Syed Zahid Ali Shah

Unfolded Protein Response Pathways in Neurodegenerative Diseases

The aggregation of disease-specific misfolded proteins resulting in endoplasmic reticulum stress is associated with early pathological events in many neurodegenerative diseases, and apoptotic signaling is initiated when the stress goes beyond the maximum threshold level of endoplasmic reticulum stress sensors. All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by signaling an adaptive pathway termed as unfolded protein response (UPR). Recently, the focus of research shifted from work on specific proteins as pathogenesis in these neurodegenerative diseases towards a more specific generic pathway known as UPR. ER is a major organelle for protein quality control, and cellular stress disrupts normal functioning of ER. The UPR acts as a protective mechanism during endoplasmic reticulum stress, but persistent long-term stress triggers UPR-mediated apoptotic pathways ultimately leading to cell death. Here in this review, we will briefly summarize the molecular events of endoplasmic reticulum stress-associated UPR signaling pathways and their potential therapeutic role in neurodegenerative diseases.

A central role for calcineurin in protein misfolding neurodegenerative diseases

Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca2+) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca2+ in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca2+ storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca2+/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.

Death Receptor 6 and Caspase-6 Regulate Prion Peptide-Induced Axonal Degeneration in Rat Spinal Neurons

Axonal degeneration is a hallmark of many neurodegenerative disorders including transmissible spongiform encephalopathies (TSE). However, the full complement of axonal degeneration triggers is not fully understood. In an in vitro prion model, we observed that treatment of rat spinal neurons with the prion peptide, PrP106-126, activated death receptor 6 (DR6, also known as TNFRSF21), caspase-6, caspase-3, and induced axonal degeneration. Knockdown of DR6 by siRNA blocked caspase-6 and caspase-3 activation and axonal degeneration. We also found that cleaved caspase-3 is only enriched in cell bodies, but cleaved caspase-6 is expressed in both cell bodies and axons. Axonal degeneration was prevented by preincubation of neurons with a caspase-6 inhibitor or siRNA of caspase-6. Our findings suggest that both DR6 and caspase-6 play important roles in axonal degeneration and caspase-6 acts downstream of DR6. We also observed that nicotinamide nucleotide adenylyltransferase 1 protein (Nmnat1), which had been reported to protect neurons from degeneration, alleviated axonal degeneration without blocking caspase-6 activation, suggesting that Nmnat acts downstream or parallel to caspase-6 activation. Our results indicate that PrP106-126 triggered axonal degeneration of the spinal cord neurons, DR6 is a key regulator of axonal degeneration, and the signaling pathway of DR6/caspase-6 mediates axonal degeneration induced by the prion fragment. Our findings raise the hope of targeting the DR6 as a potential therapeutic strategy in prion-related neurodegenerative diseases.

The Role of Unfolded Protein Response and Mitogen-Activated Protein Kinase Signaling in Neurodegenerative Diseases with Special Focus on Prion Diseases

Prion diseases are neurodegenerative pathologies characterized by the accumulation of a protease-resistant form of the cellular prion protein named prion protein scrapie (PrPSc) in the brain. PrPSc accumulation in the endoplasmic reticulum (ER) result in a dysregulated calcium (Ca2+) homeostasis and subsequent initiation of unfolded protein response (UPR) leading to neuronal dysfunction and apoptosis. The molecular mechanisms for the transition between adaptation to ER stress and ER stress-induced apoptosis are still unclear. Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that rule the signaling of many extracellular stimuli from plasma membrane to the nucleus. However the identification of numerous points of cross talk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress in prion diseases. Indeed the MAPK signaling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses including misfolded protein response stress. In this article, we review the UPR signaling in prion diseases and discuss the triad of MAPK signaling pathways. We also describe the role played by MAPK signaling cascades in Alzheimer’s (AD) and Parkinson’s disease (PD). We will also overview the mechanisms of cell death and the role of MAPK signaling in prion disease progression and highlight potential avenues for therapeutic intervention.

