In vivo and in vitro determination of cell death markers in neurons.

Mitochondria are key regulators of cellular death. The mitochondrial membranes contain essential enzyme complexes for maintaining metabolic homeostasis and meeting the energy requirements of the cell (Tait and Green, Nat Rev Mol Cell Biol 11:621-632, 2010 and Galluzzi et al., Apoptosis 12:803-813, 2007). Thus, any perturbation of outer or inner mitochondrial membranes can lead to disruptions in the normal fluxes of key ions and metabolic proteins (i.e., ADP/ATP exchange), leading to eventual cellular death. In addition to maintaining cellular viability, mitochondria play a critical role in the initiation of programmed cell death. As initiators of the cell death process, key mitochondrial proteins [Cytochrome C (Cyt C) one of the most well-studied among them] are released from the intermembrane space during early cell death events eventually leading to caspase activation. Release of Cyt C is a crucial step during cellular death (Tait and Green, Nat Rev Mol Cell Biol 11:621-632, 2010). Therefore, the measurement of Cyt C release can give vital information about cell death signaling. Immunolabeling against Cyt C can give an easy readout of mitochondrial integrity as well, allowing for simultaneous identification of mitochondrial viability (and/or damage) and initiation of intracellular death processes. In this chapter, we use Cyt C as a dual marker of mitochondrial integrity and cell death and review several protocols to measure Cyt C localization into intact mitochondria and its release into the cytosol. The goal is to offer an array of assays that, combined, provide both qualitative and quantitative analysis of the relationship between mitochondrial viability and activation of an intracellular cell death process. Immunofluorescence, Western blot, and ELISA measurements of Cyt C as are discussed in detail.

Dysregulation of axonal transport and motorneuron diseases.

MNDs (motorneuron diseases) are neurodegenerative disorders in which motorneurons located in the motor cortex, in the brainstem and in the spinal cord are affected. These diseases in their inherited or sporadic forms are mainly characterized by motor dysfunctions, occasionally associated with cognitive and behavioural alterations. Although these diseases show high variability in onset, progression and clinical symptoms, they share common pathological features, and motorneuronal loss invariably leads to muscle weakness and atrophy. One of the most relevant aspect of these disorders is the occurrence of defects in axonal transport, which have been postulated to be either a direct cause, or a consequence, of motorneuron degeneration. In fact, due to their peculiar morphology and high energetic metabolism, motorneurons deeply rely on efficient axonal transport processes. Dysfunction of axonal transport is known to adversely affect motorneuronal metabolism, inducing progressive degeneration and cell death. In this regard, the understanding of the fine mechanisms at the basis of the axonal transport process and of their possible alterations may help shed light on MND pathological processes. In the present review, we will summarize what is currently known about the alterations of axonal transport found to be either causative or a consequence of MNDs.

17-AAG increases autophagic removal of mutant androgen receptor in spinal and bulbar muscular atrophy.

Several types of motorneuron diseases are linked to neurotoxic mutant proteins. These acquire aberrant conformations (misfolding) that trigger deleterious downstream events responsible for neuronal dysfunction and degeneration. The pharmacological removal of misfolded proteins might thus be useful in these diseases. We utilized a peculiar motorneuronal disease model, spinobulbar muscular atrophy (SBMA), in which the neurotoxicity of the protein involved, the mutant androgen receptor (ARpolyQ), can be modulated by its ligand testosterone (T). 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) has already been proven to exert beneficial action in SBMA. Here we demonstrated that 17-AAG exerts its pro-degradative activity on mutant ARpolyQ without impacting on proteasome functions. 17-AAG removes ARpolyQ misfolded species and aggregates by activating the autophagic system. We next analyzed the 17-AAG effects on two proteins (SOD1 and TDP-43) involved in related motorneuronal diseases, such as amyotrophic lateral sclerosis (ALS). In these models 17-AAG was unable to counteract protein aggregation.

A role of small heat shock protein B8 (HspB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signaling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motor neuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.

