The Path To Progress Preclinical Studies Of Age-Related Neurodegenerative Diseases: A Perspective On Rodent And HiPSC-Derived Models Part 2
Jul 09, 2024
In addition to transgenic models developed by individual academic labs, the National Institutes of Health (NIH) is currently leading the Accelerating Medicines Partnership-AD (AMP-AD) Target Discovery and Preclinical Validation Project, which is a consortium of multi-institutional and multi-disciplinary grants that collectively intend to expedite the drug-discovery pipelines for AD (https:// www.nia.nih.gov/research/dn/amp-ad-target-discovery-and-preclinicalvalidation-project).
With the rapid development of biotechnology, transgenic technology has been gradually applied to the biological field. Transgenic technology is a genetic engineering technology that introduces foreign genes into organisms to make them show specific characteristics. However, there has been controversy over the relationship between transgenic models and memory.
Some researchers believe that transgenic models can have a positive effect on memory. For example, some transgenic animal experiments have shown that by changing certain genes, animals' learning ability and memory can be improved. These experimental results show that transgenic technology can potentially improve human cognitive ability.
Although these research results are very interesting, we also need to be cautious because the results after these experimental processes are not the result of human promotion or hasty application. We need to conduct more research on this technology to understand its potential risks to the environment and humans, and to measure its possible social impact. At the same time, we must also admit that the improvement of memory does not only rely on genes but also on physical health and healthy living habits can achieve a good memory state through normal channels.
In short, we should look forward to the positive aspects of transgenic technology and properly regulate and evaluate it. Through the rational use of transgenic technology, we may change our genome, which will help us solve some medical and environmental problems while promoting human progress in many fields, including memory improvement. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.
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Particularly relevant to this review is the arm of AMP-AD, named the Model Organism Development and Evaluation for LOAD (MODEL-AD) initiative. MODEL-AD aims to generate novel transgenic mouse lines that accurately model AD phenotypes and serve as an improved platform for the drug-development pipeline and more translatable and biologically relevant models for preclinical DMT validation.
The MODEL-AD initiative is comprised of the Center at Indiana University School of Medicine, The Jackson Laboratory, Sage Bionetworks, the University of Pittsburgh School of Medicine, and a center at the University of California Irvine.
Together, this consortium intends to establish the next generation of in vivo AD models based on human data in a rigorous and highly controlled fashion. In doing so, it aims to align the pathophysiological features of AD models with corresponding stages of clinical disease using translatable biomarkers as a means of increasing the translatability of preclinical findings.57
To date, 54 AD mouse models have been generated, including fAD, APOE, APP, MAPT/tau mice, as well as LOAD models based on Ab accumulation and other previously identified LOAD-related variants (https://www.model-ad.org/strain-table/), which are available through the JAX/IU/Pitt AD Precision Models Center.
Chemical-Toxin Models of AD
In addition to the more recent transgenic models, AD rodent models were historically based upon exposure to toxic compounds that cause selective neurodegeneration leading to cognitive and behavioral impairments similar to those observed in AD patients.
Whereas a variety of toxin-exposure models have been employed in the study of both AD and PD, AD models have largely been replaced by the more recent transgenic models discussed above.
Chemical-based models of AD induce AD-like pathology by using either compounds that selectively and reversibly disrupt cholinergic neuron activity or toxins that permanently disrupt cellular homeostasis processes leading to cell death, such as okadaic acid and various classes of heavy metals 58 (Table 2). Although these models produce some neurological and behavioral deficits that resemble AD symptoms, including impaired learning and memory, they largely lack AD pathological lesions (i.e., the Ab plaques and NFTs).
Animal models that focus explicitly on the disruption of cholinergic neurons are based on the now somewhat dated cholinergic hypothesis of AD.59 In such models, muscarinic receptor antagonists, for example, the compound scopolamine, are delivered to targeted brain regions, such as the HP, to block endogenous acetylcholine activity and induce cognitive impairments in learning and memory.60
Other chemical intervention models of AD involve the local or peripheral administration of neurotoxins leading to nonspecific neuronal cell death61–64 (Table 2).
Transgenic Models of PD
As is the case for AD, transgenic mouse models of PD enable the study of clinical disease features induced through manipulation of genetic loci associated with Mendelian forms of the disease, and hundreds of unique lines are now commercially available for purchase.
