The survival motor neuron (SMN) gene is the putative disease gene for human spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by progressive degeneration of lower motor neurons. Two copies of the gene, centromeric and telomeric, are present in the same 5q13 chromosomal region in humans. However, only the telomeric gene is affected in SMA. The SMN gene(s) encode(s) a novel protein of unknown function. To gain insights into the role of SMN in neurons, we have identified the SMN gene ortholog in the rat, and investigated SMN expression in the CNS of rat, monkey and humans by immunocytochemistry and in situ hybridization experiments. Antibodies against the SMN amino-terminus specifically recognized a single protein identical to the in vitro translation products of human and rat SMN cDNAs. The SMN gene transcript and product were widely but unevenly expressed throughout cerebral and spinal cord areas. The SMN protein was localized mainly in the cytoplasm of specific neuronal systems, and it was particularly expressed in lower motor neurons of newborn and adult animals. Likewise, a strong hybridization signal was detected in lamina IX of the spinal ventral horn. These results support the relevance of SMN for the motor neuron function and the pathogenetic role of the SMN gene in the neuronal degeneration associated with SMA.
The subcellular localization of the survival motor neuron (SMN) protein, encoded by the spinal muscular atrophy determining gene, was investigated in motor neurons of the developing and adult rat spinal cord by light and electron microscopy immunocytochemistry. The experiments were carried out with a panel of anti-SMN antibodies, all recognizing an SMN-specific protein band at 39 kDa in HeLa cells and rat spinal cord protein extracts. SMN protein expression decreased during postnatal spinal cord development, but it remained unchanged in distribution and intensity in motor neurons at all ages examined. SMN protein was mainly organized in immunoreactive aggregates sparse in the nucleoplasm and cytoplasm of both mature and developing motor neurons, and it was more rarely localized within the endoplasmic reticulum and in apposition to the external mitochondrial membrane. Most strikingly, the SMN protein was found in association with cytoskeletal elements in spinal dendrites and axons, where it was particularly evident during postnatal development. The present findings suggest that SMN protein may be transported via axoplasmic flow in maturing neurons. Given the RNA-binding activity of SMN, the SMN protein could be involved in the transport of specific mRNAs in axons and dendrites of motor neurons. The reduced transport of specific mRNAs within motor neurons during development could play a role in the motoneuronal degeneration and impaired axonal sprouting observed in spinal muscular atrophy.
In order to investigate the existence of anatomical subdivisions within the thalamic reticular nucleus (Rt), the distribution of reticular neurons expressing the calcium binding protein calretinin was investigated in the rat by means of immunocytochemistry. Calretinin immunoreactive (Cr-ir) neurons were mainly distributed in the lateral and ventral regions, and along the medial border of the Rt rostral pole. Caudal to the rostral pole, many neurons were Cr-ir in the more dorsal part of the rostral two-thirds (the "dorsal cap") of the Rt. Fewer Cr-ir neurons were present more caudally along the lateral and medial borders, and in the caudalmost part of the nucleus, related to the acoustic thalamus. The distribution of Cr-ir neurons in the rostral Rt was compared with that of neurons projecting to the ipsilateral and contralateral anterior, intralaminar, midline, and mediodorsal nuclei, or to the contralateral rostral Rt. The retrograde transport of Fluorogold revealed a remarkably precise topography of the rostral Rt: different reticular areas were found to project to different thalamic nuclei, or to different rostrocaudal or mediolateral portions of the same thalamic nucleus, with a limited degree of overlap. The double-labeling experiments demonstrated that the reticular neurons projecting to the ipsilateral anterodorsal, midline, mediodorsal, and anterior intralaminar nuclei frequently expressed calretinin; by contrast, the majority of the reticular commissural neurons did not express the protein, with the exception of neurons projecting to the contralateral mediodorsal and midline nuclei. The ipsilaterally projecting calretinin-positive neurons were frequently located along the medial edge of the rostral pole and in the dorsal cap of the nucleus, segregated from the commissural calretinin-negative neurons. The combined analysis of calretinin expression patterns and tract tracing data provided further insight in the anatomical organization of the thalamic reticular nucleus, suggesting a different neurophysiological role for the ipsilaterally vs. the contralaterally projecting reticular neurons in the modulation of the synaptic activity of the dorsal thalamus.
To further characterize the communication between the thalami of the two hemispheres, a connection linking the rostral reticular nuclei of the two thalamic sides was investigated in the rat by retrograde and anterograde tracing. The rostral reticular nucleus can be divided into a medial region, with densely packed fusiform neurons, and a lateral region, with less densely packed, polymorphic neurons. After injections of Fluorogold (FG) in the medial region, retrogradely labeled, small fusiform neurons were found in the corresponding contralateral region. The retrograde labeling data were confirmed by the anterograde-tracing experiments. Thin, beaded axons, anterogradely labeled after injection of biocytin or biotinylated dextranamine in the medial region, innervate the corresponding region in the contralateral reticular nucleus. The present data suggest the existence of a commissural pathway specifically devoted to the crosstalk between the rostral reticular nuclei of the two thalamic sides. The commissural gamma aminobutyric acid (GABA)-ergic input on the GABAergic neurons of the rostral reticular nucleus could modulate the generation of sleep spindles. The reticuloreticular pathway may, moreover, synchronize the diffuse modulatory effect of the rostral reticular nucleus on nonprimary cortical areas through the bilateral projections of the nucleus to the ventromedial, intralaminar, and anterior thalamic nuclei.
Abstract:The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only~2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron-specific kinesin is up-regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule-based transport during the maturation of neuronal cells in vivo. Key Words: Microtubule-based motors-Conventional kinesin isoforms-Quantitative protein expression-Immunocytochemical localization-Brain development.
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