ST-segment elevation in a patient with cardiac lymphoma

Netherlands Heart Journal -     

Mutant ataxin-3 promotes the autophagic degradation of parkin

There is growing evidence that autophagy plays a key role in neurodegenerative diseases. For instance, stimulating autophagy is neuroprotective both in vitro and in vivo in models of trinucleotide-repeat diseases such as Machado-Joseph disease (MJD). Similarly, proteins associated with familial forms of Parkinson disease (PD) such as parkin and PINK1 converge on the autophagy pathway. Yet, despite these shared mechanisms, it is not clear whether or how these disorders are related at a molecular level. We reported that the mutant form of ataxin-3, the protein responsible for MJD, promotes the autophagic degradation of parkin. Given that the loss of parkin function leads to PD, we propose that the increased turnover of parkin triggered by mutant ataxin-3 may explain some of the parkinsonian features observed in MJD. Moreover, the findings suggest that an increased clearance of parkin in MJD could mitigate the otherwise beneficial effects of autophagy in neurodegeneration.     

Ataxin-3 deubiquitination is coupled to Parkin ubiquitination via E2 ubiquitin-conjugating enzyme

We reported previously that parkin, a Parkinson disease-associated E3 ubiquitin-ligase interacts with ataxin-3, a deubiquitinating enzyme associated with Machado-Joseph disease. Ataxin-3 was found to counteract parkin self-ubiquitination both in vitro and in cells. Moreover, ataxin-3-dependent deubiquitination of parkin required the catalytic cysteine 14 in ataxin-3, although the precise mechanism remained unclear. We report here that ataxin-3 interferes with the attachment of ubiquitin (Ub) onto parkin in real-time during conjugation but is unable to hydrolyze previously assembled parkin-Ub conjugates. The mechanism involves an ataxin-3-dependent stabilization of the complex between parkin and the E2 Ub-conjugating enzyme, which impedes the efficient charging of the E2 with Ub. Moreover, within this complex, the transfer of Ub from the E2 is diverted away from parkin and onto ataxin-3, further explaining how ataxin-3 deubiquitination is coupled to parkin ubiquitination. Taken together, our findings reveal an unexpected convergence upon the E2 Ub-conjugating enzyme in the regulation of an E3/deubiquitinating enzyme pair, with important implications for the function of parkin and ataxin-3, two proteins responsible for closely related neurodegenerative diseases.     

Long-Term Potentiation in Isolated Dendritic Spines

Background

In brain, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation can induce long-lasting changes in synaptic α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor (AMPAR) levels. These changes are believed to underlie the expression of several forms of synaptic plasticity, including long-term potentiation (LTP). Such plasticity is generally believed to reflect the regulated trafficking of AMPARs within dendritic spines. However, recent work suggests that the movement of molecules and organelles between the spine and the adjacent dendritic shaft can critically influence synaptic plasticity. To determine whether such movement is strictly required for plasticity, we have developed a novel system to examine AMPAR trafficking in brain synaptosomes, consisting of isolated and apposed pre- and postsynaptic elements.

Methodology/Principal Findings

We report here that synaptosomes can undergo LTP-like plasticity in response to stimuli that mimic synaptic NMDAR activation. Indeed, KCl-evoked release of endogenous glutamate from presynaptic terminals, in the presence of the NMDAR co-agonist glycine, leads to a long-lasting increase in surface AMPAR levels, as measured by [³H]-AMPA binding; the increase is prevented by an NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). Importantly, we observe an increase in the levels of GluR1 and GluR2 AMPAR subunits in the postsynaptic density (PSD) fraction, without changes in total AMPAR levels, consistent with the trafficking of AMPARs from internal synaptosomal compartments into synaptic sites. This plasticity is reversible, as the application of AMPA after LTP depotentiates synaptosomes. Moreover, depotentiation requires proteasome-dependent protein degradation.

