Alexey Bogush

Atorvastatin-induced activation of Alzheimer's α secretase is resistant to standard inhibitors of protein phosphorylation-regulated ectodomain shedding

Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta‐ and intra‐membranous proteases. Release or ‘shedding’ of the large APP ectodomain can occur via one of two competing pathways, the α‐ and β‐secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The α‐secretase pathway cleaves within the amyloidogenic Aβ domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the α‐ and β‐secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen‐activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of α‐secretase‐released, soluble APP (sAPPα). However, statin‐induced stimulation of sAPPα release was not antagonized by inhibitors of either PKC or ERK, or by the co‐expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPα secretion. These results suggest that statins may regulate α‐secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as‐yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A ‘phospho‐proteomic’ analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin‐regulated APP ectodomain shedding is underway.

Cell-autonomous alteration of dopaminergic transmission by wild type and mutant (ΔE) TorsinA in transgenic mice

Early onset torsion dystonia is an autosomal dominant movement disorder of variable penetrance caused by a glutamic acid, i.e. DeltaE, deletion in DYT1, encoding the protein TorsinA. Genetic and structural data implicate basal ganglia dysfunction in dystonia. TorsinA, however, is diffusely expressed, and therefore the primary source of dysfunction may be obscured in pan-neuronal transgenic mouse models. We utilized the tyrosine hydroxylase (TH) promoter to direct transgene expression specifically to dopaminergic neurons of the midbrain to identify cell-autonomous abnormalities. Expression of both the human wild type (hTorsinA) and mutant (DeltaE-hTorsinA) protein resulted in alterations of dopamine release as detected by microdialysis and fast cycle voltammetry. Motor abnormalities detected in these mice mimicked those noted in transgenic mice with pan-neuronal transgene expression. The locomotor response to cocaine in both TH-hTorsinA and TH-DeltaE-hTorsinA, in the face of abnormal extracellular DA levels relative to non-transgenic mice, suggests compensatory, post-synaptic alterations in striatal DA transmission. This is the first cell-subtype-specific DYT1 transgenic mouse that can serve to differentiate between primary and secondary changes in dystonia, thereby helping to target disease therapies.

Motor neuron impairment mediated by a sumoylated fragment of the glial glutamate transporter EAAT2.

Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT‐1) is cleaved by caspase‐3 at its cytosolic carboxy‐terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post‐translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1‐G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE‐SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2‐derived proteolytic fragment. Moreover, in a co‐culture binary system, expression of CTE‐SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase‐3 activation in motor neuron‐derived NSC‐34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE‐SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE‐SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.

Nordihydroguaiaretic acid increases glutamate uptake in vitro and in vivo: Therapeutic implications for amyotrophic lateral sclerosis

Synaptic accumulation of glutamate causes neuronal death in many neurodegenerative pathologies including amyotrophic lateral sclerosis. Drugs capable of increasing glutamate uptake could therefore be therapeutically effective. We screened in a cell-based assay a library of 1040 FDA-approved drugs and nutrients for compounds that could enhance glutamate uptake. Nordihydroguaiaretic acid (NDGA), an anti-inflammatory drug that inhibits lipoxygensases, potently enhanced glutamate uptake in MN-1 cells. Given subcutaneously at 1 mg/day for 30 days in mice, NDGA increased glutamate uptake in spinal cord synaptosomes persistently throughout the treatment. However, when administered following the same regimen to the SOD1-G93A transgenic mouse model of ALS at disease onset, NDGA did not extend survival of these mice. We found that NDGA failed to sustain increased glutamate uptake in the SOD1-G93A mice despite an initial upregulation measured during the first 10 days of treatment. SOD1-G93A mice displayed a progressive increase in spinal cord expression levels of the efflux transporter P-glycoprotein beginning at disease onset. This increase was not mediated by the NDGA treatment because it was measured in untreated SOD1-G93A mice. Since P-glycoproteins control the extrusion of a broad range of toxins and xenobiotics and are responsible for drug resistance in many diseases including cancer and brain diseases such as epilepsy, we propose that the failure of NDGA in maintaining glutamate uptake upregulated in SOD1-G93A mice and its therapeutic inefficacy are due to acquired pharmacoresistance mediated by the increased expression of P-glycoprotein.

