Using CRISPR-Cas9 to connect aging processes with new treatments for brain diseases

A fundamental biological process, aging, raises the risk of illness and death by causing a deterioration in both physical and functional abilities. In terms of neurodegenerative illnesses, it is the most important risk factor (Azam et al., 2021). According to projections, the number of Americans over 65 will increase from 53 million in 2018 to 88 million by 2050, highlighting the growing prevalence of age-related illnesses. Neurodegenerative diseases stand out among these because of their significant effects on life expectancy and quality of life. Neurodegeneration is influenced by age in a number of ways. It is uncertain if aberrant protein aggregates like Aβ, hyperphosphorylated tau, and α-synuclein are directly linked to cognitive impairment, yet they are frequently found in older people's brain tissue (Azam et al., 2021; Bourdenx et al., 2017). Neuroplasticity is also impacted by aging, increasing the brain's susceptibility to genetic and environmental factors (Azam et al., 2021; Schaefers and Teuchert-Noodt, 2016).

The apolipoprotein E (APOE) ε4 allele, for instance, is a key gene that predisposes people to late-onset AD, and people with it exhibit structural brain changes well before cognitive symptoms appear (Azam et al., 2021; Erikson et al., 2016; Matteini et al., 2016).

Neurodegeneration and aging constitute a continuum, with the onset and course of neurodegenerative illnesses being influenced by the characteristics of aging, such as loss of proteostasis (López-Otín et al., 2013), mitochondrial malfunction, and genomic instability (Azam et al., 2021). According to molecular research, aging of the brain may be an accelerated type of neurodegeneration, especially in extremely elderly people (Azam et al., 2021; Wyss-Coray, 2016). Additionally, developmental environmental exposures like trauma or poisons may have long-term consequences, making people more susceptible to neurodegenerative illnesses in later life (Nabi and Tabassum, 2022). The burden of neurodegenerative illnesses will rise sharply as the population ages, calling for immediate improvements in treatment approaches. To create interventions that can extend the lifespan of old individuals, it is essential to comprehend the biological processes of aging and how they interact with neurodegeneration. Addressing the underlying mechanisms of aging and neurodegeneration holds potential for more effective and long-lasting remedies, even though present treatments concentrate on managing symptoms.

Medication is frequently used to treat neurodegenerative diseases with the goal of reducing symptoms and delaying the course of the illness. To manage these issues, the following are some standard therapy options (Cascione et al., 2020; Lipton, 2004; Hallett and Standaert, 2004; Armstrong and Okun, 2020; Caraci et al., 2020; Song C. et al., 2022; Sudhakar and Richardson, 2019):

Inhibitors of acetylcholinesterase: For AD medications including galantamine, rivastigmine, and donepezil are commonly recommended to treat dementia-related symptoms. By blocking acetylcholinesterase, which facilitates better nerve-cell communication, these drugs may stabilize cognitive function and prevent future cognitive loss in AD patients.

Antagonists of NMDA receptors: Excessive NMDA receptor activation can lead to neurodegeneration in conditions like stroke, dementia, and neuropathic pain. By preventing glutamate, a neurotransmitter that can harm brain cells when it is hyperactive, memantine, a common NMDA receptor antagonist, helps control the medical indications of PD and AD (Lipton, 2004; Hallett and Standaert, 2004).

Agonists of dopamine: Dopamine agonists, which imitate the brain's dopamine function, are crucial in the cure of PD because they help control symptoms such bradykinesia, rigidity, and tremors. Movement, memory, and other processes depend on dopamine, and agonists help make up for the loss of dopamine-producing neurons in Parkinson's disease. Antipsychotic Drugs: These medications, which are frequently recommended for Parkinson's and Alzheimer's illnesses, can reduce neuropsychiatric symptoms like agitation, delusions, and hallucinations (Caraci et al., 2020). However, its use needs to be closely watched because of the possible adverse effects and elevated mortality risk.

Immunomodulatory substances: The goal of immunomodulatory therapies is to lessen inflammation in the brain, which is assumed to contribute to neurodegenerative illnesses (Song C. et al., 2022). Specific treatments aim to improve the brain's ability to eliminate amyloid-β (Aβ), such as vaccinations and antibodies that target Aβ. In order to fight neurodegeneration, other researchers concentrate on the tau protein as a possible therapeutic target. Presently, immunotherapies are mainly in the developmental stage, although a few, such as lecanemab and Donanemab, have recently received approval for Alzheimer's disease, but hold a promising future (Espay et al., 2024).

