Claudia Manzoni

Synthetic amyloid-β oligomers impair long-term memory independently of cellular prion protein

Inability to form new memories is an early clinical sign of Alzheimer’s disease (AD). There is ample evidence that the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Aβ are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Aβ−mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Aβ1–42 oligomers impaired consolidation of the long-term recognition memory, whereas mature Aβ1–42 fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Aβ antibody. It has been suggested that the cellular prion protein (PrPC) mediates the impairment of synaptic plasticity induced by Aβ. We confirmed that Aβ1–42 oligomers interact with PrPC, with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Aβ1–42 oligomers are responsible for cognitive impairment in AD and that PrPC is not required.

A Recessive Mutation in the APP Gene with Dominant-Negative Effect on Amyloidogenesis

β-Amyloid precursor protein (APP) mutations cause familial Alzheimers disease with nearly complete penetrance. We found an APP mutation [alanine-673→valine-673 (A673V)] that causes disease only in the homozygous state, whereas heterozygous carriers were unaffected, consistent with a recessive Mendelian trait of inheritance. The A673V mutation affected APP processing, resulting in enhanced β-amyloid (Aβ) production and formation of amyloid fibrils in vitro. Co-incubation of mutated and wild-type peptides conferred instability on Aβ aggregates and inhibited amyloidogenesis and neurotoxicity. The highly amyloidogenic effect of the A673V mutation in the homozygous state and its anti-amyloidogenic effect in the heterozygous state account for the autosomal recessive pattern of inheritance and have implications for genetic screening and the potential treatment of Alzheimers disease.

Inhibition of LRRK2 kinase activity stimulates macroautophagy.

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinsons disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.

LRRK2 and Human Disease: A Complicated Question or a Question of Complexes?

The kinase LRRK2 is genetically linked to diverse pathologies, including Parkinson’s and Crohn’s diseases. Leucine-rich repeat kinase 2 (LRRK2) is linked to various diseases, including Parkinson’s disease, cancer, and leprosy. Data from LRRK2 knockout mice has highlighted a possible role for LRRK2 in regulating signaling pathways that are linked to the pathogenesis of Crohn’s disease. Here, we examine how LRRK2’s role as a signaling hub in the cell could lead to diverse pathologies.

Conformational plasticity of the Gerstmann-Sträussler-Scheinker disease peptide as indicated by its multiple aggregation pathways

The existence of several prion strains and their capacity of overcoming species barriers seem to point to a high conformational adaptability of the prion protein. To investigate this structural plasticity, we studied here the aggregation pathways of the human prion peptide PrP82-146, a major component of the Gerstmann-Sträussler-Scheinker amyloid disease. By Fourier transform infrared (FT-IR) spectroscopy, electron microscopy, and atomic force microscopy (AFM), we monitored the time course of PrP82-146 fibril formation. After incubation at 37 degrees C, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular beta-sheet infrared bands. At a critical oligomer concentration, the emergence of a new FT-IR band allowed to detect fibril formation. A different intermolecular beta-sheet interaction of the peptides in oligomers and in fibrils is, therefore, detected by FT-IR spectroscopy, which, in addition, suggests a parallel orientation of the cross beta-sheet structures of PrP82-146 fibrils. By AFM, a wide distribution of PrP82-146 oligomer volumes--the smallest ones containing from 5 to 30 peptides--was observed. Interestingly, the statistical analysis of AFM data enabled us to detect a quantization in the oligomer height values differing by steps of approximately 0.5 nm that could reflect an orientation of oligomer beta-strands parallel with the sample surface. Different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. Thermal aggregation of PrP82-146 was also investigated by FT-IR spectroscopy, which indicated for these aggregates an intermolecular beta-sheet interaction different from that observed for oligomers and fibrils. Unexpectedly, random aggregates, induced by solvent evaporation, were found to display a significant alpha-helical structure as well as several beta-sheet components. All these results clearly point to a high plasticity of the PrP82-146 peptide, which was found to be capable of undergoing several aggregation pathways, with end products displaying different secondary structures and intermolecular interactions.

