Introduction

Parkinson's disease (PD) is a basal ganglia disorder caused by progressive and selective degeneration of the neurons. This degeneration consequently depletes the dopamine levels in the striatum, which results in motor dysfunction and loss of cognitive functions in late-stage PD. Dopamine is a neurotransmitter that regulates the fine motor activities, higher cognitive functions, and feelings of pleasure and rewards. Moreover, dopamine is the metabolic precursor of norepinephrine, another crucial neurotransmitter in the brain. Dopamine is also involved in controlling synaptic transmission, axonal excitability, and dendritic integration [1]. This catecholamine is metabolized and stored in the specialized tyrosine hydroxylase (TH)-positive nerve cells, the dopaminergic neurons. A set of identically derived dopaminergic projections forms a dopaminergic pathway, and each pathway innervates into a specific region of the brain. The mammalian brain consists of various major and minor dopaminergic pathways [1, 2]. Among the 10 different pathways, only four are prominent. The nigrostriatal, mesolimbic, mesocortical, and tuberoinfundibular pathways are the key pathways, and in PD pathology, the degeneration selectively occurs in the nigrostriatal system. The soma or cell bodies of the nigrostriatal dopaminergic neurons are embedded in the substantia nigra (SN), and the axons are innervated into the striatum. Thus, the idiopathic loss of SN neurons depletes the striatal dopamine level.

Numerous studies on genetics, post-mortem human brain, and animal and cellular models have revealed the role of redundant proteins, damaged mitochondria, apoptosis, autophagy, and neuroinflammation in disease pathogenesis [3,4,5]. However, these cellular pathologies are also common to other neurodegenerative diseases [6, 7]. Moreover, the enhanced susceptibility of the SN dopaminergic neurons to endogenous insults is not well understood. It has been opined that selective SN neuronal death could be due to Lewy body (LB) toxicity [8]. LBs are heterogenous protein aggregates rich in fibrillar α-synuclein and ubiquitinated proteins. Nonetheless, cytoskeletal and neuronal synaptic vesicle trafficking proteins are also present in the LBs formed in the degenerating nigral neurons and astrocytes [9]. Researchers have accepted the role of LB toxicity in early-onset PD after the publication of reports on genetic predisposition in genes (SNCA) encoding α-synuclein. This theory gained even more attention when aberrant α-synuclein-mediated PD pathology was found to be disseminated in the healthy dopaminergic neuronal graft through synaptic leakage [10]. Since α-synuclein is a synaptic protein and is abundant near the terminals, the extensive axonal arborization of the nigrostriatal neuron increases the α-synuclein levels and, therefore, enhances its susceptibility to degeneration.

The SN dopaminergic neurons are long, thin, and unmyelinated and have an extensive arborized axon and a high synapse to neuronal soma ratio. This unique cellular architecture of the SN dopaminergic neurons differentiates them from other dopamine-containing neurons, including those in the ventral tegmental area (VTA) [8, 11]. The high density of synapses connected to a neuron's cell body demands increased energy for proper functioning. Simultaneously, the narrow width of the axon and the associated terminals allows a controlled supply of mitochondria, several essential proteins, and vesicular structures to the presynaptic terminals through axonal trafficking [12, 13]. Therefore, the SN neuron's demand–supply equilibrium remains at the edge, and the bioenergetic demand is considerably high at the synaptic site [14]. To match the elevated bioenergetic demand at the synapse, mitochondrial dynamics, anterograde transportation, and its biogenesis increase in the SN neurons [15,16,17]. The mitochondria present in the neurons govern several neurophysiological processes, including the active axonal trafficking of cargos, mobilization of synaptic vesicles, calcium ion (Ca2+) homeostasis, and neurotransmission. Dysregulation of the mitochondrial dynamics disturbs several intraneuronal functions [18,19,20]. Moreover, the elevated bioenergy at the synaptic terminal results in enhanced oxidative stress and dopamine oxidation in the neuron if the delicate supply-chain equilibrium is disturbed [13]. The dysregulation of the mitochondrial dynamics leads to protein accumulation, excessive Ca2+ release into the cytosol, and disturbance of synaptic events [13, 19]. Furthermore, the high cytosolic level of free Ca2+, which is caused by the low intrinsic calcium buffering capacity of the SN neuron, is equally responsible for its preferential vulnerability [8]. Thus, the cellular architecture and the neurophysiology of the PD-related dopaminergic neurons are the reasons for the increased vulnerability.