Like AD, the majority of clinical PD cases are classified as sporadic, with heritable forms of the disease accounting for less than 1% of all cases.89 Genetic alterations in the SNCA, LRRK2, and UCH-L1 genes are linked to autosomal-dominant forms of PD, whereas alternations in the PRKN, PINK1, and DJ-1 genes are associated with the autosomal-recessive forms90 (Table 1).
The two genes most commonly targeted in transgenic PD models are SNCA and LRRK2. 91,92 Mutations in these genes cause familial PD and were also associated with the sporadic form of PD via GWAS.93
Additionally, the role of SNCA overexpression in familial (tri/duplication cases) and sporadic PD has been well established.94
To date, most transgenic models manipulating SNCA not only introduce the familial missense mutations but also employ an overexpression strategy by inserting multiple copies of the human or mouse SNCA gene to mimic increased a-synuclein (a-syn) production, either alone or in combination, with a familial missense mutation.95–97
A missense mutation in LRRK2 is the most commonly reported mutation associated with familial PD,98 and several transgenic mouse lines have been generated based on a variety of known LRRK2 mutations.
The GS2019S mutation, for example, results in a gain-of-function mutation with enhanced kinase activity.99 Other models, such as the R1441C/G, T1348N, and A2016T mutations, report changes in a-syn expression or aggregation and in some cases, nominal motor deficits; however, the precise deficits and clinical features vary substantially among models.89,92,100
In addition to the SNCA and LRRK2 genes, multiple other transgenic lines have been generated in which other PD-associated genes are targeted (Table 1). Whereas many of these models successfully alter the expression and aggregation of a-syn, most exhibit little to no neurodegeneration, and consequently, motor functions remain relatively unimpaired92 (Table 1).
Careful analysis of a-syn aggregates produced in many of these transgenic lines has further revealed that the inclusions formed do not resemble the naturally occurring Lewy bodies (LBs) observed in postmortem PD brains but instead, display a unique structural composition.101
The failure of transgenic models to reproduce both the molecular and neurodegenerative aspects of PD is likely due to a combination of several factors. Chief among these concerns is that the nature and extent of involvement of familial-linked genes, and the more common sporadic form of the disease remains unclear, calling into question the relevance of models based on manipulation of these genes to the vast majority of PD cases.
Another limitation is that the transgenic models result in overexpression of the transgene that does not mimic physiological levels and thus, may introduce artifacts. Moreover, the sporadic form of the disease likely involves alterations in multiple genes, with susceptibility being further impacted by environmental components, suggesting that monogenic transgenic models may have limited utility.
Overall, many of the same issues plaguing the AD transgenic models hold for models of PD: there is a current lack of understanding of the precise role of the familial-linked genes in PD etiology and the extent to which they contribute to the sporadic forms of the disease.
These issues have made it exceedingly difficult to produce a transgenic PD model, which faithfully reproduces a-syn accumulation and LB formation, in turn, leading to a pattern of neurodegeneration and behavioral impairments that match those observed in clinical populations.
The current transgenic models recapitulate only some of these features and as a result, have proven to hold little predictive validity for assessing the effectiveness of potential DMTs. In contrast, chemical-toxin models of PD are still widely utilized due to their ability to faithfully reproduce the selective DA neurodegeneration characteristic of PD.
Chemical-Toxin Models of PD
Over the last half-century, a wide variety of toxic compounds have been employed to model the pattern of neurodegeneration observed in PD. Out of these, four chemical injury models have emerged as the most widely utilized for preclinical research, and their effects have been extensively characterized.
These include 6-hydroxydopamine (6-OHDA), a catecholamine analog; 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), a synthetic opioid analog; rotenone, a pesticide; and paraquat, a herbicide102 (Table 2). Another nontransgenic PD model is based on the injection of preformed fibrils (PFFs; recombinant monomeric a-syn proteins converted into the fibrillar aggregate form) into the brains of animals, which triggers hyperphosphorylation of endogenous a-syn proteins and elicits LB-like pathology in the SN pars compacta (SNpc)87,88 (Table 2).
