Alexander Shtifman

Increased intraneuronal resting [Ca2+] in adult Alzheimer’s disease mice

Neurodegeneration in Alzheimer’s disease (AD) has been linked to intracellular accumulation of misfolded proteins and dysregulation of intracellular Ca2+. In the current work, we determined the contribution of specific Ca2+ pathways to an alteration in Ca2+ homeostasis in primary cortical neurons from an adult triple transgenic (3xTg‐AD) mouse model of AD that exhibits intraneuronal accumulation of β‐amyloid proteins. Resting free Ca2+ concentration ([Ca2+]i), as measured with Ca2+‐selective microelectrodes, was greatly elevated in neurons from 3xTg‐AD and APPSWE mouse strains when compared with their respective non‐transgenic neurons, while there was no alteration in the resting membrane potential. In the absence of the extracellular Ca2+, the [Ca2+]i returned to near normal levels in 3xTg‐AD neurons, demonstrating that extracellular Ca2+contributed to elevated [Ca2+]i. Application of nifedipine, or a non‐L‐type channel blocker, SKF‐96365, partially reduced [Ca2+]i. Blocking the ryanodine receptors, with ryanodine or FLA‐365 had no effect, suggesting that these channels do not contribute to the elevated [Ca2+]i. Conversely, inhibition of inositol trisphosphate receptors with xestospongin C produced a partial reduction in [Ca2+]i. These results demonstrate that an elevation in resting [Ca2+]i, contributed by aberrant Ca2+entry and release pathways, should be considered a major component of the abnormal Ca2+ homeostasis associated with AD.

Amyloid-β protein impairs Ca2+ release and contractility in skeletal muscle

Inclusion body myositis (IBM), the most common muscle disorder in the elderly, is partly characterized by dysregulation of β-amyloid precursor protein (βAPP) expression and abnormal, intracellular accumulation of full-length βAPP and β-amyloid epitopes. The present study examined the effects of β-amyloid accumulation on force generation and Ca(2+) release in skeletal muscle from transgenic mice harboring human βAPP and assessed the consequence of Aβ(1-42) modulation of the ryanodine receptor Ca(2+) release channels (RyRs). β-Amyloid laden muscle produced less peak force and exhibited Ca(2+) transients with smaller amplitude. To determine whether modification of RyRs by β-amyloid underlie the effects observed in muscle, in vitro Ca(2+) release assays and RyR reconstituted in planar lipid bilayer experiments were conducted in the presence of Aβ(1-42). Application of Aβ(1-42) to RyRs in bilayers resulted in an increased channel open probability and changes in gating kinetics, while addition of Aβ(1-42) to the rabbit SR vesicles resulted in RyR-mediated Ca(2+) release. These data may relate altered βAPP metabolism in IBM to reductions in RyR-mediated Ca(2+) release and muscle contractility.

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)

Calcium dyshomeostasis in β-amyloid and tau-bearing skeletal myotubes

The relative scarcity of inclusion-affected muscle cells or markers of cell death in inclusion body myositis (IBM) is in distinction to the specific and early intracellular deposition of several Alzheimers Disease (AD)-related proteins. The current study examined the possible correlation between myotube β-amyloid and/or Tau accumulations and a widespread mishandling of intracellular muscle calcium concentration that could potentially account for the unrelenting weakness in affected patients. Cultured myogenic cells (C2C12) expressed β-amyloid-42 (Aβ42) and fetal Tau peptides, as human transgenes encoded by herpes simplex virus, either individually or concurrently. Co-expression of Aβ42 in C2C12 myotubes resulted in hyperphosphorylation of Tau protein that was not observed when Tau was expressed alone. Resting calcium concentration and agonist-induced RyR-mediated Ca2+ release were examined using calcium-specific microelectrodes and Fluo-4 epifluorescence, respectively. Co-expression of Aβ42 and Tau cooperatively elevated basal levels of myoplasmic-free calcium, an effect that was accompanied by depolarization of the plasma membrane. Sarcoplasmic reticulum (SR) calcium release, induced by KCl depolarization, was not affected by Aβ42 or Tau. In contrast, expression of Aβ42, Tau, or Aβ42 together with Tau resulted in enhanced sensitivity of ryanodine receptors to activation by caffeine. Notably, expression of β-amyloid, alone, was sufficient to result in an increased sensitivity to direct activation by caffeine. Current results indicate that amyloid proteins cooperate to raise resting calcium levels and that these effects are associated with a passive SR Ca2+ leak and Tau hyperphosphorylation in skeletal muscle.

Altered Ca2+ homeostasis in the skeletal muscle of DJ-1 null mice

Loss-of-function mutations in DJ-1 are associated with early-onset of Parkinsons disease. Although DJ-1 is ubiquitously expressed, the functional pathways affected by it remain unresolved. Here we demonstrate an involvement of DJ-1 in the regulation of Ca(2+) homeostasis in mouse skeletal muscle. Using enzymatically dissociated flexor digitorum brevis muscle fibers from wild-type (wt) and DJ-1 null mice, we examined the effects of DJ-1 protein on resting, cytoplasmic [Ca(2+)] ([Ca(2+)](i)) and depolarization-evoked Ca(2+) release in the mouse skeletal muscle. The loss of DJ-1 resulted in a more than two-fold increase in resting [Ca(2+)](i). While there was no alteration in the resting membrane potential, there was a significant decrease in depolarization-evoked Ca(2+) release from the sarcoplasmic reticulum in the DJ-1 null muscle cells. Consistent with the role of DJ-1 in oxidative stress regulation and mitochondrial functional maintenance, treatments of DJ-1 null muscle cells with resveratrol, a mitochondrial activator, or glutathione, a potent antioxidant, reversed the effects of the loss of DJ-1 on Ca(2+) homeostasis. These results provide evidence of DJ-1s association with Ca(2+) regulatory pathways in mouse skeletal muscle, and suggest the potential benefit of resveratrol to functionally compensate for the loss of DJ-1.

