Neurorestorative strategies for Alzheimer's disease
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.162057
Source of Support: None, Conflict of Interest: None
Alzheimer's disease (AD) is characterized by beta-amyloid plaques and neurofibrillary tangles that cause devastating cognitive and memory deficits. AD is known to be associated with neuronal death and synaptic loss. Thus, methods to retard the progression of the disease and to promote neuro-regeneration are vital for the prevention of advancement of AD. The recent trend is to decipher the molecular mechanisms of AD, and further aim at neuro-restorative mechanisms such as neuro-protection, neuro-modulation, and neuro-regeneration. In this review, we provide an overview of the recent studies describing various neuro-restorative strategies for AD and mainly focus on stem cell and neuro-modulation therapies. Furthermore, we briefly refer to the other neurorestorative treatments including medications, bioengineering, and gene therapies for AD. Although most of them remain in an experimental phase, neuro-restorative strategies may have the potential for clinical use in the management of this debilitative disease.
Keywords: Alzheimer′s disease; neuromodulation; neurorestorative strategies; stem cell treatment
Alzheimer's disease (AD) is the most prevalent, irreversible, neurodegenerative disorder that is clinically characterized by progressive cognitive, physical, and behavioral impairment, leading to characteristic devastating cognitive and memory deficits. AD is known to be associated with neuronal cell death, synaptic loss, and corresponding impairment in cognition. A German physician and neuropathologist, Dr. Alois Alzheimer in 1906, first identified the condition. He reported the presence of progressive accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain of a 55-year-old woman who showed symptoms of severe dementia with the characteristic pathological features.  Senile plaques are composed of Aβ deposits, which are a part of the neurons surrounding a group of proteins. NFTs are aggregations of hyperphosphorylated tau, a microtubule-associated protein within the aging neurons. These are two prominent pathological features of AD. Yet, the exact mechanisms leading to this disease are still under investigation. Inherited mutations in some genes and several non-genetic risk factors, such as brain trauma, infections, cardiovascular diseases, mitochondrial dysfunction, aging and age-dependent oxidative stress, have been listed in the etiology of AD. The current studies also indicate that blood flow restriction, inflammation, and free radicals are also implicated in the causation of this disease. AD is the most common cause of dementia in the elderly population. Unfortunately, 50-70% of these patients are confirmed to be suffering from AD during the postmortem examination. Aging is a major influencing factor for increasing the risk of developing AD. The prevalence of AD roughly doubles after every 5 years of age, beginning at 65 years.  AD affects 5-8% of individuals aged 65-74 years. Moreover, it affects 30% of the people aged greater than 80 years. It also affects about 35 million people worldwide, and this number is expected to quadruple by 2050 corresponding to the aging population. 
Alzheimer's disease is often incurable due to the complex nature of the underlying mechanisms of cholinergic neuronal death and brain atrophy. Current treatments, recent studies, and clinical trials have failed to modify the incidence and clinical course of AD. Thus, the need for development of novel and innovative medical treatment is of paramount importance. Both drugs and non-pharmacological therapy are used in the current treatment of AD. Over the past decade, the potential use of neural stem cells (NSCs) to treat the cognitive dysfunction has attracted substantial interest. In particular, cellular transplantation provides a novel therapeutic strategy with tremendous potential. In this review, we focus on these recently developed neuro-regenerative and neuro-restorative strategies for AD and attempt to provide an overall perspective on the advances in the treatment for AD.