Proteasomal and autophagic degradative activities in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA or Kennedy's disease) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons in the bulbar region of the brain and in the anterior horns of the spinal cord. The disease has been associated to an expansion of a CAG triplet repeat present in the first coding exon of the androgen receptor (AR) gene. SBMA was the first identified member of a large class of neurodegenerative diseases now known as CAG-related diseases, which includes Huntington's disease (HD), several types of spinocerebellar ataxia (SCAs), and dentatorubral and pallidoluysian atrophy (DRPLA). The expanded CAG tract is translated to an aberrantly long polyglutamine tract (ARpolyQ) in the N-terminal region of the AR protein. The elongated polyQ tract seems to confer a neurotoxic gain-of-function to the mutant AR, possibly via the generation of aberrant conformations (misfolding). Protein misfolding is thought to be a trigger of neurotoxicity, since it perturbs a wide variety of motor neuronal functions. The first event is the accumulation of the ARpolyQ into ubiquitinated aggregates in a ligand (testosterone) dependent manner. The mutant ARpolyQ also impairs proteasome functions. The autophagic pathway may be activated to compensate these aberrant events by clearing the mutant ARpolyQ from motor neuronal cells. This review illustrates the mechanisms at the basis of ARpolyQ degradation via the proteasomal and autophagic systems.

The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS).

Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are characterized by the presence of misfolded proteins, thought to trigger neurotoxicity. Some familial forms of ALS (fALS), clinically indistinguishable from sporadic ALS (sALS), are linked to superoxide dismutase 1 (SOD1) gene mutations. It has been shown that the mutant SOD1 misfolds, forms insoluble aggregates and impairs the proteasome. Using transgenic G93A-SOD1 mice, we found that spinal cord motor neurons, accumulating mutant SOD1 also over-express the small heat shock protein HspB8. Using motor neuronal fALS models, we demonstrated that HspB8 decreases aggregation and increases mutant SOD1 solubility and clearance, without affecting wild-type SOD1 turnover. Notably, HspB8 acts on mutant SOD1 even when the proteasome activity is specifically blocked. The pharmacological blockage of autophagy resulted in a dramatic increase of mutant SOD1 aggregates. Immunoprecipitation studies, performed during autophagic flux blockage, demonstrated that mutant SOD1 interacts with the HspB8/Bag3/Hsc70/CHIP multiheteromeric complex, known to selectively activate autophagic removal of misfolded proteins. Thus, HspB8 increases mutant SOD1 clearance via autophagy. Autophagy activation was also observed in lumbar spinal cord of transgenic G93A-SOD1 mice since several autophago-lysosomal structures were present in affected surviving motor neurons. Finally, we extended our observation to a different ALS model and demonstrated that HspB8 exerts similar effects on a truncated version of TDP-43, another protein involved both in fALS and in sALS. Overall, these results indicate that the pharmacological modulation of HspB8 expression in motor neurons may have important implications to unravel the molecular mechanisms involved both in fALS and in sALS.

Estrogen receptor beta and the progression of prostate cancer: role of 5alpha-androstane-3beta,17beta-diol.

Prostate cancer (PC) develops in response to an abnormal activation of androgen receptor induced by circulating androgens and, in its initial stages, is pharmacologically controlled by androgen blockade. However, androgen ablation therapy often allows androgen-independent PC development, generally characterized by increased invasiveness. We previously reported that 5alpha-androstane-3beta,17beta-diol (3beta-Adiol) inhibits the migration of PC cell lines via the estrogen receptor beta (ERbeta) activation. Here, by combining in vitro assays and in vivo imaging approaches, we analyzed the effects of 3beta-Adiol on PC proliferation, migration, invasiveness, and metastasis in cultured cells and in xenografts using luciferase-labeled PC3 (PC3-Luc) cells. We found that 3beta-Adiol not only inhibits PC3-Luc cell migratory properties, but also induces a broader anti-tumor phenotype by decreasing the proliferation rate, increasing cell adhesion, and reducing invasive capabilities in vitro. All these 3beta-Adiol activities are mediated by ERbeta and cannot be reproduced by the physiological estrogen, 17beta-estradiol, suggesting the existence of different pathways activated by the two ERbeta ligands in PC3-Luc cells. In vivo, continuous administration of 3beta-Adiol reduces growth of established tumors and counteracts metastasis formation when PC3-Luc cells are engrafted s.c. in nude mice or are orthotopically injected into the prostate. Since 3beta-Adiol has no androgenic activity, and cannot be converted to androgenic compounds, the effects here described entail a novel potential application of this agent against human PC.

ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl-2.

In mutant superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS), accumulation of misfolded mutant SOD1 in spinal cord mitochondria is thought to cause mitochondrial dysfunction. Whether mutant SOD1 is toxic per se or whether it damages the mitochondria through interactions with other mitochondrial proteins is not known. We previously identified Bcl-2 as an interacting partner of mutant SOD1 specifically in spinal cord, but not in liver, mitochondria of SOD1 mice and patients. We now show that mutant SOD1 toxicity relies on this interaction. Mutant SOD1 induces mitochondrial morphological changes and compromises mitochondrial membrane integrity leading to release of Cytochrome C only in the presence of Bcl-2. In cells, mouse and human spinal cord with SOD1 mutations, the binding to mutant SOD1 triggers a conformational change in Bcl-2 that results in the uncovering of its toxic BH3 domain and conversion of Bcl-2 into a toxic protein. Bcl-2 carrying a mutagenized, non-toxic BH3 domain fails to support mutant SOD1 mitochondrial toxicity. The identification of Bcl-2 as a specific target and active partner in mutant SOD1 mitochondrial toxicity suggests new therapeutic strategies to inhibit the formation of the toxic mutant SOD1/Bcl-2 complex and to prevent mitochondrial damage in ALS.

The role of the polyglutamine tract in androgen receptor.

The androgen receptor (AR) is a ligand-activated transcription factor which is responsible for the androgen responsiveness of target cells. Several types of mutations have been found in the AR and linked to endocrine dysfunctions. Surprisingly, the polymorphism involving the CAG triplet repeat expansion of the AR gene, coding for a polyglutamine (PolyGln) tract in the N-terminal transactivation domain of the AR protein, has been involved either in endocrine or neurological disorders. For example, among endocrine-related-diseases, the PolyGln size has been proposed to be associated to prostate cancer susceptibility, hirsutism, male infertility, cryptorchidism (in conjunction with polyglycine stretches polymorphism), etc.; the molecular mechanisms of these alterations are thought to involve a modulation of AR transcriptional competence, which inversely correlates with the PolyGln length. Among neurological alterations, a decreased AR function seems to be also involved in depression. Moreover, when the polymorphic PolyGln becomes longer than 35-40 contiguous glutamines (ARPolyGln), the ARPolyGln acquires neurotoxicity, because of an unknown gain-of-function. This mutation has been linked to a rare inherited X-linked motor neuronal disorder, the Spinal and Bulbar Muscular Atrophy, or Kennedy's disease. The disorder is characterized by death of motor neurons expressing high levels of AR. The degenerating motor neurons are mainly located in the anterior horns of the spinal cord and in the bulbar region; some neurons of the dorsal root ganglia may also be involved. Interestingly, the same type of PolyGln elongation has been found in other totally unrelated proteins responsible for different neurodegenerative diseases. A common feature of all these disorders is the formation of intracellular aggregates containing the mutated proteins; at present, but their role in the disease is largely debated. This review will discuss how the PolyGln neurotoxicity of SBMA AR may be either mediated or decreased by aggregates, and will present data on the dual role played by testosterone on motor neuronal functions and dysfunctions.

