Cajal bodies in neurons

Abstract

Cajal is commonly regarded as the father of modern neuroscience in recognition of his fundamental work on the structure of the nervous system. But Cajal also made seminal contributions to the knowledge of nuclear structure in the early 1900s, including the discovery of the "accessory body" later renamed "Cajal body" (CB). This important nuclear structure has emerged as a center for the assembly of ribonucleoproteins (RNPs) required for splicing, ribosome biogenesis and telomere maintenance. The modern era of CB research started in the 1990s with the discovery of coilin, now known as a scaffold protein of CBs, and specific probes for small nuclear RNAs (snRNAs). In this review, we summarize what we have learned in the recent decades concerning CBs in post-mitotic neurons, thereby ruling out dynamic changes in CB functions during the cell cycle. We show that CBs are particularly prominent in neurons, where they frequently associate with the nucleolus. Neuronal CBs are transcription-dependent nuclear organelles. Indeed, their number dynamically accommodates to support the high neuronal demand for splicing and ribosome biogenesis required for sustaining metabolic and bioelectrical activity. Mature neurons have canonical CBs enriched in coilin, survival motor neuron protein and snRNPs. Disruption and loss of neuronal CBs associate with severe neuronal dysfunctions in several neurological disorders such as motor neuron diseases. In particular, CB depletion in motor neurons seems to reflect a perturbation of transcription and splicing in spinal muscular atrophy, the most common genetic cause of infant mortality.

Keywords: Amyotrophic lateral sclerosis; Cajal body; coilin; neurodegeneration; neurons; nucleolus; pre-mRNA splicing; snRNP; spinal muscular atrophy; survival motor neuron.

Figure 1.

Cajal's original drawing of the neuronal nucleus. (A) The scheme shows the nuclear structures identified in pyramidal neurons from the cerebral cortex (“Original drawing at the Cajal Institute, C.S.I.C., Madrid”). Panels B-I illustrate the equivalent nuclear components identified at the present time in mammalian neurons. (B) Nucleolus-attached heterochromatin (“Levi basophilic grume, c”). (C) nucleolus and CB (“argyrophilic nucleolar spherules,” a;” “ground substance, b” “accessory body, d”). (D) Fibrillar centers of the nucleolus (“argyrophilic nucleolar spherules, a”). (E) Nuclear speckles (“hyaline grumes, e”). (F) Nuclear microfoci of active chromatin immunolabeled for the acetylated histone H4 (“neutrophil grains, f”). (G) Nuclear foci of 5′-fluorouridine (5′-FU) incorporation into nascent RNA (“neutrophil grains, f”). (H) Ultrastructural characterization of nuclear foci of 5′-FU incorporation (I) Fine structure of interchromatin granule clusters in a neuron (“hyaline grumes e”). Note that Cajal's drawing shows the nucleus enclosed by 2 parallel lines. (C and E from Lafarga et al., Chromosoma 2009; G from Casafont et al., Acta Neuropathol 2011, reproduced with permission from © Springer).

Figure 2.

Schematic diagram showing the association of CBs with the nucleolus and nuclear speckles or interchromatin granule clusters (IGC). The nucleolus shows its main components: fibrillar centers (FC), dense fibrillar component (DFC), containing active rRNA genes, and the granular component (GC), the site of preribosomal particle assembly.

Figure 3.

Cajal body (“Cajal's cuerpo accesorio”) (A) Original drawing by Cajal showing 2 pyramidal neurons from the human cerebral cortex. The accessory body (a), the nucleolus (b) and nuclear speckles (c) are identified in the nucleus. The cytoplasm contains neurofibrils (“Original drawing at the Cajal Institute, C.S.I.C., Madrid”). (B) Neuron stained with Cajal's reduced silver nitrate method. The nucleolus, accessory body/CB and neurofibrils appear stained (courtesy of Dr. J.M. López-Cepero). (C) Semithin section (1 µm thick) of a sensory ganglion neuron stained with Cajal's method showing sharply defined argyrophilic nucleolar spherules and a nucleolus-attached accessory body/CB. (D) Electron micrograph of a neuronal accessory body/CB. (E) Fine structure of a silver-stained accessory body/CB from a Purkinje neuron. Silver precipitates specifically decorate the coiled threads of the CB. (F) Immunogold electron microscopy localization of coilin on the coiled threads of an accessory body/CB from a neuron. (B and E from Lafarga et al., Chromosoma (2009) reproduced with permission from © Springer; C from Lafarga el al. J Neurosci Meth (1986) reproduced with permission from © Elsevier).

Figure 4.

