Whereas the mammalian retina possesses a repertoire of factors known to establish general retinal cell types, these factors alone cannot explain the vast diversity of neuronal subtypes. In other CNS regions, the differentiation of diverse neuronal pools is governed by coordinately acting LIM-homeodomain proteins including the Islet-class factor Islet-1 (Isl1). We report that deletion of Isl1 profoundly disrupts retinal function as assessed by electroretinograms and vision as assessed by optomotor behavior. These deficits are coupled with marked reductions in mature ON-and OFF-bipolar (Ͼ76%), cholinergic amacrine (93%), and ganglion (71%) cells. Mosaic deletion of Isl1 permitted a chimeric analysis of "wild-type" cells in a predominantly Isl1-null environment, demonstrating a cell-autonomous role for Isl1 in rod bipolar and cholinergic amacrine development. Furthermore, the effects on bipolar cell development appear to be dissociable from the preceding retinal ganglion cell loss, because Pou4f2-null mice are devoid of similar defects in bipolar cell marker expression. Expression of the ON-and OFF-bipolar cell differentiation factors Bhlhb4 and Vsx1, respectively, requires the presence of Isl1, whereas the early bipolar cell marker Prox1 initially did not. Thus, Isl1 is required for engaging bipolar differentiation pathways but not for general bipolar cell specification. Spatiotemporal expression analysis of additional LIM-homeobox genes identifies a LIMhomeobox gene network during bipolar cell development that includes Lhx3 and Lhx4. We conclude that Isl1 has an indispensable role in retinal neuron differentiation within restricted cell populations and this function may reflect a broader role for other LIM-homeobox genes in retinal development, and perhaps in establishing neuronal subtypes.

The mammalian retina is comprised of six major neuronal cell types and is subdivided into more morphological and physiological subtypes. The transcriptional machinery underlying these subtype fate choices is largely unknown. The LIM-homeodomain protein, Isl1, plays an essential role in central nervous system (CNS) differentiation but its relationship to retinal neurogenesis remains unknown. We report here its dynamic spatiotemporal expression in the mouse retina. Among bipolar interneurons, Isl1 expression commences at postnatal day (P)5 and is later restricted to ON-bipolar cells. The intensity of Isl1 expression is found to segregate the pool of ON-bipolar cells into rod and ON-cone bipolar cells with higher expression in rod bipolar cells. As bipolar cell development proceeds from P5-10 the colocalization of Isl1 and the pan-bipolar cell marker Chx10 reveals the organization of ON-center bipolar cell nuclei to the upper portion of the inner nuclear layer. Further, whereas Isl1 is predominantly a ganglion cell marker prior to embryonic day (E)15.5, at E15.5 and later its expression in nonganglion cells expands. We demonstrate that these Isl1-positive, nonganglion cells acquire the expression of amacrine cell markers embryonically, likely representing nascent cholinergic amacrine cells. Taken together, Isl1 is expressed during the maturation of and is later maintained in retinal ganglion cells and subtypes of amacrine and bipolar cells where it may function in the maintenance of these cells into adulthood.

Forebrain cholinergic neurons modulate complex mammalian behaviors such as reward-related learning and cognitive functions. Although their dysfunction is implicated in various psychiatric and neurodegenerative diseases, the factors governing cholinergic neuron differentiation and diversity are mostly unknown. We tested the role of the LIM-homeobox gene Isl1 in the development of forebrain cholinergic neurons by conditionally deleting Isl1 using a Six3-cre transgene. A depletion of cholinergic interneurons in the dorsal and ventral striatum, and cholinergic projection neurons in the nucleus basalis is observed and is ascribed to an early and persistent defect in cholinergic neuron differentiation. Notably, cholinergic innervation to the neocortex is abolished, whereas that to the hippocampus is unaltered. The unique pattern of cholinergic hypoinnervation encountered is supported by the presence of cholinergic projection neurons in the medial septum, the magnocellular preoptic area, and the substantia innominata. Together, these results demonstrate the requirement for Isl1 in the development of restricted telencephalic cholinergic neurons and link the development of cholinergic neurons in anatomically disparate sites to Isl1 function.

Neuronal populations display conspicuous variability in their size among individuals, but the genetic sources of this variation are largely undefined. We demonstrate a large and highly heritable variation in neuron number within the mouse retina, affecting a critical population of interneurons, the horizontal cells. Variation in the size of this population maps to the distal end of chromosome (Chr) 13, a region homologous to human Chr 5q11.1–11.2. This region contains two genes known to modulate retinal cell number. Using conditional knock-out mice, we demonstrate that one of these genes, the LIM homeodomain gene Islet-1 ( Isl1 ), plays a role in regulating horizontal cell number. Genetic differences in Isl1 expression are high during the period of horizontal cell production, and cis -regulation of Isl1 expression within the retina is demonstrated directly. We identify a single nucleotide polymorphism in the 5′ UTR of Isl1 that creates an E-box sequence as a candidate causal variant contributing to this variation in horizontal cell number.

