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.

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.

The formation and accumulation of amyloid beta (Aβ) peptide are considered the crucial events that are responsible for the progression of Alzheimer's disease (AD). Herein, we have designed and synthesized a series of fluorescent probes by using electron acceptor−donor end groups interacting with a π-conjugating system for the detection of Aβ aggregates. The chemical structure of these probes denoted as RMs, having a conjugated π-system (C�C), showed a maximum emission in PBS (>600 nm), which is the best range for a fluorescent imaging probe. Among all these probes, RM-28 showed an excellent fluorescence property with an emission maximum of >598 nm upon binding to Aβ aggregates. RM-28 also showed high sensitivity (7.5-fold) and high affinities toward Aβ aggregates (K d = 175.69 ± 4.8 nM; K a = 0.5 × 10 7 M −1 ). It can cross the blood−brain barrier of mice efficiently. The affinity of RM-28 toward Aβ aggregates was observed in 3xTg-AD brain sections of the hippocampus and cortex region using a fluorescent imaging technique, as well as an in vitro fluorescence-based binding assay with Aβ aggregates. Moreover, RM-28 is highly specific to Aβ aggregates and does not bind with intracellular proteins like bovine serum albumin (BSA) and α-synuclein (α-Syn) aggregates. The results indicate that the probe RM-28 emerges as an efficient and veritable highly specific fluorescent probe for the detection of Aβ aggregates in both in vitro and in vivo model systems.

Brain damage is associated with spatial imbalance of cholinergic system, which makes severe impact in recovery of damaged neurons of brain. Therefore, maintenance of cholinergic system is extremely important. Here, we fabricated an injectable hydrogel with acetylcholine-functionalized graphene oxide and poly(acrylic acid). Results revealed that this hydrogel is non-cytotoxic, promotes neurite outgrowth, stabilizes microtubule networks, and enhances the expression of some key neural markers in rat cortical primary neurons. Further, this hydrogel exhibits significant potential in neuroregeneration and also promotes fast recovery of the sham injured mice brain. Moreover, we found significant enhancement of reactive astrocytes in the hippocampal dentate gyrus region of the sham injured brain, indicating its excellent potential in neural repair of the damaged brain. Finally, above results clearly indicate that this neuro-regenerative hydrogel is highly capable of maintaining the cholinergic balance through local release of acetylcholine in the injured brain, which is crucial for brain repair.

Brain injury can lead to the loss of neuronal functions and connections, along with the damage of the extracellular matrix (ECM). Thus, it ultimately results in devastating long-term damage, and recovery from this damage is a challenging task. To address this issue, we have designed a sulfo-group-functionalized injectable biocompatible peptide hydrogel, which not only mimics the ECM and supports the damaged neurons but also releases a neurotrophic factor around the injured sites of the brain in the presence of the matrix metalloproteinase 9 (MMP9) enzyme. It has also been observed that the driving force of hydrogel formation is a β-sheet secondary structure and π–π stacking interactions between Phe-Phe moieties. The hydrogel is able not only to promote neurite outgrowth of PC12-derived neurons and primary neurons cultured in its presence but also to nullify the toxic effects of anti-nerve growth factor (Anti-NGF)-induced neurons. It also promotes the expression of vital neuronal markers in rat cortical primary neurons, displays substantial potential in neuroregeneration, and also promotes fast recovery of the sham injured mice brain. Increased expression of reactive astrocytes in the hippocampal dentate gyrus region of the sham injured brain clearly suggests its tremendous ability in the neural repair of the damaged brain. Thus, we can convincingly state that our hydrogel is capable of repairing brain injury by mimicking an ECM-like environment and providing neuroprotection to the damaged neurons.

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.

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.

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.