Notwithstanding the several advances in understanding PD, including pathogenesis, selective histological loss, and the complex interplay of genetic and environmental factors, the specific cause for disease onset and progression at the cellular level remains unknown. Most of the recent investigations have focused on pathogenesis, alteration in dopaminergic neuron counts, and experimental therapeutics. However, the search for a permanent cure for the disease is not yet over. The impairment of axonal trafficking, mitochondrial dynamics, synaptic protein expression, neuronal excitability, Ca2+ homeostasis, synaptic integrity, and increased cytosolic dopamine level in the synaptic terminals, which have been reviewed here, point to the involvement of axon terminal loss before the degeneration of dopaminergic soma. However, the role of axon terminals and the initiation of the degeneration cascade at the cellular level of a dopaminergic neuron remain undeciphered. In this review, we have explored the cause of axonal degeneration and the role of impaired synaptic terminals in retrograde neurodegeneration that occurs in PD. Furthermore, we have suggested that future research should delve into the mechanism of preventing the degeneration of dopaminergic axons and their auxiliary terminals, which is likely to aid in restoring the dopamine level in the striatum and alleviate the motor complications in patients with PD.

Selective Vulnerability: Cellular Architecture and Neurotransmitter

The axon fiber of the SN dopaminergic neurons is thin, unmyelinated, and extensively ramified, with millions of synapses in the striatum (Figs. 2 and 3) [8]. The dense arborized axon terminals demand an excessive amount of energy for maintenance of their basal functions and survival. Furthermore, preservation of membrane potential, spreading of the action potential throughout the axolemma, release of dopamine from the synapses, and synaptic transmission of neural information create additional bioenergetic demands [11, 14]. The extreme demand for energy in the diffused neuronal extremities is met by densely packed mitochondria in the dopaminergic axon [12], and the regular supply of the mitochondria—as per the demand—in the neuronal extremities maintains their density therein. The increased density of the mitochondria in the neuron’s thin axon and the extremities helps cope with the tight energy budget [8]. However, because of either endogenous or exogenous insults, an imbalance in the demand and supply equilibrium of energy in the axons halts the energy-driven neuronal events and leads to dopaminergic neurodegeneration [21, 22]. Thus, the SN dopaminergic neuron's cellular morphology and mitochondrial homeostasis are peerless and contribute to its preferential vulnerability. One of the reasons for the sudden failure in the energy balance and the irreversible increase in anomalies in the SN neurons is their limited reserve capacity and the slow anterograde transportation of mitochondria to the site of need [13, 23, 24]. Therefore, in idiopathic PD and in several models of Parkinsonism, mitochondrial dysfunction is critical and is the reason for the selective degeneration [22, 25,26,27,28]. However, in addition to the SN axon terminals, mitochondrial dysfunction is also evident in the cholinergic neurons of the pedunculopontine nucleus, hippocampal nucleus, and the prefrontal cortex of the brain in patients with PD; furthermore, mitochondrial dysfunction has been reported in the unaffected regions of the brain, such as the cerebellar cortex and dentate nucleus [29,30,31].

