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Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across 6 species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varied dramatically across different brain regions in mice, cynomolgus macaques, and humans. PrP expression did not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP was lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55%, and D178N just 31%, of the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA was reflected in brain parenchyma PrP and, in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.

Retinitis pigmentosa 1 (RP1) is a common inherited retinopathy with variable onset and severity. TheRP1gene encodes a photoreceptor-specific, microtubule-associated ciliary protein containing the doublecortin (DCX) domain. Here we show that another photoreceptor-specific Rp1-like protein (Rp1L1) in mice is also localized to the axoneme of outer segments (OSs) and connecting cilia in rod photoreceptors, overlapping with Rp1.Rp1L1−/− mice display scattered OS disorganization, reduced electroretinogram amplitudes, and progressive photoreceptor degeneration, less severe and slower than inRp1−/− mice. In single rods ofRp1L1−/−, photosensitivity is reduced, similar to that ofRp1−/−. While individual heterozygotes are normal, double heterozygotes ofRp1andRp1L1exhibit abnormal OS morphology and reduced single rod photosensitivity and dark currents. The electroretinogram amplitudes of double heterozygotes are more reduced than those of individual heterozygotes combined. In support, Rp1L1 interacts with Rp1 in transfected cells and in retina pull-down experiments. Interestingly, phototransduction kinetics are normal in single rods and whole retinas of individual or doubleRp1andRp1L1mutant mice. Together, Rp1 and Rp1L1 play essential and synergistic roles in affecting photosensitivity and OS morphogenesis of rod photoreceptors. Our findings suggest that mutations inRP1L1could underlie retinopathy or modify RP1 disease expression in humans.

Mouse retina undergoes crucial changes during early postnatal development. By using Affymetrix microarrays, we analyzed gene expression profiles of wild-type 129SvEv/C57BL/6 mouse retinas at postnatal days (P) 7, 10, 14, 18, and 21 and found significantly altered expression of 355 genes. Characterization of these 355 genes provided insight into physiologic and pathologic processes of mouse retinal development during the second and third postnatal weeks, a period that corresponds to human embryogenesis between weeks 12 and 28. These genes formed 6 groups with similar change patterns. Among the genes, sixteen cause retinal diseases when mutated; most of these 16 genes were upregulated in retina during this period. Using the PathArt program, we identified the biological processes in which many of the 355 gene products function. Among the most active processes in the P7-P21 retina are those involved in neurogenesis, obesity, diabetes type II, apoptosis, growth and differentiation, and protein kinase activity. We examined the expression patterns of 58 genes in P7 and adult retinas by searching the Brain Gene Expression Map database. Although most genes were present in various cell types in retinas, many displayed high levels of expression specifically in the outer nuclear, inner nuclear, and/or ganglion cell layers. By combining our 3 analyses, we demonstrated that during this period of mouse retinal development, many genes play important roles in various cell types, multiple pathways are involved, and some genes in a pathway are expressed in coordinated patterns. Our results thus provide foundation for future detailed studies of specific genes and pathways in various genetic and environmental conditions during retinal development.

To understand the mechanisms underlying autosomal dominant progressive retinitis pigmentosa (RP) caused by the mutations of the RP1 gene and to identify molecules that play roles in the early disease process, we used Affymetrix U74Av2 microarrays to compare the gene expression profiles of retinas from Rp1-/- and Rp1+/+ mice at postnatal days (P) 7, 10, 14, 18 and 21. These profiles were independently verified by comparison with results of retinal serial analysis of gene expression, U74Av2 array studies of mouse retinas, real-time PCR and in situ hybridization. We found that the disruption of Rp1 significantly affected the expression of multiple clusters of genes whose products were involved in diverse biological pathways. The molecular responses to the disruption of Rp1 changed dramatically during development and were distinct from responses to the disruption of photoreceptor transcription factors (Crx-/- or Nrl-/-) and a phototransduction molecule (Pde6brd1). We found specific alterations of gene expression in the c-Jun N-terminal kinase (JNK) signaling cascades. Western analysis confirmed that the phosphorylation of key members in the JNK signaling cascades (i.e. JNK1, JNK2, MAP2, MKK4 and c-Jun) is reduced, whereas phospho-ERK and phospho-p38 are unchanged, in Rp1-/- retinas at P18-21. Immunostaining demonstrated that, like Rp1, phospho-JNKs and phospho-MAP2 are present in outer segments of photoreceptors. Our studies reveal unique molecular phenotypes in multiple biological pathways and the specific reduction of JNK signaling cascades in RP1 diseases, and suggest that RP1, a doublecortin-containing microtubule associated protein, and JNK signaling cascades play integral roles in photoreceptor development and maintenance. Our studies further suggest JNK-related therapeutic strategies for RP1 diseases.