Kinetic monitoring of neuronal stress response to proteostasis dysfunction.

Proteostasis dysfunction and activation of the unfolded protein response (UPR) are characteristic of all major neurodegenerative diseases. Nevertheless, although the UPR and proteostasis dysfunction has been studied in great detail in model organisms like yeast and mammalian cell lines, it has not yet been examined in neurons. In this study, we applied a viral vector-mediated expression of a reporter protein based on a UPR transcription factor, ATF4, and time-lapse fluorescent microscopy to elucidate how mouse primary neurons respond to pharmacological and genetic perturbations to neuronal proteostasis. In in vitro models of endoplasmic reticulum (ER) stress and proteasome inhibition, we used the ATF4 reporter to reveal the time course of the neuronal stress response relative to neurite degeneration and asynchronous cell death. We showed how potential neurodegenerative disease co-factors, ER stress and mutant α-synuclein overexpression, impacted neuronal stress response and overall cellular health. This work therefore introduces a viral vector-based reporter that yields a quantifiable readout suitable for non-cell destructive kinetic monitoring of proteostasis dysfunction in neurons by harnessing ATF4 signaling as part of the UPR activation.

In Vivo Assessment of Cell Death and Nigrostriatal Pathway Integrity Following Continuous Expression of C3 Transferase.

The bacterial exoenzyme C3 transferase (C3) irreversibly inhibits RhoA GTPase leading to stimulation of axonal outgrowth in injured neurons. C3 has been used successfully in models of neurotrauma and shows promise as an option to support cell survival and axonal growth of dopaminergic (DA) neurons in Parkinson's disease (PD) cell therapy. Whether the continuous expression of C3 in DA neurons is well-tolerated is unknown. To assess the potential neurotoxicity of sustained expression of C3 in DA neurons, we generated Cre recombinase-dependent adeno-associated viral vectors (AAV) for targeted C3 delivery to DA neurons of the mouse substantia nigra pars compacta (SNc). The effect of continuous expression of C3 on DA neurons was assessed by immunohistochemistry and compared to that of Enhanced Yellow Fluorescent Protein (EYFP) as negative controls. We did not find significant reduction of tyrosine hydroxylase (TH) expression levels nor the presence of cleaved activated caspase 3. Astrocytic activation as determined by GFAP expression was comparable to EYFP controls. To evaluate the impact of C3 expression on striatal terminals of the nigrostriatal pathway, we compared the rotational behavior of wildtype mice injected unilaterally with either C3 or 6-hydroxydopamine (6-OHDA). Mice injected with C3 exhibited similar ipsiversive rotations to the site of injection in comparison to control mice injected with EYFP and significantly fewer ipsiversive rotations compared to 6-OHDA lesioned mice. Non-significant difference between C3 and EYFP controls in behavioral and histological analyses demonstrate that transduced DA neurons express C3 continuously without apparent adverse effects, supporting the use of C3 in efficacy studies targeting DA neurons.

C3 Transferase Gene Therapy for Continuous RhoA Inhibition.

The identification of RhoA inhibition as a therapeutic target in neurodegenerative diseases and traumatic central nervous system (CNS) injuries has introduced a need to develop tools that effectively modulate intracellular RhoA-dependent signaling. In neurons, the bacterial exoenzyme C3 transferase irreversibly inactivates RhoA GTPase signaling to promote neuritogenesis and axon regeneration following an injury. Thus, we have adopted a gene therapy approach for the targeted inhibition of RhoA activity in the CNS by expressing C3 transferase. Herein we describe the construction of adeno-associated viral vectors for the expression of cell-permeable-C3 transferase and their functional characterization in vitro.

Detecting the functional complexities between high-density lipoprotein mimetics.

High-density lipoprotein (HDL) is a key regulator of lipid homeostasis through its native roles like reverse cholesterol transport. The reconstitution of this natural nanoparticle (NP) has become a nexus between nanomedicine and multi-disease therapies, for which a major portion of HDL functionality is attributed to its primary scaffolding protein, apolipoprotein A1 (apoA1). ApoA1-mimetic peptides were formulated as cost-effective alternatives to apoA1-based therapies; reverse-4F (r4F) is one such peptide used as part of a nanoparticle platform. While similarities between r4F- and apoA1-based HDL-mimetic nanoparticles have been identified, key functional differences native to HDL have remained undetected. In the present study, we executed a multidisciplinary approach to uncover these differences by exploring the form, function, and medical applicability of engineered HDL-mimetic NPs (eHNPs) made from r4F (eHNP-r4F) and from apoA1 (eHNP-A1). Comparative analyses of the eHNPs through computational molecular dynamics (MD), advanced microfluidic NP synthesis and screening technologies, and in vivo animal model studies extracted distinguishable eHNP characteristics: the eHNPs share identical structural and compositional characteristics with distinct differences in NP stability and organization; eHNP-A1 could more significantly stimulate anti-inflammatory responses characteristic of the scavenger receptor class B type 1 (SR-B1) mediated pathways; and eHNP-A1 could outperform eHNP-r4F in the delivery of a model hydrophobic drug to an in vivo tumor. The biomimetic microfluidic technologies and MD simulations uniquely enabled our comparative analysis through which we determined that while eHNP-r4F is a capable NP with properties mimicking natural eHNP-A1, challenges remain in reconstituting the full functionality of NPs naturally derived from humans.

DNA electrochemical biosensor for metallic drugs at physiological conditions.

Entrapment of dsSS-DNA into the polypyrrole-polyvinyl sulphonate (dsSS-DNA-PPy-PVS) film over indium-tin-oxide (ITO) coated glass has been designed to detect titanium and platinum drugs, titanocene dichloride and cisplatin. The disposable dsSS-DNA-PPy-PVS/ITO biosensor was characterized by cyclic voltammetry, attenuated total reflectance Infrared spectroscopy and atomic force microscopy. Amperometric studies by cyclic voltammetry using, dsSS-DNA-PPy PVS/ITO biosensor, demonstrated the ability of this biosensor to detect these metallic drugs in millimolar concentration by monitoring the decrease of the guanine oxidation signal as a result of the DNA damage. The concentration range detected for titanocene dichloride is 0.25 to 1.5 mM and for cisplatin is 0.06 to 1.0 mM.