Apart from the elevated mitochondrial density in the SN dopaminergic terminals, which engenders more risk, an important cause for the retrograde degeneration of the neurons is the presence of the neurotransmitter dopamine. The nascent neurotransmitter molecules are synthesized in the cytosol of the neuron. Dopamine synthesis, which occurs in two steps, is catalyzed by TH and aromatic amino acid decarboxylase (AADC) to generate L-dihydroxyphenylalanine (L-DOPA) and dopamine, respectively. L-DOPA, produced from the TH-catalyzed reaction in the presence of oxygen, serves as a substrate for AADC that forms dopamine upon decarboxylation in the cytosol [32,33,34]. Both enzymes (TH and AADC) bind to the synaptic vesicular membrane-localized vesicular monoaminergic transporter-2 (VMAT-2) protein and form a complex. Additionally, the dopamine molecules in the synaptic cleft are taken up into the cytosol through dopamine transporters (DATs) localized in the membranes of the axon terminals [35, 36]. Under normal circumstances, dopamine in the cytosol is recurrently pumped into the monoaminergic vesicles and stored therein. The translocation of dopamine into these acidic vesicles is VMAT-2-mediated and helps store dopamine in the axon terminals for future use and prevents the rapid conversion and depletion of the neurotransmitter in the cytosol [32, 37, 38]. If, somehow, dopamine fails to pass into the acidic lumen of the monoaminergic vesicles, it undergoes degradation in the cytosol and the levels of its metabolites increase.

In the axoplasm, monoamine oxidase (MAO) and catechol ortho-methyltransferase (COMT) catalyze the catabolism of dopamine. The MAO is a flavin adenine dinucleotide (FAD)-containing flavoenzyme present in the outer membrane of the mitochondria [39]. The enzyme converts the cytosolic dopamine into 3,4-dihydoxyphenylacetaldehyde and enhances oxidative stress because the oxidative deamination reaction generates hydrogen peroxide (H2O2). Furthermore, 3,4-dihydoxyphenylacetaldehyde is oxidized to form 3,4-dihydroxyphenylacetic acid (DOPAC) in the presence of aldehyde dehydrogenase. COMT methylates DOPAC and produces homovanillic acid (HVA). However, COMT can also directly methylate dopamine to produce 3-methoxytyramine (3-MT), which is also degraded to HVA in the presence of MAO and aldehyde dehydrogenase [32]. Also, the cytosolic dopamine is oxidized to dopamine o-quinone and further undergoes cyclization to form aminochrome and neuromelanin at physiological pH, which results in the production of reactive oxygen species (ROS). However, the end product of dopamine catabolism, neuromelanin, is neuroprotective [37, 40], and its presence is relatively higher in the SN neurons than in the VTA subtype. The augmented expression of neuromelanin in the neuron indicates increased dopamine oxidation in its cytoplasm. Thus, the resultant intermediate products and the ROS generated during the reaction cascade pose a significant risk to the neuron (Fig. 1). Reports have shown an increased level of oxidative stress and dopamine catabolites in the brain of patients with idiopathic PD and in animal models of PD [41, 42]. The dopamine catabolites enhance metabolic dysfunction as well as mitochondrial and lysosomal impairment [43].

Fig. 1
figure 1

Dopamine metabolism: In the presence of tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC) enzymes, amino acid tyrosine is converted to dopamine, the neurotransmitter. An intermediate L-Dopa is also synthesized in the enzyme-catalyzed reactions in the axoplasm (cytoplasm of the axon). The degradation of dopamine in the cytosol is preserved by vesicular monoaminergic transporter-2 (VMAT-2) mediated transportation into the monoaminergic vesicles, the vesicle with low pH inside. The catabolism of dopamine in relatively alkaline pH of the cytosol is catalyzed by the mitochondrial monoamine oxidase (MAO), and cytosol localized catechol methyl-ortho-transferase (COMT) enzymes to form several intermediate metabolites and reactive oxygen species (ROS) molecules. Dopamine is also oxidized directly through different cascades to generate dopamine-O-quinone, aminochrome, neuromelanin, and ROS (All chemical structures were drawn using CambridgeSoft ChemDraw, RRID:SCR_016768) [32, 37, 39, 40, 42]