The pathophysiology of AD is very complex. Aβ plaques, and NFTs are the main neuropathological hallmarks. Senile Aβ plaques are important pathophysiological changes seen in AD and thus form the essential component of the "amyloid hypothesis. This hypothesis proposes that the deposition of Aβ plaques precedes and induces the neuronal abnormalities that underlie the development of dementia.  In AD, the soluble form of Aβ has toxic effects on the neurons, including increasing the oxidative stress, precipitating the programmed cell death and lowering the cell injury threshold.  NFTs are dense intracellular protein deposits that are composed of the normal cytoskeletal protein, tau. The formation of NFTs in neurons is associated with extensive phosphorylation and cross-linking of the tau molecules. However, the initiating event is not well understood.  Many of the therapeutic strategies in AD, including neuro-restorative strategies to be discussed below, depend on treating the above mentioned mechanisms, especially decreasing the Aβ deposits. However, the pathophysiology of AD involves multiple mechanisms, that are at present, not well understood.
Medicines administered in AD have a central role in inflammatory modulation and in protection of neuro- restorative processes.  Current Food and Drug Administration-approved medications include cholinesterase inhibitors, memantine, anti-inflammatory medicaments, antioxidants, and several other medications. Memantine is an N-methyl-d-aspartate receptor antagonist that helps to prevent neuronal excitotoxicity. Parsons et al. found that memantine helped to treat dysfunction in glutaminergic transmission, while acetylcholinesterase inhibitors (AChEIs) served to increase pathologically lowered levels of the neurotransmitter, acetylcholine.  Thus, there may be an increased therapeutic benefit in administering a combination therapy that includes both memantine and AChEIs.  However, these drugs cannot reverse the natural course of AD. The damage to neuronal cells caused by over activity of the immune system (which is characterized by the increased concentrations of acute phase reactants, cytokines, and complement proteins) is overcome by anti-inflammatory agents that play a crucial role in immunomodulation in AD patients. It has also been proven that natural antioxidants possess neuro-protective effects that considerably alleviate the symptoms of neurodegenerative diseases. Furthermore, epidemiological researches exploring the risk of AD have demonstrated that antioxidants may protect against Aβ toxicity and the oxidative stress and help in reducing the progression of AD. , As mentioned above, deposits of Aβ form the pathological hallmarks of AD; therefore, any potential neuro-restorative treatment that is able to lower Aβ concentrations and help in its depletion should be effective in AD. Cathepsin B reduces the relative abundance of Aβ by limiting its deposition.  Neprilysin is another major extracellular Aβ degrading enzyme that improves the clearance rate of amyloid plaques. It has been demonstrated that injections of human neprilysin decreased Aβ in transgenic mice.  However, many of these drugs are currently in the trial phase and there is a long way to go before they may be used for therapeutic application. Other strategies to combat AD include the enhancement of mitochondrial function, as well as targeting serotonin receptors, nerve growth factors (NGFs) and receptors for advanced glycation end products. These are only theoretical strategies and a large number of confirmatory studies are still required prior to their clinical use.
Neuro-modulation involves an alteration of neurophysiological activity by providing drugs or electrical stimulation that act directly upon nerves to produce a natural biological response. Therefore, electrical stimulation or neurotransmitters produced by neuromodulators or pharmaceutical factors are considered to be effective in promoting neurological recovery in AD.
Deep brain stimulation (DBS) involves an implantable electrical stimulation technology that helps in treating neurological diseases, such as Parkinson's disease, dystonia, and some types of tremors.  As a technique of therapeutic neuro-modulation, it also has a potential role in the treatment of cognitive disability.  Recent research has shown that DBS may help in improving cognitive functions in AD. , Laxton et al. found that DBS could drive and modulate neural activity in the memory circuit and activate brain's default mode network. Besides this, glucose utilization in the brain improved after 12 months of continuous DBS.  According to the Alzheimer's disease assessment scale-cognitive subscale (ADAS-cog) and the Mini-Mental State Examination (MMSE) results, some patients showed progressive improvement, and the rate of cognitive decline slowed down at 6 and 12 months.  In a prospective study of patients with AD with mild cognitive decline, the MMSE and ADAS-cog scores stabilized compared to baseline, and the metabolism of the target region increased after a year of DBS. In addition, this research also provided evidence regarding the safety and efficacy of forniceal DBS in the hypothalamus.  Another report that presented an overview of human and animal researches investigating the curative effects of DBS on cognitive functions revealed improved neuropsychological and memory scores with a low complication rate.  Being a therapeutically effective, safe, and reversible treatment method, DBS indeed improves memory and the quality of life in carefully selected patients. Further investigations are warranted in this field.
