Little is known about molecular changes occurring within retinal ganglion cells (RGCs) before their death in glaucoma. Taking advantage of the fact that γ-synuclein (Sncg) mRNA is expressed specifically and highly in adult mouse RGCs, we show in the DBA/2J mouse model of glaucoma that there is not only a loss of cells expressing this gene, but also a downregulation of gene expression of Sncg and many other genes within large numbers of RGCs. This downregulation of gene expression within RGCs occurs together with reductions in FluoroGold (FG) retrograde transport. Surprisingly, there are also large numbers of Sncg-expressing cells without any FG labeling, and among these many that have a marker previously associated with disconnected RGCs, accumulation of phosphorylated neurofilaments in their somas. These same diseased retinas also have large numbers of RGCs that maintain the intraocular portion while losing the optic nerve portion of their axons, and these disconnected axons terminate within the optic nerve head. Our data support the view that RGC degeneration in glaucoma has two separable stages: the first involves atrophy of RGCs, whereas the second involves an insult to axons, which causes the degeneration of axon portions distal to the optic nerve head but does not cause the immediate degeneration of intraretinal portions of axons or the immediate death of RGCs.

https://www.jneurosci.org/content/jneuro/28/2/548.full.pdf

Retinal Ganglion Cells Downregulate Gene Expression and Lose Their Axons within the Optic Nerve Head in a Mouse Glaucoma Model

A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung's disease

Diagnosis of Hirschsprung's disease: a prospective, comparative accuracy study of common tests.

Intermediate filament (IF) proteins, a large family of tissue specific proteins, undergo several posttranslational modifications, with phosphorylation being the most studied modification. IF protein phosphorylation is highly dynamic and involves the head and/or tail domains of these proteins, which are the domains that impart most of the structural heterogeneity and hence presumed tissue specific functions. Although the function of IF proteins remains poorly understood, several regulatory roles for IF protein phosphorylation have been identified or are emerging. Those roles include filament disassembly and reorganization, solubility, localization within specific cellular domains, association with other cytoplasmic or membrane associated proteins, protection against physiologic stress and mediation of tissue-specific functions. Understanding the mechanistic and functional aspects of IF protein phosphorylation is providing insights not only regarding the function of this modification, but also regarding the function of IF proteins.

Implications of intermediate filament protein phosphorylation.

Reproducible visualization of neurons and glia in human brain is essential for quantitative studies of the cellular changes in neurological disease. However, immunohistochemistry in human brain specimens is often compromised because of prolonged fixation. To select cell lineage-specific antibodies for quantitative studies of neurons and the major types of glia, we used 29 different antibodies, different epitope retrieval methods, and different detection systems to stain tissue arrays of formalin-fixed human brain. The screening pointed at CD45/leukocyte common antigen (LCA), CD68(KP1), 2',3' cyclic nucleotide phosphatase (CNPase), glial fibrillary acidic protein (GFAP), HLA-DR, Ki67, neuronal nuclei (NeuN), p25alpha-antigen, and S100beta as candidates for future cell counting purposes, because these markers visualized specific neuronal and glial cell bodies. However, significant negative correlation between staining result and formalin fixation was observed by blinded scoring of staining for CD45/LCA, CNPase, GFAP, and NeuN in brain specimens fixed by immersion and stored up to 10 years in 4% formalin solution at room temperature, independent of donor sex and postmortem interval. In contrast, improved preservation of NeuN and CNPase staining, and full preservation of GFAP and CD45/LCA staining in tissue fixed by perfusion and stored for up to 3 years in 0.1% paraformaldehyde solution at 4C, indicated that immunohistochemistry can be performed in well-preserved biobank material.

F.J.W. ten Kate

Papers

Diagnosis of congenital neurogenic abnormalities of the bowel with monoclonal anti-neurofilament antibodies

Differences in phosphorylation state of neurofilament proteins in ganglionic and aganglionic bowel segments of children with Hirschsprung's disease.

Disturbed defecation after colectomy for aganglionosis investigated with monoclonal antineurofilament antibody.