Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

CSIR-Indian Institute of Chemical Biology

Nitric oxide (NO), an endogenously produced free radical species, is an extremely important signalling molecule in several biochemical processes related to neurotransmission, neuronal communication, and vasodilation, to name a few. Other than relying on endogenous synthesis, intracellular NO delivery presents an interesting challenge to fully exploit the therapeutic potential of this gaseous molecule. We have applied a self-assembling peptide conjugate strategy to devise a construct carrying a NO-release arm, which can be activated under standard redox conditions. Consequently, a tryptophan-based peptide carrier was designed, which self-assembled in the solution phase to afford soft nanospherical structures, and released NO in Neuro2a cell line, resulting in neurite outgrowth.

Self-assembling soft structures for intracellular NO release and promotion of neurite outgrowth.

Design and development of acetylcholinesterase (AChE) inhibitor has tremendous implications in the treatment of Alzheimer's disease (AD). Here, we have adopted a computational approach for the design of a peptide based AChE inhibitor from its active site. We identified an octapeptide, which interacts with the catalytic anionic site (CAS) of AChE enzyme and inhibits its activity. Interestingly, this peptide also inhibits amyloid aggregation through its interaction at the 17-21 region of amyloid-beta (Aβ) and stabilizes microtubules by interacting with tubulin as well. Eventually, in the PC12 derived neurons, it shows noncytotoxicity, promotes neurite out-growth, stabilizes intracellular microtubules, and confers significant neuroprotection even upon withdrawal of nerve growth factor (NGF). Further, results reveal that this peptide possesses good serum stability, crosses the blood-brain barrier (BBB), and maintains the healthy architecture of the primary cortical neurons. This work shows discovery of an excellent peptide-based AChE inhibitor with additional potential as a microtubule stabilizer, which will pave the way for the development of potential anti-AD therapeutics in the near future.

Peptide-Based Acetylcholinesterase Inhibitor Crosses the Blood-Brain Barrier and Promotes Neuroprotection.

Reconstitution of a complex biological structure or system following a simple and facile strategy using minimum physiochemical cues is challenging for an in-depth understanding of the system. In particular, the brain is a highly sophisticated and complex network of trillions of neurons and glial cells that controls function of our body. Understanding this complex machinery requires an innovative and simple bottom-up approach. In this venture, we report an easy and efficient strategy to culture cortical and hippocampal primary neurons from the E14-E16 embryo of Sprague-Dawley rat. This generates spontaneous neurospheres within 6-7 days of primary neuron culture of E14-E16 embryo. It further proliferates and forms radial glia-like structures, which are known to be the primary neural progenitor cells that differentiate into neurons, astrocytes, and oligodendrocytes. Interestingly, neurospheres lead to the formation of large projection neurons and radial glia, which mimic the early stage of cortical development in an in vivo system. Overall, this new, facile, strategic mixed primary neuron culture method offers a potential platform for understanding the effect of neurochemical modulators, which has tremendous future implications in the screening of neurotherapeutics.

Neurosphere Development from Hippocampal and Cortical Embryonic Mixed Primary Neuron Culture: A Potential Platform for Screening Neurochemical Modulator.

Development of potential therapeutics for Alzheimer's disease (AD) requires a multifaceted strategy considering the high levels of complexity of the human brain and its mode of function. Here, we adopted an advanced strategy targeting two key pathological hallmarks of AD: senile plaques and neurofibrillary tangles. We derived a lead short tetrapeptide, Ser-Leu-Lys-Pro (SLKP), from a dodeca-neuropeptide of amphibian (frog) brain. Results suggested that the SLKP peptide had a superior effect compared to the dodecapeptide in neuroprotection. This result encouraged us to adopt peptidomimetic approach to synthesize an SLKP peptoid. Remarkably, we found that the SLKP peptoid is more potent than its peptide analogue, which significantly inhibits Aβ fibrillization, moderately binds with tubulin, and promotes tubulin polymerization as well as stabilization of microtubule networks. Further, we found that SLKP peptoid is stable in serum, shows significant neuroprotection against Aβ mediated toxicity, promotes significant neurite outgrowth, maintains healthy morphology of rat primary cortical neurons and crosses the blood-brain barrier (BBB). To the best of our knowledge, our SLKP peptoid is the first and shortest peptoid to show significant neuroprotection and neuroregeneration against Aβ toxicity, as well as to cross the BBB offering a potential lead for AD therapeutics.

Discovery of Neuroregenerative Peptoid from Amphibian Neuropeptide That Inhibits Amyloid-β Toxicity and Crosses Blood-Brain Barrier.

