Global untargeted serum metabolomic analyses nominate metabolic pathways responsive to loss of expression of the orphan metallo ß-lactamase, MBLAC1.

The C. elegans gene swip-10 encodes an orphan metallo ß-lactamase that genetic studies indicate is vital for limiting neuronal excitability and viability. Sequence analysis indicates that the mammalian gene Mblac1 is the likely ortholog of swip-10, with greatest sequence identity localized to the encoded protein's single metallo ß-lactamase domain. The substrate for the SWIP-10 protein remains unknown and to date no functional roles have been ascribed to MBLAC1, though we have shown that the protein binds the neuroprotective ß-lactam antibiotic, ceftriaxone. To gain insight into the functional role of MBLAC1 in vivo, we used CRISPR/Cas9 methods to disrupt N-terminal coding sequences of the mouse Mblac1 gene, resulting in a complete loss of protein expression in viable, homozygous knockout (KO) animals. Using serum from both WT and KO mice, we performed global, untargeted metabolomic analyses, resolving small molecules via hydrophilic interaction chromatography (HILIC) based ultra-performance liquid chromatography, coupled to mass spectrometry (UPLC-MS/MS). Unsupervised principal component analysis reliably segregated the metabolomes of MBLAC1 KO and WT mice, with 92 features subsequently nominated as significantly different by ANOVA, and for which we made tentative and putative metabolite assignments. Bioinformatic analyses of these molecules nominate validated pathways subserving bile acid biosynthesis and linoleate metabolism, networks known to be responsive to metabolic and oxidative stress. Our findings lead to hypotheses that can guide future targeted studies seeking to identify the substrate for MBLAC1 and how substrate hydrolysis supports the neuroprotective actions of ceftriaxone.

Glial loss of the metallo ß-lactamase domain containing protein, SWIP-10, induces age- and glutamate-signaling dependent, dopamine neuron degeneration.

Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitability, thus supporting many complex behaviors. Perturbed synaptic and extrasynaptic Glu homeostasis in the human brain has been implicated in multiple neuropsychiatric and neurodegenerative disorders including Parkinson's disease, where theories suggest that excitotoxic insults may accelerate a naturally occurring process of dopamine (DA) neuron degeneration. In C. elegans, mutation of the glial expressed gene, swip-10, results in Glu-dependent DA neuron hyperexcitation that leads to elevated DA release, triggering DA signaling-dependent motor paralysis. Here, we demonstrate that swip-10 mutations induce premature and progressive DA neuron degeneration, with light and electron microscopy studies demonstrating the presence of dystrophic dendritic processes, as well as shrunken and/or missing cell soma. As with paralysis, DA neuron degeneration in swip-10 mutants is rescued by glial-specific, but not DA neuron-specific expression of wildtype swip-10, consistent with a cell non-autonomous mechanism. Genetic studies implicate the vesicular Glu transporter VGLU-3 and the cystine/Glu exchanger homolog AAT-1 as potential sources of Glu signaling supporting DA neuron degeneration. Degeneration can be significantly suppressed by mutations in the Ca2+ permeable Glu receptors, nmr-2 and glr-1, in genes that support intracellular Ca2+ signaling and Ca2+-dependent proteolysis, as well as genes involved in apoptotic cell death. Our studies suggest that Glu stimulation of nematode DA neurons in early larval stages, without the protective actions of SWIP-10, contributes to insults that ultimately drive DA neuron degeneration. The swip-10 model may provide an efficient platform for the identification of molecular mechanisms that enhance risk for Parkinson's disease and/or the identification of agents that can limit neurodegenerative disease progression.

Withaferin A Regulates LRRK2 Levels by Interfering with the Hsp90- Cdc37 Chaperone Complex.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multi-domain protein that has been found to be mutated in patients with familial and sporadic Parkinson's disease, Alzheimer's disease and Crohn's disease. While the functions of LRRK2 are still largely unclear and mutations in LRRK2 are associated with adverse gain-of-function activities such as increased kinase activity, increased levels of LRRK2 alone are associated with toxicity in neurons. Consequently, exploring mechanisms to decrease levels of LRRK2 using pharmacological inhibitors would be highly advantageous. Previous work has shown that the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Cdc37 interact with and stabilize LRRK2. In the current study, we explore the regulation of LRRK2 by withaferin A (WA), a potent inhibitor of the interaction between Hsp90 and Cdc37. We report that treatment of the microglial cell line N9 with WA causes a decrease in cellular levels of LRRK2 in a dose- and time-dependent manner. We also find that treatment with WA disrupts the interaction between Hsp90, its co-chaperone Cdc37 and LRRK2, which leads to the destabilization and decreased levels of LRRK2. Additionally, treatment with celastrol, which is also an inhibitor of the Hsp90-Cdc37 complex, decreased LRRK2 levels. Interestingly, treatment with WA in the presence of celastrol enhanced the clearance of LRRK2. Overall, our data suggest that LRRK2 levels can be regulated by targeting the Hsp90-Cdc37 complex, which may have implications in the search for therapeutic strategies for Alzheimer's disease, Parkinson's disease and other LRRK2 proteinopathies.

An optimized InCell Western screening technique identifies hexachlorophene as a novel potent TDP43 targeting drug.

TAR DNA binding protein (TDP43) is a DNA- and RNA-binding protein that is implicated in several neurodegenerative disorders termed as "TDP43 proteinopathies" including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and fronto-temporal lobe dementia (FTLD). We have developed an InCell Western (ICW) technique for screening TDP targeting drugs in 96 well plates. We tested 281 compounds and identified a novel compound hexachlorophene (referred to as B10) that showed potent reduction in TDP43 levels. The effect of B10 on TDP protein level was validated in two different cellular models: endogenous TDP43 expressing N9 microglial cells and TDP43-over-expressing HEK293 and HeLa cells. We also analyzed effect of B10 on various pathological forms of TDP such as the C25 cleaved fragment that localizes to the cytosol, insoluble high molecular weight species, and ALS-linked mutants. Our data suggest that B10 effectively reduces all forms of TDP. Overall, our data suggest that B10 could serve as a potential drug molecule for the treatment of AD, ALS and other TDP43 proteinopathies.