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Inclusion body myositis, the most common muscle disorder in the elderly, is partly characterized by abnormal expression of amyloid precursor protein (APP) and intracellular accumulation of its proteolytic fragments collectively known as β-amyloid. The present study examined the effects of β-amyloid accumulation on mitochondrial structure and function of skeletal muscle from transgenic mice (MCK-βAPP) engineered to accumulate intramyofiber β-amyloid. Electron microscopic analysis revealed that a large fraction of myofibers from 2-3-month-old MCK-βAPP mice contained numerous, heterogeneous alterations in mitochondria, and other cellular organelles. [1H-decoupled]13C NMR spectroscopy showed a substantial reduction in TCA cycle activity and indicated a switch from aerobic to anaerobic glucose metabolism in the MCK-βAPP muscle. Isolated muscle fibers from the MCK-βAPP mice also exhibited a reduction in cytoplasmic pH, an increased rate of ROS production, and a partially depolarized plasmalemma. Treatment of MCK-βAPP muscle cells with Ru360, a mitochondrial Ca2+ uniporter antagonist, reversed alterations in the plasmalemmal membrane potential (Vm) and pH. Consistent with altered redox state of the cells, treatment of MCK-βAPP muscle cells with glutathione reversed the effects of β-amyloid accumulation on Ca2+ transient amplitudes. We conclude that structural and functional alterations in mitochondria precede the reported appearance of histopathological and clinical features in the MCK-βAPP mice and may represent key early events in the pathogenesis of inclusion body myositis. [ABSTRACT FROM AUTHOR]

Abstract: Striatal degeneration occurs through unknown mechanisms in certain neurodegenerative disorders characterized by increased and sustained synaptic levels of dopamine. In the present studies, we examined the effects of treatment of SK-N-MC neuroblastoma cells with dopamine to understand the participation of dopamine D1 receptor in postsynaptic cytotoxicity. Treatment of SK-N-MC cells either with dopamine or the D1 receptor agonist SKF R-38393 resulted in a significant increase in the production of reactive oxygen species (by ∼2.75-fold) and cell death (∼50%), while antagonism of the D1 receptor with SCH 23390 significantly reversed (to ∼75% of control level) these effects. Accumulation of cAMP in dopamine treated cells (t 1/2 =1.5h) preceded changes in ionic gradient (t 1/2 =6.5h), as measured by intracellular potassium concentration and leakage of cytochrome c into the cytosol (t 1/2 =13h), suggesting a possible staging of toxic events as a result of activation of D1 receptor by dopamine. Examination of cellular metabolic properties with 13C NMR spectroscopy showed an inhibitory effect on tricarboxylic acid cycle metabolism via D1-mediated receptors after treatment with dopamine, suggesting a direct role for D1 receptor in dopamine-induced postsynaptic cell death. The present studies provide novel insight into a possible patho-physiological staging of cytotoxic events that are mediated by activation of D1 receptor. [Copyright &y& Elsevier]

α-Synuclein aggregates have been linked to the pathogenesis of Parkinson''s disease (PD), with Lewy bodies (LBs) and Lewy neurites (LNs) constituting the pathological hallmarks in the brains of patients with PD and dementia with LBs. LBs are formed by the conversion of soluble monomers of α-synuclein into insoluble aggregates. Here we report an abnormal electrophoretic mobility, at a higher molecular weight (MW) than the expected theoretical MW, of both recombinant histidine-tagged human α-synuclein, human α-synuclein expressed in SH-SY5Y human neuroblastoma cells or Ltk- fibroblasts, and rat brain α-synuclein, on SDS–PAGE polyacrylamide, but not on Nu-PAGE gradient peptide, gels, suggesting possible α-synuclein data misinterpretations associated with gel electrophoresis. These studies raise important considerations about the type of protein gel electrophoresis system suitable to study the alterations of α-synuclein associated with neurodegeneration, PD and other synucleinopathies. [Copyright &y& Elsevier]

Alzheimer disease is characterized by extracellular β-amyloid (Aβ) plaques and intracellular inclusions containing neurofibrillary tangles of phospho-Tau and intraneuronal Aβ associated with neuronal cell death. We generated a novel gene transfer animal model using lentiviral Aβ1-42 that resulted in intracellular but not extracellular Aβ accumulations in the targeted rat primary motor cortex. Expression of intracellular Aβ1-42 led to pathological changes seen in human Alzheimer disease brains, including cell death, inflammatory signs, activation of two Tau kinases, and Tau hyperphosphorylation. Promoting clearance of lentiviral Aβ1-42 reversed these effects, demonstrating that intraneuronal Aβ1-42 is a toxic peptide that lies upstream of Tau modification. These studies reveal the role of intracellular Aβ1-42 in a novel gene transfer animal model, which is a useful tool to study intraneuronal Aβ1-42-induced pathology in the absence of extracellular plaques. Targeted delivery of Aβ will allow speedy delineation of pathological mechanisms associated with specific neurodegenerative lesions. [ABSTRACT FROM AUTHOR]