Repairing of brain damage is a challenging task due to the limited regenerative potential of complex network of neurons interspersed with glial cells. To address this issue, a number of approaches are in progress. Here we discuss a fascinating approach in which reactive astrocytes generated during any brain injury can be transformed into functional neurons using a mixture of small molecules or "chemical cocktail". This "chemical cocktail" leads to the conversion of reactive astrocytes into neurons by targeting two very important transcription factors, NeuroD1 and NeuroG2. This chemical approach opens up a new direction in development of regenerative medicine.

Glial-Neuron Transformation by "Chemical Cocktail".

A biocompatible hydrogel containing a hexapeptide as a key unit has been designed and fabricated. Our design construct comprises a β-sheet-rich short hexapeptide in the center with a hydrophobic long chain and hydrophilic triple lysine unit attached at the N- and C-terminals, respectively. Thus, it is this amphiphilic nature of the molecule that facilitates gelation. It can capture solvent molecules in the three-dimensional cross-linked fibrillar networks. The amphiphilic character of the construct has been modulated to produce an excellent biocompatible soft material for the inhibition of bacterial growth by rupturing the bacterial cell membrane. This hydrogel is also stable against enzymatic degradation (proteinase K) and, most importantly, offers a biocompatible environment for the growth of normal mammalian cells due to its noncytotoxic nature as observed through the cell viability assay. From the hemolytic assay, the morphology of the human red blood cells is found to be almost intact, which suggests that the hydrogel can be used in biomedical applications. Thus, this newly designed antibacterial hydrogel can be used as both an antibacterial biomaterial and a biocompatible scaffold for mammalian cell culture.

Biocompatible Lipopeptide-Based Antibacterial Hydrogel.

A multiarm nanomedicine template has been designed following bottom-up approach, which target neuropilin-1 (Nrp-1) receptor of cancer cells. Through this venture, we discovered that cucurbit [6] uril (CB [6]) binds with tubulin close to binding pocket of vinblastine site and perturbs tubulin polymerization. To increase the specificity of gold nanoparticle (GNP) toward Nrp-1-rich cancer cells, we further modified this GNP with Nrp-1 receptor-specific short peptide (CGNKRTR). Remarkably, we found an interesting self-assembly process upon addition of curcumin into the CB [6] and peptide-functionalized GNP, leading to the formation of a spherical nanocapsule (CGNP·Cur). It can deliver and release significantly higher amounts of anticancer drug curcumin in Nrp-1-rich cancer cells. It causes microtubule depolymerization and significant tumor regression in Nrp-1 overexpressed mice melanoma model. These interesting findings show that nanocapsule has high potential to develop a powerful anticancer nanomedicine and help in its preclinical validation.

Dual-Arm Nanocapsule Targets Neuropilin-1 Receptor and Microtubule: A Potential Nanomedicine Platform.

Alzheimer's disease (AD) is a constantly recurring neurodegenerative disease that deteriorates over a period of time. In this pathology, connections between neurons become extremely damaged due to the deposition of senile plaques in the membrane region, which results in abnormal signal transduction processes. Also, the intracellular microtubule networks are disrupted in the hyperphosphorylated tau cascade of AD. Therefore, design and development of potent neuroprotective molecules that can instantaneously target multiple facets of AD pathogenesis are greatly needed to tackle this unmet medical need. Here, we have implemented a pharmacophore based in silico analysis of various neuroprotective peptides known for neurotherapeutic application in AD. Fascinatingly, we have identified an active core of these peptides and designed a library of hexapeptides. We observed that peptide "LETVNQ" (LE6) has shown significant protection ability against degeneration of neurons. Experimental evidence suggests that this peptide immensely reduced the aggregation rate of amyloid-β (Aβ) and helped in microtubule polymerization. Intriguingly, this newly designed peptide does not have any cytotoxicity toward differentiated PC12 neurons; rather it helps in neurite outgrowth. Further, LE6 helps to maintain the complex microtubule network in cells by promoting the polymerization rate of intracellular microtubules and mediates excellent protection of neurons even after removal of nerve growth factor (NGF). Finally, we observed that this LE6 peptide has substantial stability under physiological conditions and helps to retain healthy morphology of primary rat cortical neurons. This excellent piece of work identifies a potent hexapeptide, which has exceptional ability to protect neurons as well as microtubule from degeneration and may become potent therapeutics against AD pathogenesis in the future.

Varsha Gupta

Organic and Medicinal Chemistry Division

CSIR-Indian Institute of Chemical Biology

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Varsha Gupta

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Organic and Medicinal Chemistry Division

CSIR-Indian Institute of Chemical Biology

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Organic and Medicinal Chemistry Division