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Impaired Axonal Transport in PD

Axonal transport is a process in the axoplasm that is responsible for the movement and distribution of essential biomolecules and organelles to and from its nerve endings. This transport maintains the equilibrium in the supply and demand of vital organelles, such as mitochondria, and several proteins and vesicles, including synaptic vesicles, in the neuron's cell body and axon terminals. Thus, the event is crucial for the proper functioning of the neuron and is equally essential for its growth and survival. The cytoskeletal and motor proteins facilitate the continuous anterograde and retrograde trafficking of cargos in the functional neuron to accomplish efficient axonal transport. In dopaminergic neurons, morphology-regulating actin and tubulin are the major cytoskeletal proteins that form “tracks” for cargo transportation [44,45,46] and kinesin and dynein are the motor proteins that assist in the anterograde and retrograde trafficking, respectively, of several proteins, vesicular structures, mitochondria, and other organelles (Fig. 2) [47, 48]. Studies have established that altered levels of these proteins in the dopaminergic neurons cause the dysregulation of axonal transport. This dysregulation leads to neuronal dystrophy, which is characterized by bulging neurites, loss of presynaptic terminals, deficit in synaptic function, abnormal protein accumulation in the neurites, defective mitochondrial dynamics, and dysregulated homeostasis long before the dopaminergic neurodegeneration [10, 44].

Fig. 2
figure 2

Axonal trafficking in the functional dopaminergic neuron [A] and in degenerating neuron in PD [B]. The cytoskeleton proteins (actin and tubulin) form “tracks” to transport proteins and organelles to and from the neuron's cell body. The kinesin and the dynein are two motor proteins for anterograde and retrograde trafficking, respectively. Impaired axonal transport in PD is marked by a higher ratio of obstructed protein inclusion and dysfunctional mitochondria in the deteriorating dopaminergic neurons. Failure in the trafficking of essential proteins, mitochondria, lysosomes and vesicular structures at the distal ends causes retrograde axonal loss (All structures are made with Inkscape Software, RRID:SCR_014479) [10, 45,46,47,48,49, 53]

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Several reports have shown that axonal transport is significantly impaired in sporadic PD and that it precedes the early decline in dopaminergic neurons [49, 50]. A similar finding has also suggested impaired axonal transport, which leads to early degeneration in a retrograde manner in familial PD [10] and toxin-based PD models [51, 52]. Alterations in the expressions of motor proteins (kinesin and dynein) and cytoskeletal protein (tubulin) impair the axonal transport [46, 49, 53]. Disruption of the anterograde trafficking of the mitochondria into the axonal projections as per their energy demand results in energy deficit and the loss of synapses [10]. The exact reason for the onset of impairment in axonal trafficking is yet unknown; however, a few PD-related proteins (DJ-1, leucine-rich repeat kinase-2 (LRRK-2), and α-synuclein) have been linked to the process of axonal transport, and mutations in the corresponding genes cause axonal transport abruption [10, 46]. Moreover, hyperserotonergic neurotransmission, a state of neuropathology in the brain of patients with PD, also disrupts the regular axonal transportation in the dopaminergic neuron because serotonin plays an inhibitory role in axonal growth and regeneration [54].