Dysfunction of the autophagy/lysosomal degradation pathway is a shared feature of the genetic synucleinopathies

The past decade has witnessed huge advances in our understanding of the genetics underlying Parkinsons disease. Identifying commonalities in the biological function of genes linked to Parkinsons provides an opportunity to elucidate pathways that lead to neuronal degeneration and eventually to disease. We propose that the genetic forms of Parkinsons disease largely associated with α‐synuclein‐positive neuropathology (SNCA, LRRK2, and GBA) are brought together by involvement in the autophagy/lysosomal pathway and that this represents a unifying pathway to disease in these cases.—Manzoni, C., Lewis, P. A., Dysfunction of the autophagy/lysosomal degradation pathway is a shared feature of the genetic synucleinopathies. FASEB J. 27, 3424–3429 (2013). www.fasebj.org

Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation

Highlights • Mutations in the ROC, COR and Kinase domain of LRRK2 alter the autophagic response to starvation.• LC3-I/II ratio following starvation is altered by mutations, as well as p62 and WIPI2 positive puncta.• This occurs independently of any alteration in downstream targets of mTORC1.

Cellular processes associated with LRRK2 function and dysfunction

Mutations in the leucine‐rich repeat kinase 2 (LRRK2)‐encoding gene are the most common cause of monogenic Parkinsons disease. The identification of LRRK2 polymorphisms associated with increased risk for sporadic Parkinsons disease, as well as the observation that LRRK2‐Parkinsons disease has a pathological phenotype that is almost indistinguishable from the sporadic form of disease, suggested LRRK2 as the culprit to provide understanding for both familial and sporadic Parkinsons disease cases. LRRK2 is a large protein with both GTPase and kinase functions. Mutations segregating with Parkinsons disease reside within the enzymatic core of LRRK2, suggesting that modification of its activity impacts greatly on disease onset and progression. Although progress has been made since its discovery in 2004, there is still much to be understood regarding LRRK2′s physiological and neurotoxic properties. Unsurprisingly, given the presence of multiple enzymatic domains, LRRK2 has been associated with a diverse set of cellular functions and signalling pathways including mitochondrial function, vesicle trafficking together with endocytosis, retromer complex modulation and autophagy. This review discusses the state of current knowledge on the role of LRRK2 in health and disease with discussion of potential substrates of phosphorylation and functional partners with particular emphasis on signalling mechanisms. In addition, the use of immune cells in LRRK2 research and the role of oxidative stress as a regulator of LRRK2 activity and cellular function are also discussed.

Gerstmann-Sträussler-Scheinker Disease Amyloid Protein Polymerizes According to the “Dock-and-Lock” Model

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired prion diseases such as Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues ∼82–146, which when synthesized as a peptide, readily forms fibrils featuring GSS amyloid. The present study employed surface plasmon resonance (SPR) to characterize the binding events underlying PrP82–146 oligomerization at the first stages of fibrillization, according to evidence suggesting a pathogenic role of prefibrillar oligomers rather than mature amyloid fibrils. We followed in real time the binding reactions occurring during short term (seconds) addition of PrP82–146 small oligomers (1–5-mers, flowing species) onto soluble prefibrillar PrP82–146 aggregates immobilized on the sensor surface. SPR data confirmed very efficient aggregation/elongation, consistent with the hypothesis of nucleation-dependent polymerization process. Much lower binding was observed when PrP82–146 flowed onto the scrambled sequence of PrP82–146 or onto prefibrillar Aβ42 aggregates. As previously found with Aβ40, SPR data could be adequately fitted by equations modeling the “dock-and-lock” mechanism, in which the “locking” step is due to sequential conformational changes, each increasing the affinity of the monomer for the fibril until a condition of irreversible binding is reached. However, these conformational changes (i.e. the locking steps) appear to be faster and easier with PrP82–146 than with Aβ40. Such differences suggest that PrP82–146 has a greater propensity to polymerize and greater stability of the aggregates.

Genetic and phenotypic characterization of complex hereditary spastic paraplegia

High-throughput next-generation sequencing can identify disease-causing mutations in extremely heterogeneous disorders. Kara et al . investigate a series of 97 index cases with complex hereditary spastic paraplegia (HSP). They identify SPG11 defects in 30 families, as well as mutations in other HSP genes and genes associated with disorders including Parkinson’s disease.