Lautaro Francisco Belfiori

Introduction: Parkinson’s disease (PD) is the second most common neurodegenerative disorder, increasing both in terms of prevalence and incidence. To date, only symptomatic treatment is available, highlighting the need to increase knowledge on disease etiology in order to develop new therapeutic strategies. Hemizygosity for the gene Engrailed-1 (En1), encoding a conserved transcription factor essential for the programming, survival, and maintenance of midbrain dopaminergic neurons, leads to progressive nigrostriatal degeneration, motor impairment and depressive-like behavior in SwissOF1 (OF1-En1^+/−). The neurodegenerative phenotype is, however, absent in C57Bl/6j (C57-En1^+/−) mice. En1^+/− mice are thus highly relevant tools to identify genetic factors underlying PD susceptibility.Methods: Transcriptome profiles were defined by RNAseq in microdissected substantia nigra from 1-week old OF1, OF1- En1^+/−, C57 and C57- En1^+/− male mice. Differentially expressed genes (DEGs) were analyzed for functional enrichment. Neurodegeneration was assessed in 4- and 16-week old mice by histology.Results: Nigrostriatal neurodegeneration was manifested in OF1- En1^+/− mice by increased dopaminergic striatal axonal swellings from 4 to 16 weeks and decreased number of dopaminergic neurons in the SNpc at 16 weeks compared to OF1. In contrast, C57- En1^+/− mice had no significant increase in axonal swellings or cell loss in SNpc at 16 weeks. Transcriptomic analyses identified 198 DEGs between OF1- En1^+/− and OF1 mice but only 52 DEGs between C57- En1^+/− and C57 mice. Enrichment analysis of DEGs revealed that the neuroprotective phenotype of C57- En1^+/− mice was associated with a higher expression of oxidative phosphorylation-related genes compared to both C57 and OF1- En1^+/− mice.Discussion: Our results suggest that increased expression of genes encoding mitochondrial proteins before the onset of neurodegeneration is associated with increased resistance to PD-like nigrostriatal neurodegeneration. This highlights the importance of genetic background in PD models, how different strains can be used to model clinical and sub-clinical pathologies and provides insights to gene expression mechanisms associated with PD susceptibility and progression.

1. 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 (±79%) in dopaminergic striatal axonal swellings from 4 to 16 weeks and a loss (±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 upregulation 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. 2. Significance statement: Most PD cases are idiopathic and caused by a complex interplay between genetic variants and environmental risk factors. However, the underlying mechanisms remain elusive. Here we show that En1 hemizygosity leads to progressive nigrostriatal degeneration with a loss of dopaminergic neurons in OF1- En1 ^+/- but that C57- En1 ^+/- mice only exhibit early signs of nigrostriatal pathology and do not progress to a PD-like phenotype over time. We identified differences in gene expression related to oxidative phosphorylation before the onset of neurodegeneration to be associated to the differential susceptibility to En1 ^+/- induced PD-like pathology. Our work shows how gene expression changes modulate vulnerability to dopaminergic neurodegeneration in the En1 ^+/- mouse and reveals putative molecular mechanisms behind the onset and progression of PD.

BACKGROUND: Parkinson’s disease (PD) is characterized by alpha-synuclein (α-Syn) pathology, neurodegeneration and neuroinflammation. HLA variants associated with PD and α-Syn specific circulating CD4+ T lymphocytes in PD patients highlight the importance of antigen presentation in PD etiology. The class II transactivator (CIITA) is the major regulator of MHCII expression. Reduced Ciita levels significantly increase α-Syn pathology, nigrostriatal neurodegeneration and behavioral deficits in α-Syn seed-induced rat PD models. OBJECTIVE: To characterize immune profiles associated with enhanced PD-like pathology observed in rats expressing lower Ciita levels (DA.VRA4) compared to the background (DA) strain. METHODS: To model PD, we combined rAAV-mediated α-Syn overexpression in the substantia nigra with striatal injection of α-Syn pre-formed fibrils (PFF). Immune profiles in brain and blood were analyzed by flow cytometry and multiplexed ELISA in naïve rats, 4- and 8 weeks post rAAV injection. RESULTS: Flow cytometry showed Ciita -dependent regulation of MHCII on microglia, brain macrophages and circulating myeloid cells. The MHCII-dependent microglial response peaked at 4 weeks post rAAV injection, whereas the MHCII levels in circulating myeloid cells peaked at 8 weeks. There was no major infiltration of macrophages or T lymphocytes into the CNS in response to α-Syn and only subtle Ciita - and/or α-Syn-dependent changes in the T lymphocyte compartment. Lower Ciita levels were consistently associated with higher TNF levels in serum. CONCLUSIONS: These results suggest that Ciita regulates susceptibility to PD-like pathology through minor but detectable changes in resident and peripheral immune cells and TNF levels, and indicate that mild immunomodulatory therapies could have therapeutic effects in PD.

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.

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.

The accumulation and spreading of protein tau in the human brain are major features of neurodegenerative disorders known as tauopathies. In addition to several subcellular abnormalities, tau aggregation within neurons seems capable of triggering endo

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 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 of unknown function associated with papillary renal cell carcinoma. HPD encodes 4-hydroxyphenylpyruvate dioxygenase, a key enzyme in tyrosine degradation whose deficiency causes autosomal recessive Tyrosinemia type 3, characterized by mental retardation. Interestingly, lines with a partial deficiency 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