Gene therapy: By injecting new or altered genes into the brain, gene therapy is a cutting-edge method of treating neurodegenerative illnesses. Although for now gene therapies are considered an experimental approach but, in the future, this approach offers hope for long-term illness management and possible cures by correcting underlying genetic abnormalities, providing neuroprotection, and stimulating neurorestoration (Sudhakar and Richardson, 2019).

Pharmacological therapies for neurodegenerative diseases are challenging due to the limited efficacy of current drugs, progressive disease course and, more importantly, heterogeneity across motor, cognitive, and psychiatric domains. Despite ongoing research, there remains no definitive cure for these conditions, and the existing therapeutic options primarily focus on symptomatic relief. However, promising prospects for the future are emerging, fueled by advances in understanding the pathophysiological mechanisms underlying such medical conditions for the discovery of undiscovered pharmacological agents. Recent curative approaches have addressed the hallmark pathologies of neurological implications by concentrating on anti-amyloid and anti-tau therapies. Although these therapies have some potential, they cannot stop the development of neuronal degeneration or brain atrophy. Additionally, new pathways for intervention have been made possible by the development of advanced gene-modifying approaches like CRISPR/Cas9. Because they provide new opportunities for the selective genetic material modifications to rectify pathogenic processes at the molecular level, these cutting-edge techniques have shown promise in the treatment of a variety of aging disorders, containing AD, PD, and HD (Rahimi et al., 2024; Lamptey et al., 2022).

AD one of the leading causes of progressive neurological illness that is currently a leading cause of death and disability, behind cancer and heart disease. Memory impairments and cognitive decline are common features of the disease, which eventually affect language, behavior, movement, reasoning, judgment, and memory before resulting in dementia and mortality (Querfurth and LaFerla, 2010). The cleavage of amyloid precursor protein (APP) generates Aβ peptides, which accumulate to form amyloid plaques–abnormal deposits implicated in the pathogenesis of AD (Querfurth and LaFerla, 2010; Selkoe and Hardy, 2016). Neurofibrillary tangles (NFTs) are another constituent of these plaques, which, together with Aβ are responsible for the AD symptoms. These are the two neuropathological features that most commonly define AD. The classical hallmark of AD is thought to be the link between these two pathologies. According to researchers, the conventional Aβ hypothesis offered a fundamental basis for the creation of possible AD modulating treatments that target and stop the production of Aβ and encourage the removal of harmful proteins from the brain, including Aβ (Du et al., 2018). There have been several unsuccessful trials to create disease-modulating therapies utilizing in vivo disease models of the illness, nevertheless. As a result, the CRISPR/Cas9 technique has become more well-known in the field of AD in recent years because of its low consumption and brief trial period. For activities including creating AD models, detecting harmful genes through screening, and implementing targeted therapy, it is currently widely used.

A tiny proportion of AD cases are familial, also referred to as familial AD or FAD, even though most cases are sporadic (Bekris et al., 2010). These instances arise due to pathogenic variants inherited in an autosomal dominant manner affecting one of the following loci: the gene encoding amyloidogenic precursor protein (APP), presenilin-1 (PSEN1), or presenilin-2 (PSEN2). The main cause of FAD is PSEN1 mutations, which usually cause symptoms to appear earlier than those caused by mutations in the other two genes (Bekris et al., 2010). The majority of PSEN1 mutations result in an elevated production of the more aggregation-prone Aβ42 compared to Aβ40. The development of Aβ plaques in the brain, a defining feature of Alzheimer's disease, is known to be facilitated by this aberrant Aβ42 synthesis (Bateman et al., 2012; Sherrington et al., 1995). Recent research suggests that autosomal dominant mutations may be successfully corrected using the CRISPR/Cas9 technology. This nucleic acid alteration tool's potential for genetic modification is further supported by reports that it has effectively corrected similar kinds of mutations. Later research has shown that employing the CRISPR-Cas9 genome editing technique modifies cell models derived from PSEN2 N141I variant-carrying patient fibroblasts can effectively reverse related electrical activity disruptions and reestablish a physiological Aβ42 to Aβ40 ratio (Sherrington et al., 1995; Haass and Selkoe, 2007; De Strooper, 2007). These findings were further supported by other research investigations that used iPSCs obtained from patients and CRISPR/Cas9 to fix the genetic alterations in the PSEN gene in FAD. Furthermore, another study showed that the background of endogenous γ-secretase was abolished when PSEN1 genes were knocked out in N2a cells using the Type II CRISPR/Cas9 method. Additionally, they found that the synthesis of Aβ42 and Aβ40 was reduced when the PSEN1 mutation-derived recombinant protein was introduced (Pimenova and Goate, 2020).

