Kenneth M. Rosen

The Insulin/Akt Signaling Pathway Is Targeted by Intracellular β-Amyloid

Intraneuronal beta-amyloid (Abeta(i)) accumulates early in Alzheimers disease (AD) and inclusion body myositis. Several organelles, receptor molecules, homeostatic processes, and signal transduction components have been identified as sensitive to Abeta. Although prior studies implicate the insulin-PI3K-Akt signaling cascade, a specific step within this or any essential metabolic or survival pathway has not emerged as a molecular target. We tested the effect of Abeta42 on each component of this cascade. In AD brain, the association between PDK and Akt, phospho-Akt levels and its activity were all decreased relative to control. In cell culture, Abeta(i) expression inhibited both insulin-induced Akt phosphorylation and activity. In vitro experiments identified that beta-amyloid (Abeta), especially oligomer preparations, specifically interrupted the PDK-dependent activation of Akt. Abeta(i) also blocked the association between PDK and Akt in cell-based and in vitro experiments. Importantly, Abeta did not interrupt Akt or PI3K activities (once stimulated) nor did it affect more proximal signal events. These results offer a novel therapeutic strategy to neutralize Abeta-induced energy failure and neuronal death.

Intraneuronal β-Amyloid Expression Downregulates the Akt Survival Pathway and Blunts the Stress Response

Early events in Alzheimers disease (AD) pathogenesis implicate the accumulation of β-amyloid (Aβ) peptide inside neurons in vulnerable brain regions. However, little is known about the consequences of intraneuronal Aβ on signaling mechanisms. Here, we demonstrate, using an inducible viral vector system to drive intracellular expression of Aβ42 peptide in primary neuronal cultures, that this accumulation results in the inhibition of the Akt survival signaling pathway. Induction of intraneuronal Aβ42 expression leads to a sequential decrease in levels of phospho-Akt, increase in activation of glycogen synthase kinase-3β, and apoptosis. Downregulation of Akt also paralleled intracellular Aβ accumulation in vivo in the Tg2576 AD mouse model. Overexpression of constitutively active Akt reversed the toxic effects of Aβ through a mechanism involving the induction of heat shock proteins (Hsps). We used a small-interfering RNA approach to explore the possibility of a link between Akt activity and Hsp70 expression and concluded that neuroprotection by Akt could be mediated through downstream induction of Hsp70 expression. These results suggest that the early dysfunction associated with intraneuronal Aβ accumulation in AD involve the associated impairments of Akt signaling and suppression of the stress response.

Differential Effects of Mitochondrial Heat Shock Protein 60 and Related Molecular Chaperones to Prevent Intracellular β-Amyloid-induced Inhibition of Complex IV and Limit Apoptosis

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been reported in Alzheimer disease tissue and in cultured cells that overexpress amyloid precursor protein. Mitochondrial dysfunction contributes to neurodegeneration in Alzheimer disease partly through formation of reactive oxygen species and the release of sequestered molecules that initiate programmed cell death pathways. The heat shock proteins (HSP) are cytoprotective against a number of stressors, including accumulations of misfolded proteins and reactive oxygen species. We reported on the property of Hsp70 to protect cultured neurons from cell death caused by intraneuronal β-amyloid. Here we demonstrate that Hsp60, Hsp70, and Hsp90 both alone and in combination provide differential protection against intracellular β-amyloid stress through the maintenance of mitochondrial oxidative phosphorylation and functionality of tricarboxylic acid cycle enzymes. Notably, β-amyloid was found to selectively inhibit complex IV activity, an effect selectively neutralized by Hsp60. The combined effect of HSPs was to reduce the free radical burden, preserve ATP generation, decrease cytochrome c release, and prevent caspase-9 activation, all important mediators of β-amyloid-induced neuronal dysfunction and death.

