Julie E. Tetzlaff

Formation of toxic oligomeric α-synuclein species in living cells

Background Misfolding, oligomerization, and fibrillization of α-synuclein are thought to be central events in the onset and progression of Parkinsons disease (PD) and related disorders. Although fibrillar α-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells. Methodology/Principal Findings Here we used bimolecular fluorescence complementation (BiFC) to directly visualize α-synuclein oligomerization in living cells, allowing us to study the initial events leading to α-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting α-synuclein aggregation affect the process over time. Stabilization of α-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of α-synuclein oligomers. Introduction of PD-associated mutations in α-synuclein did not affect oligomer formation but the biochemical properties of the mutant α-synuclein oligomers differ from those of wild type α-synuclein. Conclusions/Significance This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of α-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies.

CHIP Targets Toxic α-Synuclein Oligomers for Degradation

α-Synuclein (αSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric αSyn intermediates may represent the most toxic αSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on αSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced αSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic αSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of αSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric αSyn may contribute to the successful development of drug therapies that target oligomeric αSyn by mimicking or enhancing the powerful effects of CHIP.

Evidence that 5-HT2A receptors in the hypothalamic paraventricular nucleus mediate neuroendocrine responses to (-)DOI.

The present study determined whether the serotonin2A (5-HT2A) receptors in the hypothalamic paraventricular nucleus mediate the neuroendocrine responses to a peripheral injection of the 5-HT2A/2Creceptor agonist (−)DOI [(−)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]. The 5-HT2A receptor antagonist MDL100,907 ((±)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol), the 5-HT2C receptor antagonist SB-242084 (6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline), or vehicle were microinjected bilaterally through a chronically implanted double-barreled cannula into the hypothalamic paraventricular nucleus 15 min before a peripheral injection of (−)DOI in conscious rats. (−)DOI significantly elevated plasma levels of oxytocin, prolactin, ACTH, corticosterone, and renin. Neither the 5-HT2A receptor antagonist nor the 5-HT2Creceptor antagonist, injected alone, altered the basal levels of these hormones. MDL100,907 (0.748, 7.48, and 18.7 nmol) dose dependently inhibited the (−)DOI-induced increase in all of the hormones except corticosterone. In contrast, SB-242084 (10 nmol) did not inhibit (−)DOI-increased hormone levels. To confirm the presence of 5-HT2A receptors in the hypothalamic paraventricular nucleus, 5-HT2A receptors were mapped using immunohistochemistry. Densely labeled magnocellular neurons were observed throughout the anterior and posterior magnocellular subdivisions of the hypothalamic paraventricular nucleus. Moderately to densely labeled cells were also observed in parvicellular regions. Thus, it is likely that 5-HT2A receptors are present on neuroendocrine cells in the hypothalamic paraventricular nucleus. These data provide the first direct evidence that neuroendocrine responses to a peripheral injection of (−)DOI are predominantly mediated by activation of 5-HT2A receptors in the hypothalamic paraventricular nucleus.

Dopamine-Induced Conformational Changes in Alpha-Synuclein

Background Oligomerization and aggregation of α-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinsons disease [1]. However, α-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of α-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7]. Methodology/Principal Findings Here, we show that α-synuclein adopts a variety of conformations in primary neuronal cultures using fluorescence lifetime imaging microscopy (FLIM). Importantly, we found that dopamine, but not dopamine agonists, induced conformational changes in α-synuclein which could be prevented by blocking dopamine transport into the cell. Dopamine also induced conformational changes in α-synuclein expressed in neuronal cell lines, and these changes were also associated with alterations in oligomeric/aggregated species. Conclusion/Significance Our results show, for the first time, a direct effect of dopamine on the conformation of α-synuclein in neurons, which may help explain the increased vulnerability of dopaminergic neurons in Parkinsons disease.

Functional recovery after facial nerve crush is delayed in severe combined immunodeficient mice

The goal of the current study was to determine if T and B lymphocytes play a role in functional recovery after peripheral nerve injury. The time course of behavioral recovery following facial nerve crush injury at the stylomastoid foramen was established in scid mice which lack functional T and B cells and reconstituted scid mice as compared to wild-type mice. The average time necessary for recovery of full eye blink reflex and vibrissae movements in wild-type mice was 10.3+/-0.2 and 9.9+/-0.34 days, respectively. In contrast, recovery of full eye blink reflex and vibrissae movements took 14.8+/-0.54 and 12.3+/-0.41 days, respectively, in scid mice. Reconstitution of scid mice with whole splenocytes resulted in functional recovery times similar to wild-type, with eye blink reflex recovery and vibrissae movement being 10.5+/-0.3 and 10.0+/-0.0 days, respectively. These results suggest that the delayed behavioral recovery time observed in scid mice may be due to the absence of T and B lymphocytes.

Motoneuron injury and repair: New perspectives on gonadal steroids as neurotherapeutics.

In this review, we will summarize recent work from our laboratory on the role of gonadal steroids as neuroprotective agents in motoneuron viability following cell stress. Three motoneuron models will be discussed: developing axotomized hamster facial motoneurons (FMNs); adult axotomized mouse FMNs; and immortalized, cultured mouse spinal motoneurons subjected to heat shock. New work on two relevant motoneuron proteins, the survival of motor neuron protein, and neuritinor candidate plasticity-related gene 15, indicates differential steroid regulation of these two proteins after axotomy. The concept of gonadal steroids as cellular stress correction factors and the implications of this for acute neurological injury situations will be presented as well.

Exogenous androgen treatment delays the stress response following hamster facial nerve injury.

Following injury or stress of any type, cells undergo a stress response, involving the cessation of general protein synthesis and the up‐regulation of heat shock proteins (HSP), which have been implicated in promoting cell survival and repair. In a variety of neuronal injury models, including the hamster facial motoneurone (FMN) model, steroid hormones augment regeneration and are neuroprotective. We have previously shown that facial nerve axotomy induces expression of HSP70 (HSP70) and/or up‐regulates constitutively expressed HSP70 (HSC70) mRNA in axotomised hamster FMN and that testosterone propionate (TP) treatment reduces this response. These previous studies were unable to differentiate between HSC70 mRNA and HSP70 mRNA. Therefore, an objective of the present study was to determine which HSP (HSC70 or HSP70) was being up‐regulated by axotomy and reduced by TP. Axotomy increased HSC70 protein in axotomised and non‐axotomised FMN, relative to untreated baseline hamsters. Interestingly, TP transiently delayed the stress‐induced up‐regulation of HSC70 protein in axotomised FMN compared to axotomised FMN from non‐TP treated controls. A potential explanation for this delay may involve the TP‐induced liberation of HSP from the androgen receptor, which would provide the injured cell with an immediately available pool of protective HSP. An hypothesis is presented suggesting that this TP‐induced delay of stress‐induced HSC70 up‐regulation might allow for the diversion of cellular energy away from HSP synthesis and towards the synthesis of proteins required for regeneration and survival.