The Path To Progress Preclinical Studies Of Age-Related Neurodegenerative Diseases: A Perspective On Rodent And HiPSC-Derived Models Part 3

Similar viral vector approaches have been assessed in various models of PD, with the earliest renditions employing AAV2/2 or LVs.123–126 These vectors injected into adult rat brains were used to deliver WT, A30P, or A53T mutants of human a-syn for disease induction. 

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The authors demonstrated efficient expression of a-syn in nigral DA neurons, accompanied by molecular and cellular pathologies and nigral DA degeneration that evolved gradually over time. 

Nonetheless, the first generation of rAAV2/2 vectors used in these studies displayed progressive neuronal loss, although the neurodegeneration of the tyrosine-hydroxylase-positive neurons, as well as the time course, was quite variable (25%–80% and 6 weeks to 1 year, respectively).123,124 

In contrast, LVs encoding WT-, A30P-, or A53T-mutated a-syn were capable of inducing neuronal cell loss in rats in a more delayed and more consistent manner (25%–40% and 5 months, respectively).126 Both studies demonstrated a progressive loss in neurite length and swollen perikarya in remaining DA neurons. 

Furthermore, massive cytoplasmic accumulations of a-syn were found in both cell bodies and neurites. These results have been replicated recently in multiple studies that utilized both viral platforms.127–131 

LVs have also been utilized in a variety of mice PD models. For example, Lauwers and colleagues125 showed that injection of LV carrying WT or A30P mutant of a-syn in the striatum, amygdala, or SN of mice was capable of inducing neurodegenerative changes associated with PD in a time-dependent manner and included a-independent neuritic enlargement and cytoplasmic inclusions. 

Further, this study demonstrated that nigral overexpression of A30P-a-syn resulted in about 25% cell loss at 10–12 months.125 Delivery of A30P, E57K, and E35K to the SN of the rat using LV demonstrated a similar extent in DA neuron loss (about 50%) compared with the WT (30%), whereas the faster fibril-forming mutant A53T used in the same study did not show a significant decrease in DA cell number. 

Interestingly, the overall neuropathological features observed in mice (i.e., onset and severity) appeared less severe compared to the rats, although these differences may be an artifact of viral purity or production titers of the injected virus and/or other related technical discrepancies.

Important Considerations for AD and PD Viral Vector Models

The development of novel serotypes, including AAV2/1, AAV2/5, AAV2/6, AAV2/8, AAVrh10, DJ, and DJ8, has broadened cellular targets and improved AAV transduction efficiencies. 

The AAVs serotyped with these new capsids have been tested in rats and nonhuman primates to overexpress a-syn.132–137 Overexpression of the S129A form delivered via AAV2/5 and AAV2/6 consistently displays enhanced toxicity in the neurodegeneration process.134,138 

These experiments also demonstrated that careful dosing of the overexpression transgene is crucial, as even control vectors carrying only fluorescent reporters delivered at high titers can demonstrate nonspecific toxic effects.135 

This caveat should be taken into careful consideration when designing rescue experiments based on AAV. For example, it has been shown that the therapeutic AAV harboring small interfering RNA (siRNA) targeting SNCA resulted in a high level of toxicity and caused a significant loss of nigrostriatal DA neurons. This study further highlighted the limitations of AAV applications in animal studies. 

However, it cannot be excluded that the neurotoxicity was caused by the robust reduction of SNCA levels in rat models, as a-syn plays a crucial biochemical role in DA neurons, and its robust reduction could decrease cell viability.139 Taken together, this underscores the importance of developing a tool for fine-tuning the expression of disease-associated genes for designing gene therapy approaches. 

In this regard, the more recent CRISPR-dCas9-based system may present a more advantageous system for modulating gene expression in a precise and fine-tuned fashion. 

Along this line, we recently developed the LV-CRISPR-Cas9 system to achieve precise and fine-tuned regulation of SNCA gene expression. The study provided proof of concept that the manipulation of gene expression (e.g., reversing overexpression) through epigenome editing is a valuable therapeutic strategy for neurological disorders, such as PD,140 caused by gene dysregulation. 

As such, the viral vector PD models using LV and AAV for the overexpression of a-syn protein display PD pathology associated with DA neurodegeneration, and the use of novel AAV serotypes improves a-syn expression in DA neurons compared to AAV2/2. 

Overall, both AAVs and LVs are capable of inducing transgene expression and demonstrate a high level of tropism for DA neurons, resulting in similar levels of neurodegeneration. Further improvement of these viral systems will be required to circumvent a slow progression of the cellular PD phenotypes and relatively low levels of behavioral impairments.

In Vitro Cell-Culture Models Overview of Current In Vitro AD and PD Models: Limitations and Opportunities

Over the past four decades, a variety of AD and PD in vitro models have been developed to examine the interactions between key aspects of their relative disease pathophysiology and subsequently utilized for preclinical therapeutic screening. 

Current AD and PD in vitro models primarily aim at recapitulating molecular and cellular disease hallmarks, such as mitochondrial dysfunction, oxidative stress, cell survival, and the presence of intracellular and/or extracellular protein aggregates. For example, the most prominent and widely used cellular models of AD are based on induction of Ab40
42 aggregates or tau hyperphosphorylation and aggregation141,142 (Table 1). 

Similarly, in vitro models of PD are based on the aforementioned toxin-induced mitochondrial dysfunction (e.g., MPTP, rotenone, paraquat, 6-OHDA) and disrupted proteostasis (e.g., thapsigargin, ionomycin, tunicamycin), a-syn overexpression, and expression of the mutated PD familial genes (e.g., a-syn, Parkin, PINK1) 92 (Table 2). 

