-GC1 is pivotal for the operations of phototransduction and, most likely, of the synaptic activity. The phototransduction- and the synapse-linked domains are separate, and they are located in the intracellular region of ROS-GC1. These domains sense Ca(2+) signals via Ca(2+)-binding proteins. These proteins are ROS-GC activating proteins, GCAPs. GCAPs control ROS-GC1 activity through two opposing regulatory modes. In one mode, at nanomolar concentrations of Ca(2+), the GCAPs activate the cyclase and as the Ca(2+) concentrations rise, the cyclase is progressively inhibited. This mode operates in phototransduction via two GCAPs: 1 and 2. The second mode occurs at micromolar concentrations of Ca(2+) via S100beta. Here, the rise of Ca(2+) concentrations progressively stimulates the enzyme. This mode is linked with the retinal synaptic activity. In both modes, the final step in Ca(2+) signal transduction involves ROS-GC dimerization, which causes the cyclase activation. The identity of the dimerization domain is not known. A heterozygous, triple mutation -E786D, R787C, T788M- in ROS-GC1 has been connected with autosomal cone-rod dystrophy in a British family. The present study shows the biochemical consequences of this mutation on the phototransduction- and the synapse-linked components of the cyclase. (1) It severely damages the intrinsic cyclase activity. (2) It significantly raises the GCAP1- and GCAP2-dependent maximal velocity of the cyclase, but this compensation, however, is not sufficient to override the basal cyclase activity. (3) It converts the cyclase into a form that only marginally responds to S100beta. The mutant produces insufficient amounts of the cyclic GMP needed to drive the machinery of phototransduction and of the retinal synapse at an optimum level. The underlying cause of the breakdown of both types of machinery is that, in contrast to the native ROS-GC1, the mutant cyclase is unable to change from its monomeric to the dimeric form, the form required for the functional integrity of the enzyme. The study defines the CORD in molecular terms, at a most basic level identifies a region that is critical in its dimer formation, and, thus, discloses a single unifying mechanistic theme underlying the complex pathology of the disease.

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells., Author Summary Hereditary spastic paraplegias (HSPs) are inherited disorders characterized by progressive weakness and spasticity of the legs. In HSP patients, nerve fibers connecting cortical motoneurons with spinal cord neurons are progressively lost. HSP subtype 15 (SPG15) is caused by mutations in ZFYVE26, and is characterized by additional cerebellar symptoms. We show that the Zfyve26 protein is broadly expressed in the brain. At the subcellular level Zfyve26 localizes to an intracellular compartment in the endocytic pathway from the plasma membrane to lysosomes, which is part of the degradative system of the cell. Closely resembling the human disease, mice deficient for Zfyve26 develop a progressive spastic gait disorder with cerebellar symptoms and degeneration of both neurons of the motor cortex and Purkinje cells in the cerebellum. Importantly, this degeneration is characterized by the intracellular accumulation of abnormal deposits, which stain positive for the lysosomal marker Lamp1. As Zfyve26 has been shown to interact with the newly identified adaptor complex AP5, which is supposed to be involved in cargo trafficking in the endolysosomal compartment, endolysosomal dysfunction may be caused by a targeting defect upon disruption of Zfyve26. As highly specialized neurons like cortical motoneurons and cerebellar Purkinje cells degenerate, these neurons appear to be particularly dependent on proper endolysosomal function.