Repetitive transcranial magnetic stimulation (rTMS) generates an electric current that induces cortical modulation and brings about long-lasting neuronal plasticity changes using a noninvasive technique.  It has been reported to improve the language ability, memory capacity, and executive functions for several weeks after a short period of treatment. , The rTMS combined with cognitive training seems to be a beneficial, effective and promising modality for AD treatment.  A second method, however, has drawn public attention in more recent years. Transcranial direct current stimulation (tDCS) induces neural modulation of cortical excitability and its favorable effects outlast the period of stimulation. It has been proven that tDCS is not only helpful in treating cognitive dysfunction but also helps in reducing the P300 latency, which is acknowledged to be abnormally increased in patients suffering from AD.  The neuro-modulation methods, including DBS, rTMS, and tDCS have been applied in patients with AD with certain curative benefits. However, the sample size being studied is usually small, and the time points of stimulation, the duration of treatment, and the safety of the procedure are still unknown. Moreover, large-scale clinical trials are required to prove its efficacy before neuro-modulation can be incorporated as a conventional treatment for AD.
Bioengineering involves neurogenesis and angiogenesis, axonal regeneration, and neuronal-replacement in treating neurological disorders.  So far, mesenchymal stem cells (MSCs) have been most frequently used in this endeavor and have shown the greatest benefit in clinical trials.  A continuous decrease in Aβ deposit aggregation might be a promising approach in treating AD. Thus, genetically modified human MSCs or neural stem cells (NSCs) that secrete the Aβ-degrading enzyme, neprilysin, administered along with plasmin, cathepsin B, and human nerve growth factor (NGF), have all been utilized in genetically modified mice in whom AD had been induced. , Remarkably, these strategies have helped in restoring memory functions, in stimulating cholinergic functions, and in enhancing synaptic plasticity in animal models. , Investigators have also found that brain-derived neurotrophic factor (BDNF) gene delivery reverses the neuronal-synapse loss, regulates gene expression and restores learning and memory ability in amyloid-transgenic mice. 
Some studies in gene therapy focus on protease-expressing NSCs. The administration of the neprilysin gene expressing viruses to genetically modified mouse models has resulted in a reduction of Aβ and a reversal of neuronal atrophy in the hippocampus.  Furthermore, Chen et al. suggest that intraventricular injection of the human neprilysin gene expressing fibroblasts into transgenic mouse models has contributed to a surprising decrease in the amount of Aβ plaques.  A research has shown that NGF expressing human NSCs transplanted into hippocampus could improve the memory capacity and may differentiate into neurons and astrocytes. Besides, it has also been confirmed that NSCs are overexpressing foreign genes like HB1.F3.ChAT, a finding that has proven its effectiveness in treating AD. 
Although bioengineering and gene therapy directly address the pathogenesis of AD and seem to be effective in reversing the course of AD, current studies are restricted to animal models. It will take some time before their clinical applicability is seen.
Widespread nonspecific neuronal death in the brain of patients with AD makes stem cell-based therapy for restoration incredibly challenging. Stem cell therapy is characterized by the differentiation potential of implanted cells into multiple cellular lineages, based on their proliferative capacity, neurotrophin secretion, promotion of neovascularization, anti-inflammatory activity, and neurotrophic, and neuro-protective activity.  The therapeutic strategies of stem cell implantation involve inducing activation of endogenous stem cells and regenerating the injured cells through stem cell transplantation.