Mitochondrial Dysfunction in Skeletal Muscle of Amyloid Precursor Protein-overexpressing Mice

Background: Intracellular accumulation of β-amyloid is a key step in pathogenesis of the inclusion body myositis (IBM). Results: Intramyofiber accumulation of β-amyloid in MCK-βAPP mice leads to structural and functional mitochondrial abnormalities. Conclusion: Mitochondrial abnormalities precede IBM-related histological and motor deficits in MCK-βAPP mice. Significance: The diminished mitochondrial function may play a key role during the β-amyloid mediated pathogenesis in IBM. 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.

INTRACELLULAR β-AMYLOID ACCUMULATION LEADS TO AGE-DEPENDENT PROGRESSION OF Ca2+ DYSREGULATION IN SKELETAL MUSCLE

Intramyofiber accumulation of β‐amyloid fragments (Aβ) is a pathologic hallmark of inclusion‐body myositis (IBM), a progressive skeletal muscle disorder. We investigated the temporal pattern of alterations in the resting cytoplasmic [Ca2+] ([Ca2+]i) as well as the depolarization‐evoked Ca2+ release from the sarcoplasmic reticulum in skeletal muscle from transgenic mice expressing human βAPP (MCK‐βAPP). MCK‐βAPP mice show an age‐dependent increase in [Ca2+]i along with a reduction in depolarization‐evoked Ca2+ release, which appear well before the other reported aspects of IBM, such as inclusion formation, inflammation, centralized nuclei, atrophy, and skeletal muscle weakness. In the young MCK‐βAPP animals the increase in resting [Ca2+]i can be attributed largely to Ca2+ influx through nifedipine‐sensitive Ca2+ channels. In the adult MCK‐βAPP mice, in addition to the nifedipine‐sensitive pathway, there is also a substantial contribution by the intracellular compartments to the increase in [Ca2+]i. These results suggest that β‐amyloid‐induced disuption of Ca2+ handling may represent an early event in the pathogenesis of IBM. Muscle Nerve, 2010

TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells

Background: Ionizing radiation can induce DNA damage in nonirradiated (N-IR) cells via nontargeted effects (NTE). Results: TNF-α and IL-1α mediate NTE in N-IR bone marrow-derived EPCs, and neutralizing TNF-α diminishes NTE in WT and p55 knock-out BM-EPCs. Conclusion: TNF-TNFR2/p75 signaling alters accumulation of inflammatory cytokines that attenuate NTE in N-IR EPCs. Significance: TNFR2/p75 may represent a gene target for mitigation of delayed RBR in BM-EPCs. TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1–5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3–5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general.

Divergent Modification of Low-Dose 56Fe-Particle and Proton Radiation on Skeletal Muscle

It is unknown whether loss of skeletal muscle mass and function experienced by astronauts during space flight could be augmented by ionizing radiation (IR), such as low-dose high-charge and energy (HZE) particles or low-dose high-energy proton radiation. In the current study adult mice were irradiated whole-body with either a single dose of 15 cGy of 1 GeV/n 56Fe-particle or with a 90 cGy proton of 1 GeV/n proton particles. Both ionizing radiation types caused alterations in the skeletal muscle cytoplasmic Ca2+ ([Ca2+]i) homeostasis. 56Fe-particle irradiation also caused a reduction of depolarization-evoked Ca2+ release from the sarcoplasmic reticulum (SR). The increase in the [Ca2+]i was detected as early as 24 h after 56Fe-particle irradiation, while effects of proton irradiation were only evident at 72 h. In both instances [Ca2+]i returned to baseline at day 7 after irradiation. All 56Fe-particle irradiated samples revealed a significant number of centrally localized nuclei, a histologic manifestation of regenerating muscle, 7 days after irradiation. Neither unirradiated control or proton-irradiated samples exhibited such a phenotype. Protein analysis revealed significant increase in the phosphorylation of Akt, Erk1/2 and rpS6k on day 7 in 56Fe-particle irradiated skeletal muscle, but not proton or unirradiated skeletal muscle, suggesting activation of pro-survival signaling. Our findings suggest that a single low-dose 56Fe-particle or proton exposure is sufficient to affect Ca2+ homeostasis in skeletal muscle. However, only 56Fe-particle irradiation led to the appearance of central nuclei and activation of pro-survival pathways, suggesting an ongoing muscle damage/recovery process.

Erratum: TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells (Journal of Biological Chemistry (2014) 289 (14178-14193) DOI:10.1074/jbc.A114.567743)

Sharath P. Sasi, Jin Song, Daniel Park, Heiko Enderling, J. Tyson McDonald, Hannah Gee, Brittany Garrity, Alexander Shtifman, Xinhua Yan, Kenneth Walsh, Mohan Natarajan, Raj Kishore, and David A. Goukassian The following grant information needs to be updated. National Aeronautic and Space Administration Grant NNJ10ZSA001N (to D. A. G.) has been updated to NNX11AD22G. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 45, p. 27014, November 6, 2015