Springer Science and Business Media LLC

It is unclear to what extent pre-clinical studies in genetically homogeneous animal models of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder, can be informative of human pathology. The disease modifying effects in animal models of most therapeutic compounds have not been reproduced in patients. To advance therapeutics in ALS, we need easily accessible disease biomarkers which can discriminate across the phenotypic variants observed in ALS patients and can bridge animal and human pathology. Peripheral blood mononuclear cells alterations reflect the rate of progression of the disease representing an ideal biological substrate for biomarkers discovery. We have applied TMTcalibrator™, a novel tissue-enhanced bio fluid mass spectrometry technique, to study the plasma proteome in ALS, using peripheral blood mononuclear cells as tissue calibrator. We have tested slow and fast progressing SOD1G93A mouse models of ALS at a pre-symptomatic and symptomatic stage in parallel with fast and slow progressing ALS patients at an early and late stage of the disease. Immunoassays were used to retest the expression of relevant protein candidates. The biological features differentiating fast from slow progressing mouse model plasma proteomes were different from those identified in human pathology, with only processes encompassing membrane trafficking with translocation of GLUT4, innate immunity, acute phase response and cytoskeleton organization showing enrichment in both species. Biological processes associated with senescence, RNA processing, cell stress and metabolism, major histocompatibility complex-II linked immune-reactivity and apoptosis (early stage) were enriched specifically in fast progressing ALS patients. Immunodetection confirmed regulation of the immunosenescence markers Galectin-3, Integrin beta 3 and Transforming growth factor beta-1 in plasma from pre-symptomatic and symptomatic transgenic animals while Apolipoprotein E differential plasma expression provided a good separation between fast and slow progressing ALS patients. These findings implicate immunosenescence and metabolism as novel targets for biomarkers and therapeutic discovery and suggest immunomodulation as an early intervention. The variance observed in the plasma proteomes may depend on different biological patterns of disease progression in human and animal model.

The protein aggregation that occurs in neurodegenerative diseases is classically thought to occur as an undesirable, nonfunctional byproduct of protein misfolding. This model contrasts with the biology of RNA binding proteins, many of which are linked to neurodegenerative diseases. RNA binding proteins use protein aggregation as part of a normal regulated, physiological mechanism controlling protein synthesis. The process of regulated protein aggregation is most evident in formation of stress granules. Stress granules assemble when RNA binding proteins aggregate through their glycine rich domains. Stress granules function to sequester, silence and/or degrade RNA transcripts as part of a mechanism that adapts patterns of local RNA translation to facilitate the stress response. Aggregation of RNA binding proteins is reversible and is tightly regulated through pathways, such as phosphorylation of elongation initiation factor 2α. Microtubule associated protein tau also appears to regulate stress granule formation. Conversely, stress granule formation stimulates pathological changes associated with tau. In this review, I propose that the aggregation of many pathological, intracellular proteins, including TDP-43, FUS or tau, proceeds through the stress granule pathway. Mutations in genes coding for stress granule associated proteins or prolonged physiological stress, lead to enhanced stress granule formation, which accelerates the pathophysiology of protein aggregation in neurodegenerative diseases. Over-active stress granule formation could act to sequester functional RNA binding proteins and/or interfere with mRNA transport and translation, each of which might potentiate neurodegeneration. The reversibility of the stress granule pathway also offers novel opportunities to stimulate endogenous biochemical pathways to disaggregate these pathological stress granules, and perhaps delay the progression of disease.

Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants. In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7.

The ability of oligodendrocyte progenitor cells (OPCs) to give raise to myelin forming cells during developmental myelination, normal adult physiology and post-lesion remyelination in white matter depends on factors which govern their proliferation, migration and differentiation. Tissue plasminogen activator (tPA) is a serine protease expressed in the central nervous system (CNS), where it regulates cell fate. In particular, tPA has been reported to protect oligodendrocytes from apoptosis and to facilitate the migration of neurons. Here, we investigated whether tPA can also participate in the migration of OPCs during CNS development and during remyelination after focal white matter lesion. OPC migration was estimated by immunohistological analysis in spinal cord and corpus callosum during development in mice embryos (E13 to P0) and after white matter lesion induced by the stereotactic injection of lysolecithin in adult mice (1 to 21 days post injection). Migration was compared in these conditions between wild type and tPA knock-out animals. The action of tPA was further investigated in an in vitro chemokinesis assay. OPC migration along vessels is delayed in tPA knock-out mice during development and during remyelination. tPA enhances OPC migration via an effect dependent on the activation of epidermal growth factor receptor. Endogenous tPA facilitates the migration of OPCs during development and during remyelination after white matter lesion by the virtue of its epidermal growth factor-like domain.

The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo. We generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence. Zebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP. These novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.