Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wildtype SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of posttranslational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57-Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril. Familial amyotrophic lateral sclerosis (FALS)3 caused by mutations in the SOD1 gene shows similarities to other members of the large class of neurodegenerative disorders that are characterized by gradual aggregation of specific proteins and subsequent cell death in characteristic regions of the central nervous system. Some prominent examples are amyloid-␤ deposits in Alzheimer disease, ␣-synuclein and hyperphosphorylated Tau protein deposits in Parkinson disease, prion protein deposits in the transmissible spongiform encephalopathies, and huntingtin deposits in Huntington disease (1). In the case of SOD1-linked FALS, protein deposits have been identified in affected tissues from patients and from ALS-SOD1 transgenic mice consisting largely of aggregated copper,zinc superoxide dismutase protein (Cu,Zn-SOD, SOD1 protein).Our current understanding of this class of diseases is that the implicated proteins misfold, aggregate, and ultimately deposit as extracellular plaques or intracellular inclusions in the afflicted regions of the CNS. Although the exact structures contributing to these heterogeneous protein aggregates are unknown, such diseases are nevertheless usually referred to as "amyloid diseases" because fibrillar properties characteristic of cross-␤-sheet amyloid are often detected in them and because each of the isolated proteins implicated in causing disease shows a...

Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. These sensors, rendered highly lipophilic to anchor the conjugated pi-wire molecular framework in the membrane, offer several favorable functional parameters including fast response kinetics and high sensitivity to membrane potential changes. The impact of VSDs has, however, been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a non-genetic molecular platform for cell- and molecule-specific targeting of synthetic voltage sensitive dyes in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of voltage sensitive dyes by dynamic encapsulation, and high-affinity ligands to target the construct to specific neuronal cells utilizing only native components of the neurotransmission machinery at physiological expression levels. Dichloropane, a monoamine transporter ligand, enables targeting of dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the target proteins. We envision that modularity of our platform will enable its application to a variety of molecular targets and sensors, as well as lipophilic drugs and signaling modulators. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.

Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. These sensors, rendered highly lipophilic to anchor the conjugated pi-wire molecular framework in the membrane, offer several favorable functional parameters including fast response kinetics and high sensitivity to membrane potential changes. The impact of VSDs has, however, been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a non-genetic molecular platform for cell- and molecule-specific targeting of synthetic voltage sensitive dyes in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of voltage sensitive dyes by dynamic encapsulation, and high-affinity ligands to target the construct to specific neuronal cells utilizing only native components of the neurotransmission machinery at physiological expression levels. Dichloropane, a monoamine transporter ligand, enables targeting of dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the target proteins. We envision that modularity of our platform will enable its application to a variety of molecular targets and sensors, as well as lipophilic drugs and signaling modulators. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.