Current strategies for treating patients with AD mainly focus on cholinergic neurotransmission, Aβ clearance as well as delivering NGF to the targeted region of the brain. However, these therapeutic methods can only alleviate symptoms associated with AD to some extent but cannot alter the degeneration and loss of neurons in brains of patients with AD; thus, they cannot cure the disease.  Hence, newer strategies are needed to alter the pathophysiology in order to obtain long-term benefits. Enhancement of neurogenesis may be an effective treatment. Several neuroprotective factors that can promote neurogenesis have been used for treatment of AD in animal models. It was shown that administration of encapsulated vascular endothelial growth factor helped in reversing behavioral deficits, reducing Aβ deposits and promoting angiogenesis, as well as reducing neuronal loss in APP/PS1 mice, a kind of transgenic model of AD.  In addition, Anitua et al. found that the intranasal delivery of plasma that was rich in growth factors was able to activate neuronal progenitor cells, enhance hippocampal neurogenesis, and reduce Aβ-induced neuro-degeneration in APP/PS1 mice.  Other factors, such as NGF, BDNF, and transforming growth factor, are also known to potentiate NSC generation as well as cell signal mediator function in several neurodegenerative processes, both in vitro and in vivo models.  Although endogenous NSCs can be induced, and the newly generated neurons can partially compensate for the neuronal loss in AD, they are not permanent substitutes for the damaged neurons. Moreover, administration of these drugs always needs a craniotomy and repeated injection. Hence, these limitations call for the introduction of exogenous NSCs in AD treatment.
Currently, cell-based therapy has been demonstrated to be a promising approach for many kinds of diseases including AD. Stem cell-derived neurons have demonstrated a great potential in integrating with the neural networks of the brain. Thus, the effective cell treatment of AD require the stem cells to exhibit targeted migration toward the damaged regions of the brain and then differentiate into multiple subtypes of neural cells.  Stem cells include NSCs, embryonic stem cells (ESCs), MSCs, and induced pleuripotent stem cells (iPSCs). It has also been proven that symptoms of AD could be alleviated by transplanting MSCs derived from the human umbilical cord into the brains in transgenic mouse models where Aβ-aggregation has been observed.  Furthermore, human umbilical cord blood-derived MSCs (UCB-MSC) are currently in phase I clinical trial. It is worth noting that NSC transplantation can reduce neuronal loss and improve cognitive function by increasing acetylcholine levels, and secrete neurotrophic factors to enhance/regulate synaptic plasticity in animal models of AD. ,,
Neural stem cells
Neural stem cell therapy for AD is an offshoot of the general concept of transplanting NSC in many neurological disorders. The neural progenitor and stem cells of the adult brain may be stimulated locally, by trophic factors or cytokines, to repair and restore the degenerated or injured nerve pathways. Intravenous administration of the neural progenitor and stem cells is a noninvasive procedure for transplantation, and it seems to be a relatively safe method of treatment.  Park et al. genetically modified human NSCs to encode choline acetyltransferase gene and found that it had a lesion-tropic property and improved cognitive function and reversed learning deficits in rat models with hippocampal injury by increasing the acetylcholine levels.  It was demonstrated that genetically-modified NSCs expressing neprilysin that significantly reduced Aβ and increased synaptic density in two kinds of transgenic mice. In addition, Aβ plaque loads were reduced not only in the subiculum and hippocampus adjacent to the engrafted NSCs, but also within the medial septum and amygdala.  Hence, adult NSCs offer novel opportunities for cell therapy and help to repair and restore degenerated and injured nerve pathways. They also provide novel opportunities for pharmacological interventions that help to compensate and even reverse deficits in AD.
