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Alzheimer’s Disease (AD) is a neurodegenerative disease, one of the most common forms of dementia that causes the degeneration of neuronal cells and is characterised by progressive cognitive, behavioural and physical impairments. AD covers almost 75% of all dementia cases and has a global prevalence of 24 million, that is predicted to rise every 20 years (Mayeux & Stern, 2012). Age, is considered one of the most important factors that can distinguish two groups in AD, the early-onset (EOAD) and late-onset (LOAD)(Cacace, Sleegers & Van Broeckhoven, 2016) EOAD occurs in patients that are below the age of 65 years and cover almost 10% of all AD cases, where as, LOAD occurs in patients that are over 65 years old covering the highest prevalence amongst the cases of AD, especially, in North America and Western Europe (Mayeux & Stern, 2012);(Pierce, Bullain & Kawas, 2017) .
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EOAD and LOAD are considered to have similar pathological features. EOAD is mostly defined by the genetic heritability. Several gene mutations play a role in the pathology, which are the Amyloid precursor protein mutation (APP), Presenilin 1 (PS1), Presenilin 2 (PS2). LOAD, is the most common form of AD and is mostly caused by the aging-related biological process (Cacace, Sleegers & Van Broeckhoven, 2016). Nevertheless, both EOAD and LOAD share a genetic characteristic which is the apolipoprotein E (APOE), ε4 allele. APOE is considered to be another fundamental risk factor in AD, which is mainly involved in the clinical manifestation of the disease (Tellechea et al., 2018) (van der Flier et al., 2011) .
The UN Aging Program and the US Centers for Disease Control and Prevention have predicted that the number of elderly people is expected to reach 1 billion in approximately 15 years. It is also important to mention that countries with poor development are expected to have higher prevalence with older people suffering from Alzheimer’s disease than the elderly people in developing nations. Concomitantly, AD is expected to pose a worldwide public health burden and will have an impact in the elderly care systems in countries across the world(Qiu, Kivipelto & von Strauss, 2009).
1.1.2 AD Pathophysiology
Amyloid beta and Neurofibrillary tangles
AD mainly affects the cerebral cortex and hippocampus in the brain. The extracellular accumulation of amyloid plaques formed by amayloid-β peptide and the intraneuronal accumulation of neurofibrillary tangles (NFTs) formed by tau protein are the fundamental neuropathologies of AD. Amyloid precursor protein (APP) precludes the accumulation of amyloidogenic Aβ peptide resulting in amyloid plaques, which are formed in the extracellular space of the brain. However, APP an integral transmembrane protein cleaved by β-, α- or γ- secretases, which result in two proteolytic pathways, the amyloidogenic and non-amyloidogenic pathway (Figure 1) (Nicolas & Hassan, 2014). The activity of α- secretase, in the non-amyloidogenic pathway, leads to the release of sAPPα and carboxy-terminus fragment (CTFα), where as, and γ- secretase cleaves CTFα, resulting in APP intracellular cytoplasmic domain
(AICD) and soluble peptide p3. On the other hand, β- secretase (BACE1), in the amyloidogenic pathway leads to the formation of sAPPβ and carboxy-terminus fragment β CTFβ, . CTFβ is then cleaved by γ- secretase resulting in the formation of amyloid β oligomer and domain AICD. Therefore, Aβ fragments aggregate into plaques and induce toxicity (Zolezzi et al., 2014). The microtubule soluble protein tau, facilitates the stabilization and microtubule assembly of neurons intracellularly. When tau protein is hyperphosorylated it results in the accumulation of neurofibrillary tangles and plaque neurites (Grundke-Iqbal et al., 1986) Despite the two fundamental pathophysiological mechanisms, AD combines more mechanisms, such as, neuronal cell death, mitochondrial dysfunction and changes in synaptic plasticity(Crews & Masliah, 2010). Impaired mitochondrial biogenesis causes the degeneration of neuronal cells which results in, the synaptic damage, oxidative stress by the interaction with signal pathways, cognitive decline and plays a role in the detoxification of Reactive oxygen species (ROS) (Sweeney & Song, 2016).
