Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are characterized by the presence of misfolded proteins, thought to trigger neurotoxicity. Some familial forms of ALS (fALS), clinically indistinguishable from sporadic ALS (sALS), are linked to superoxide dismutase 1 (SOD1) gene mutations. It has been shown that the mutant SOD1 misfolds, forms insoluble aggregates and impairs the proteasome. Using transgenic G93A-SOD1 mice, we found that spinal cord motor neurons, accumulating mutant SOD1 also over-express the small heat shock protein HspB8. Using motor neuronal fALS models, we demonstrated that HspB8 decreases aggregation and increases mutant SOD1 solubility and clearance, without affecting wild-type SOD1 turnover. Notably, HspB8 acts on mutant SOD1 even when the proteasome activity is specifically blocked. The pharmacological blockage of autophagy resulted in a dramatic increase of mutant SOD1 aggregates. Immunoprecipitation studies, performed during autophagic flux blockage, demonstrated that mutant SOD1 interacts with the HspB8/Bag3/Hsc70/CHIP multiheteromeric complex, known to selectively activate autophagic removal of misfolded proteins. Thus, HspB8 increases mutant SOD1 clearance via autophagy. Autophagy activation was also observed in lumbar spinal cord of transgenic G93A-SOD1 mice since several autophago-lysosomal structures were present in affected surviving motor neurons. Finally, we extended our observation to a different ALS model and demonstrated that HspB8 exerts similar effects on a truncated version of TDP-43, another protein involved both in fALS and in sALS. Overall, these results indicate that the pharmacological modulation of HspB8 expression in motor neurons may have important implications to unravel the molecular mechanisms involved both in fALS and in sALS.

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By light microscopic immunocytochemistry it has been previously shown that approximately equal to 70% of the neurons in rat dorsal root ganglia are labeled with an antiserum for glutamate conjugated to hemocyanin; the smaller among these neurons are also positive for substance P. By using a postembedding ImmunoGold method and electron microscopy, it is shown here that synaptic terminals in the superficial laminae of the spinal cord of rats selectively stain for the same glutamate antiserum. Immunolabeling is in small dome-shaped and in large scalloped synaptic terminals. Scalloped terminals are of two types. One type consists of dark terminals with many agranular vesicles of different size and a few large granular vesicles; these are probably endings of unmyelinated and small myelinated primary afferent fibers. The other type consists of light terminals with small agranular vesicles homogeneous in size with neurofilaments and many mitochondria; these are probably endings of larger myelinated primary afferent fibers. By means of double-labeling electron microscopic immunocytochemistry with colloidal gold particles of two different sizes, it is also shown here that substance P is present in only the dark type of glutamate-labeled scalloped terminals. The primary afferent origin of the terminals labeled by the antisera for glutamate and for substance P is demonstrated by a triple-labeling strategy: immunocytochemistry for both antisera on sections from rats in which dorsal rhizotomy or dorsal root ganglion injection of horseradish peroxidase conjugated to wheat germ agglutinin was performed. It is proposed that glutamate is the neurotransmitter in primary afferents mediating input from different peripheral receptor classes, including nociceptors. Effects of glutamate and substance P on spinal dorsal horn neurons may result from co-release of these two mediators from the same dorsal root afferent terminal.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by motoneuron loss. Some familial cases (fALS) are linked to mutations of superoxide dismutase type-1 (SOD1), an antioxidant enzyme whose activity is preserved in most mutant forms. Owing to the similarities in sporadic and fALS forms, mutant SOD1 animal and cellular models are a useful tool to study the disease. In transgenic mice expressing either wild-type (wt) human SOD1 or mutant G93A-SOD1, we found that wtSOD1 was present in cytoplasm and in nuclei of motoneurons, whereas mutant SOD1 was mainly cytoplasmic. Similar results were obtained in immortalized motoneurons (NSC34 cells) expressing either wtSOD1 or G93A-SOD1. Analyzing the proteasome activity, responsible for misfolded protein clearance, in the two subcellular compartments, we found proteasome impairment only in the cytoplasm. The effect of G93A-SOD1 exclusion from nuclei was then analyzed after oxidative stress. Cells expressing G93A-SOD1 showed a higher DNA damage compared with those expressing wtSOD1, possibly because of a loss of nuclear protection. The toxicity of mutant SOD1 might, therefore, arise from an initial misfolding (gain of function) reducing nuclear protection from the active enzyme (loss of function in the nuclei), a process that may be involved in ALS pathogenesis.

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Apoptosis is a form of naturally occurring cell death that plays a fundamental role during development and is characterized by internucleosomal DNA fragmentation. In this study we used specific in situ labeling of DNA breaks (Gavrieli et al. [1992] J. Cell. Biol. 119:493-501) to analyze the distribution of apoptotic cells in rat cerebral cortex and thalamus at different developmental stages from embryonic day 16 to adulthood. Control experiments and electron microscopy confirmed that the reaction product was confined to the nucleus of selected cells. Plotting and counting of labeled nuclei in counterstained paraffin sections showed that apoptosis occurred mainly during the first postnatal week and was absent in embryonic and adult samples. In the cortex, the number of apoptotic cells progressively increased from birth to the first postnatal week, with a peak between postnatal (P) day 5 and P8, and subsequently decreased. At the time of maximal expression of apoptosis, labeled nuclei were present mainly in layer VIb and underlying white matter and at the border between cortical plate and layer I. Only a few apoptotic cells were found scattered in the thalamus, without a particular concentration in selected areas, but with a peak at P5. Differences in the number of apoptotic cells between cortex and thalamus suggest that apoptotic cell death may have a different functional significance in the two brain areas.