When Swedish APP (APPswe) mutations were eliminated using Type II CRISPR/Cas9 technology, the expression of Aβ protein decreased, according to a distinct study conducted on patient-derived fibroblasts (György et al., 2018). This mutation, often referred to be Swedish KM670/671NL APP, raises the amounts of Aβ protein by increasing enzymatic cleavage through β-secretase. Furthermore, Guyon led a team of researchers that introduced a novel mutation in 2021 by modifying the APP gene using this technique (Guyon et al., 2021). In both mammalian cell lines (SH-SY5Y cells and HEK293T cells) containing the APP gene, having the amine group removed from the cytosine one and the cytosine two R groups. They changed the alanine codon to threonine (Guyon et al., 2021). According to their reported findings, they succeeded in the insertion of the A673T mutation in every 53 out of 100 HEK293T cells, along with a unique mutation in the amino acid residue number 674, which was replaced from E to K. This alteration further reduced the accumulation of Aβ peptide (Guyon et al., 2021).

It is now known that the APOE (apolipoprotein E) gene increases a person's risk of developing sporadic AD (SAD). In AD patients, the APOE allele primarily originates from an individual's central nervous system, containing an astrocyte-rich area. There are several variations of the APOE gene, such as E2, E3, and E4. Among these variations, the strongest genetic risk factor for SAD is the APOE4 type (Lozupone et al., 2023). To ascertain the function of APOE4, Lin and associates used hiPSC and the Type II CRISPR system in 2018. The research-driven outcomes of their study provided the conclusion that, depending on the kind of cell, APOE4 had different effects on Aβ metabolism (Lin et al., 2018). Additionally, Wadhwani et al. (2019) investigated possible APOE4 treatment targets in 2019. They corrected the E4 allele to the E3/E3 genotype in iPSCs from two AD patients using the Type II CRISPR system. The findings demonstrated that E3 neurons exhibited lowered numbers of phosphorylated tau protein (Wadhwani et al., 2019). Their study thus also suggests that E3 neurons may be more resistant to ionomycin-driven cellular toxicity in comparison to the E4 neurons.

Without any conflict, PD has now taken the place of the second most common neurodegenerative disorder (Jankovic, 2008; Kalia and Lang, 2015). Like AD, this disease also primarily occurs due to aging and affected individuals are of 55-plus aged. Impaired body movements are a defining feature of these diverse neurodegenerative disorders with medical implications (Jankovic, 2008; Kalia and Lang, 2015). As we all know, dopamine is one of the most important neurotransmitters found in humans, playing a crucial role in controlling normal muscular movements. These signal transmitter molecules are produced in the section of the brain known as the substantia nigra pars compacta (and abbreviated as SNPC) (Kalia and Lang, 2015). In PD, the SNPC region fails to produce adequate levels of dopamine, which, over time, as the individual ages, continues to decrease. Now that the dopamine levels are continuously declining in the patient, normal muscular movements turn out to be impaired, resulting in visible abnormal movements (Kalia and Lang, 2015; Surmeier et al., 2017; Dorsey et al., 2018). According to Blesa and Przedborski (2014), this sudden decrease in dopamine count in the striatum is significantly reduced. This reduction impairs the motor circuit's ability to function, which ultimately causes PD symptoms. From reported symptoms and available literature, we know that such symptoms may include resting tremors (unwilling shaking gestures without any apparent will), bradykinesia (slowness in locomotory speed and actions), and rigidity (increased resistance to arm and leg movement) (Blesa and Przedborski, 2014; Hacker et al., 2012).