Neocortical expression of mutant huntingtin is not required for alterations in striatal gene expression or motor dysfunction in a transgenic mouse

Huntingtons disease (HD) is an autosomal-dominant neurodegenerative disease caused by an expanded polyglutamine tract in the ubiquitously expressed huntingtin protein. Clinically, HD is characterized by motor, cognitive and psychiatric deficits. Striking degeneration of the striatum is observed in HD with the medium spiny neurons (MSNs) being the most severely affected neuronal subtype. Dysfunction of MSNs is marked by characteristic changes in gene expression which precede neuronal death. The ubiquitous expression of the huntingtin protein raises the question as to whether the selective vulnerability of the MSN is cell-autonomous, non-cell-autonomous, or a combination thereof. In particular, growing evidence suggests that abnormalities of the cortex and corticostriatal projections may be primary causes of striatal vulnerability. To examine this issue, we developed transgenic mice that, within the forebrain, selectively express a pathogenic huntingtin species in the MSNs, specifically excluding the neocortex. These mice develop a number of abnormalities characteristic of pan-cellular HD mouse models, including intranuclear inclusion bodies, motor impairment, and changes in striatal gene expression. As this phenotype develops in the presence of normal levels of brain-derived neurotrophic factor and its major striatal receptor, tropomyosin-related kinase B, these data represent the first demonstration of in vivo cell-autonomous transcriptional dysregulation in an HD mouse model. Furthermore, our findings suggest that therapies targeted directly to the striatum may be efficacious at reversing some of the molecular abnormalities present in HD.

Sumoylation of the astroglial glutamate transporter EAAT2 governs its intracellular compartmentalization

EAAT2 is a predominantly astroglial glutamate transporter responsible for the majority of synaptic glutamate clearance in the mammalian central nervous system (CNS). Its dysfunction has been linked with many neurological disorders, including amyotrophic lateral sclerosis (ALS). Decreases in EAAT2 expression and function have been implicated in causing motor neuron excitotoxic death in ALS. Nevertheless, increasing EAAT2 expression does not significantly improve ALS phenotype in mouse models or in clinical trials. In the SOD1‐G93A mouse model of inherited ALS, the cytosolic carboxy‐terminal domain is cleaved from EAAT2, conjugated to SUMO1, and accumulated in astrocytes where it triggers astrocyte‐mediated neurotoxic effects as disease progresses. However, it is not known whether this fragment is sumoylated after cleavage or if full‐length EAAT2 is already sumoylated prior to cleavage as part of physiological regulation. In this study, we show that a fraction of full‐length EAAT2 is constitutively sumoylated in primary cultures of astrocytes in vitro and in the CNS in vivo. Furthermore, the extent of sumoylation of EAAT2 does not change during the course of ALS in the SOD1‐G93A mouse and is not affected by the expression of ALS‐causative mutant SOD1 proteins in astrocytes in vitro, indicating that EAAT2 sumoylation is not driven by pathogenic mechanisms. Most interestingly, sumoylated EAAT2 localizes to intracellular compartments, whereas non‐sumoylated EAAT2 resides on the plasma membrane. In agreement, promoting desumoylation in primary astrocytes causes increased EAAT2‐mediated glutamate uptake. These findings could have implications for optimizing therapeutic approaches aimed at increasing EAAT2 activity in the dysfunctional or diseased CNS. GLIA 2014;62:1241–1253

Small Peptides against the Mutant SOD1/Bcl-2 Toxic Mitochondrial Complex Restore Mitochondrial Function and Cell Viability in Mutant SOD1-Mediated ALS

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of familial cases (fALS). Mitochondria are one of the targets of mutant SOD1 (mutSOD1) toxicity. We previously demonstrated that at the mitochondria, mutSOD1 forms a toxic complex with Bcl-2, which is then converted into a toxic protein via a structural rearrangement that exposes its toxic BH3 domain (Pedrini et al., 2010). Here we now show that formation of this toxic complex with Bcl-2 is the primary event in mutSOD1-induced mitochondrial dysfunction, inhibiting mitochondrial permeability to ADP and inducing mitochondrial hyperpolarization. In mutSOD1-G93A cells and mice, the newly exposed BH3 domain in Bcl-2 alters the normal interaction between Bcl-2 and VDAC1 thus reducing permeability of the outer mitochondrial membrane. In motor neuronal cells, the mutSOD1/Bcl-2 complex causes mitochondrial hyperpolarization leading to cell loss. Small SOD1-like therapeutic peptides that specifically block formation of the mutSOD1/Bcl-2 complex, recover both aspects of mitochondrial dysfunction: they prevent mitochondrial hyperpolarization and cell loss as well as restore ADP permeability in mitochondria of symptomatic mutSOD1-G93A mice.

