Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10(-136)) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to themacroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

Multiple insults to the brain lead to neuronal cell death, thus raising the question to what extent can lost neurons be replenished by adult neurogenesis. Here we focused on the hippocampus and especially the dentate gyrus (DG), a vulnerable brain region and one of the two sites where adult neuronal stem cells (NSCs) reside. While adult hippocampal neurogenesis was extensively studied with regard to its contribution to cognitive enhancement, we focused on their underestimated capability to repair a massively injured, nonfunctional DG. To address this issue, we inflicted substantial DG-specific damage in mice of either sex either by diphtheria toxin-based ablation of >50% of mature DG granule cells (GCs) or by prolonged brain-specific VEGF overexpression culminating in extensive, highly selective loss of DG GCs (thereby also reinforcing the notion of selective DG vulnerability). The neurogenic system promoted effective regeneration by increasing NSCs proliferation/survival rates, restoring a nearly original DG mass, promoting proper rewiring of regenerated neurons to their afferent and efferent partners, and regaining of lost spatial memory. Notably, concomitantly with the natural age-related decline in the levels of neurogenesis, the regenerative capacity of the hippocampus also subsided with age. The study thus revealed an unappreciated regenerative potential of the young DG and suggests hippocampal NSCs as a critical reservoir enabling recovery from catastrophic DG damage.

The domestic dog segregates a significant number of inherited progressive retinal diseases, several of which mirror human retinal diseases and which are collectively termed progressive retinal atrophy (PRA). In 2014, a novel form of PRA was reported in the Swedish Vallhund breed, and the disease was mapped to canine chromosome 17. The causal mutation was not identified, but expression analyses of the retinas of affected Vallhunds demonstrated a 6-fold increased expression of the MERTK gene compared to unaffected dogs. Using 24 retinopathy cases and 97 controls with no clinical signs of retinopathy, we replicated the chromosome 17 association in Swedish Vallhunds from the UK and aimed to elucidate the causal variant underlying this association using whole genome sequencing (WGS) of an affected dog. This revealed a 6-8 kb insertion in intron 1 of MERTK that was not present in WGS of 49 dogs of other breeds. Sequencing and BLASTN analysis of the inserted segment was consistent with the insertion comprising a full-length intact LINE-1 retroelement. Testing of the LINE-1 insertion for association with retinopathy in the UK set of 24 cases and 97 controls revealed a strong statistical association (P-value 6.0 x 10(-11)) that was subsequently replicated in the original Finnish study set (49 cases and 89 controls (P-value 4.3 x 10(-19)). In a pooled analysis of both studies (73 cases and 186 controls), the LINE-1 insertion was associated with a similar to 20-fold increased risk of retinopathy (odds ratio 23.41, 95% confidence intervals 10.99-49.86, P-value 1.3 x 10(-27)). Our study adds further support for regulatory disruption of MERTK in Swedish Vallhund retinopathy; however, further work is required to establish a functional overexpression model. Future work to characterise the mechanism by which this intronic mutation disrupts gene regulation will further improve the understanding of MERTK biology and its role in retinal function.

Cerebral Dopamine Neurotrophic Factor (CDNF) shows beneficial effects in rodent models of Parkinson?s and Alzheimer?s disease. The brain is a challenging target for protein therapy due to its exclusive blood?brain barrier. Hence, the therapeutic protein should be delivered directly to the brain parenchyma. Implantation of encapsulated mammalian cells that constantly secrete CDNF is a potential approach for targeted and long-term protein delivery to the brain. In this study, we generated several CDNF-secreting cell clones derived from human retinal pigment epithelial cell line ARPE-19, and studied CDNF secretion from the clones maintained as monolayers and in polymeric microcapsules. The secretion of wild type (wt) CDNF transgene was low and the majority of the produced protein remained intracellular, locating mainly to the endoplasmic reticulum (ER). The secretion of wtCDNF decreased to even lower levels when the clones were in a non-dividing state, as in the microcapsules. Both codon optimization and deletion of the putative ER-retrieval signal (four last amino acids: KTEL) improved CDNF secretion. More importantly, the secretion of KTEL-deleted CDNF remained constant in the non-dividing clones. Thus, cells expressing KTEL-deleted CDNF, in contrast to wtCDNF, can be considered for cell encapsulation applications if the KTEL-deleted CDNF is proven to be biologically active in vivo.

Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITP alpha, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITP alpha lipid exchange cycle: (i) interaction of PITP alpha with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITP alpha to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITP alpha; (iv) the trajectory of PtdIns or PtdCho into and through the lipidbinding pocket is chaperoned by sets of PITP alpha residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITP alpha PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family.

Cross-sectional area (CSA) decreases and fat infiltration increases in epaxial muscles of Dachshunds with intervertebral disc disease (IVDD), but less is known about large breed dogs with IVDD. The aim here was to investigate thoracolumbar epaxial muscle CSA and fat infiltration in large breed dogs with compressive IVDD and acute non-compressive nucleus pulposus extrusion (ANNPE) or fibrocartilaginous embolism (FCE). This retrospective study included large breed dogs with MRI-confirmed IVDD (n = 17) and ANNPE or FCE (n = 13). The CSA and fat infiltration of the thoracolumbar M. longissimus and Mm. multifidi were assessed from T1-weighted transverse MR images using Osirix. The CSA was significantly smaller in dogs with compressive IVDD than in dogs with non-compressive ANNPE or FCE for Mm. multifidi (p = 0.015), M. longissimus (p = 0.070), and these two muscles combined (p = 0.016). Fat infiltration in all muscle measurements was significantly higher in dogs with compressive IVDD than in dogs with non-compressive ANNPE or FCE (all P < 0.050). A significant positive correlation existed between age, duration of clinical signs, and fat infiltration, suggesting more fat infiltration in older dogs with more chronic signs. These signs of muscle atrophy are likely caused by denervation and secondary disuse due to chronic spinal cord compression and prolonged duration of clinical signs.