Whereas these cell-culture models have been invaluable in progressing our understanding of pertinent neurobiology, they have not yet resulted in viable DMTs aimed at prevention, delayed onset, or slowed disease progression, calling into question their relevance and translational validity. 

Several clear limitations in these models are important to acknowledge to improve current preclinical practices. 

For example, models based on overexpression and knockout/down of pertinent disease-associated genes are notoriously variable in the quantitative changes to protein levels and the methods used to induce changes to gene expression (i.e., viral-mediated, breeding-based, constitutive, or transient expression) that may lead to contradictory data and inferences.92 

This lack of consistency in eliciting disease-associated phenotypes introduces a caveat to preclinical studies using these models to test potential DMTs. Moreover, variability in cellular phenotypes and methodology observed in preclinical NDD models (e.g., fold change of protein overexpression or knockdown) strongly suggests a greater genetic heterogeneity that is not able to be fully explored in current cell models. 

This is further complicated by the prevalence of cell models based on genetic variants identified in familial cohorts, despite familial variants representing a small minority of overall NDD cases.109,143 

Due to the uncovered genetic causes, it remains difficult to accurately represent sporadic NDD cohorts or disease subtypes with non-Mendelian genetics in preclinical models, creating a significant barrier in elucidating pertinent disease mechanisms, identifying potential therapeutic targets, and drug development. 

Given these challenges, researchers have been compelled to evaluate these models to identify areas of improvement and appraise the opportunities presented with novel cell-culture techniques. 

In recent years, we have built upon these models, incorporating novel gene-editing technology and evaluating important genetic disease associations obtained in GWASs in improved tissue-culture models.144,145 

The increased incorporation of hiPSC technology represents a major progression in the field of NDD in vitro modeling and provides previously unavailable opportunities to utilize disease-relevant cell types derived from patients to explore disease pathogenesis in the context of the human genome. 

The full spectrum of advantages and limitations of hiPSC-derived NDD models, particularly those about AD and PD, are explored in full in the next section.

hiPSC-Derived Models of NDDs: Versatile and Genetically "Faithful"

Modern advances in cell-culture technology represent one of the most exciting breakthroughs in the research of NDDs, providing opportunities for improved disease modeling, drug screening, and personalized medicine. 

Until recently, postmortem brain examination was considered the gold-standard human-based biological material for obtaining insights into pathological processes of human neurodegenerative conditions. 

However, modern stem cell technologies, including the analysis of patient-specific neurons and glial cells, have opened up new avenues for investigating the pathological mechanisms of NDDs and have been increasingly harnessed in the molecular dissection of NDDs with enhanced genetic complexity, such as AD and PD. 

Through a simple, noninvasive biopsy of patient skin samples, in addition to a variety of alternative biological samples, the generation of hiPSCs enables researchers to reveal molecular pathways and disease mechanisms that underly NDDs in a way that was impossible with previous cell-culture methods. 

At present, the most prevalent donor cells are fibroblasts, which are utilized in over 80% of all published reprogramming experiments;146 however, hiPSCs can be obtained from a variety of sources, including embryonic cord stem cells, umbilical cord blood, corneal epithelial cells, and blood cells, such as peripheral blood mononuclear cells147–149 (Figure 1). 

Consequently, hiPSC-derived models represent a versatile alternative to existing cellular disease models and are obtained from patient somatic tissues that are coerced into the pluripotency through use of a variety of reprogramming tools, such as plasmids, vectors (e.g., episomal), and viral transduction (e.g., adenovirus, Sendai virus, lentivirus; these methods are comprehensively reviewed in Brouwer et al.150 and Malik and Rao151). 

These cells are self-renewing and offer an alternative to embryonic stem cells (ESCs), which are accompanied by ethical considerations and social concerns. There are currently several hiPSC repositories that collect and distribute hiPSC lines derived from patients with NDDs and health age-matched controls to centralize samples and make them readily available to researchers (Table S2). 

One of the major applications of hiPSCs is the differentiation into specific cell types, which have consequently been utilized in a variety of biological contexts, including neurodevelopmental152 and neurodegeneration studies,144 ex vivo transplantation,153,154 disease modeling, target validation, and drug discovery.155 

To date, several cellular differentiation protocols have been developed that generate not only generic neurons but also specific neuronal cell-type populations, including excitatory, cholinergic, DAs, and inhibitory GABAergic neurons.156–158 A variety of specific glial subpopulation cells have also been derived from hiPSCs, including astrocytes,159–161 oligodendrocytes,162,163 and microglia.164–166 

Several novel gene-editing techniques, including CRISPR-Cas9, zinc-finger nucleases (ZFN), and transcription activator-like effector nucleases (TALENS), introduced an additional dimension to the hiPSC-derived system-the creation of isogenic models that possess the same genetic background and only differ in the chosen mutation.167 

The diversity of cell differentiation protocols has facilitated mechanistic studies, and the availability of isogenic lines has supported genetic analyses of gene function and its pathogenic role in NDDs. hiPSC-derived models are suitable for these investigations, as they exhibit key disease-related features. 

For example, fAD- and sAD-derived hiPSC neuronal models carrying PS1 and PS2 mutations (A246E and N14II, respectively)168 and APP duplication mutation169 display important AD biochemical features, including Ab secretion, increased Ab42:Ab40 ratio, and elevated hyperphosphorylated tau. 

It is important to note that the use of hiPSC models that generate Ab-related phenotypes represents a more accurate means of exploring the role of Ab expression in AD neurodegeneration when compared to traditional ectopic expression models or models that require supplement and/or induction of Ab aggregates, as these adopt the "natural" occurring pathological processes related to Ab deposits.

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