Defective Mitochondrial Dynamics in the Axon

The SN dopaminergic neurons are top-to-toe energy-demanding cells. The neurons require energy for axonal transport and synaptic release and for maintaining basal cell functions, growth, and survival. The unique morphology of the SN neurons—dense axon terminal arborization with millions of synaptic contacts—requires additional energy when compared with the less vulnerable VTA dopaminergic neurons to maintain their synaptic events and adequate axonal transport as per the demand. The elevated bioenergetic demand is fulfilled by transporting more mitochondria to the axon terminals. In the dopaminergic neuron, the mitochondria perform many intraneuronal functions such as Ca2+ buffering, dopamine synthesis and degradation, neurotransmitter translocation, synaptic vesicle mobilization, and bidirectional axonal transport [48, 55, 56]. However, the narrow axonal width of the SN neurons imposes cytoplasmic space restrictions and controls the motility of the trafficking mitochondria [12]. Therefore, in the SN neurons, mitochondrial homeostasis is at the edge. However, the dynamic properties of the mitochondria, such as fission, fusion, trafficking, biogenesis, and degradation, are significantly higher in the SN neurons than in the VTA counterpart for maintaining their health [12, 57]. A disruption in any of these dynamic mitochondrial events disturbs the others and is associated with mitochondrial dysfunction, a critical pathology reported in PD [27, 48, 58].

Experimental studies have suggested that several endogenous and exogenous insults influence the defective mitochondrial dynamic processes in the axon terminals during early-stage PD [59,60,61]. For being functional, neurons depend on the regular supply and distribution of functional mitochondria in their axon and terminals. Simultaneously, the dysfunctional mitochondria need to be eliminated from the synaptic terminals for their ultimate degradation via mitophagy [62, 63]. The equilibrium between the supply of healthy mitochondria and the clearance of aberrant mitochondria is crucial for maintaining the morphology, plasticity, and functionality of the synapses. Several experimental and idiopathic PD studies have stated that defects in the mitochondrial dynamics appear early, well before neurodegeneration [27, 28, 64]. In familial PD cases, mutations in a few genes associated with disease onset have been linked to the mitochondria and have been shown to play a crucial role in regulating the mitochondrial dynamics in the neurons [61, 65]. Defects in the dynamics distort the synapse morphology and reduce the postsynaptic dendritic spines, which lead to a loss of plasticity [55, 66]. Moreover, abnormal mitochondrial dynamics cause cyclic oxidative stress, increased energy demand at the synaptic contact, and dysregulated Ca2+ buffering at the synaptic site. These events culminate in the untimely degeneration of the dopaminergic neurons originating from their synaptic sites [24, 61].

Early Impairment of Synaptic Homeostasis

The synapses are often distantly located from the cell body of the neurons owing to axonal extension. Synapse is the functional junction and comprises the presynaptic bouton, which contains neurotransmitters packaged in vesicles, and the postsynaptic neuronal membrane, which consists of target receptors that bind the released neurotransmitters to exchange the presynaptic neural information. Between them, there is a synaptic cleft that isolates and holds the presynaptic and postsynaptic components. The homeostasis and sustained functionality of the neuronal synapses, including the SN dopaminergic neurons, depend on several synaptic proteins residing in the presynaptic boutons, mitochondrial influx and elevated bioenergetics in the presynaptic boutons that fuel effective and sustained neurotransmission, and the postsynaptic membrane-receptor proteins that bind the neurotransmitter and transmit the neural information to the postsynaptic neurons.

A study has established the loss of synaptic sites and its association with the onset of motor symptoms in PD [67]. A similar study has suggested that synaptic decay and loss of auxiliary axonal fibers in the striatum occur earlier than the degeneration of the cell bodies in the nigral compartment. Thus, the demise of the synaptic terminals occurs before that of the cell bodies in the SN dopaminergic neurons [68, 69]. However, the reason behind the early loss of synaptic terminals in PD is largely obscure.

The presynaptic boutons contain several proteins called synaptic proteins, which are involved in maintaining the survivability, plasticity, and functionality of the synapse. Members of these proteins also regulate vesicular packaging and exocytosis of the neurotransmitters. In the SN dopaminergic neurons, members of the synaptic protein family belong to both types, i.e., membrane-bound and soluble cytosolic proteins (Table 1) [70,71,72]. These proteins regulate the Ca2+ level, dopamine release, growth and alignment of the dendrites, synaptic density, and the plasticity of the synapses [70]. Impairment in the genes encoding the synaptic proteins and their abnormal expression cause synaptic dysfunction in PD-linked dopaminergic neurons. The abnormal expression of the synaptic proteins alters neurotransmission and results in synaptic dysfunction but does not necessarily lead to synaptic degeneration (Fig. 3). However, abnormalities in a few synaptic proteins and/or genes encoding them, including α-synuclein, LRRK-2, auxilin, synaptojanin-1 (Synj-1), and endophilin A1, have been linked to Parkinsonism [70, 73, 74]. Moreover, elevated levels of the synaptic proteins neurogranin and the 25-kDa synaptosomal-associated protein (SNAP25) have been observed in the cerebrospinal fluid of patients with PD [75].