The protective role of cells and the fudnementals of neuroinflammation
Despite the Amyloid beta peptides and neurofibrillary tangles aggregation that are the key fundamental pathologies in Alzheimer’s disease, neuroinflammation is considered to play an important role in the progression of the AD. Astrocytes, microglia and neurons are the neuroinflammatory mediators that exert the neuroprotective action for AD. Aβ plaques and neurofibrillary tangles are considered to be the trigger for the activation of microglia and astrocyte.
The first line of defence in the brain’s innate immune system are considered to be the microglia cells. Concomitantly, microglia cells not only can act as neuroprotective factors but also can provoke neurodegeneration. Microglia consist the 10% of the cells in the nervous system and attack under pathological stimuli, migrate and target injured areas in the brain in order to mediate cellular clearance (Heneka & O’Banion, 2007). Moreover, they release proinflammatory mediators and also undergo morphological changes, resulting in a vicious cycle that is owed to their immunostimulation against neurodegenerative mechanisms. APP processing, is considered to be a major immunostimulator for the activation of microglia, resulting in the activation of nuclear factor kappa B-dependent pathway (NFκB)a s well as in other proteins involved in inflammatory mechanisms (Heneka & O’Banion, 2007)(Dickson et al., 1993) .
Astrocytes, by maintaining the nutrient that neurons require and their association with the neuronal synapses, play an important role in the CNS and the pathology of AD. Astrocytes maintain the Aβ clearance, with phagocytosis, from the chemotactic molecules that are secreted from Aβ peptides. Concomitantly they are also associated with inflammation, by maintaining neurotoxicity of NO when expressing inducible nitric ocide synthase iNOS(Heneka & O’Banion, 2007).
Neurons are considered to be a source of secretion of several cytokines such as IL-β, IL-6 and TNF-α. Neurons confer their own anti-inflamamtory activity for the destruction of AD. iNOS dependent and independent formation it has been reported that is expressed in degerative neurons in brains with AD. As a result this causes the neuronal dysfunction in in vivo and in vitro cases in AD(Heneka & O’Banion, 2007).
APP processing is influenced by the neurodegenerative pathways from several proinflammatory cytokines that are excreted from microglia, astrocytes and neurons
AD therapeutic strategies
Amyloid β processing is one of the key mechanisms in AD for research in drug development and treatment in order to reduce its processing by inhibiting β or γ secretase or enhance α-secretase activity. Specifically, the deletion of BACE1 can prevent the deposition of amyloid plaques in adult mice with AD and also can result in the prevention of neurodegeneration (Hu et al., 2018). Additionally, presenilin 1 has been a target for the inhibition of γ-secretase activity in AD(De Strooper et al., 1998). Moreover, APP is considered a fundamental factor in neurodegeneration from the neuroprotective activity of sAPPa (Zhang et al., 2011)
1.2 Targeting fundamental pathological mechanisms in AD with PGC-1a and Necdin
1.2.1 PGC-1α in AD
Peroxisome proliferator activator receptor gamma (PPAR-γ), belongs to the family of nuclear receptors (NRs) and is involved in the regulation of energy dependent processes, such as glucose metabolism, cellular inflammation and differentiation(Desvergne & Wahli, 1999). PPAR-γ is considered to be a neuroprotective agonist in many neurodegenerative diseases. Most importantly, in vivo studies have shown that the delivery of PPARγ ligands result in the reduction of the pathology and mediate neuropathological mechanisms in AD(Heneka & Landreth, 2007) (Kaundal & Sharma, 2010) (Heneka et al., 2005). PGC-1α is a transcriptional coactivator for the PPAR-γ, that, regulates the genes involved in energy metabolism, has a major role in the suppression in reactive oxygen species (ROS) and is considered the master regulator of mitochondrial biogenesis(Katsouri, Blondrath & Sastre, 2012). PGC-1a precludes domains with diverse functions. The N-terminus domain encloses transcriptional factor- binding sites, such as the nuclear hormone receptor-interacting motif (LXXLL). The C-terminal domain contains RNA-binding motif (RMM) and the serine-arginine-rich (RS) domain(Puigserver & Spiegelman, 2003). Studies have shown that, PGC-1α expression has a protective role in AD, by decreasing the Aβ aggregation with its mitochondrial activity and also reduces neurodegeneration, is specific areas in the brain (Katsouri et al., 2016) .