These toxin-based models can induce, with varying degrees, a-syn aggregation and selective neurodegeneration of DA neurons in the nigrostriatal pathway, a notable shortcoming of their transgenic model counterparts.
The pathways involved in triggering cell death and the extent of DA neuron loss, however, vary substantially among models, which have been reviewed in detail elsewhere.103 Ultimately, the result of DA cell loss in PD is that motor nuclei of the striatum are no longer able to maintain normal physiological function, causing a wide range of impaired motor abilities.
However, loss of nigrostriatal DA neurons is, in and of itself, not a model of PD per se but instead mimics a more general Parkinsonism characterized by a broader spectrum of motor symptoms.104
To this point, whereas some toxin-based models, including PFF, chronic MPTP, and rotenone administration, can induce a-syn aggregates and LB-like formation, they are distinct from the characteristic LB inclusions in PD, and the overall distribution of these aggregates does not reflect the pattern observed in PD brains.102
These models are also limited in their ability to model the progressive nature of PD and the spread of the pathology to other brain regions, as PD patients also suffer neurodegeneration in subcortical and eventually, cortical regions in the later stages of the disease.105
In summary, toxin exposure-based models demonstrate some similarities to the pathologies observed in PD; however, the mechanism of cell death induced by the toxins is likely distinct from those underlying neuronal loss in PD.106,107 Of note, typical dosing schedules for chemical-toxin models of PD occur over days to weeks, whereas the course of PD develops over decades and progresses over years.
These limitations suggest that toxin exposure-based models aimed at inducing selective DA neuron death in the nigrostriatal pathway present an incomplete picture of PD and provide an explanation as to why efforts to identify DMTs in such models have failed to be effective in clinical trials.12
Viral Vector Models for PD and AD Advantages of Viral Vector Systems
Viral vector delivery systems offer several advantages that are difficult to achieve with other approaches.108 First, viral vector systems provide robust control over the temporal expression of the gene of interest.
Second, aging is the primary risk factor of AD-PD spectrum disorders, and the possibility of delivering the vector at any point in the animal's lifespan represents a major advantage for study design. Third, viral vectors support local transgene delivery and expression, thereby allowing for accurate and specific targeting of brain regions of interest.
Fourth, precise dosage manipulations can be readily achieved with the use of viral vectors. Fifth, the model of interest can be created in multiple animal species, ranging from small rodents to large nonhuman primates.
Sixth, different variations of genes can easily be made to obtain a phenotype of interest. Seventh, the versatility and flexibility of different vector platforms used for viral-mediated gene transfer into the central nervous system are vast.
The viral
systems, including recombinant adeno-associated vectors (UAVs)
and lentiviral vectors (LVs), efficiently and robustly support short or long-term gene expression, respectively, both locally and globally.
Last, the viral vector-based systems are significantly more economical, both effort and cost-wise, relative to other disease models.
AD and PD Viral Vector Models: AAVs and LVs
The viral vector approach to model AD and PD phenotypes in vivo has been utilized by several different groups using either rAAVs or LVs. Since their first use at the beginning of the millennium, a variety of viral-based models have been developed and utilized for AD.
The initial AAV-tauopathy models exploited AAV2/2 to express human tau P301L protein in the brains of mice and rats.109,110 In these studies, the AAV-tau-P301L vector was injected into the basal forebrain of adult rats leading to the increased levels of the tau for at least 8 months post-transduction. Significantly, hyperphosphorylated tau and aggregates resembling NFTs were found at 3to 4 weeks posttransduction.110,111
These studies provided the first proof of concept that injection of AAV-P301L tau could result in persistent expression of the protein and its aggregation in mice and rats.
Consistently, injection of AAV-wild-type (WT) or triple-mutant APP resulted in the formation of Ab plaques but no overt neurodegeneration. These findings are in contrast with the above observations that AAV-WT tau or AAV-P301L tau was capable of causing significant neurodegeneration of HP pyramidal neurons.112
In more recent experiments, AAV2/5 was used to deliver WT tau and the GFP control vector into the dorsal HP of aged Fischer 344 rats.113 Both viruses were robustly expressed in HP neurons and led to aberrant axonal structure, axonal degeneration, and fragmentation of tau in HP axons compared to control GFP-expressing axons that appeared normal.