Conclusions/Significance

Together, the results indicate that the minimal machinery required for LTP is present and functions locally within isolated dendritic spines.     

Long-term culture of hepatocytes from human adults

A long-term primary human hepatocyte culture retraining liver-specific functions is important and essential for basic research and for the future development of hepatocyte-based applications. We established a normal hepatocyte culture system from excess normal tissues obtained from adult liver cancer patients who received partial liver resection. Hepatocytes were isolated after perfusion and enzymatic disaggregation, and were first maintained in hormonally defined media on a Matrigel matrix, and then transferred to collagen sandwich gel. The hepatocytes formed clusters on the Matrigel matrix and increased in size and numbers with time of culture and eventually grew into spheroids of variable sizes. After being transferred to collagen gel, the cells migrated outward from spheroids to form a monolayer with cuboidal or polygonal cell shapes with granular cytoplasm and continued to proliferate. Cellular functions specific for hepatocytes were analyzed using immunoblot assay for proteins specifically secreted by the liver cells on different days of culture. The cells secreted albumin, transferrin and -fetoprotein consistently for more than 100 days, to a maximum of 150 days. Thus, we have established a long-term culture of hepatocytes from human adults, which will be useful for basic studies of liver physiology such as metabolism and morphogenesis, as well as for other applications in the study of infectious hepatitis, pharmacology, pharmacokinetics, and toxicology.     

Lanosterol metabolism and sterol regulatory element binding protein (SREBP) expression in male germ cell maturation

Expression of genes involved in cholesterol biosynthesis in male germ cells is insensitive to the negative cholesterol feedback regulation, in contrast to cholesterol level-sensitive/sterol regulatory element binding protein (SREBP)-dependent gene regulation in somatic cells. The role of sterol regulatory element binding proteins in spermatogenic cells was an enigma until recently, when a soluble, 55 kDa cholesterol-insensitive form of SREBP2 (SREBP2gc) was discovered [Mol. Cell. Endocrinol. 22 (2002) 8478], being translated from a germ cell-specific SREBP2 mRNA. Our RT-PCR results also show that SREBP2 as well as SREBP1c mRNAs are detectable in prepubertal and postpubertal male germ cells while SREBP1a is not detected. Surprisingly, three SREBP2 immunoreactive proteins (72, 63 and 55 kDa), that are not present in mouse liver nuclei, reside in testis nuclei of prepubertal and adult mice. The 55 kDa protein is likely SREBP2gc, the other two isoforms are novel. HPLC measurements in liver and testes of fasted prepubertal and postpubertal mice showed no significant difference in cholesterol level. However, FF-MAS and lanosterol/testis-meiosis activating sterol (T-MAS) intermediates that are detectable mainly in testes, increase in fasted postpubertal mice which coincides well with the elevated level of 68 kDa SREBP2. Similar to SREBP2gc, the two novel SREBP2 immunoreactive proteins seem to be insensitive to the level of cholesterol.     

BAG5 inhibits parkin and enhances dopaminergic neuron degeneration

Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, are the major cause of early-onset Parkinson's disease (PD). Decreases in parkin activity may also contribute to neurodegeneration in sporadic forms of PD. Here, we show that bcl-2-associated athanogene 5 (BAG5), a BAG family member, directly interacts with parkin and the chaperone Hsp70. Within this complex, BAG5 inhibits both parkin E3 ubiquitin ligase activity and Hsp70-mediated refolding of misfolded proteins. BAG5 enhances parkin sequestration within protein aggregates and mitigates parkin-dependent preservation of proteasome function. Finally, BAG5 enhances dopamine neuron death in an in vivo model of PD, whereas a mutant that inhibits BAG5 activity attenuates dopaminergic neurodegeneration. This contrasts with the antideath functions ascribed to BAG family members and suggests a potential role for BAG5 in promoting neurodegeneration in sporadic PD through its functional interactions with parkin and Hsp70.