1.2.2 Necdin in AD
Necdin is a melanoma antigen family protein expressed abundantly in postmitotic neurons derived from proliferative stem cells and in neuronal precursor cells throughout the Central nervous system (CNS) and Peripheral nervous system (PNS), studies have shown that is located in fibroblasts and microglia(Takagi, 1996). Moreover, necdin regulates the neuronal cell proliferation and apoptosis through interacting with considerable nuclear proteins such as p53 which is a tumor suppressor gene product, E2Fs, that is a transcription factor promoting the progression of cell-cycle and retinoblastoma protein (Rb), which is a growth suppressor factor, replaced by necdin, indicating that necdin mimics the role of Rb in neuron-specific cells(Taniura et al., 1998). When necdin is deficient in neuronal cells leads to the reduction of mitochondrial dysfunction and causes the degeneration of neuron. However, the coexistence of necdin and PGC-1α are increasing the activity of mitochondrial biogenesis. When necdin is bound to the N-terminal region of PGC-1α, can act as a strong stabilizer of PGC-1α and concomitantly, inhibits ubiquitin-dependent degradation of PGC-1α. Moreover, necdin exhibits a neuroprotective role, when is overexpressed in dopaminergic neurons in vivo (Hasegawa et al., 2016)
1.3 Gene Therapy, a therapeutic approach for AD
1.3.1 Gene Therapy
Gene therapy can be defined as a therapeutic delivery of a transgene of interest by a viral vector that infects host cells, where the gene of interest can be expressed. However, gene therapy is not only a system where the replacement of a gene of interest is accomplished, but also it has been used for nucleic acid transfer for the prevention and treatment of incurable diseases. This kind of therapy is considered as an improvement of pre-existing therapies (Lundstrom, 2018). Gene therapy systems such as viral and non-viral synthetic vectors have nowadays been established. A variety of viral vectors have been developed in order to transfer the desired therapeutic gene into the tissue of interest. Many challenges have arisen for the application of gene therapy in the Central Nervous System (CNS) that include neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s (Sakuma, Barry & Ikeda, 2012a).
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Adeno-associated viruses (AAV) and retroviruses are the most common vectors used ‘in gene therapy (Yazdani et al., 2018). Recently, Retroviruses and especially lentivectors have now been the leading therapies in the pathway to treat or prevent human genetic diseases (Escors & Breckpot, 2010).
1.3.2 Lentiviruses in Gene Therapy
Lentiviruses belong to the family of retroviridae. They have a positive single-stranded RNA genome and consist of gag, pol, and env, representing the structural genes and the accessory proteins Nef, Vif, Vpu and Vpr (Escors & Breckpot, 2010). A retroviral vector system can be derived from primates, such as Human and simian immunodeficiency virus (HIV, SIV) and non-primates, such as bovine and feline immunodeficiency virus (BIV,FIV)(Nanou & Azzouz, 2009). Lentiviruses have many characteristic features that distinguished them from retroviruses. They have the ability to stably integrate into the host genome, ensuring long term expression for stable cell line generation, which makes it beneficial to target the CNS in order to treat neurodegenerative diseases. Additionally, lentiviruses have broad tissue tropism making them effective vehicles, they do not generate immunogenic proteins and they can deliver transgene fragments as large as 9kb (Wong et al., 2006). Also the fact that Lentiviruses, can be divided into non-dividing and dividing cells, made it possible to utilize recombinant lentiviruses in gene therapy (Wong et al., 2006).