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1-and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identi¢ed reproducible changes in gene expression in the brains of both 10-week-old Cln1-and Cln3-knockout mice as compared to wild-type controls, and con¢rmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations a¡ect the expression of di¡erent, non-overlapping sets of genes. The possible signi¢cance of these changes and the pathological mechanisms underlying NCL diseases are discussed. ß

Purpose To evaluate ocular hypertension (OHT) after Ozurdex injection to determine the incidence of OHT, therapy of OHT, and any associative factors such as diagnosis, underlying glaucoma and therapy, or sequential Ozurdex injection(s). Methods Retrospective consecutive case series with patients receiving one or more intravitreal Ozurdex implantations at a tertiary care academic center. OHT was defined as a single measurement of ≥30 mmHg, or an increase of ≥10 mmHg from baseline. Results Ninety-four injections in 52 patients (59 eyes) were reviewed. Forty eyes received a single injection, and 19 eyes received multiple injections. OHT developed in 14 patients (26.9%). Thirteen patients (25%) had pre-existing glaucoma or glaucoma suspicion, and six of these developed OHT. Glaucoma eye drops were initiated following 13 injections (13.8%). Invasive glaucoma surgery was required in three patients (3.2%): all had glaucoma or glaucoma suspicion (one case was related to neovascular glaucoma and unlikely related to steroid response after Ozurdex). There was no difference in relative IOP increase (i.e. difference between final follow-up or subsequent intravitreal injection versus baseline) between single versus multiple Ozurdex injections (P = 0.883). Conclusions 26.9% of patients who received Ozurdex developed OHT. Glaucoma or glaucoma suspicion factors were present in all patients who required invasive glaucoma surgery. A greater proportion of patients who received multiple injections had an IOP elevation, but the relative IOP increase was not significant.

Batten disease, or juvenile neuronal ceroid lipofuscinosis (JNCL), results from mutations in the CLN3 gene. This disorder presents clinically around the age of five years with visual deficits progressing to include seizures, cognitive impairment, motor deterioration, hallucinations, and premature death by the third to forth decade of life. The motor deficits include coordination and gait abnormalities, myoclonic jerks, inability to initiate movements, and spasticity. Previous work from our laboratory has identified an early reduction in catechol-O-methyltransferase (COMT), an enzyme responsible for the efficient degradation of dopamine. Alterations in the kinetics of dopamine metabolism could cause the accumulation of undegraded or unsequestered dopamine leading to the formation of toxic dopamine intermediates. We report an imbalance in the catabolism of dopamine in three month Cln3 -/-mice persisting through nine months of age that may be causal to oxidative damage within the striatum at nine months of age. Combined with the Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript previously reported inflammatory changes and loss of post-synaptic D1α receptors, this could facilitate cell loss in striatal projection regions and underlie a general locomotion deficit that becomes apparent at twelve months of age in Cln3 -/-mice. This study provides evidence for early changes in the kinetics of COMT in the Cln3 -/-mouse striatum, affecting the turnover of dopamine, likely leading to neuron loss and motor deficits. These data provide novel insights into the basis of motor deficits in JNCL and how alterations in dopamine catabolism may result in oxidative damage and localized neuronal loss in this disorder.

The neuronal ceroid-lipofuscinoses (NCL) are the most common group of progressive neurodegenerative diseases in children, with an incidence as high as one in 12,500 live births. The main features of this disease are failure of psychomotor development, impaired vision, seizures, and premature death. Many biochemical and physiological studies have been initiated to determine the cellular defect underlying the disease, although only a few traits have been truly associated with the disorders. One of the paradox's of the NCL-diseases is the characteristic accumulation of autofluorescent hydrophobic material in the lysosomes of neurons and other cell types. However, the accumulation of this lysosomal storage material, which no doubt contributes to the neurologic disease, does not apparently lead to disease outside the CNS, and how these cellular alterations relate to the neurodegeneration in NCLs is unknown. Mutations have been identified in six distinct genes/proteins, namely CLN1, which encodes PPT1, a protein thiolesterase; CLN2, which encodes TPP1, a serine protease; and CLN3, CLN5, CLN6, and CLN8, which encode novel transmembrane proteins. Mutation in any one of these CLN-proteins results in a distinct type of NCL-disease. However, there are many shared similarities in the pathology of these diseases. The most obvious connection between PPT1, TPP1, CLN3, CLN5, CLN6, and CLN8 is their subcellular localization. To date, three of the four proteins whose subcellular localization has been confirmed, namely PPT1, TPP1, and CLN3, reside in the lysosome. We review the function of the CLN-proteins and discuss the possibility that a disruption in a common biological process leads to an NCL-disease.