Table 1 List of few critical synaptic proteins, corresponding genes in humans, their physiological functions at or near the synapse, and their relevance in Parkinsonism
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Fig. 3
figure 3

Presynaptic homeostasis in the functional dopaminergic neuron [A] and presynaptic impairment in PD [B]. The presynaptic bouton of an operational dopaminergic neuron shows a proper morphology and consists of abundant mitochondria, functional lysosomes and synaptic vesicles packed with neurotransmitters [A]. Early synaptic alteration in PD is marked with a change in the morphology of the bouton, increased cytosolic dopamine, protein inclusion/aggregates, dysfunctional mitochondria and lysosomes and reduction in the synaptic structures in the presynaptic bouton [B] (All structures are made with Inkscape Software, RRID:SCR_014479) [10, 53, 78,79,80,81, 128]

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The exact mechanism of synaptic dysfunction, which is caused by abnormalities in the proteins and leads to synaptic loss, is an enigmatic question. The autopsy of PD-affected brains has revealed aggregates of α-synuclein, a protein involved in regulating the synaptic functions [71], in the presynaptic terminals [76]. Such reports are also standard in experimental studies [77]. The aberrant α-synuclein is propagated intercellularly to neighboring neurons, which results in the transfection of the deleterious cascade in a prion-like manner. The presence of the pathological α-synuclein in deteriorating neurons and ingrafted tissues received by patients with PD supports the prion hypothesis of disease spread [78,79,80]. The aberrant α-synuclein impairs the dopamine release [79], abruptly snares the essential cellular components at an inappropriate location, including the mitochondria and lysosomes, and obstructs the normal axonal trafficking [10, 53]. Furthermore, the protein binds to other presynaptic proteins such as cysteine-string protein α (CSP α)—a protein involved in maintaining synaptic integrity—and heat shock protein cognate 70 (Hsc 70)—a chaperone protein for α-synuclein turnover—thereby hindering their functions [81].

Breakdown of Ca2+ Homeostasis in the Axon Terminals

The abundance and homeostasis of Ca2+ in the cytoplasm of dopaminergic neurons are vital for the function and survival of these neurons. Ca2+ regulates many physiological processes, including membrane excitability, dopamine release, and synaptic plasticity [82]. The divalent cation is pumped into the cytosol from intracellular and extracellular sources in a nigrostriatal dopaminergic neuron. The central intracellular Ca2+ reserve is the endoplasmic reticulum (ER), and the opening of voltage-gated Ca2+-channel (Cav) proteins, which are located on the ER membrane, releases these cations into the cytoplasm. Simultaneously, Ca2+ from the extracellular source enters through the Cav proteins situated on the axonal membrane [83, 84]. However, the dopaminergic neuron's action potential modulates the Ca2+ influx into the cytosol from its reservoirs, i.e., the intracellular and the extracellular sources. The Ca2+ entry, its cytosolic oscillation, and the electric activity of the neuron play a decisive role in dopamine release within the dorsal striatum. Additionally, the secondary messenger molecule maintains the basal dopamine levels for the next neurotransmission cycle [85].