For the recombination of a lentivirus vector the essential genes gag, pol, rev, envelope are used for the transient transfection along with the interested transgene, due to their robust infection ability. HIV vectors have been modified from their pathogenic viral form to a transgene-carrying viral form, in order to be safe to use (Sakuma, Barry & Ikeda, 2012b) . In an effort to reduce biosafety risk generations of recombinant lentiviral packaging systems have been developed, such as second- and third- generation. Lentiviral constructs are based mostly on third- generation systems. The third generation system requires four plasmids, a packaging plasmid, that contains gag and pol, a regulatory plasmid that contains the regulatory gene rev, an envelope plasmid carrying the envelope gene and a plasmid which is carrying the transgene plasmid (Figure 2). The vector plasmid is modified in a way that will lack the U3 region from the 5’LTR, the 3’LTR will be partially deleted in order to reduce the production of competent viruses. CMV is a strong promoter that is inserted in front of each plasmid for the expression of the vector.
Figure 2: 3rd Generation lentiviral system. Packaging plasmid encodes gag-pol genes, envelope encoding plasmid represents viral glycoprotein, regulatory plasmid encodes rev gene and vector plasmid encoding the transgene. All plasmids have a strong promoter CMV.
188.8.131.52 Pseudotyping lentiviral vectors
Viral tropism is the specificity that the virus exhibits when interacts with its viral envelope glycoprotein in specific receptors that are located and expressed on the surface of the targeted cell, in a differentiated tissue. While lentiviral vectors can be enclosed in heterologous envelopes they have been developed in a way that can be produced with different envelopes depending on their desired tropism. This procedure is called pseudotyping. Lentiviral vectors have been pseudotyped with a wide range of viral glycoproteins which also have an impact in the stability and efficiency of packaging as well as on the titres (Wong et al., 2004). Rabies glycoprotein is considered a major presynaptic connection for the entire nervous system (Kim et al., 2016). It has been shown that lentiviral vectors pseudotyped with RV-G of rabies virus exhibit neuronal tropism and axonal retrograde transport (Mazarakis et al., 2001).
1.3.3 Application of lentiviral vectors in in vitro and in vivo systems in AD
The various models that have been exploited in order to study Alzheimer’s disease are classifiedinto in vitro and in vivo systems. AD being an age related disease with molecular-related neurodegenerative mechanisms requires a variety of techniques to detect or even to prevent it. for the study of neurodegenerative diseases, targeting neuronal cell lines for experimental gene transfer, are attracting the lentiviral vectors. The use of lentiviruses has been investigated in in vitro systemsin order for several neuronal cell lines, such as astrocytes, adult neuronal stem cells and glioma cells to be transduced. The transduction in the CNS appeared to be efficient using internal promoters such as CMV when pseudotyped lentiviruses are used to transduce specific areas in the CNS (Jakobsson & Lundberg, 2006). Another system that has been exploited in vitro, is the brain organoids (ref). Brain organoids are miniature brain-like tissues that are derived from stem cells. This system makes the investigation and modelling of neurodegenerative diseases such as AD, more realistic than cells in dishes.
The first in vivo transduction of lentiviral vectors in the CNS was reported by Blomer in 1998, using an LV expressing the transgene Bcl-xL (Blömer et al., 1998). In later years and until recently LV-mediated gene delivery in the brains of animal models has been reported.
Separate into in vitro and in vivo. Discuss APP23 mice. Discuss advantages and disadvantages of in house systems.
Aim and objectives
The aim of this project was to generate a third generation pseudotyped-glycoprotein lentiviral vector expressing proliferator-activated receptor γ coactivator 1α (PGC-1α) and or necdin, in high titres in order to be used for therapeutic approach in Alzheimer’s Disease in in vitro and in in vivo systems. In order to archive this aim, transfection with four separate plasmids was performed, using the third generation system, gag-pol, rev, the envelope of rabies glycoprotein CVS-B2C strain and the additional carrying the reporter gene of interest which are the green fluorescent protein (eGFP), a bi-cistronic PGC-1α with necdin (LV-NP2A- PGC-1α) and a mono-cistronic LV-PGC-1α. Lentiviral vectors, expressing PGC-1α and eGFP, were used for in vivo work in APP23 mice in order to target and observe the expression of those vectors in the frontal cortex and hippocampus as well as investigate their therapeutic approaches.
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