Cajal bodies in neurons. (A) Dissociated neuronal bodies from a sensory ganglion co-stained for nucleic acids with propidium iodide (PI) and for coilin clearly revealed the distribution of CBs free in the nucleoplasm and associated with the nucleolus. (B) Electron micrograph of a typical neuronal nucleus illustrating a typical nucleolus with numerous fibrillar centers and a CB free in the nucleoplasm. (C-E) Confocal images of double immunostained for coilin in combination with SMN (C), Gemin2 (D) and TMG-cap of spliceosomal snRNPs (E) in mammalian neurons demonstrate the colocalization of these molecular constituents in the CB. (F) Dissociated neurons from the supraoptic nucleus silver stained with Cajal's procedure showing the specific staining of nucleoli and CBs. (G) Immunogold electron microscopy detection of 5′-FU incorporation sites in the nucleolus (No) and euchromatin domains after a 30-min pulse of of 5′-FU. Gold particles are absent from the inner side of the CB. (H) In situ hybridization for the c-fos intron I pre-mRNA (red) in combination with coilin immunolabeling (green) revealed the spatial association of a CB with a gene loci of c-fos following a osmotic stress in supraoptic neurons.

Figure 5.

Loss of CBs in coilin knockout neurons. (A, B) Sensory ganglion neurons from wild-type and coilin knockout mice were silver stained with Cajal's procedure. Note the staining of the nucleolus and CBs in the wild-type neuron and the absence of CBs in the knockout cell. (C, D) Coilin immunostaining in wild-type and knockout sensory ganglion neurons shows typical CBs in the wild-type neurons and the lack of coilin staining in the knockout cells.

Figure 6.

Association of the CB with the neuronal nucleolus. (A) Double immunostaining for coilin and fibrillarin illustrates the close association of CBs with nucleoli in a sensory ganglion neuron. (B) Electron micrograph of a nucleolus (No) attached CB. Note the association of the CB with the dense fibrillar component of the nucleolus. (C) Electron micrograph of 2 nucleoli physically linked with a CB. (inset) Similar confocal picture co-stained for fibrillarin and coilin. (D) Double immunogold electron microscopy labeling for coilin and SMN (arrowheads) of a nucleolus-attached CB. (E) Electron micrograph of a CB immunogold labeled for coilin connecting the nucleolus (No) with an interchromatin granule cluster (IGC). Note the presence of an amorphous material associated with the CB (asterisk). (F) Confocal picture of a neuronal nucleus illustrating the distribution of nuclear speckles, immunostained for the TMG-cap, and their association with CBs immunolabeled for coilin. (G) Electron micrograph showing the association of an interchromatin granule cluster (IGC) with a CB. (H) High voltage electron micrograph from a resinless preparation of neuronal nucleus illustrates the spatial association of an interchromatin granule cluster (IGC) with a CB. (E from Lafarga et al., J. Neurocytol 1998, reproduced with permission from © Springer).

Figure 7.

Reorganization of Cajal bodies in Purkinje cells of the pcd mice and motor neurons from SMA. (A-C) Confocal microscopy images from squash preparations of Purkinje cells from wild type (A) and pcd mice (B, C) co-stained for fibrillarin and coilin (A, B) or single immunostained for coilin (C). (A) Fibrillarin and coilin colocalize in a CB from a control Purkinje cell. (B, C) At advanced stages of Purkinje cell degeneration, with nucleolar segregation and fragmentation, coilin appears redistributed as a thin ring surrounding the segregated masses of fibrillarin or inside the nucleolus (No). (D) In wild type Purkinje cells, CBs show the typical morphology of coiled threads immunogold labeled for coilin. (E) CB with an irregular morphology and loosely arranged threads in a degenerating Purkinje cell. (F) Gold particles of coilin immunoreactivity are also observed surrounding an electrondense mass presumably corresponding to a segregated portion of the dense fibrillar component of the nucleolus. (G-I) Double immunostained for coilin and SMN on dissociated motor neurons from control and SMA samples. (G) In a control neuron coilin concentrates in several large CBs and numerous mini-CBs where it colocalizes with SMN (inset). (H) This motor neuron from an SMA patient shows a large CB immunolabeled for coilin and SMN and numerous coilin-positive and SMN-negative mini-CBs (inset). (I) SMA motor neuron with an eccentric nucleus, intranucleolar accumulation of coilin and numerous coilin microfoci free of SMN. (inset) Detail of a coilin microfocus with immunogold electron microscopy. (J-L) Motor neurons from a wild type mouse (J) and an SMA mouse model (K and L). (J, K) Co-staining for coilin and propidium iodide (PtdIns) illustrates the typical organization of CBs in the control neuron (J) and the redistribution of coilin as perinucleolar caps in the SMA motor neuron (K). (L) Intranucleolar localization of coilin (No) in an SMA motor neuron. (A; B, D-F, from Baltanas et al., Brain Pathol 2011, reproduced with permission from © John Wiley and Sons; G-I from Tapia et al. Histochem Cell Biol 2012, reproduced with permission from © Springer.