1. Protein aggregation, Protein misfolding and Neuronal Interactions: The pathological hallmark of Alzheimer’s disease is the neural deposition of self-assembled fibrillar aggregates of the 39-42 residue long peptides Amyloid beta (Aβ).We have employed FCS to measure diffusional transport (and therefore the hydrodynamic size) of protein aggregates and quantify them by using a new method for analyzing FCS data in such complex systems (more...).
We have investigated the aggregation of amyloid beta (implicated in Alzheimer's disease) and are currently inerested in different variants of amyloid beta (more...).
The conformation of the peptide is an important degree of freedom of the peptide which is intricately linked to both its aggregation behaviour and its toxicity. It is possible that each of the major class of the aggregates has a specific conformation. Biophysical methods so far has only uncovered the conformation of the fibrillar aggregates, which are the end-products of the aggregation process. Since the intermediates are the likely bio-active species, it is important to understand the conformational transition from the monomeric to the oligomeric state.The Alzheimer's Aβ, which is unstructured in the monomeric state, go on to form characteristic beta-hairpin like structures in the fibrillar aggregates through a ‘misfolding’ step. On the way of transition from the monomeric peptide to fibrils Aβ forms a wide variety of species of different sizes and some of them are related to the toxicity. The hairpin like ‘misfolding’ of the peptide seems to a critical phenomenon for the aggregation and may also be strongly related to the toxicity (more...)
We have also used FCS to investigate oligomerization of amyloid beta on membranes and alpha synuclein in the cytoplasm of PC-12 and HEK cells respectively, in order to understand the basic mechanism of toxicity of these aggregates on specific type of neurons(more...).
2. Neurotransmitter dynamics: Intra- and inter-cellular transport of neurotransmitters is fundamental to the process of neuronal communication, and this is also affected in amyloid-induced toxicity. Yet, these processes have typically evaded direct detection in a living neuron, as the neurotransmitters themselves have remained invisible. Many of the most important neurotransmitters (such as serotonin, dopamine and nor-epinephrine, some of which are also important in protein-aggregation related diseases) exhibit ultraviolet fluorescence in solution. We had earlier established a multiphoton microscopy technique to achieve label-free imaging of neurotransmitters, and our lab provided the first direct measurement of neurotransmitter dynamics in live neurons. Our work has now made it possible to understand the effect of various drugs, such as cocaine and amphetamines, at a single cell level in live brain tissue We have shown that a significant fraction of neuronal communication can take place through somatic (as against synaptic) exocytosis. We have also showed that it is possible to make a neurotransmitter map of a live whole brain-section, and to compare the activity in different areas of the brain (more...). This technique has attracted the attention of several scientists, who are interested in different roles of serotonin in the living systems. Together with them, we have applied this technique for various other applications, such as monitoring the differentiation of human embryonic stem cells or embryogenesis (more...). In addition, we have extended this technique using an “Optical Parametric Oscillator” to visualize the neurotransmitter dopamine (a deeper ultraviolet chromophore). The combination of this technique with those developed for studying aggregation should allow us to quantify dopamine depletion as a function of alpha-synuclein aggregation, a process that some believe is at the core of the events that lead to Parkinson’s disease (more...). 3. Instrumentation:Our laboratory has engineered the art of building cutting edge instrument and modifying commercial ones to suit experimental requirements. Some of them have been perfected for measurements which were not possible earlier. Briefly,a) A 76 MHz Ti-Sa 100 fs pulsed laser has been coupled with a confocal scanning microscope to enable us to do three-photon imaging of serotonin in live neurons. The existing set up has been modified for two-photon imaging of dopamine, the first one of its kind. A fiber based non-invasive in-vivo neurotransmitter imaging setup is also envisioned in the near future.b) A home-built fiber detection based FCS instrument capable of single molecule detection (PPB of RhB is ~240 KHz) is used to measure size distribution of aggregating proteins. Dual channel FCS for cross-correlation analysis and dual objective FCS for increased sensitivity are being developed at present.c) A home-built fiber excitation based FCS instrument has been used to measure size distribution of aggregating proteins. This set up promises to be a diagnostic tool to study aggregation process in brains of live specimens.A greater detail would be found here.