Regulatory Mechanisms of Endoplasmic Reticulum Resident IP3 Receptors

Dysregulated calcium signaling and accumulation of aberrant proteins causing endoplasmic reticulum stress are the early sign of intra-axonal pathological events in many neurodegenerative diseases, and apoptotic signaling is initiated when the stress goes beyond the maximum threshold level of endoplasmic reticulum. The fate of the cell to undergo apoptosis is controlled by Ca2+ signaling and dynamics at the level of the endoplasmic reticulum. Endoplasmic reticulum resident inositol 1,4,5-trisphosphate receptors (IP3R) play a pivotal role in cell death signaling by mediating Ca2+ flux from the endoplasmic reticulum into the cytosol and mitochondria. Hence, many prosurvival and prodeath signaling pathways and proteins affect Ca2+ signaling by directly targeting IP3R channels, which can happen in an IP3R-isoform-dependent manner. Here, in this review, we summarize the regulatory mechanisms of inositol triphosphate receptors in calcium regulation and initiation of apoptosis during unfolded protein response.

Early Minocycline and Late FK506 Treatment Improves Survival and Alleviates Neuroinflammation, Neurodegeneration, and Behavioral Deficits in Prion-Infected Hamsters

Prion infections of the central nervous system (CNS) are characterized by initial reactive gliosis followed by overt neuronal death. Gliosis is likely to be caused initially by the deposition of misfolded, proteinase K-resistant, isoforms (termed PrPSc) of the normal cellular prion protein (PrPc) in the brain. Proinflammatory cytokines and chemokines released by PrPSc-activated glia and stressed neurons may also contribute directly or indirectly to the disease development by enhancing gliosis and inducing neurotoxicity. Recent studies have illustrated that early neuroinflammation activates nuclear factor of activated T cells (NFAT) in the calcineurin signaling cascade, resulting in nuclear translocation of nuclear factor kappa B (NF-κB) to promote apoptosis. Hence, useful therapeutic approaches to slow down the course of prion disease development should control early inflammatory responses to suppress NFAT signaling. Here we used a hamster model of prion diseases to test, for the first time, the neuroprotective and NFAT-suppressive effect of a second-generation semisynthetic tetracycline derivative, minocycline, versus a calcineurin inhibitor, FK506, with known NFAT suppressive activity. Our results indicate that prolonged treatment with minocycline, starting from the presymptomatic stage of prion disease was more effective than FK506 given either during the presymptomatic or symptomatic stage of prion disease. Specifically, minocycline treatment reduced the expression of the astrocyte activation marker glial fibrillary acidic protein and of the microglial activation marker ionized calcium-binding adapter molecule-1, subsequently reducing the level of proinflammatory cytokines interleukin 1β and tumor necrosis factor-α. We further found that minocycline and FK506 treatment inhibited mitogen-activated protein kinase p38 phosphorylation and NF-κB nuclear translocation in a caspase-dependent manner, and enhanced phosphorylated cyclic adenosine monophosphate response element-binding protein and phosphorylated Bcl2-associated death promoter levels to reduce cognitive impairment and apoptosis. Taken together, our results indicate that minocycline is a better choice for prolonged use in prion diseases and encourage its further clinical development as a possible treatment for this disease.

Role of the Retromer Complex in Neurodegenerative Diseases

The retromer complex is a protein complex that plays a central role in endosomal trafficking. Retromer dysfunction has been linked to a growing number of neurological disorders. The process of intracellular trafficking and recycling is crucial for maintaining normal intracellular homeostasis, which is partly achieved through the activity of the retromer complex. The retromer complex plays a primary role in sorting endosomal cargo back to the cell surface for reuse, to the trans-Golgi network (TGN), or alternatively to specialized endomembrane compartments, in which the cargo is not subjected to lysosomal-mediated degradation. In most cases, the retromer acts as a core that interacts with associated proteins, including sorting nexin family member 27 (SNX27), members of the vacuolar protein sorting 10 (VPS10) receptor family, the major endosomal actin polymerization-promoting complex known as Wiskott-Aldrich syndrome protein and scar homolog (WASH), and other proteins. Some of the molecules carried by the retromer complex are risk factors for neurodegenerative diseases. Defects such as haplo-insufficiency or mutations in one or several units of the retromer complex lead to various pathologies. Here, we summarize the molecular architecture of the retromer complex and the roles of this system in intracellular trafficking related the pathogenesis of neurodegenerative diseases.

