Miguel Bronfman

Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by β -amyloid fibrils

Alzheimers disease (AD) is a progressive neurodegenerative disorder, which is probably caused by the cytotoxic effect of the amyloid β-peptide (Aβ). We report here molecular changes induced by Aβ, both in neuronal cells in culture and in rats injected in the dorsal hippocampus with preformed Aβ fibrils, as an in vivo model of the disease. Results indicate that in both systems, Aβ neurotoxicity resulted in the destabilization of endogenous levels of β-catenin, a key transducer of the Wnt signaling pathway. Lithium chloride, which mimics Wnt signaling by inhibiting glycogen synthase kinase-3β promoted the survival of post-mitotic neurons against Aβ neurotoxicity and recovered cytosolic β-catenin to control levels. Moreover, the neurotoxic effect of Aβ fibrils was also modulated with protein kinase C agonists/inhibitors and reversed with conditioned medium containing the Wnt-3a ligand. We also examined the spatial memory performance of rats injected with preformed Aβ fibrils in the Morris water maze paradigm, and found that chronic lithium treatment protected neurodegeneration by rescuing β-catenin levels and improved the deficit in spatial learning induced by Aβ. Our results are consistent with the idea that Aβ-dependent neurotoxicity induces a loss of function of Wnt signaling components and indicate that lithium or compounds that mimic this signaling cascade may be putative candidates for therapeutic intervention in Alzheimers patients.

Peroxisome Proliferator-activated Receptor γ Up-regulates the Bcl-2 Anti-apoptotic Protein in Neurons and Induces Mitochondrial Stabilization and Protection against Oxidative Stress and Apoptosis

Peroxisome proliferator-activated receptor γ (PPARγ) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARγ agonists preventβ-amyloid (Aβ)-induced neurodegeneration in hippocampal neurons, and PPARγ is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARγ agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Aβ-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARγ are resistant to Aβ-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 μm H2O2. Conversely, cells expressing a dominant negative mutant of PPARγ show increased Aβ-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARγ present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARγ-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARγ results in increased sensitivity to Aβ and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARγ protective effects. PPARγ prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARγ supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.

Fatty acid oxidation by human liver peroxisomes

Abstract A cyanide insensitive fatty acid oxidation system is detected in human liver and is shown to be localized in peroxisomes by subcellular fractionation in Metrizamide continuous density gradients. Fatty acyl-CoA oxidase, its characteristic enzyme, acts maximally on C 12 –C 18 saturated fatty acids and on oleoyl-CoA and requires FAD. These results, together with the already established properties of the system in rat liver, support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans.

Protein kinase C inhibits amyloid β peptide neurotoxicity by acting on members of the Wnt pathway

Current evidence supports the notion that the amyloid β‐peptide (Aβ) plays a major role in the neurotoxicity observed in the brain in Alzheimers disease. However, the signal transduction mechanisms involved still remain unknown. In the present work, we analyzed the effect of protein kinase C (PKC) on some members of the Wnt signaling pathway and its implications for Aβ neurotoxicity. Activation of PKC by phorbol 12‐myristate 13‐acetate protected rat hippocampal neurons from Aβ toxicity. This effect was accomplished by inhibition of glycogen synthase kinase‐3β (GSK‐3β) activity, which led to the accumulation of cytoplasmic β‐catenin and transcriptional activation via β‐catenin/T‐cell factor/lymphoid enhancer factor‐1 (TCF/LEF1) of Wnt target genes, which in the present study were engrailed‐1 (en‐1) and cyclin D1 (cycD1). In contrast, inhibition of Ca2+‐dependent PKC isoforms activated GSK‐3β and offered no protection from Aβ neurotoxicity. Wnt‐3a and lithium salts, classical activators of the Wnt pathway, mimicked PKC activation. Our results suggest that regulation of members of the Wnt signaling pathway by Ca2+‐dependent PKC isoforms may be important in controlling the neurotoxic process induced by Aβ.

Rosiglitazone Treatment Prevents Mitochondrial Dysfunction in Mutant Huntingtin-expressing Cells: POSSIBLE ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ (PPARγ) IN THE PATHOGENESIS OF HUNTINGTON DISEASE*

Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the PPAR family of transcription factors. Synthetic PPARγ agonists are used as oral anti-hyperglycemic drugs for the treatment of non-insulin-dependent diabetes. However, emerging evidence indicates that PPARγ activators can also prevent or attenuate neurodegeneration. Given these previous findings, the focus of this report is on the potential neuroprotective role of PPARγ activation in preventing the loss of mitochondrial function in Huntington disease (HD). For these studies we used striatal cells that express wild-type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin protein at physiological levels. Treatment of mutant cells with thapsigargin resulted in a significant decrease in mitochondrial calcium uptake, an increase in reactive oxygen species production, and a significant decrease in mitochondrial membrane potential. PPARγ activation by rosiglitazone prevented the mitochondrial dysfunction and oxidative stress that occurred when mutant striatal cells were challenged with pathological increases in calcium. The beneficial effects of rosiglitazone were likely mediated by activation of PPARγ, as all protective effects were prevented by the PPARγ antagonist GW9662. Additionally, the PPARγ signaling pathway was significantly impaired in the mutant striatal cells with decreases in PPARγ expression and reduced PPARγ transcriptional activity. Treatment with rosiglitazone increased mitochondrial mass levels, suggesting a role for the PPARγ pathway in mitochondrial function in striatal cells. Altogether, this evidence indicates that PPARγ activation by rosiglitazone attenuates mitochondrial dysfunction in mutant huntingtin-expressing striatal cells, and this could be an important therapeutic avenue to ameliorate the mitochondrial dysfunction that occurs in HD.