Cell-Free Embryonic Stem Cell Extract–Mediated Derivation of Multipotent Stem Cells From NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair

The oocyte-independent source for the generation of pluripotent stem cells is among the ultimate goals in regenerative medicine. We report that on exposure to mouse embryonic stem cell (mESC) extracts, reversibly permeabilized NIH3T3 cells undergo dedifferentiation followed by stimulus-induced redifferentiation into multiple lineage cell types. Genome-wide expression profiling revealed significant differences between NIH3T3 control and ESC extract–treated NIH3T3 cells including the reactivation of ESC-specific transcripts. Epigenetically, ESC extracts induced CpG demethylation of Oct4 promoter, hyperacetylation of histones 3 and 4, and decreased lysine 9 (K-9) dimethylation of histone 3. In mouse models of surgically induced hindlimb ischemia or acute myocardial infarction transplantation of reprogrammed NIH3T3 cells significantly improved postinjury physiological functions and showed anatomic evidence of engraftment and transdifferentiation into skeletal muscle, endothelial cell, and cardiomyocytes. These data provide evidence for the generation of functional multipotent stem-like cells from terminally differentiated somatic cells without the introduction of retroviral mediated transgenes or ESC fusion.

Parkin Protects against Mitochondrial Toxins and β-Amyloid Accumulation in Skeletal Muscle Cells

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-amyloid. 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.

Aβ42 generation is toxic to endothelial cells and inhibits eNOS function through an Akt/GSK-3β signaling-dependent mechanism

The application of beta-amyloid (Abeta) is cytotoxic to endothelial cells, promotes vasoconstriction and impairs nitric oxide (NO) generation or action. However, there is no information on the effect of intracellular Abeta on endothelial cell biology, although recent studies indicate that neuronal Abeta drives Alzheimers disease pathogenesis. Since the serine-threonine kinase Akt is crucial to both neuronal and endothelial cell survival as well as eNOS activation, we investigated the effects of Abeta expression on Akt-signaling in cultured endothelial cells. Virally-encoded Abeta42 was proapoptotic and inhibitory to Akt phosphorylation in human umbilical vein endothelial cells (HUVECs). Toxicity was characterized by mitochondrial dysfunction, DNA condensation and activation of caspase-3. Substrates downstream of Akt action, GSK-3beta and eNOS, are underphosphorylated in the presence of Abeta. Constitutive activation of Akt reversed Abeta-induced toxicity and stimulated caspase-3 activity, suggesting that inhibition of Akt signaling is functionally significant. These Abeta effects were mediated, in part, through the derepression of GSK-3beta activation and correlated with reductions in NO production. We conclude that intracellular production of Abeta42 is cytotoxic to endothelial cells and that disruption of the Akt/GSK-3beta cell signaling pathway is involved.

Parkin Reverses Intracellular β-Amyloid Accumulation and Its Negative Effects on Proteasome Function

The significance of intracellular β‐amyloid (Aβ42) accumulation is increasingly recognized in Alzheimers disease (AD) pathogenesis. Aβ removal mechanisms that have attracted attention include IDE/neprilysin degradation and antibody‐mediated uptake by immune cells. However, the role of the ubiquitin‐proteasome system (UPS) in the disposal of cellular Aβ has not been fully explored. The E3 ubiquitin ligase Parkin targets several proteins for UPS degradation, and Parkin mutations are the major cause of autosomal recessive Parkinsons disease. We tested whether Parkin has cross‐function to target misfolded proteins in AD for proteasome‐dependent clearance in SH‐SY5Y and primary neuronal cells. Wild‐type Parkin greatly decreased steady‐state levels of intracellular Aβ42, an action abrogated by proteasome inhibitors. Intracellular Aβ42 accumulation decreased cell viability and proteasome activity. Accordingly, Parkin reversed both effects. Changes in mitochondrial ATP production from Aβ or Parkin did not account for their effects on the proteasome. Parkin knock‐down led to accumulation of Aβ. In AD brain, Parkin was found to interact with Aβ and its levels were reduced. Thus, Parkin is cytoprotective, partially by increasing the removal of cellular Aβ through a proteasome‐dependent pathway.

