Surface-enhanced
Raman spectroscopy (SERS)-based protein analysis
is a promising alternative to existing early stage diagnoses. However,
SERS research conducted thus far accompanies challenges such as nonuniformity
of plasmonic nanostructures, irregular coating of analytes, and denaturation
of proteins, which seriously limit the practicability of suggested
approaches. Here, we introduce a carboxylic acid-functionalized and
graphitic nanolayer-coated three-dimensional SERS substrate (CGSS)
fabricated by sequential nanotransfer printing. The substrate consists
of well-defined, uniform gold nanowire arrays for effective Raman
signal enhancement and a strong protein-immobilization layer. With
an enhancement factor (EF) of 5.5 × 105, on par with
the highest ever reported values, the CGSS allows the detection of
protein conformational changes and the determination of protein concentration
via Raman measurements. Exploiting the CGSS, we successfully measured
the SERS spectra of Alzheimer’s biomarkers, tau protein and
amyloid β, based on which secondary structural changes were
analyzed quantitatively.
Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson’s disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 cKO) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and upon MPTP treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the SNpc or dopamine-dependent motor deficits over the 24-month lifespan. These mice were just as susceptible to MPTP as control mice. However, compared to control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the SNpc. We also utilized an inducible Ndufs4 knockout mouse strain (Ndufs4 iKO) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 iKO mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell- autonomous dopaminergic neuron death during the normal lifespan of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or non-autonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron death during aging nor does it contribute to dopamine neuron toxicity in the MPTP model of Parkinson’s disease. These findings suggest the existence of alternative mechanisms of dopaminergic neuron death independent of mitochondrial complex I inhibition.
The development of treatment for neurodegenerative diseases (NDs) such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis is facing medical challenges due to the increasingly aging population. However, some pharmaceutical companies have ceased the development of therapeutics for NDs, and no new treatments for NDs have been established during the last decade. The relationship between ND pathogenesis and risk factors has not been completely elucidated. Herein, we review the potential involvement of transient receptor potential (TRP) channels in NDs, where oxidative stress and disrupted Ca 2+ homeostasis consequently lead to neuronal apoptosis. Reactive oxygen species (ROS) -sensitive TRP channels can be key risk factors as polymodal sensors, since progressive late onset with secondary pathological damage after initial toxic insult is one of the typical characteristics of NDs. Recent evidence indicates that the dysregulation of TRP channels is a missing link between disruption of Ca 2+ homeostasis and neuronal loss in NDs. In this review, we discuss the latest findings regarding TRP channels to provide insights into the research and quests for alternative therapeutic candidates for NDs. As the structures of TRP channels have recently been revealed by cryo-electron microscopy, it is necessary to develop new TRP channel antagonists and reevaluate existing drugs.
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