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

Microtubules play a crucial role in maintaining the shape and function of neurons. During progression of Alzheimer's disease (AD), severe destabilization of microtubules occurs, which leads to the permanent disruption of signal transduction processes and memory loss. Thus, microtubule stabilization is one of the key requirements for the treatment of AD. Taxol, a microtubule stabilizing anticancer drug, has been considered as a potential anti-AD drug but was never tested in AD patients, likely because of its' toxic nature and poor brain exposure. However, other microtubule-targeting agents such as epothilone D (BMS-241027) and TPI-287 (abeotaxane) and NAP peptide (davunetide) have entered in AD clinical programs. Therefore, the taxol binding pocket of tubulin could be a potential site for designing of mild and noncytotoxic microtubule stabilizing molecules. Here, we adopted an innovative strategy for the development of a peptide based microtubule stabilizer, considering the taxol binding pocket of β-tubulin, by using alanine scanning mutagenesis technique. This approach lead us to a potential octapeptide, which strongly binds to the taxol pocket of β-tubulin, serves as an excellent microtubule stabilizer, increases the expression of acetylated tubulin, and acts as an Aβ aggregation inhibitor and neuroprotective agent. Further, results revealed that this peptide is nontoxic against both PC12 derived neurons and primary cortical neurons. We believe that our strategy and discovery of peptide-based microtubule stabilizer will open the door for the development of potential anti-AD therapeutics in near future.

Crafting of Neuroprotective Octapeptide from Taxol-Binding Pocket of β-Tubulin.

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.

Aβ peptide and hyper-phosphorylated microtubule associated protein (Tau) aggregation causes severe damage to both the neuron membrane and key signal processing microfilament (microtubule) in Alzheimer's disease (AD) brains. To date, the key challenge is to develop nontoxic, proteolytically stable amyloid inhibitors, which can simultaneously target multiple pathways involved in AD. Various attempts have been made in this direction; however, clinical outcomes of those attempts have been reported to be poor. Thus, we choose development of peptoid (N-substituted glycine oligomers)-based leads as potential AD therapeutics, which are easy to synthesize, found to be proteolytically stable, and exhibit excellent bioavailability. In this paper, we have designed and synthesized a new short peptoid for amyloid inhibition from 30-34 hydrophobic pocket of amyloid beta (Aβ) peptide. The peptoid selectively binds with 17-21 hydrophobic region of Aβ and inhibits Aβ fibril formation. Various in vitro assays suggested that our AI peptoid binds with tubulin/microtubule and promotes its polymerization and stability. This peptoid also inhibits AChE-induced Aβ fibril formation and provides significant neuroprotection against toxicity generated by nerve growth factor (NGF) deprived neurons derived from rat adrenal pheochromocytoma (PC12) cell line. Moreover, this peptoid shows serum stability and is noncytotoxic to primary rat cortical neurons.

Genesis of Neuroprotective Peptoid from Aβ30-34 Inhibits Aβ Aggregation and AChE Activity.

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.

Microtubules play a crucial role in maintenance of structure, function, axonal extensions, cargo transport, and polarity of neurons. During neurodegenerative diseases, microtubule structure and function get severely damaged due to destabilization of its major structural proteins. Therefore, design and development of molecules that stabilize these microtubule networks have always been an important strategy for development of potential neurotherapeutic candidates. Toward this venture, we designed and developed a tyrosine rich trisubstituted triazine molecule (TY3) that stabilizes microtubules through close interaction with the taxol binding site. Detailed structural investigations revealed that the phenolic protons are the key interacting partners of tubulin. Interestingly, we found that this molecule is noncytotoxic in PC12 derived neurons, stabilizes microtubules against nocodazole induced depolymerization, and increases expression of acetylated tubulin (Ac-K40), an important marker of tubulin stability. Further, results show that TY3 significantly induces neurite sprouting as compared to the untreated control as well as the two other analogues (TS3 and TF3). It also possesses anti-Aβ fibrillation properties as confirmed by ThT assay, which leads to its neuroprotective effect against amyloidogenic induced toxicity caused through nerve growth factor (NGF) deprivation in PC12 derived neurons. Remarkably, our results reveal that it reduces the expression of TrkA (pY490) associated with NGF deprived amyloidogenesis, which further proves that it is a potent amyloid β inhibitor. Moreover, it promoted the health of the rat primary cortical neurons through higher expression of key neuronal markers such as MAP2 and Tuj1. Finally, we observed that it has good serum stability and has the ability to cross the blood-brain barrier (BBB). Overall, our work indicates the importance of phenolic -OH in promoting neuroprotection and its importance could be implemented in the development of future neurotherapeutics.

Power of Tyrosine Assembly in Microtubule Stabilization and Neuroprotection Fueled by Phenol Appendages.

Amyloid-beta (Aβ) peptide misfolds into fibrillary aggregates (β-sheet) and is deposited as amyloid plaques in the cellular environment, which severely damages intraneuronal connections leading to Alzheimer's disease (AD) pathogenesis. Furthermore, neurons are rich in tubulin/microtubules, and the intracellular network of microtubules also gets disrupted by the accumulation of Aβ fiber in the brain. Hence, development of new potent molecules, which can simultaneously inhibit Aβ fibrillations and stabilize microtubules, is particularly needed for the efficient therapeutic application in AD. To address these issues, here we introduced an innovative fusion strategy to design and develop next generation anti-AD therapeutic leads. This unexplored fusion strategy entails design and development of a potent nonapeptide by taking into account both the hydrophobic core (17-21) of Aβ peptide and the taxol binding region of β-tubulin. In vitro results suggest that this newly designed peptide interacts at the taxol binding region of β-tubulin with a moderate binding affinity and promotes microtubule polymerization. It has the ability to bind at the hydrophobic core (17-21) of Aβ, responsible for its aggregation, and prevent amyloid fibril as well as plaque formation. In addition, it interacts at the CAS site (catalytic anionic site) of acetylcholinesterase (AChE) and significantly inhibits AChE induced Aβ fibrillation, stimulates neurite branching, and provides stability to intracellular microtubules and extensive protection of neurons against nerve growth factor (NGF) deprived neuron toxicity. Moreover, this newly designed peptide shows good stability in serum obtained from humans and efficiently permeates the blood-brain barrier (BBB) without showing any toxicity toward differentiated PC12 neurons as well as primary rat cortical neurons. This excellent feature of protecting the neurons by stabilizing the microtubules without showing any toxicity toward neurons will make this peptide a potent therapeutic agent of AD in the near future.

Potential Neuroprotective Peptide Emerged from Dual Neurotherapeutic Targets: A Fusion Approach for the Development of Anti-Alzheimer's Lead.

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.

Juhee Khan

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

Juhee Khan

.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