Background The neurophysiological correlates of gastrointestinal symptoms (GISs) in Parkinson's disease (PD) are not well understood. It has been proposed that in patients with a gastrointestinal origin of PD dopaminergic neurodegeneration would be more symmetric. Objectives The aim is to assess the associations between GISs and asymmetry of nigrostriatal dopaminergic neurodegeneration in PD. Methods Ninety PD patients were assessed using motor and GIS scales and I-123-FP-CIT SPECT. We calculated the asymmetry index and the predominant side of motor symptoms and dopamine transporter (DAT) imaging defect and assessed their association with GISs. Results There were no significant differences in GISs between symmetric and asymmetric dopaminergic defect. Left predominant defect was related to more GIS and higher constipation scores. Conclusions GISs were associated with left predominant reduction in putaminal DAT binding but not asymmetry per se. It remains open whether left-sided DAT deficit is related to more pronounced GI involvement or symptom perception in PD. (c) 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Genetic mutations underlying neurodegenerative disorders impair ribosomal DNA (rDNA) transcription suggesting that nucleolar dysfunction could be a novel pathomechanism in polyglutamine diseases and in certain forms of amyotrophic lateral sclerosis/frontotemporal dementia. Here, we investigated nucleolar activity in pre-symptomatic digenic models of Parkinson's disease (PD) that model the multifactorial aetiology of this disease. To this end, we analysed a novel mouse model mildly overexpressing mutant human alpha-synuclein (hA53T-SNCA) in a PTEN-induced kinase 1 (PINK1/ PARK6) knockout background and mutant mice lacking both DJ-1 (also known as PARK7) and PINK1. We showed that overexpressed hA53T-SNCA localizes to the nucleolus. Moreover, these mutants show a progressive reduction of rDNA transcription linked to a reduced mouse lifespan. By contrast, rDNA transcription is preserved in DJ-1/PINK1 double knockout (DKO) mice. mRNA levels of the nucleolar transcription initiation factor 1A (TIF-IA, also known as RRN3) decrease in the substantia nigra of individuals with PD. Because loss of TIF-IA, as a tool to mimic nucleolar stress, increases oxidative stress and because DJ-1 and PINK1 mutations result in higher vulnerability to oxidative stress, we further explored the synergism between these PD-associated genes and impaired nucleolar function. By the conditional ablation of TIF-IA, we blocked ribosomal RNA (rRNA) synthesis in adult dopaminergic neurons in a DJ-1/PINK1 DKO background. However, the early phenotype of these triple knockout mice was similar to those mice exclusively lacking TIF-IA. These data sustain a model in which loss of DJ-1 and PINK1 does not impair nucleolar activity in a pre-symptomatic stage. This is the first study to analyse nucleolar function in digenic PD models. We can conclude that, at least in these models, the nucleolus is not as severely disrupted as previously shown in DA neurons from PD patients and neurotoxin-based PD mouse models. The results also show that the early increase in rDNA transcription and nucleolar integrity may represent specific homeostatic responses in these digenic pre-symptomatic PD models.

Purpose To examine whether real-world clinical patients with macular oedema (MO) receiving intravitreal antivascular endothelial growth factor (VEGF) therapy have a higher mortality compared with a matched reference population. Methods A population-based, retrospective cohort study of 26 386 patients from Finland, from January 1, 2001, to December 31, 2017. Index patients were identified through the Caring Epidemiology Project database, receiving at least one intravitreal anti-VEGF injection for wet age-related macular degeneration (AMD, n = 2243, 48.61%), diabetic MO (n = 744, 16.12%), MO due to retinal vascular occlusion (n = 589, 12.77%), or other MO (n = 1038, 22.5%). For each individual treated with intravitreal injection (n = 4614), five age- , sex- , calendar year- and hospital district- matched control individuals (n = 21 772) were chosen. Baseline data of chronic conditions were available. All-cause and cause-specific mortality was analysed using Cox ' s proportional hazards model. Results In general, the anti-VEGF treated patients had a higher prevalence of systemic conditions, including diabetes (60.1% vs. 46.8%, p <0.001), chronic hypertension (38.4% vs. 34.6%, p <0.001), in hospital-treated ischaemic heart disease (23.1% vs. 21.5%, p = 0.014), and glaucoma (11.1% vs. 6.3%, p <0.001) than controls. There was no difference in all-cause mortality between the anti-VEGF treated patients and matched controls (p = 0.62). In unadjusted Kaplan-Meier analysis of wet AMD subgroup, all-cause mortality was lower in anti-VEGF treated patients than matched controls (p = 0.015), but adjusted Cox ' s proportional hazards model showed no difference in the risk of all-cause mortality (HR 0.85, 95% CI 0.66-1.09). Conclusions Intravitreal anti-VEGF therapy was not associated with an increase in the risk of mortality in patients with MO compared with age- and sex-matched controls.