Biology subjects

Hulleman, J. D.

Publications and source records attributed to Hulleman, J. D..

6 recordsLinked to original sources

bioRxiv2024

Genetic removal of Nlrp3 protects against sporadic and R345W Efemp1-induced basal laminar deposit formation

Chronic, unresolved inflammation has long been speculated to serve as an initiating and propagating factor in numerous neurodegenerative diseases, including a leading cause of irreversible blindness in the elderly, age-related macular degeneration (AMD). Intracellular multiprotein complexes called inflammasomes in combination with activated caspases facilitate production of pro-inflammatory cytokines such as interleukin 1 beta. Specifically, the nucleotide-binding oligomerization (NOD)-like receptor protein 3 (NLRP3) has received heightened attention due to the wide range of stimuli to which it can respond and its potential involvement in AMD. In this study, we directly tested the role of Nlrp3 and its downstream effector, caspase 1 (Casp1) in mediating early AMD-like pathology (i.e., basal laminar deposits [BLamDs]) in wild-type (WT) mice and the Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) mouse model (p.R345W mutation in Efemp1). Compared to aged-matched controls, R345W+/+ knockin mice demonstrated increased Muller cell gliosis, subretinal Iba-1+ microglial cells, higher Nlrp3 immunoreactivity in the retina, as well as significant transcriptional upregulation of complement component 3, Nlrp3, pro-Il1b, pro-caspase-1, and tissue inhibitor of matrix metalloproteinase 3 in the retinal pigmented epithelium (RPE)/choroid. These findings were accompanied by an age-related increase in BLamD formation in the R345W+/+ mice. Genetic elimination of either Nlrp3 or Casp1 significantly reduced both the size and coverage of BLamDs in the R345W+/+ background, highlighting an important and underappreciated pathway that could affect ML/DHRD onset and progression. Moreover, Nlrp3 knockout reduced spontaneous, idiopathic BLamDs in WT mice, suggesting translatability of our findings not only to rare inherited retinal dystrophies, but also potentially to AMD itself.

immunology↗

bioRxiv2024

Dominant nonsense mutations in efemp1 alter vertebral and craniofacial characteristics in adult zebrafish

Heritable Disorders of Connective Tissues (HDCT) are a heterogenous, pleiotropic group of conditions that broadly affect connective tissues. EFEMP1 is a member of the fibulin family of extracellular matrix (ECM) glycoproteins which is expressed in various human tissues. Individuals with EFEMP1 variants have recently been identified and appear to have Marfan-like characteristics. Clinical phenotypes of these individuals include hernias, advanced bone age, tall stature, myopia, joint laxity, and thin skin. EFEMP1-associated HDCTs have been identified in individuals with biallelic and monoallelic variants. There is an urgent need to better understand the role of EFEMP1 in regulating connective tissues including bone, and the pathophysiological mechanisms underlying the spectrum of genotype-phenotype relationships seen in EFEMP1-associated HDCTs. To investigate the role of EFEMP1 in developing and adult bone, we used CRISPR-based editing to generate two efemp1 zebrafish alleles encoding for premature termination codons (PTCs) predicted to delete or severely alter the fibulin-type domain. Both alleles exhibited similar phenotypes in juvenile and adult fish. In juvenile fish, we did not identify changes in body size or vertebral development. In adults, we found significant changes in body length, bone microarchitecture, and craniofacial measurements in both heterozygous and homozygous mutant fish. These results expand our understanding of the role of efemp1 in the skeleton and highlight the potential for dominant nonsense variants to play a role in manifestation of clinical phenotypes in EFEMP1-associated HDCTs.

genetics↗

bioRxiv2023

GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology

Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.

neuroscience↗

bioRxiv2022

Development of a new DHFR-based destabilizing domain with enhanced basal turnover and applicability in mammalian systems

Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion proteins abundance. Yet in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of ~1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of FACS sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as C12, contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated a significant 2.9-fold reduction in basal levels compared to the conventional ecDHFR DD YFP. This domain was similarly responsive to TMP with respect to dose-response and maximal stabilization, indicating an overall enhanced dynamic range. Interestingly, both computational and wet-lab experiments identified the W74R and T113S mutations of C12 as the main contributors towards its basal destabilization. Yet, the combination of all the C12 mutations were required to maintain both its enhanced degradation and TMP stabilization. We further demonstrate the utility of C12 by fusing it to I{kappa}B and Nrf2, two stress-responsive proteins that have previously been challenging to regulate. In both instances, C12 significantly enhanced the basal turnover of these proteins and improved the dynamic range of regulation post stabilizer addition. These advantageous features of the C12 ecDHFR DD variant highlight its potential for replacing the conventional N-terminal ecDHFR DD, and overall improving the use of destabilizing domains, not only as a chemical biology tool, but for gene therapy avenues as well.

biochemistry↗

bioRxiv2022

A loss-of-function cysteine mutant in fibulin-3 (EFEMP1) forms aberrant extracellular disulfide-linked homodimers and alters extracellular matrix composition

Fibulin-3 (F3 or EFEMP1) is a disulfide-rich, secreted glycoprotein necessary for maintaining extracellular matrix (ECM) and connective tissue integrity. Two studies have identified distinct autosomal recessive F3 mutations in individuals with Marfan Syndrome-like phenotypes. Herein we characterized how one of these mutations, c.163T>C; p.Cys55Arg (C55R), disrupts F3 secretion, quaternary structure, and function by forming unique extracellular disulfide-linked homodimers. Dual cysteine mutants suggest that the C55R-induced disulfide species forms because of new availability of Cys70 on adjacent F3 monomers. Surprisingly, mutation of single cysteines located near C55 (i.e., C29, C42, C48, C61, C70, C159, and C171) also produced similar extracellular disulfide-linked dimers, suggesting that this is not a phenomenon isolated to the C55R mutant. To assess C55R functionality, F3 knockout (KO) retinal pigmented epithelial (RPE) were generated, followed by reintroduction of wild-type (WT) or C55R F3. F3 KO cells produced lower levels of the ECM remodeling enzyme, matrix metalloproteinase 2 and reduced formation of collagen VI ECM filaments, both of which were partially rescued by WT F3 overexpression. However, C55R F3 was unable to compensate for these same ECM-related defects. Our results highlight the unique behavior of particular cysteine mutations in F3 and uncover potential routes to restore C55R F3 loss-of-function.

biochemistry↗

bioRxiv2019

Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein

-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinsons disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models as well as their biochemical properties relevant to aSyn pathobiology. We report that mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

neuroscience↗