Virulent Mycobacterium bovis Beijing Strain Activates the NLRP7 Inflammasome in THP-1 Macrophages

Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals, including humans. Macrophages are the first line of host defense. They secrete proinflammatory cytokines, such as interleukin-1 beta (IL-1β), in response to mycobacterial infection, but the underlying mechanisms by which human macrophages are activated and release IL-1β following M. bovis infection are poorly understood. Here we show that the ‘nucleotide binding and oligomerization of domain-like receptor (NLR) family pyrin domain containing 7 protein’ (NLRP7) inflammasome is involved in IL-1β secretion and caspase-1 activation induced by M. bovis infection in THP-1 macrophages. NLRP7 inflammasome activation promotes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha (TNF-α), Chemokine (C-C motif) ligand 3 (CCL3) and IL-1β mRNAs. Thus, the NLRP7 inflammasome contributes to IL-1β secretion and induction of pyroptosis in response to M. bovis infection in THP-1 macrophages.

Role of the AMPK pathway in promoting autophagic flux via modulating mitochondrial dynamics in neurodegenerative diseases: Insight into prion diseases

Neurons are highly energy demanding cells dependent on the mitochondrial oxidative phosphorylation system. Mitochondria generate energy via respiratory complexes that constitute the electron transport chain. Adenosine triphosphate depletion or glucose starvation act as a trigger for the activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK is an evolutionarily conserved protein that plays an important role in cell survival and organismal longevity through modulation of energy homeostasis and autophagy. Several studies suggest that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. Mild mitochondrial dysfunction leads to activated AMPK signaling, but severe endoplasmic reticulum stress and mitochondrial dysfunction may lead to a shift from autophagy towards apoptosis and perturbed AMPK signaling. Hence, controlling mitochondrial dynamics and autophagic flux via AMPK activation might be a useful therapeutic strategy in neurodegenerative diseases to reinstate energy homeostasis and degrade misfolded proteins. In this review article, we discuss briefly the role of AMPK signaling in energy homeostasis, the structure of AMPK, activation mechanisms of AMPK, regulation of AMPK, the role of AMPK in autophagy, the role of AMPK in neurodegenerative diseases, and finally the role of autophagic flux in prion diseases.

Inflammasomes-dependent regulation of IL-1β secretion induced by the virulent Mycobacterium bovis Beijing strain in THP-1 macrophages

Mycobacterium bovis is the causative agent of tuberculosis in cattle. Infection of macrophages with M. bovis leads to the activation of the “nucleotide binding and oligomerization, leucine-rich repeat and pyrin domains-containing protein 3” (NLRP3) and “absent in melanoma 2” (AIM2) inflammasomes, which in turn triggers release of the proinflammatory cytokine interleukin-1β (IL-1β) that contributes to bacterial clearance and plays a crucial role in the host defense. However, NLRP3 and AIM2 inflammasome activation is influenced by several factors and how IL-1β secretion by M. bovis-infected macrophages is regulated via the inflammasome pathway remains unclear. Here we found that IL-1β secretion and pro-IL-1β protein accumulation were inhibited in THP-1 macrophages upon exposure to the virulent M. bovis Beijing strain in the presence of high K+ concentrations, cycloheximide (a protein synthesis inhibitor) and PR-619 (a deubiquitinating enzyme inhibitor). Scavenging reactive oxygen species (ROS) induced by N-acetylcysteine reduced IL-1β release independent of the mitochondrial permeability transition. Collectively, our results suggest that IL-1β secretion by M. bovis-infected THP-1 macrophages is reduced by high extracellular K+ concentration, inhibition of new protein synthesis, deubiquitination, and ROS generation.