Diacylglycerol activation of protein kinase C is modulated by long-chain acyl CoA

The activity of rat brain protein kinase C, measured in the presence of diacylglycerol, phosphatidylserine and Ca+2, was found to be greatly increased by micromolar amounts of long chain acyl-CoAs, using two different assay systems (lipids added as sonicated dispersion or as mixed micelles with Triton X-100). The potentiation phenomenon required the presence of both diacylglycerol and phosphatidylserine; it was observed at low and saturating concentrations of these effectors, and it was inhibited at high, non physiological Ca+2 concentrations. Under similar conditions, fatty acids alone or coenzyme A were ineffective. The data strongly suggest that acyl-CoAs at the intracellular concentration levels, are important in the modulation of protein kinase C, after activation of the enzyme by the phospholipase C/phosphatidylinositol pathway.

Inhibition of Nuclear Factor-κB Enhances the Capacity of Immature Dendritic Cells to Induce Antigen-Specific Tolerance in Experimental Autoimmune Encephalomyelitis

Autoimmune disorders develop as a result of deregulated immune responses that target self-antigens and cause destruction of healthy host tissues. Because dendritic cells (DCs) play an important role in the maintenance of peripheral immune tolerance, we are interested in identifying means of enhancing their therapeutic potential in autoimmune diseases. It is thought that during steady state, DCs are able to anergize potentially harmful T cells bearing T cell receptors that recognize self-peptide-major histocompatibility complexes. The tolerogenic capacity of DCs requires an immature phenotype, which is characterized by a reduced expression of costimulatory molecules. On the contrary, activation of antigen-specific naive T cells is enhanced by DC maturation, a process that involves expression of genes controlled by the transcription factor nuclear factor (NF)-κB. We evaluated the capacity of drugs that inhibit NF-κB to enhance the tolerogenic properties of immature DCs in the experimental autoimmune encephalomyelitis (EAE) model. We show that andrographolide, a bicyclic diterpenoid lactone, and rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, were able to interfere with NF-κB activation in murine DCs. As a result, treated DCs showed impaired maturation and a reduced capacity to activate antigen-specific T cells. Furthermore, NF-κB-blocked DCs had an enhanced tolerogenic capacity and were able to prevent EAE development in mice. The tolerogenic feature was specific for myelin antigens and involved the expansion of regulatory T cells. These data suggest that NF-κB blockade is a potential pharmacological approach that can be used to enhance the tolerogenic ability of immature DCs to prevent detrimental autoimmune responses.

Wnt signaling involvement in β-amyloid-dependent neurodegeneration

Abstract Alzheimer’s disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-β peptide (Aβ). We have observed that Aβ-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer’s patients.

Potentiation of diacylglycerol-activated protein kinase C by acyl-coenzyme A thioesters of hypolipidaemic drugs.

Acyl-Coenzyme A thioesters of the hypolipidaemic and cancerinogenic peroxisome proliferators clofibric acid, nafenopin, ciprofibrate, bezafibrate and tibric acid were found to greatly increase the activity of rat brain protein kinase C. Maximal activation required the simultaneous presence of Ca+2, phosphatidylserine and diolein, thus differentiating their action from that of other tumor promoters such as phorbol esters. Under similar conditions the unesterified drugs were comparatively ineffective. Similar results were obtained using the rat liver enzyme. The data suggest that acylcoenzyme A thioesters of hypolipidaemic drugs, may play a role in the induction of liver tumors by these compounds, through the potentiation of protein kinase C.

PPAR γ activators induce growth arrest and process extension in B12 oligodendrocyte‐like cells and terminal differentiation of cultured oligodendrocytes

Peroxisome proliferator‐activated receptors (PPARs) are key transcription factors in the control of lipid homeostasis and cell differentiation, but little is known about their function in oligodendrocytes, the major lipid‐synthesizing cells in the central nervous system (CNS). Using the B12 oligodendrocyte‐like cell line and rat spinal cord‐derived oligodendrocytes, we evaluated the importance of PPARγ in the maturation process of these cells. B12 cells express all PPAR isoforms (α, β/δ, and γ), as assessed by RT‐PCR, Western‐blot, and transactivation assays. B12 cells respond specifically to PPARγ agonists by arresting cell proliferation and extending cell processes, events that are blocked by the PPARγ antagonist GW9662. In addition, alkyl‐dihydroxyacetone phosphate synthase (ADAPS), a key peroxisomal enzyme involved in the synthesis of myelin‐rich lipid plasmalogens, is increased in PPARγ agonist‐treated B12 cells. In contrast with B12 cells, both immature and mature isolated spinal cord oligodendrocytes presented a high and similar expression level of ADAPS, as assessed by immunocytochemistry. However, as in B12 cells, isolated spinal cord oligodendrocytes were also found to respond specifically to PPARγ agonists with a four‐fold increase in the number of mature cells. Our data suggest a relevant role for PPARγ in oligodendrocyte lipid metabolism and differentiation.