Ivelisse Sánchez

Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders

The expansion of a CAG repeat coding for polyglutamine in otherwise unrelated gene products is central to eight neurodegenerative disorders including Huntingtons disease. It has been well documented that expanded polyglutamine fragments, cleaved from their respective full-length proteins, form microscopically visible aggregates in affected individuals and in transgenic mice. The contribution of polyglutamine oligomers to neurodegeneration, however, is controversial. The azo-dye Congo red binds preferentially to β-sheets containing amyloid fibrils and can specifically inhibit oligomerization and disrupt preformed oligomers. Here we show that inhibition of polyglutamine oligomerization by Congo red prevents ATP depletion and caspase activation, preserves normal cellular protein synthesis and degradation functions, and promotes the clearance of expanded polyglutamine repeats in vivo and in vitro. Infusion of Congo red into a transgenic mouse model of Huntingtons disease, well after the onset of symptoms, promotes the clearance of expanded repeats in vivo and exerts marked protective effects on survival, weight loss and motor function. We conclude that oligomerization is a crucial determinant in the biochemical properties of expanded polyglutamine that are central to their chronic cytotoxicity.

Caspase-8 Is Required for Cell Death Induced by Expanded Polyglutamine Repeats

We show here that caspase-8 is required for the death of primary rat neurons induced by an expanded polyglutamine repeat (Q79). Expression of Q79 recruited and activated caspase-8. Inhibition of caspase-8 blocked polyglutamine-induced cell death. Coexpression of Q79 with the caspase inhibitor CrmA, a dominant-negative mutant of FADD (FADD DN), Bcl-2, or Bcl-xL, but not an N-terminally tagged Bcl-xL, prevented the recruitment of caspase-8 and inhibited polyglutamine-induced cell death. Furthermore, Western blot analysis revealed the presence of activated caspase-8 in the insoluble fraction of affected brain regions from Huntingtons disease (HD) patients but not in those from neurologically unremarkable controls, suggesting the relocation and activation of caspase-8 during the pathogenesis of HD. These results suggest an essential role of caspase-8 in HD-related neural degenerative diseases.

A Convoluted Way to Die

The current picture of sympathetic neuronal cell death pathway is as follows (Figure 1(Figure 1). Upon removal of NGF, neurons induce at least four proteins that may play critical roles in apoptotic signal transduction: BIM, DP5/Hrk, and two transcription factors of the AP-1 family—c-Jun and c-Fos. Because c-Jun is necessary but maybe not sufficient to induce BIM expression, c-Jun and another factor(s) may act together to regulate the expression of BIM, DP5/Hrk, or other BH3-like proteins. c-Fos is unlikely to be involved as its induction is a very late event. The mechanism by which neurons activate c-Jun is at least partially through JNK. Activation of JNK is critical for the development of competence-to-die, which may act by releasing the cytoplasmic “brake” for caspase activation. Upregulated BIM, DP5/Hrk, etc., may act to neutralize the neuronal antiapoptotic arsenals such as BCL-2 and BCL-xL, thereby facilitating BAX aggregation and mitochondrial insertion. Thus, the neuronal apoptotic pathway is built in such a way to assure that only when both proapoptotic signaling pathways are activated is the green light to caspase activation turned on to be swiftly followed by neuronal death.The existing information showed that certain apoptotic pathways, such as death receptor–induced cell death, are incredibly direct: there are very few signal transduction steps that go from an extracellular death signal to the activation of caspases, which results in the quick final demise of a cell. In contrast, neurons seem to have evolved an intricate and convoluted way to die. Why? One reasonable answer is that most cell types in our body are programmed to live for a short time and are turned over rather quickly. Therefore, they are highly disposable—from the organism point of view, it is much better to eliminate a compromised cell than try to mend it. Neurons, on the other hand, are much more precious—throughout our lives, we maintain roughly the same set of neurons as when we are born, if we are fortunate enough to live free of neurodegenerative diseases. Furthermore, mature neurons are nonproliferative and, therefore, it is unlikely that a defective neuron may overproliferate and turn cancerous. Thus, the organism seems to have adapted an entirely different policy for neurons: to keep them alive any way it can.What does this complicated neuronal apoptosis pathway tell us about the therapeutic opportunity of neurodegenerative diseases? Many studies have demonstrated that apoptotic mechanisms contribute to both acute and chronic neurodegenerative diseases (Yuan and Yankner, 2000xYuan, J and Yankner, B.A. Nature. 2000; 407: 802–809Crossref | PubMed | Scopus (1267)See all References(Yuan and Yankner, 2000). Inhibition of caspase activation has shown beneficial effects in a number of animal models of neurodegenerative models. Convoluted neuronal cell death pathways may be good news to us: as multiple controlling points in the neuronal death pathways may provide additional flexibility in choosing the best therapeutic points. One word of caution, however, is that it remains to be seen whether activation of a single neuronal cell death pathway, which is not sufficient for neurons to die in short-term culture, can be ignored in the long term and whether a “half-dead” neuron can be fully functional. Nevertheless, we have made tremendous progress in understanding the mechanisms of neuronal cell death in the last decade, and this growing body of information will very likely help us to develop novel treatments of neurodegenerative diseases in the next decade.