The SN neuron has a unique slow and broad action potential that maximizes the Ca2+ entry into the cytoplasm from its reserves [8]. The additional feature of the SN dopaminergic neuron that distinguishes it from the other neuronal types is its low intrinsic calcium buffering ability. The inflow of Ca2+, when released from the reserves, floods the nearby cytosol. Therefore, calcium-binding proteins in the neuron, such as calbindin, interact with the available free cytosolic Ca2+ and rapidly buffer the situation until the surplus Ca2+ is instead pumped back into the reservoirs [86]. In the SN neurons, the presence of the calcium buffering protein is low. Thus, the surplus free Ca2+ interacts with other proteins, alters the cytosolic pH, and inhibits the cellular functions. Furthermore, the Cav aligned to the plasma membrane of the SN neurons allows more Ca2+ to enter the cytosol than the Cav of the VTA neurons [87, 88]. Ca2+ long-lasting presence in the cytosol synergizes with the PD-linked risk factors including ageing, environmental agents, and genetics, and induces mitochondrial dysfunction, which leads to oxidative stress [89]. A report has suggested imbalance in Cav activity in the dopaminergic terminals of PD models [90] and altered expression of Cav and calcium-binding proteins in early- and late-stage PD, which establishes the key role of calcium ions in the disease [91, 92]. The aberrant α-synuclein in the presynaptic terminals of the degenerating neurons causes dysregulation of Ca2+ and also induces Ca2+-mediated neuronal death [93]. Gene mutations and abnormal expressions of PD-associated proteins also dysregulate the homeostasis of Ca2+, the secondary signaling molecule [94, 95].

Relevance of Axonal Degeneration in PD

The dopaminergic neurons of the nigrostriatal pathway connect the SN nucleus to the striatum compartment, the pathway which is defective in PD. Of course, both SN and striatal tissue deteriorate as the disease progresses, but from where does the degeneration initiate? Does the neuronal soma of the SN neuron degenerate before losing its extended axon and arborized terminals, or does the dysfunction begin in the axon terminals/axon and propagate in a retrograde manner? Many believe that the degeneration co-occurs in the SN dopaminergic soma and axon terminals, whereas others are unclear of the degeneration pattern. Thus, the underlying facts are yet to be established.

Studies have indicated that the motor deficits are visible in PD only after approximately 70% of the striatal dopamine has been depleted [25, 41]; hence, the demise of the presynaptic terminals governing the neurotransmitter release must begin much earlier than the appearance of the disease symptoms. Therefore, the loss of striatal axon terminals outweighs the loss of cell bodies of the SN dopaminergic neurons during the disease progression because the axon terminals participate earlier in the degenerative cascade [68]. Furthermore, the flaws in the presynaptic terminals of the degenerating neurons confirm the prior involvement of the axon terminals during progressive degeneration. The presynaptic terminals become a damping corner of aberrant proteins, damaged mitochondria, and dysregulated Ca2+ [24]. The accumulation of aberrant proteins and the breakdown of the synaptic bioenergetics—due to defective mitochondria—hinder the transmission of impulses from the neuron's functional junction. These aberrations need to be urgently cleared form the deteriorating neuron to be revived and regain its functionality. However, the central clearance machinery for defective proteins and organelles exists in the neuron's cell body, and the defective axon trafficking of the deteriorating neurons obstructs the retrograde cargo transportation. In addition, dopaminergic neurons are post-mitotic cells that forever remain in their resting phase (G0 phase) and never redistribute their damaged proteins and organelles to the daughter cells [96]. Thus, in the absence of redispersal and proper trafficking, these abnormalities remain intracellularly in millions of tiny presynaptic axon terminals and cell bodies and are disproportionately overshared in the axon terminals when compared with the cell body of the neurons. Moreover, the terminals exhibit synaptic density and structural alterations, which is yet another link to disease progression [69]. Apart from PD, chronic exposure to various psychostimulant addictions also impairs the dopaminergic terminals. These addictive drugs increase cytosolic dopamine levels, block DAT reuptake, modulate neuronal firing, alter neurotransmission, and induce changes in synaptic morphology [97,98,99].