Mutation of SOD1 in ALS: a gain of a loss of function.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by motoneuron loss. Some familial cases (fALS) are linked to mutations of superoxide dismutase type-1 (SOD1), an antioxidant enzyme whose activity is preserved in most mutant forms. Owing to the similarities in sporadic and fALS forms, mutant SOD1 animal and cellular models are a useful tool to study the disease. In transgenic mice expressing either wild-type (wt) human SOD1 or mutant G93A-SOD1, we found that wtSOD1 was present in cytoplasm and in nuclei of motoneurons, whereas mutant SOD1 was mainly cytoplasmic. Similar results were obtained in immortalized motoneurons (NSC34 cells) expressing either wtSOD1 or G93A-SOD1. Analyzing the proteasome activity, responsible for misfolded protein clearance, in the two subcellular compartments, we found proteasome impairment only in the cytoplasm. The effect of G93A-SOD1 exclusion from nuclei was then analyzed after oxidative stress. Cells expressing G93A-SOD1 showed a higher DNA damage compared with those expressing wtSOD1, possibly because of a loss of nuclear protection. The toxicity of mutant SOD1 might, therefore, arise from an initial misfolding (gain of function) reducing nuclear protection from the active enzyme (loss of function in the nuclei), a process that may be involved in ALS pathogenesis.

Aggregation and proteasome: the case of elongated polyglutamine aggregation in spinal and bulbar muscular atrophy.

Aggregates, a hallmark of most neurodegenerative diseases, may have different properties, and possibly different roles in neurodegeneration. We analysed ubiquitin-proteasome pathway functions during cytoplasmic aggregation in polyglutamine (polyQ) diseases, using a unique model of motor neuron disease, the SpinoBulbar Muscular Atrophy. The disease, which is linked to a polyQ tract elongation in the androgen receptor (ARpolyQ), has the interesting feature that ARpolyQ aggregation is triggered by the AR ligand, testosterone. Using immortalized motor neurons expressing ARpolyQ, we found that a proteasome reporter, YFPu, accumulated in absence of aggregates; testosterone treatment, which induced ARpolyQ aggregation, allowed the normal clearance of YFPu, suggesting that aggregation contributed to proteasome de-saturation, an effect not related to AR nuclear translocation. Using AR antagonists to modulate the kinetic of ARpolyQ aggregation, we demonstrated that aggregation, by removing the neurotoxic protein from the soluble compartment, protected the proteasome from an excess of misfolded protein to be processed.

The androgen derivative 5alpha-androstane-3beta,17beta-diol inhibits prostate cancer cell migration through activation of the estrogen receptor beta subtype.

Prostate cancer growth depends, in its earlier stages, on androgens and is usually pharmacologically modulated with androgen blockade. However, androgen-ablation therapy may generate androgen-independent prostate cancer, often characterized by an increased invasiveness. We have found that the 5alpha-reduced testosterone derivative, dihydrotestosterone (the most potent natural androgen) inhibits cell migration with an androgen receptor-independent mechanism. We have shown that the dihydrotestosterone metabolite 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), a steroid which does not bind androgen receptors, but efficiently binds the estrogen receptor beta (ERbeta), exerts a potent inhibition of prostate cancer cell migration through the activation of the ERbeta signaling. Very surprisingly, estradiol is not active, suggesting the existence of different pathways for ERbeta activation in prostate cancer cells. Moreover, 3beta-Adiol, through ERbeta, induces the expression of E-cadherin, a protein known to be capable of blocking metastasis formation in breast and prostate cancer cells. The inhibitory effects of 3beta-Adiol on prostate cancer cell migration is counteracted by short interfering RNA against E-cadherin. Altogether, the data showed that (a) circulating testosterone may act with estrogenic effects downstream in the catabolic process present in the prostate, and (b) that the estrogenic effect of testosterone derivatives (ERbeta-dependent) results in the inhibition of cell migration, although it is apparently different from that linked to estradiol on the same receptor and may be protective against prostate cancer invasion and metastasis. These results also shed some light on clinical observations suggesting that alterations in genes coding for 3beta-hydroxysteroid dehydrogenases (the enzymes responsible for 3beta-Adiol formation) are strongly correlated with hereditary prostate cancer.