Biology subjects

Kofler, J.

Publications and source records attributed to Kofler, J..

3 recordsLinked to original sources

bioRxiv2023

White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells

White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. GWAS identified TRIM47 at 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found selectively expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially-induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we identified the highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription and vacuole formation. Together, we confirm that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation. SUMMARY STATEMENTTRIM47, top genetic risk factor for white matter hyperintensity formation, is a negative regulator of autophagy in brain endothelial cells and implicates a novel cellular mechanism for age-related cerebrovascular changes.

cell biology↗

bioRxiv2023

An oligodendrocyte silencer element underlies the pathogenic impact of lamin B1 structural variants

The role of non-coding regulatory elements and how they might contribute to tissue type specificity of disease phenotypes is poorly understood. Autosomal Dominant Leukodystrophy (ADLD) is a fatal, adult-onset, neurological disorder that is characterized by extensive CNS demyelination. Most cases of ADLD are caused by tandem genomic duplications involving the lamin B1 gene (LMNB1) while a small subset are caused by genomic deletions upstream of the gene. Utilizing data from recently identified families that carry LMNB1 gene duplications but do not exhibit demyelination, ADLD patient tissues, CRISPR modified cell lines and mouse models, we have identified a novel silencer element that is lost in ADLD patients and that specifically targets overexpression to oligodendrocytes. This element consists of CTCF binding sites that mediate three-dimensional chromatin looping involving the LMNB1 and the recruitment of the PRC2 repressor complex. Loss of the silencer element in ADLD identifies a previously unknown role for silencer elements in tissue specificity and disease causation.

genetics↗

bioRxiv2023

Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity

BackgroundThe accumulation of amyloid beta (A{beta}) peptides in fibrils is prerequisite for Alzheimers disease (AD). Our understanding of the proteins that promote A{beta} fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. MethodsTo investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of A{beta} immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic A{beta}42 flies, and A{beta}42 seeded cultured neurons. ResultsAmyloid fibrils are primarily composed by A{beta}42 and unexpectedly harbor A{beta}38 but generally lack A{beta}40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 17 new amyloid-associated proteins. Notably, we confirmed 126 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic A{beta}42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Eight RNAi lines suppressed and 11 enhanced A{beta}42 toxicity. ConclusionsThese discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.

neuroscience↗