Embryonic stem cells
Embryonic stem cells are stem cells derived from the undifferentiated inner mass cells of a human embryo that are able to differentiate into derivatives of the three primary germ layers. Killian et al. discovered that human ESC (hESC) transplantation suppressed pathways for proper metabolism of AD-associated molecules, suggesting that ESCs may be useful for AD treatment.  In addition, it was revealed that ESCs restored hippocampal function by markedly decreasing escape latency and exploratory time, indicating that ESCs transplantation may be an effective choice for improving cognitive function caused by Aβ injury.  Although there has been no report pointing out the direct therapeutic benefits of hESCs in AD, multiple researches have supported the potential therapeutic effects of hESCs. More importantly, ethical issues should be considered before hESCs are used in clinical trials of AD because these cells are derived from preimplantation human embryos.
Mesenchymal stem cells
Bone and cartilage formation in the embryo involves the progeny of a small number of cells called MSCs. The most commonly used MSCs in clinical trials are umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) and autologous bone marrow MSCs (BM-MSCs) isolated from individuals. Lee et al. found that soluble CCL5 (a chemokine) derived from BM-MSCs, attenuated microglia immune responses with a reduction in Aβ deposition and memory impairment in APP/PS1 mice.  It was also reported that BM-MSCs could produce an acute reduction in Aβ deposits and facilitate changes in key proteins required for synaptic transmission, when administrated at a young age in mice with AD, at which stage, the young mice displayed neuropathological, but not cognitive features of AD.  Besides the BM-MSCs, umbilical cord blood (UCB)-MSCs are also considered to be protective for AD. Nikolic et al. found that multiple low-dose infusions of UCB-MSCs markedly reduced Aβ plaques in the AD mouse model, which was associated with suppression of the CD40-CD40L interaction.  Moreover, previous studies showed that transplantation of UCB-MSCs, which was induced to differentiate into neuron-like cells, improved cognitive function, increased synapsin I level, and significantly reduced Aβ deposition in APP/PS1 mice by a mechanism associated with activating M2-like microglia and modulating neuroinflammation.  Hence, transplantation of MSCs may be an effective therapy for AD, but till date, most evidences are obtained from animal models and further clinical trials are urgently needed.
Induced pleuripotent stem cells
Induced pleuripotent stem cells (iPSC) are mature and genetically reprogrammed cells so that they return to their embryonic state. Because iPSCs can differentiate into all cell types, including neural cells, providing iPSC-derived cell replacement in AD (by employing AD patient-specific iPSC-derived neurons) is a promising approach to treat this disease.  It was reported that AD-iPSC-derived neurons exhibited lower levels of Aβ, higher tau phosphorylation, and active glycogen synthase kinase 3β (aGSK-3β) and β-secretase inhibitors that caused a significant reduction in pTau and aGSK-3β levels.  It suggests the potential therapeutic effects of iPSCs for AD but further investigations are required to prove this statement.
Since newer stem cells are being continuously developed for AD therapy, the selection of appropriate stem cells is very important. Although ESCs have the potential for differentiation, the risk of tumorigenesis as well as the ethical issues involved have significantly limited their clinical applications. The rarity of their source and the complexity of management of AD utilizing stem cells, are limitations encountered in the clinical applicability of NSCs despite the absence of tumorigenesis and immunogenicity while using them. MSCs can be induced to differentiate into neurons. This can compensate for the limitations prevalent in the use of ESCs and NSCs. MSCs are considered as the stem cells with the greatest potential to be beneficial in AD therapy. The evidence, however, mainly comes from animal studies and clinical trials are lacking to confirm their safety and reliability. iPSCs do not have ethical issues associated with ESCs but cannot avoid new tumor formation.
Treatment strategies for AD have revolved around retarding the progression of the disease rather than restoring the damaged neural cells. However, the recent trend is to decipher the molecular mechanisms responsible for this devastating disease. The treatment is further aimed at improving the neuro-restorative mechanisms such as neuro-protection, neuro-modulation, neuro-plasticity, and neuro-regeneration. In this review, we mainly focused on the current researches that are in practice attempting to combat the progressive downhill course of AD. For every existing treatment, there are many more in the horizon, and hopefully, AD will be conquered in the not-too-distant future.