In another model, the tau P301L expressed from the human synapsin I promoter was delivered by the AAV2/9 vector into the lateral entorhinal cortex, which allowed the authors to detect multiple forms of phosphorylated and aggregated tau variants before the loss of perforant path synapses and neurodegeneration.114
These data support the usefulness of AAV-based approaches in AD, particularly in modeling early pathological changes, such as axonal degeneration and degradation. Several studies used viral-based models aimed at inducing other pathological aspects of LOAD.115,116 For instance, AAV-BRI-Ab42 and AAV-BRI-Ab40 vectors were used for controlled expression and secretion of Ab peptides in rodent HP.116
Notably, AAV-BRIAb42-injected animals demonstrated that these vectors were able to induce plaque formation, but this was not seen with rAAV2/1-BRIAb40 expression.
In another study, AAV2/2 was used to deliver Ab40 and Ab42, a C-terminal fragment of APP containing the Ab peptides (C100), and a V717F mutant of C100 to the HP and cerebellum of mice.
Interestingly, Ab42 and V717F mutants demonstrated greater induction of microgliosis and disruption of the blood-brain barrier (BBB) compared to the Ab40 forms but did not activate plaque formation, astrocyte induction, or neurodegeneration. The use of AAV and LV technology to express various forms of Ab and its precursors will continue to be an essential method of studying their role in LOAD.117
More recently, LV has been used to express TDP-43, Ab42, or both in the motor cortex of rats, which uncovered that the loss of TDP-43 implicates microglia as a cause of the synaptic degeneration observed in LOAD.118
Furthermore, lenti-TDP-43 triggered alterations in the APP processing associated with Ab42 production and induced caspase activity and neuroinflammation, which was similarly observed with the lenti-Ab42.
The combined expression of both TDP-43 and Ab42 protein resulted in an outcome similar to TDP-43 alone but with the added feature of neuronal loss, suggesting a possible synergistic effect between the two factors. Another study using LVs to express TDP-43 in the motor cortex found altered amino acid metabolism, oxidative stress, and neuronal death.119
These results together demonstrate the utility of LV-based AD model systems to elucidate key pathogenic factors related to protein dysfunction implicated in AD, whereas Ab and tau have been extensively tested in LOAD, consistent with the long-held notion that Ab is a key suspect in AD pathogenesis. Nevertheless, the continuing failures in drug trials aimed at lowering amyloid levels demand refocusing the effort on other potential culprits.
Multiple recent studies demonstrated that APOEε4 may contribute to toxicity associated with the AD phenotype, and to date, APOE remains the most significant and replicable genetic risk factor for AD.120
Given the role of APOEε4 in AD pathogenesis and the potential protective effect of the ε2 isoform, Hu and colleagues121 generated mouse models using AAV8-APOE isoforms driven by GFAP promoter specifically expressed in astrocytes in all brain regions, which resulted in an overall increase in APOE levels throughout the mouse brain.
The viral-mediated overexpression of APOEε4 in the APOEε4- TR mice increased poorly lipidated APOE lipoprotein particles and decreased APOE-associated cholesterol in APOEε4-TR mice. Conversely, APOEε2 overexpression in APOEε4-TR mice enhanced APOE lipidation and associated cholesterol.
Furthermore, overexpression of APOEε4 elevated the levels of endogenous Ab, whereas APOEε2 overexpression trended to lower endogenous Ab. Based on these data, authors suggest that increasing APOEε2 in APOEε4 carriers is a beneficial strategy to treat AD, whereas increasing APOEε4 in APOEε4 carriers is harmful. In another study, direct intracerebral injection was performed using AAV to express APOEε2.
The study reported that APOEε2 overexpression markedly reduced brain-soluble (including oligomeric) and -insoluble Ab levels, as well as amyloid burden, in two mouse models of brain amyloidosis in which pathology is dependent on either the expression of a mouse or human APOEε4 isoforms.122
Zhao et al.122 further showed that a widespread reduction of brain Ab can be achieved through a single injection of the virus via intra-thalamic delivery of AAV expressing APOEε2.
Collectively, these data suggest that AAV gene delivery of APOEε2, using an AAV, can rescue the detrimental effects of APOEε4 on brain amyloid pathology; thus, this approach may represent a viable therapeutic avenue for treating or preventing LOAD.
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