No abstract

The accumulation of amyloid β peptide (Aβ) in the brain of Alzheimer’s disease (AD) patients begins many years before clinical onset. Such process has been proposed to be pathogenic through the toxicity of Aβ soluble oligomers leading to synaptic dysfunction, phospho-tau aggregation and neuronal loss. Yet, a massive accumulation of Aβ can be found in approximately 30% of aged individuals with preserved cognitive function. Therefore, within the frame of the “amyloid hypothesis”, compensatory mechanisms and/or additional neurotoxic or protective factors need to be considered and investigated. Here we describe a modifier genetic screen in Drosophila designed to identify genes that modulate toxicity of Aβ42 in the CNS. The expression of Aβ42 led to its accumulation in the brain and a moderate impairment of negative geotaxis at 18 days post-eclosion (d.p.e) as compared with genetic or parental controls. These flies were mated with a collection of lines carrying chromosomal deletions and negative geotaxis was assessed at 5 and 18 d.p.e. Our screen is the first to take into account all of the following features, relevant to sporadic AD: (1) pan-neuronal expression of wild-type Aβ42; (2) a quantifiable complex behavior; (3) Aβ neurotoxicity associated with progressive accumulation of the peptide; and (4) improvement or worsening of climbing ability only evident in aged animals. One hundred and ninety-nine deficiency (Df) lines accounting for ~6300 genes were analyzed. Six lines, including the deletion of 52 Drosophila genes with human orthologs, significantly modified Aβ42 neurotoxicity in 18-day-old flies. So far, we have validated CG11796 and identified CG17249 as a strong candidate (whose human orthologs are HPD and PRCC, respectively) by using RNAi or mutant hemizygous lines. PRCC encodes proline-rich protein PRCC (ppPRCC) of unknown function associated with papillary renal cell carcinoma. HPD encodes 4-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme in tyrosine degradation whose Df causes autosomal recessive Tyrosinemia type 3, characterized by mental retardation. Interestingly, lines with a partial Df of HPD ortholog showed increased intraneuronal accumulation of Aβ42 that coincided with geotaxis impairment. These previously undetected modifiers of Aβ42 neurotoxicity in Drosophila warrant further study to validate their possible role and significance in the pathogenesis of sporadic AD.

Initial seizures observed in young rats during the 60 min after administration of pilocarpine (Pilo) were delayed and attenuated by pretreatment with a non-convulsive dose of methionine sulfoximine (MSO). We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was administered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (short period) or 15 min before sacrifice (long period). Their conversion to Glu and Gln in the hippocampus and entorhinal cortex was followed using [13C] gas chromatography-mass spectrometry. Release of in vitro loaded Glu surrogate, [3H]d-Asp from ex vivo brain slices was monitored in continuously collected superfusates. [3H]d-Asp uptake was tested in freshly isolated brain slices. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]d-Asp release, but did not affect [3H]d-Asp uptake. The results indicate that MSO at a non-convulsive dose delays the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.

No abstract

Engrailed 1 (EN1) is a conserved transcription factor essential for programming, survival, and maintenance of midbrain dopaminergic neurons. En1-hemizygosity (En1+/-) leads to a spontaneous Parkinson's disease-like (PD-like) progressive nigrostriatal degeneration as well as motor impairment and depressive-like behavior in SwissOF1 (OF1-En1+/-) mice. This phenotype is absent in C57Bl/6j (C57-En1+/-) mice. Here we studied PD-like phenotypes and early transcriptome profiles in OF1 wild-type (WT) and OF1-En1+/- male mice and compare to that of C57 WT and C57-En1+/- male mice. To detect transcriptional changes prior to dopaminergic cell loss, we performed RNA-seq of 1-week old mice substantia nigra pars compacta (SNpc). Histology and stereology were used to assess dopaminergic nigrostriatal pathology in 4 and 16 weeks old mice. OF1-En1+/- mice showed an increase (+-61617;79%) in dopaminergic striatal axonal swellings from 4 to 16 weeks and a loss (+-61617;23%) of dopaminergic neurons in the SNpc at 16 weeks compared to OF1 WT. Axonal swellings were also present in C57-En1+/- mice but did not increase over time. 52 differentially expressed genes (DEGs) were observed between the C57-WT and the C57-En1+/- mice, while 198 DEGs were observed in the OF1 strain. Enrichment analysis revealed that the neuroprotective phenotype of C57-En1+/- mice was associated with an overexpression of oxidative phosphorylation-related genes compared to both C57 WT and to OF1- En1+/- mice. These results highlight the importance of considering genetic background in PD models and provide valuable insight on how expression of mitochondrial proteins before the onset of neurodegeneration is associated to vulnerability of nigrostriatal dopaminergic neurons.

Parkinson's disease (PD) is a heterogeneous disorder characterized by intraneuronal inclusions of alpha-synuclein (α-Syn), a strong neuroinflammatory component and neurodegeneration. Human genetic association studies have shown that variants affecting quantity and quality of major histocompatibility complex II (MHCII) have implications in PD susceptibility and it was recently shown that PD patients have α-Syn specific T lymphocytes in circulation. The class II transactivator (Ciita) is the major regulator of MHCII expression and reduced Ciita expression has been shown to increase α-Syn induced neurodegeneration and pathology in vivo. Here we show, using flow cytometry in an α-Syn overexpression model combined with α-Syn pre-formed fibrils (PFF), that congenic rats with naturally occurring differences in Ciita expression have altered local and peripheral immune populations. Lower Ciita levels are associated with increased percentages of microglia and circulating myeloid cells being MHCII+ but with lower levels of MHCII on individual cells. Additionally, lower Ciita levels was associated to higher TNF levels in serum, trends of higher CD86 levels in circulating myeloid population and a lower CD4/CD8 T lymphocyte ratio. Taken together, these results indicate that Ciita regulates serum TNF levels and baseline immune populations which could mediate an increased susceptibility to PD-like pathology.

Pretreatment with non-convulsive dose of methionine sulfoximine (MSO) attenuated lithi-um-pilocarpine-induced (Li-Pilo) seizures in young rats [1]. We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was admin-istered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (onset) or 15 min before sacrifice (final phase). Their conversion to Glu and Gln in hippocampus and entorhinal cortex was followed us-ing [13C] gas chromatography-mass spectrometry. Release of in vitro loaded [3H]D-Asp from ex vi-vo brain slices was measured in continuously collected superfusates. Protein and mRNA expres-sion were measured by Western Blot and real-time PCR techniques, respectively. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]D-Asp release and reduced by ~50% the synaptic vesicular Glu transporter VGLUT1 protein, but not mRNA content in the hippo-campus. The results indicate that MSO at non-convulsive dose mitigates the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.