Egr-1 Induces DARPP-32 Expression in Striatal Medium Spiny Neurons via a Conserved Intragenic Element

DARPP-32 (dopamine and adenosine 3′, 5′-cyclic monophosphate cAMP-regulated phosphoprotein, 32 kDa) is a striatal-enriched protein that mediates signaling by dopamine and other first messengers in the medium spiny neurons. The transcriptional mechanisms that regulate striatal DARPP-32 expression remain enigmatic and are a subject of much interest in the efforts to induce a striatal phenotype in stem cells. We report the identification and characterization of a conserved region, also known as H10, in intron IV of the gene that codes for DARPP-32 (Ppp1r1b). This DNA sequence forms multiunit complexes with nuclear proteins from adult and embryonic striata of mice and rats. Purification of proteins from these complexes identified early growth response-1 (Egr-1). The interaction between Egr-1 and H10 was confirmed in vitro and in vivo by super-shift and chromatin immunoprecipitation assays, respectively. Importantly, brain-derived neurotrophic factor (BDNF), a known inducer of DARPP-32 and Egr-1 expression, enhanced Egr-1 binding to H10 in vitro. Moreover, overexpression of Egr-1 in primary striatal neurons induced the expression of DARPP-32, whereas a dominant-negative Egr-1 blocked DARPP-32 induction by BDNF. Together, this study identifies Egr-1 as a transcriptional activator of the Ppp1r1b gene and provides insight into the molecular mechanisms that regulate medium spiny neuron maturation.

Sumoylation of critical proteins in amyotrophic lateral sclerosis: emerging pathways of pathogenesis.

Emerging lines of evidence suggest a relationship between amyotrophic lateral sclerosis (ALS) and protein sumoylation. Multiple studies have demonstrated that several of the proteins involved in the pathogenesis of ALS, including superoxide dismutase 1, fused in liposarcoma, and TAR DNA-binding protein 43 (TDP-43), are substrates for sumoylation. Additionally, recent studies in cellular and animal models of ALS revealed that sumoylation of these proteins impact their localization, longevity, and how they functionally perform in disease, providing novel areas for mechanistic investigations and therapeutics. In this article, we summarize the current literature examining the impact of sumoylation of critical proteins involved in ALS and discuss the potential impact for the pathogenesis of the disease. In addition, we report and discuss the implications of new evidence demonstrating that sumoylation of a fragment derived from the proteolytic cleavage of the astroglial glutamate transporter, EAAT2, plays a direct role in downregulating the expression levels of full-length EAAT2 by binding to a regulatory region of its promoter.

Phosphatidylinositide 3-kinase and protein kinase C zeta mediate retinoic acid induction of DARPP-32 in medium size spiny neurons in vitro

Mature striatal medium size spiny neurons express the dopamine and cAMP‐regulated phosphoprotein, 32 kDa (DARPP‐32), but little is known about the mechanisms regulating its levels, or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP‐32 mRNA and/or protein levels include retinoic acid (RA), brain‐derived neurotrophic factor, and estrogen (E2). We now demonstrate that RA regulates DARPP‐32 mRNA and protein in primary striatal neuronal cultures. Furthermore, DARPP‐32 induction by RA in vitro requires phosphatidylinositide 3‐kinase, but is independent of tropomyosin‐related kinase B, cyclin‐dependent kinase 5, and protein kinase B. Using pharmacologic inhibitors of various isoforms of protein kinase C (PKC), we also demonstrate that DARPP‐32 induction by RA in vitro is dependent on PKC zeta (PKCζ). Thus, the signal transduction pathways mediated by RA are very different than those mediating DARPP‐32 induction by brain‐derived neurotrophic factor. These data support the presence of multiple signal transduction pathways mediating expression of DARPP‐32 in vitro, including a novel, important pathway via which phosphatidylinositide 3‐kinase regulates the contribution of PKCζ.