operating-biocontainment-procedures-facility-for-laboratory-mice-with

Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science ...

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin

Cluster of Excellence, NeuroCure

German Center for Neurodegenerative Diseases (DZNE) Berlin

The family of lysosome-associated membrane proteins (LAMP) includes the ubiquitously expressed LAMP1 and LAMP2, which account for half of the proteins in the lysosomal membrane. Another member of the LAMP family is LAMP3, which is expressed only in certain cell types and differentiation stages. LAMP3 expression is linked with poor prognosis of certain cancers, and the locus where it is encoded was identified as a risk factor for Parkinson's disease (PD). Here, we investigated the role of LAMP3 in the two main cellular degradation pathways, the proteasome and autophagy. LAMP3 mRNA was not detected in mouse models of PD or in the brain of human patients. However, it was strongly induced upon proteasomal inhibition in the neuroblastoma cell line SH-SY5Y. Induction of LAMP3 mRNA following proteasomal inhibition was dependent on UPR transcription factor ATF4 signaling and induced autophagic flux. Prevention of LAMP3 induction enhanced apoptotic cell death. In summary, these data demonstrate that LAMP3 regulation as part of the UPR contributes to protein degradation and cell survival during proteasomal dysfunction. This link between autophagy and the proteasome may be of special importance for the treatment of tumor cells with proteasomal inhibitors.

Loss of lysosome-associated membrane protein 3 (LAMP3) enhances cellular vulnerability against proteasomal inhibition.

Senescent cells, which are cells in a post-proliferative state, show an increased number of dysfunctional mitochondria and oxidatively damaged and aggregated proteins. The mitochondrial-lysosomal axis theory of aging proposes that the autophago-lysosomal system is unable to cope with the rising amount of damaged organelles and proteins. We used human umbilical vein endothelial cells (HUVEC) as in vitro model system to determine which part/s of the autophago-lysosomal pathway become deficient by aging. Senescent HUVEC contained a much larger population of autophagosomes and lysosomes compared to young cells. Transcriptome analysis comparing young and old cells demonstrated several age-related changes of autophagy gene expression. One reason for the observed increase of autophagosomes was an impairment of the autophagic flux in senescent cells due to reduced V-ATPase activity required for acidification of the lysosomes and thus functionality of lysosomal hydrolases. The hypothesis that reduced mitochondrial ATP production underlies low V-ATPase activity was supported by addition of exogenous ATP. This procedure rescued the lysosomal acidification and restored the autophagic flux. Thus, we propose impaired lysosomal acidification due to ATP shortage which may result from mitochondrial dysfunction as a mechanism underlying the accumulation of dysfunctional cellular constituents during aging.

Age-related dysfunction of the autophago-lysosomal pathway in human endothelial cells.

Hereditary Parkinson's disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCA in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As an exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension, and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was also detected for other microtubule and microfilament factors, namely MAP2, MARK1, MAP1A, KIF1A, 4.1N, 4.1G, and ADD2. While the MAP1B heavy chain supports regeneration and growth cones, its light chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2, VPS13D, and VPS13C in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in  that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly,  gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.

SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes.

Deposition of amyloid beta (Aβ) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aβ pathology and glial cell-mediated neuroinflammation.

Article	Year
Loss of lysosome-associated membrane protein 3 (LAMP3) enhances cellular vulnerability against proteasomal inhibition. European journal of cell biology\| PubMed ID: 25681212	2015
Age-related dysfunction of the autophago-lysosomal pathway in human endothelial cells. Pflugers Archiv : European journal of physiology\| PubMed ID: 31222491	2019
SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes. International journal of molecular sciences\| PubMed ID: 31277379	2019
Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model. Journal of neuroinflammation\| PubMed ID: 35780157	2022

Marina Jendrach

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