Thus, the axonal projections, not the cell bodies, are the initial players in the SN dopaminergic neurodegeneration; subsequently, their loss, which propagates toward the cell body, leads to the appearance and progression of clinical manifestations. Unfortunately, very few studies have reported the axonal origin of the dopaminergic neurons' disintegration in PD [3, 62, 100, 101]. If loss of axon terminals occurs early in PD and is responsible for the progression of the motor symptoms, targeting the cell bodies for a neuroprotective approach is non-directional. However, despite the mounting evidence for the predominant role of the axon terminals in PD, to date, researchers have rarely studied the axons and the cell body of a neuron separately and have continued to focus on the death of the entire neuron structure. The strategy to cure the disease has been to either target the neuronal soma or replace the defective ones with exogenous healthy neurons. Similarly, experimental strategies for neuroprotection have focused on preventing the demise of the cell bodies and not axonal loss. Until now, the protective strategies have revolved around targeting different pathways of programmed cell death, but the apoptotic mechanism of cell death is applied only in the neuronal soma. The mechanism of axonal degeneration is utterly different from programmed cell death, and much of the degenerative cascade is indescribable [3]. Investigating the pathological changes only in neuronal soma could well be the reason for our failure in developing clinical neuroprotective and restorative therapeutics despite experimental successes.

The Future Perspective of Axon Neurobiology

The proper functioning of the nigrostriatal system depends on the structurally polarized SN dopaminergic neuron. In the polarized architecture, the soma overshares the cellular machinery for its maintenance. Simultaneously, the axon projections rely on bidirectional axonal transport for their survival and for maintaining their functionality. Unfortunately, in PD, the axonal trafficking in the related neurons deteriorates with the progression of the disease. The progressive deterioration in bidirectional trafficking is due to the gradual accumulation of redundant proteins and damaged organelles. At the initial stage of the disease, neuronal debris is formed at the synaptic site but is successively accumulated in the axon and cell body because of the obstruction of axonal trafficking, retardation in the clearance machinery, and advancement of the disease. Thus, unlike other cell types, the neuron's architectural components—neuronal soma and axon projections—should be studied separately and the neuropathological changes should be classified discretely in the ramified terminals and soma of the degenerating PD-related neurons. The neurobiological alterations should be separately measured per unit area/volume in both the neuronal compartments. Designing such studies could help investigate the comparative alterations in the soma and terminals and identify early-stage PD biomarkers residing at or near the synaptic sites and causing synaptic dysfunction. Furthermore, spatial autophagy in the terminals and synaptic sites, which involve synaptic proteins, could be induced to digest the synaptic junks locally before they exponentially multiply in the axon and hinder the axonal transport [102,103,104]. Since dopaminergic neurons are non-dividing cells, correcting the neuronal aberrations and digesting the cellular debris through site-specific autophagy, particularly at an early stage, are the sole strategies available.

Although neuronal soma restoration is crucial for axonal growth, maintenance, and survival, it is not sufficient to prevent the clinical progression of PD. Ignoring the axons' decisive relevance in formulating neuroprotective strategies could be due to the obstacles in the experimental demonstration of axons and their auxiliary fibers in the brain tissue. Studying axons requires high-quality tissue preservation and special techniques. However, if studying axonal destruction, regrowth, and anatomical organization becomes feasible in the future with advanced techniques, attempts to develop therapeutic strategies by correcting the synaptic autophagy at the pre-symptomatic stage may be more effective. Moreover, while the lost neuronal tissues are replaced by the exogenous implant in the grafting technique, in the new approach, the surviving neurons can be stimulated to regrow their axons and the associated auxiliary fibers for proper anatomical arrangement and reintegration into the surviving circuitry. Thus, if the approach for correcting the spatial clearance machinery of the axon terminals to design neuroprotective and restorative drugs could be made feasible in the future, it could offer a better direction in devising novel therapeutic strategies for PD.