Characterization of the Photophysical, Thermodynamic, and Structural Properties of the Terbium(III)-DREAM Complex.

DREAM (also known as K(+) channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of KV4 channels in neuronal cells as well as a novel Ca(2+)-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimer's disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb(3+), are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb(3+). We also observe that unlike Tb(3+)-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (τaverage ∼ 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb(3+) binds to at least three sites with high affinity (Kd = 1.8 µM in the presence of Ca(2+)) and displaces bound Ca(2+) through an entropically driven mechanism (ΔH ∼ 12 kcal mol(-1), and TΔS ∼ 22 kcal mol(-1)). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb(3+), like Ca(2+), triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca(2+) binding, Tb(3+) binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry-mass spectrometry reveal that replacement of Ca(2+) with Tb(3+) preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM-protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.

Towards functional restoration for persons with limb amputation: A dual-stage implementation of regenerative agonist-antagonist myoneural interfaces.

While amputation has traditionally been viewed as a failure of therapy, recent developments in amputation surgery and neural interfacing demonstrate improved functionality and bidirectional communication with prosthetic devices. The agonist antagonist myoneural interface (AMI) is one such bi-directional neural communication model comprised of two muscles, an agonist and an antagonist, surgically connected in series within the amputated residuum such that contraction of one muscle stretches the other. By preserving agonist-antagonist muscle dynamics, the AMI allows proprioceptive signals from mechanoreceptors within both muscles to be communicated to the central nervous system. Preliminary human evidence suggests that AMIs have the capacity to provide high fidelity control of a prosthetic device, force feedback, and natural proprioception. However, AMIs have been implemented only in planned amputations and require healthy distal tissues, whereas the majority of amputations occur in patients who do not have healthy distal tissues. Through the use of a dual-stage surgical procedure which leverages existent tissues, this study proposes a revision model for implementation of the AMI in patients who are undergoing traumatic amputation or have already undergone a standard amputation. This paper validates the resulting AMI's physiology, revealing robust viability and mechanical and electrophysiological function. We demonstrate the presence of H-waves in regenerative grafts, indicating the incorporation of the AMI into physiological reflexive loops.

Closed-loop functional optogenetic stimulation.

Optogenetics has been used to orchestrate temporal- and tissue-specific control of neural tissues and offers a wealth of unique advantages for neuromuscular control. Here, we establish a closed-loop functional optogenetic stimulation (CL-FOS) system to control ankle joint position in murine models. Using the measurement of either joint angle or fascicle length as a feedback signal, we compare the controllability of CL-FOS to closed-loop functional electrical stimulation (CL-FES) and demonstrate significantly greater accuracy, lower rise times and lower overshoot percentages. We demonstrate orderly recruitment of motor units and reduced fatigue when performing cyclical movements with CL-FOS compared with CL-FES. We develop and investigate a 3-phase, photo-kinetic model to elucidate the underlying mechanisms for temporal variations in optogenetically activated neuromusculature during closed-loop control experiments. Methods and insights from this study lay the groundwork for the development of closed-loop optogenetic neuromuscular stimulation therapies and devices for peripheral limb control.

Optogenetic Peripheral Nerve Immunogenicity.

Optogenetic technologies have been the subject of great excitement within the scientific community for their ability to demystify complex neurophysiological pathways in the central (CNS) and peripheral nervous systems (PNS). The excitement surrounding optogenetics has also extended to the clinic with a trial for ChR2 in the treatment of retinitis pigmentosa currently underway and additional trials anticipated for the near future. In this work, we identify the cause of loss-of-expression in response to transdermal illumination of an optogenetically active peroneal nerve following an anterior compartment (AC) injection of AAV6-hSyn-ChR2(H134R) with and without a fluorescent reporter. Using Sprague Dawley Rag2-/- rats and appropriate controls, we discover optogenetic loss-of-expression is chiefly elicited by ChR2-mediated immunogenicity in the spinal cord, resulting in both CNS motor neuron death and ipsilateral muscle atrophy in both low and high Adeno-Associated Virus (AAV) dosages. We further employ pharmacological immunosuppression using a slow-release tacrolimus pellet to demonstrate sustained transdermal optogenetic expression up to 12 weeks. These results suggest that all dosages of AAV-mediated optogenetic expression within the PNS may be unsafe. Clinical optogenetics for both PNS and CNS applications should take extreme caution when employing opsins to treat disease and may require concurrent immunosuppression. Future work in optogenetics should focus on designing opsins with lesser immunogenicity.