Another distinguishing feature is the existence of intra-cytoplasmic Lewy bodies (LB), which are mainly made up of ubiquitin and α-synuclein (Blesa et al., 2012). Although mutations in the α-synuclein gene have only been associated with rare familial cases of Parkinson's disease, it is important to remember that all Lewy bodies include α-synuclein. The remaining 10% of PD patients have familial PD brought on by mutations in particular genes such as PRKN/PARK2, SNCA, PINK1, UCH-L1, LRRK2, PARK7, MAPT/STH, and DJ-1, GBA. Around 90% of patients having PD have no known etiology (idiopathic). These mutations might possibly be linked to sporadic Parkinson's disease (PD) (Cota-Coronado et al., 2020; Nalls et al., 2019). The SNCA gene is intimately associated with α-synuclein expression (Ferreira and Massano, 2017). SNCA gene mutation, particularly at amino acid residue number 53, is one of the most significant risk factors of PD. At this position, originally lies Alanine residue, which gets replaced by Threonine due to the occurrence of a missense mutation. Although SNCA contains several mutations, A53T stands out due to its correlation with Parkinson's disease (Spira et al., 2001). According to a study done in the year 2022 by Yoon et al. (2022), the increase in α-synuclein protein and reactive microgliosis caused by it or PD-linked motor neuron indications may be considerably improved by employing the newly developed CRISPR-Cas9 technique by targeting this mutation within the SNCA gene.

Several attempts have been made to test whether this technique is effective against PD. An example of such an effort is Kantor et al. (2018), for utilized CRISPR-Cas9 technology; their team chose a lentiviral vector. They succeeded and reported that using the CRISPR-Cas9 technique, mRNA linked to the SNCA gene was precisely downregulated, limiting its protein expression (Kantor et al., 2018). Further attempts were made by Chen et al. (2021), who sought to identify the mechanism that works behind the functioning of the SNCA gene within the nucleus of a cell. For this purpose, their group obtained human-induced pluripotent stem cells from patients diagnosed with PD having the A53T and SNCA-triplication dominant mutations, along with their version of the same gene, which was obtained upon modification using the CRISPR-Cas9 technique. In their findings based on preclinical models, it was reported that the absence of this gene leads to the development of resistance against Lewy body pathology (Chen V. et al., 2020; Chen X. et al., 2020). Then Zhou et al. (2015) tried to test the same using porcine models obtained from a domestically grown environment. Then, a combinatory technique encompassing CRISPR-Cas9 and somatic cell nuclear transfer (abbreviated as SCNT) was used to determine the effects of PARK2 and PINK1 genes (Zhou et al., 2015). Both of these genes were then knocked out with a success rate of approximately 38%. In a separate notable study focusing on nigral dopaminergic neurons (DN), CRISPR/Cas-mediated ablation targeted the ATP13A2 (PARK9), DJ-1 (PARK7), and PRKN (PARKIN) genes. Integrated transcriptomic and proteomic analyses across these isogenic cell models consistently identified oxidative stress as a common dysregulated pathway (Ahfeldt et al., 2020). In another more recent study, it has been reported that loss-of-function mutations in DNAJC6, which encodes the HSP40 co-chaperone auxilin, are associated with early-onset PD pathogenesis. To elucidate the functional consequences of such genetic alterations, CRISPR-Cas9-mediated genome editing was applied to human embryonic stem cells (hESCs). Transcript profiling and experimental validation suggested that disruptions in DNAJC6 dependent endocytic processes impair WNT-LMX1A signaling during the development of dopaminergic neurons found within the midbrain (mDA). Such impairments cause the reduced LMX1A expression throughout the process of neurogenesis, which may consequently lead to the generation of developmentally compromised mDA neurons. mDA neurons are known to exhibit increased pathogenic vulnerabilities (Wulansari et al., 2021; Nouri Nojadeh et al., 2023).

An autosomal dominant pattern of inheritance leads to a single genetic mutation that causes Huntington's disease (HD), a progressive neurological illness. According to Bates et al. (2015), it is the most common hereditary neurodegenerative illness, and it is caused by a pathogenic mutation [CAG trinucleotide repeat expansion, encoding glutamine (Q)] (Paulsen, 2011). As a result, the huntingtin protein's N-terminal domain develops an extended polyglutamine strand (Kim et al., 2001; Orr and Zoghbi, 2007). A wide range of molecular and cellular processes in the brain are disrupted by this mutant protein, which leads to clinical symptoms like a continuous decrease in decision-making capabilities, accompanied by multiple disorders such as chorea, dystonia, non-coordination, and psychiatric disorders (Waldvogel et al., 2015; Byun et al., 2022). HD is a suitable candidate for gene therapy since it is caused by a single genetic alteration, accompanied by the deposition of faulty protein aggregates. Nowadays, with advancements in the CRISPR/Cas9 technique, it is also pronounced as one gene one therapy and has the potential to inhibit the expression of the faulty HTT gene (Shin et al., 2016).