A novel function of Ataxin-1 in the modulation of PP2A activity is dysregulated in the spinocerebellar ataxia type 1

An expansion of glutamines within the human ataxin-1 protein underlies spinocerebellar ataxia type 1 (SCA1), a dominantly inherited neurodegenerative disorder characterized by ataxia and loss of cerebellar Purkinje neurons. Although the mechanisms linking the mutation to the disease remain unclear, evidence indicates that it involves a combination of both gain and loss of functions of ataxin-1. We previously showed that the mutant ataxin-1 interacts with Anp32a, a potent and selective PP2A inhibitor, suggesting a role of PP2A in SCA1. Herein, we found a new function of ataxin-1: the modulation of Pp2a activity and the regulation of its holoenzyme composition, with the polyglutamine mutation within Atxn1 altering this function in the SCA1 mouse cerebellum before disease onset. We show that ataxin-1 enhances Pp2a-bβ expression and down-regulates Anp32a levels without affecting post-translational modifications of Pp2a catalytic subunit (Pp2a-c) known to regulate Pp2a activity. In contrast, mutant Atxn1 induces a decrease in Y307-phosphorylation in Pp2a-c, known to enhance its activity, while reducing Pp2a-b expression and inhibiting Anp32a levels. qRT-PCR and chromatin immunoprecipitation analyses show that ataxin-1-mediated regulations of the Pp2a-bβ subunit, specifically bβ2, and of Anp32a occur at the transcriptional level. The Pp2a pathway alterations were confirmed by identified phosphorylation changes of the known Pp2a-substrates, Erk2 and Gsk3β. Similarly, mutant ataxin-1-expressing SH-SY5Y cells exhibit abnormal neuritic morphology, decreased levels of both PP2A-Bβ and ANP32A, and PP2A pathway alterations, all of which are ameliorated by overexpressing ANP32A. Our results point to dysregulation of this newly assigned function of ataxin-1 in SCA1 uncovering new potential targets for therapy.

Characterization of Alu and recombination-associated motifs mediating a large homozygous SPG7 gene rearrangement causing hereditary spastic paraplegia

Spastic paraplegia type 7 (SPG7) is one of the most common forms of autosomal recessive hereditary spastic paraplegia (AR-HSP). Although over 77 different mutations have been identified in SPG7 patients, only 9 gross deletions have been reported with only a few of them being fully characterized. Here, we present a detailed description of a large homozygous intragenic SPG7 gene rearrangement involving a 5144-base pair (bp) genomic loss (c. 1450-446_1779 + 746 delinsAAAGTGCT) encompassing exons 11 to 13, identified in a Spanish AR-HSP family. Analysis of the deletion junction sequences revealed that the 5′ breakpoint of this SPG7 gene deletion was located within highly homologous Alu sequences where the 3′ breakpoint appears to be flanked by the core crossover hotspot instigator (chi)-like sequence (GCTGG). Furthermore, an 8-bp (AAAGTTGCT) conserved sequence at the breakpoint junction was identified, suggesting that the most likely mechanism for the occurrence of this rearrangement is by Alu microhomology and chi-like recombination-associated motif-mediated multiple exon deletion. Our results are consistent with non-allelic homologous recombination and non-homologous end joining in deletion mutagenesis for the generation of rearrangements. This study provides more evidence associating repeated elements as a genetic mechanism underlying neurodegenerative disorders, highlighting their importance in human diseases.

Rare Neurodegenerative Diseases: Clinical and Genetic Update

More than 600 human disorders afflict the nervous system. Of these, neurodegenerative diseases are usually characterised by onset in late adulthood, progressive clinical course, and neuronal loss with regional specificity in the central nervous system. They include Alzheimers disease and other less frequent dementias, brain cancer, degenerative nerve diseases, encephalitis, epilepsy, genetic brain disorders, head and brain malformations, hydrocephalus, stroke, Parkinsons disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS or Lou Gehrigs Disease), Huntingtons disease, and Prion diseases, among others. Neurodegeneration usually affects, but is not limited to, the cerebral cortex, intracranial white matter, basal ganglia, thalamus, hypothalamus, brain stem, and cerebellum. Although the majority of neurodegenerative diseases are sporadic, Mendelian inheritance is well documented. Intriguingly, the clinical presentations and neuropathological findings in inherited neurodegenerative forms are often indistinguishable from those of sporadic cases, suggesting that converging genomic signatures and pathophysiologic mechanisms underlie both hereditary and sporadic neurodegenerative diseases. Unfortunately, effective therapies for these diseases are scarce to non-existent. In this chapter, we highlight the clinical and genetic features associated with the rare inherited forms of neurodegenerative diseases, including ataxias, multiple system atrophy, spastic paraplegias, Parkinsons disease, dementias, motor neuron diseases, and rare metabolic disorders.