Antiangiogenesis Mediates Cisplatin-Induced Peripheral Neuropathy Attenuation or Reversal by Local Vascular Endothelial Growth Factor Gene Therapy Without Augmenting Tumor Growth

Background—Toxic neuropathies induced by cisplatin and other chemotherapeutic agents are important clinical problems because of their high incidence, their lack of effective treatment, and the fact that neuropathy represents a dose-limiting factor for these therapies. The pathogenic basis for toxic neuropathies induced by chemotherapeutic agents has not been completely elucidated. Methods and Results—We investigated the hypothesis that experimental toxic neuropathy results from an antiangiogenic effect of these drugs, resulting in destruction of the vasa nervorum, and accordingly that the neuropathy could be prevented or reversed by locally administered VEGF gene transfer without augmenting tumor growth. In an animal model of cisplatin-induced neuropathy, nerve blood flow was markedly attenuated, and there was a profound reduction in the number of vasa nervorum associated with marked endothelial cell apoptosis, resulting in a severe peripheral neuropathy with focal axonal degeneration characteristic of ischemic neuropathy. After intramuscular gene transfer of naked plasmid DNA encoding VEGF-1 in animals with an established neuropathy, vascularity and blood flow returned to levels similar to those of control rats, peripheral nerve function was restored, and histological nerve architecture was normalized. Gene therapy administered in parallel with cisplatin chemotherapy completely attenuated endothelial cell apoptosis and inhibited destruction of nerve vasculature, deterioration of nerve function, and axonal degeneration. In a rat tumor model, VEGF gene transfer administered locally did not alter tumor growth or vascularity. Conclusions—These findings implicate microvascular damage as the basis for toxic neuropathy induced by cisplatin and suggest that local angiogenic gene therapy may constitute a novel prevention or treatment for this disorder without augmenting tumor growth or vascularization.

Transgenic expression of β-APP in fast-twitch skeletal muscle leads to calcium dyshomeostasis and IBM-like pathology

Intracellular deposition of the β‐amyloid (Aβ) peptide is an increasingly recognized pathological hallmark associated with neurodegeneration and muscle wasting in Alzheimers disease (AD) and inclusion body myositis (IBM), respectively. Previous reports have implicated dysregulation of β‐amyloid precursor protein (αAPP) expression in IBM. Accumulation of full‐length αAPP, its various proteolytic derivatives including A, and phospho‐tau into vacuolated inclusions is an early pathogenic event. We previously reported on a statistical tendency favoring fast twitch fiber involvement in IBM, reminiscent of the tissue specific patterns of misfolded protein deposition seen in neurodegenerative diseases. To test this principle, we generated an animal model in which human wild‐type (WT) βAPP expression was limited to postnatal type II skeletal muscle. Hemizygous transgenic mice harboring increased levels of holoβAPP751 and Aβ in skeletal muscle fibers became significantly weaker with age compared with nontransgenic littermates and exhibited typical myopathic features. A subpopulation of dissociated muscle fibers from transgenic mice exhibited a 2‐fold increase in resting calcium and membrane depolarization compared with nontransgenic littermates. Taken together, these data indicate that overexpression of human βAPP in fast twitch skeletal muscle of transgenic mice is sufficient for the development of some features characteristic of IBM, including abnormal tau histochemistry. The increase in resting calcium and depolarization are novel findings, suggesting both a mechanism for the weakness and an avenue for therapeutic intervention in IBM.—Moussa, C. E‐H., Fu, Q., Kumar, P., Shtifman, A., Lopez, J. R., Allen, P. D., LaFerla, F., Weinberg, D., Magrane, J., Aprahamian, T., Walsh, K., Rosen, K. M., Querfurth, H. W. Transgenic expression of ‐APP in fast‐twitch skeletal muscle leads to calcium dyshomeostasis and IBM‐like pathology. FASEB J. 20, E1570 –E1578 (2006)

AMPA Receptor-Dependent Clustering of Synaptic NMDA Receptors Is Mediated by Stargazin and NR2A/B in Spinal Neurons and Hippocampal Interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.