Shin and associates carried out a study to increase allele specificity. They employed a customized allele-selective CRISPR/Cas9 approach based on SNPs that modify the Pro-tospacer Adjacent Motif (PAM) (Shin et al., 2016). This approach combined a thorough understanding of the huntingtin (HTT) gene haplotype structure with a focus on patient-specific CRISPR/Cas9 locations. According to them, the objective is to specifically deactivate the mutated HTT allele and limit its conversion into a specific diplopic gene (Shin et al., 2016). Furthermore, Suzuki and colleagues devised a method known as homology-independent targeted integration (HITI), which employs CRISPR to effectively eliminate nucleic acids in a growing population of cells observed through in vitro/vivo methods (Suzuki et al., 2016). By enhancing visual function, HITI was demonstrated to be effective in a rat retinitis pigmentosa model, a disorder that results in retinal degeneration (Suzuki et al., 2016). Yang et al. (2017) showed in another study that polyglutamine expansion-driven toxic environment within neuronal cells within the mature brain may be successfully and permanently eliminated by employing CRISPR. They came to such conclusions upon performing their studies on the HD140Q-KI mouse model, where HTT was eliminated. CRISPR/Cas9 in HD140Q-KI mice to deplete HTT in a non-allele-specific way (Yang et al., 2017). According to them, the experimental group showed a significant improvement in motor dysfunction and a notable decrease in reactive astrocytes (Yang et al., 2017). Additionally, a study by Ekman et al. (2019) showed that neurotoxic inclusion development was reduced by two times when CRISPR-Cas9 was used for targeting the altered HTT gene in an R6/2 mouse model strain. The mouse model used by them carried exon 1, which is found on the HTT gene, having 115–150 CAG repetitions. In the same mice, this also resulted in a longer lifespan and improvement in some of the motor neuro defects (Nouri Nojadeh et al., 2023; Xie et al., 2019). Such outcomes show the potential of CRISPR-Cas9 as a tool for HD, reaffirming its applicability to address other similar neurodegenerative diseases.

Also, named as Lou Gehrig's disease (given upon a famous athlete), it affects the human motor system and progresses quickly. According to van den Bos et al. (2019), this disorder is brought on by the central nervous system's motor neuron. Dysfunction in motor neurons is the most observed pathological hallmark affecting the whole-body movement and is the most frequently observed type of motor neuron disease (MND). As the disease progresses, patients witness multiple malformations encompassing lack of strength, severe muscle loss (atrophy), partially or complete lack of movement (or paralysis), and finally failure in respiration due to weakened diaphragm muscles, becoming the most common cause of death because of this dysfunction (Oskarsson et al., 2018). There are two forms of ALS: the first one that runs in families, familial ALS (fALS), which makes up only a small portion of the observed cases (approximately 10%), while the other one, which is developed during an individual's lifetime due to unclear reasons (Boylan, 2015). C9orf72, SOD1, TARDBP, and FUS are the most common pathogenic genes linked to ALS (Bursch et al., 2019). Furthermore, some of the most frequently observed reasons for ALS patients, both inherited (40%) and sporadic (5-6%), are the recurrence in noncoding regions of the hexanucleotide sequence of the gene C9ORF72 (DeJesus-Hernandez et al., 2011; Majounie et al., 2012). To eliminate HRE from the C9ORF72 chromosomal locus, Meijboom's team used an adeno-linked viral vector method for the transfer of the CRISPR-Cas9 system. Various models were used to successfully demonstrate this purpose, including murine models, patient-derived iPSC motor neurons, organoids, and primary cortical neurons. All-important markers C9-ALS/FTD (such as RNA foci, poly-dipeptides, and haploinsufficiency) used in ALS detection and treatments were observed to lower signifying positive results. Treating these illnesses with this therapy approach is hopeful (Meijboom et al., 2022).

In a work by Deng et al. (2021), they edited transgenes (hSOD1-G93A) linked to Lou Gehrig's disease using transgenic mice with CRISPR/Cas9. In two other mouse model studies with hSOD1-G93A (G1H and G1L) mutations, it was shown that the gene-editing approach is beneficial in targeting and correcting the mutation. Another work addressed the SOD1 E100G mutation by using the CRISPR/Cas9 technique. iPSCs from an ALS patient having these mutations underwent targeted gene repair (Yun and Ha, 2020). Later, the iPSCs developed into motor neurons. In addition, Chen et al. (2021) described a quick, easy, and effective method for CRISPR-Cas9-induced point mutations associated with ALS in human iPSCs without the need for antibiotic selection.