To investigate chaperone-mediated autophagy in the pathogenesis of Parkinson disease (PD).
The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra
Brain-derived neurotrophic factor (BDNF), and full-length and truncated tyrosin kinase B receptor (TrkB) protein expression were examined by Western blotting and immunohistochemistry in the frontal cortex and hippocampus of individuals affected by long-lasting severe Alzheimer disease (AD) and age-matched controls. Since preliminary processing studies in the brains of rats have shown loss of immunoreactivity depending on the postmortem delay in tissue processing and on the type, duration, and temperature of the fixative solution, only human samples obtained up to 6 hours (h) after death for biochemical and morphological studies and fixed by immersion in 4% paraformaldehyde for 24 h for morphological studies were included in the present series. Decreased BDNF and full-length TrkB expression accompanied by increased truncated TrkB expression, as revealed by Western blotting, was observed in the frontal cortex of patients with AD. Immunohistochemistry disclosed reduced BDNF and full-length TrkB immunoreactivity in neurons. BDNF decrease was equally observed in tangle-bearing and non-tangle-bearing neurons, as revealed with double-labeling immunohistochemistry to BDNF and phosphorylated tau or phosphorylated neurofilament epitopes. Full-length TrkB immunoreactivity was largely decreased in tangle-bearing neurons, whereas only moderate decreases occurred in neurons with granulovacuolar degeneration. Strong BDNF immunoreactivity was observed in dystrophic neurites surrounding senile plaques, whereas strong TrkB expression occurred in reactive glial cells, including those surrounding senile plaques. Finally, truncated TrkB immunoreactivity was observed in individual neurons and in reactive glial cells in the cerebral cortex and white matter in AD. These results show decay in the expression of BDNF and TrkB in AD neurons, accompanied by altered BDNF, and full-length and truncated TrkB expression in dystrophic neurites and reactive glial cells, respectively, in this disease. The present results demonstrate selective decline of the BDNF/TrkB neurotrophic signaling pathway in the frontal cortex and hippocampus in AD and provide supplemental data that may be relevant in discussing the suitability of the use of BDNF as a therapeutic agent in patients with AD.
Focal ischemia by middle cerebral artery occlusion (MCAO) results in necrosis at the infarct core and activation of complex signal pathways for cell death and cell survival in the penumbra. Recent studies have shown activation of the extrinsic and intrinsic pathways of caspase-mediated cell death, as well as activation of the caspase-independent signaling pathway of apoptosis in several paradigms of focal cerebral ischemia by transient MCAO to adult rats and mice. The extrinsic pathway (cell-death receptor pathway) is initiated by activation of the Fas receptor after binding to the Fas ligand (Fas-L); increased Fas and Fas-L expression has been shown following focal ischemia. Moreover, focal ischemia is greatly reduced in mice expressing mutated (nonfunctional) Fas. Increased expression of caspase-1, -3, -8, and -9, and of cleaved caspase-8, has been observed in the penumbra. Activation of the intrinsic (mitochondrial) pathway following focal ischemia is triggered by Bax translocation to and competition with Bcl-2 and other members of the Bcl-2 family in the mitochondria membrane that is followed by cytochrome c release to the cytosol. Bcl-2 over-expression reduces infarct size. Cytochrome c binds to Apaf-1 and dATP and recruits and cleaves pro-caspase-9 in the apoptosome. Both caspase-8 and caspase-9 activate caspase-3, among other caspases, which in turn cleave several crucial substrates, including the DNA-repairing enzyme poly(ADP-ribose) polymerase (PARP), into fragments of 89 and 28 kDa. Inhibition of caspase-3 reduces the infarct size, further supporting caspase-3 activation following transient MCAO. In addition, caspase-8 cleaves Bid, the truncated form of which has the capacity to translocate to the mitochondria and induce cytochrome c release. The volume of brain infarct is greatly reduced in Bid-deficient mice, thus indicating activation of the mitochondrial pathway by cell-death receptors following focal ischemia. Recent studies have shown the mitochondrial release of other factors; Smac/DIABLO (Smac: second mitochondrial activator of caspases: DIABLO: direct IAP binding protein with low pI) binds to and neutralizes the effects of the X-linked inhibitor of apoptosis (XIAP). Finally, apoptosis-inducing factor (AIF) translocates to the mitochondria and the nucleus following focal ischemia and produces peripheral chromatin condensation and large-scale DNA strands, thus leading to the caspase-independent cell death pathway of apoptosis. Delineation of the pro-apoptotic and pro-survival signals in the penumbra may not only increase understanding of the process but also help to rationalize strategies geared to reducing brain damage targeted at the periphery of the infarct core.
Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.
An imbalance of tau isoforms containing either three or four microtubule-binding repeats causes frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) in families with intronic mutations in the MAPT gene. Here we report equivalent imbalances at the mRNA and protein levels and increased total tau levels in the brains of subjects with Huntington's disease (HD) together with rod-like tau deposits along neuronal nuclei. These tau nuclear rods show an ordered filamentous ultrastructure and can be found filling the neuronal nuclear indentations previously reported in HD brains. Finally, alterations in serine/arginine-rich splicing factor-6 coincide with tau missplicing, and a role of tau in HD pathogenesis is evidenced by the attenuation of motor abnormalities of mutant HTT transgenic mice in tau knockout backgrounds.
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