Mutations in the ubiquitin ligase-encoding Parkin gene have been implicated in the pathogenesis of autosomal recessive Parkinson disease. Outside of the central nervous system, Parkin is prominently expressed in skeletal muscle. We have found accumulations of Parkin protein in skeletal muscle biopsies taken from patients with inclusion body myositis, a degenerative disorder in which intramyofiber accumulations of the β-amyloid peptide are pathognomonic. In comparing primary cultures of skeletal muscle derived from parkin knock-out and wild-type mice, we have found the absence of parkin to result in greater sensitivity to mitochondrial stressors rotenone and carbonyl cyanide 3-chlorophenylhydrazone, without any alteration in sensitivity to calcium ionophore or hydrogen peroxide. Utilizing viral expression constructs coding for the Alzheimer disease and inclusion body myositis-linked β-amyloid precursor protein and for its metabolic byproducts Aβ42 and C100, we found that parkin knock-out muscle cells are also more sensitive to the toxic effects of intracellular Aβ. We also constructed a lentiviral system to overexpress wild-type Parkin and have shown that boosting the levels of parkin expression in normal skeletal muscle cultures provides substantial protection against both mitochondrial toxins and overexpressed β-amyioid. Correspondingly, exogenous Parkin significantly lowered Aβ levels. These data support the hypothesis that in myocytes parkin has dual properties in the maintenance of skeletal muscle mitochondrial homeostasis and in the regulation of Aβ levels. [ABSTRACT FROM AUTHOR]

The importance of E3 ubiquitin ligases, involved in the degradation of misfolded proteins or promotion of protein-protein interaction, is increasingly recognized in neurodegeneration. TDP-43 is a predominantly nuclear protein, which regulates the transcription of thousands of genes and binds to mRNA of the E3 ubiquitin ligase Parkin to regulate its expression. Wild type and mutated TDP-43 are detected in ubiquitinated forms within the cytosol in several neurodegenerative diseases. We elucidated the mechanisms of TDP-43 interaction with Parkin using transgenic A315T mutant TDP-43(TDP43-Tg) mice, lentiviral wild type TDP-43, and Parkin gene transfer rat models. TDP-43 expression increased Parkin mRNA and protein levels. Lentiviral TDP-43 increased the levels of nuclear and cytosolic protein, whereas Parkin co-expression mediated Lys-48 and Lys-63-linked ubiquitin to TDP-43 and led to cytosolic co-localization of Parkin with ubiquitinated TDP-43. Parkin and TDP-43 formed a multiprotein complex with HDAC6, perhaps to mediate TDP-43 translocation. In conclusion, Parkin ubiquitinates TDP- 43 and facilitates its cytosolic accumulation through a multiprotein complex with HDAC6. [ABSTRACT FROM AUTHOR]

Abstract: Alzheimer’s disease (AD) is a neurodegenerative aging disorder characterized by extracellular Aβ plaques and intraneuronal neurofibrillary tangles. We conducted longitudinal studies to examine the effects of Aβ on brain amino acid metabolism in lentiviral Aβ1–42 gene transfer animals and transgenic AD mice. We also performed lentiviral parkin gene delivery to determine the effects of Aβ clearance in AD models. Aβ1–42 activated mTOR signaling, and increased 4E-BP phosphorylation. Aβ1–42 increased the synthesis of glutamate and aspartate, but not glutamine, leucine and isoleucine, but an increase in leucine and isoleucine levels was concurrent with diminution of neurotransmitters. Additionally, Aβ1–42 attenuated mitochondrial tricarboxylic acid (TCA) cycle activity and decreased synthesis of its by-products. Glutamate levels increased prior to lactate accumulation, suggesting oxidative stress. Importantly, parkin reversed the effects of Aβ1–42 on amino acid levels, prevented TCA cycle impairment and protected against glutamate toxicity. Cortical atrophy was observed in aged 3xTg-AD mice, while parkin expression was associated with reduced atrophy. Similarly, Aβ1–42 resulted in significant cell loss, pronounced astrogliosis and cortical atrophy and parkin reduced astrogliosis and reversed Aβ1–42 effects on cell loss and cortical atrophy. Taken together these data suggest that parkin prevents amyloid-induced alteration of brain metabolism and may be used as a therapeutic target to limit neuronal loss in AD. [Copyright &y& Elsevier]

Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with loss of motor neurons in the brain and spinal cord. ALS is occasionally diagnosed with frontotemporal lobar dementia with ubiquitin-positive inclusions (FTLD-U). Alzheimer''s disease (AD) is the most common type of age-associated dementia. Abnormal levels of aggregated Tar-DNA binding protein-43 (TDP-43) are detected in the majority of patients with ALS, FTLD and AD. We observed a significant increase (200%) in the levels of TDP-43 in cortical autopsies of late stage AD patients. Lentiviral expression of Aβ1–42 in the rat motor cortex led to an increase in TDP-43 pathology, including up-regulation of the mature ~44kDa protein, identical to the pathological changes seen in AD. Furthermore, expression of Aβ1–42 was associated with TDP-43 phosphorylation and accumulation in the cytosol. Clearance of Aβ with parkin prevented TDP-43 pathology. TDP-43 modifications were also observed in 3xTransgenic AD (3xTg-AD) compared to wild type mice, but these changes were attenuated in parkin-injected hippocampi, even in the presence of Tau pathology, suggesting that TDP-43 pathology is triggered by Aβ, independent of Tau. Increased levels of casein kinase (CK1 and CK2), which are associated with TDP-43 phosphorylation, were also observed in Aβ1–42 expressing brains. These data indicate an overlap in TDP-43 pathology between AD and ALS-FTLD and suggest that Aβ triggers modifications of TDP-43. [Copyright &y& Elsevier]