**4. Role of clearance mechanisms**

Emerging data support the view that dysregulation of alpha-synuclein clearance machinery might represent the key feature leading to the intraneuronal accumulation of this protein. These mechanisms include the ubiquitin-proteasome system and the autophagic-lysosomal pathways; the autophagic pathways involved in the degradation of alpha-synuclein are chaperone-mediated autophagy and macroautophagy (Webb et al 2000). Anyway, the accumulation of alpha-synuclein does not represent the only negative consequence of clearence systems failure.

#### **4.1 Impairment of the ubiquitin-proteasomal system**

Increasing evidences indicate that the ubiquitin–proteasome pathway is defective in Parkinson's disease. The finding that Lewy bodies are ubiquitin-positive aggregates has

Alpha-Synuclein, Oxidative Stress and

**4.2 Autophagy dysfunction** 

embryonic tissues (Nixon et al 2006).

constituted by specific carrier proteins.

preventing early onset of massive neuronal loss.

**4.2.1 Impairment of chaperone-mediated autophagy** 

et al 2010).

Autophagy Failure: Dangerous Liaisons in Dopaminergic Neurodegeneration 121

Chronic oxidative stress alters the subunits of proteasome, so that even reactive oxygen

Autophagy is a finely regulated intracellular process that mediates lysosomal degradation of proteins and organelles. Its function allows the clearance of substrates characterized by alterations limiting their physiologic function or responsible for a cytotoxic effect. This degradative process exerts a cytoprotective role that is probably dependent on the clearance of toxic intracellular structures and the catabolism of substrates in order to obtain energy during starvation. Anyway, in particular situations autophagy seems to mediate a specific pathway of programmed cell death; this function requires a strong activation of autophagy and until now, in vivo, has been identified only during involutional physiologic processes in

In mammalian cells autophagy encompasses three main processes: microautophagy, macroautophagy and chaperone-mediated autophagy. Microautophagy is a constitutive, non selective process consisting on endocytosis of small amounts of cytoplasm into lysosomes through invagination of lysosomal membrane. Macroautophagy and chaperonemediated autophagy are inducible processes: the first one allows lysosomal degradation of organelles and proteins after their sequestration within a double-membrane-limited vacuole called autophagosome; the second one is a selective device for degradation of aberrant proteins, which are directly transported into the lysosomal lumen by a translocation system

Recent research has revealed the existence of a dysfunction in autophagy pathways in the cerebral regions involved in neurodegenerative processes. A decrease in the activity of both autophagic-lysosomal pathway and the ubiquitin-proteasome system has been reported during aging in every tissue, included neurons (Martinez-Vicente et al 2005). This deficit might be a major responsible for the intracellular accumulation of misfolding proteins and aberrant mitochondria, representing factors leading to oxidative stress and neuronal damage. Therefore, autophagy impairment might be a primum movens of the neurodegenerative processes and, at the same time, a limiting factor that is necessary for

In the last years the pathogenetic role of altered clearance machinery has become a central subject of research in Parkinson's disease: indeed, the impairment of chaperone-mediated autophagy and macroautophagy, together with the dysfunction of ubiquitin-proteasome system, seems to give a major contribute to the development and progression of nigral degeneration. The loss of function of these degradative pathways might produce a deleterious effect through induction of the pathogenetic processes more directly responsible for neuronal death, such as the accumulation and oligomerization of alpha-synuclein, the persistence of damaged mitochondria and the consequent production of reactive oxygen species. In turn, these key mediators of dopaminergic neurodegeneration can further worsen the impairment of clearance machinery and, in particular, of autophagic pathways (Cuervo

Chaperone mediated autophagy mediates the translocation of proteins containing a Lys-Phe-Glu-Arg-Gln (KFERQ) motif into lysosomes. This process requires the presence of three

species might be responsible for proteasome impairment in Parkinson's disease.

suggested that a dysfunction in proteasome might contribute to the accumulation and aggregation of alpha-synuclein and other neurotoxic proteins. The first confirmation of this hypothesis has derived from the identification of hereditary forms of Parkinson's disease linked to two genes within the ubiquitin-proteasome system: parkin and UCHL1.

Parkin (PARK2) is an ubiquitin E3 ligase, an enzyme that catalyzes the addition of ubiquitin chains to substrate proteins which must be degraded by the proteasome. Mutations in parkin gene are responsible for almost half of autosomal recessive cases of hereditary Parkinson's disease (Kitada et al 1998); most of the pathogenetic mutations are associated with a defect in the E3 ubiquitin ligase activity of the protein. Furthermore, parkin is responsible for K63 ubiquitination of alpha-synuclein; this function is not involved in the degradation of substrate proteins through proteasome but favors protein aggregation and generation of insoluble inclusions. Mutations in the parkin gene prevent the formation of Lewy bodies in the substantia nigra, thus suggesting that the K63 ubiquitin ligase function of parkin is essential for the fibrillization of alpha-synuclein and formation of insoluble inclusions; the degeneration of dopaminergic neurons deriving from parkin loss of function supports the hypothesis that Lewy bodies might contrast the toxic effects of the protofibrillar protein.

Ubiquitin C-terminal hydrolase L1 (UCHL1/PARK5) is an enzyme that cleaves peptideubiquitin bonds and recycles ubiquitin monomers. Variants of UCHL1 gene have been associated to familial forms of Parkinson's disease. The I93M mutation was identified in a family with probable autosomal dominant Parkinson's disease; in vitro studies have demonstrated that this mutation results in partial loss of UCHL1 hydrolytic activity. In addition to the hydrolase activity, UCHL1 exerts a dimerization-dependent ubiquitin ligase activity that promotes alpha-synuclein aggregation. The single nucleotide polymorphism S18Y, which has been found to decrease the susceptibility to sporadic disease, seems to be associated with reduced ligase activity.

A direct proof of proteasome dysfunction in Parkinson's disease has been the finding of structural and functional alterations in the 20S proteasome subunit in the substantia nigra of patients with sporadic disease. The pathogenetic relevance of proteasome impairment has been reinforced by the observation that administration of proteasomal inhibitors to animals can produce the neuropathological and motor manifestations of Parkinson's disease, including selective nigral cell loss, Lewy-bodies-like inclusions and typical clinical signs.

Two mechanisms are known to be responsible for a dysfunction in the ubiquitin-proteasome system: aging is associated with a physiologic decrease of proteasome efficiency and ubiquitination activity; oxidative species and alpha-synuclein protofibrils exert a deleterious effects on proteasome subunits.

Alpha-synuclein oligomers have demonstrated to inhibit proteasome function through direct interaction with 20S subunit; this effect might favor further accumulation of alphasynuclein, which in turn may worsen proteasome impairment.

The mechanisms behind proteasome inhibition by oligomeric alpha-synuclein require further investigation in order to be clarified. Synphilin-1 has been associated to the pathogenesis of Parkinson's disease since its identification as an alpha-synuclein-interacting protein, a component of Lewy bodies and a substrate of the E3 ligase parkin. This protein has been found to interact with the protein S6 ATPase, which exerts a regulatory function on proteasome. Furthermore, recent studies indicate that synphilin-1 exerts an inhibitory effect on proteasome activity partially due to the interaction with alpha-synuclein (Alvarez-Castelao et al 2010); mutations in synphilin-1 gene have been detected in patients with sporadic Parkinson's disease.

Chronic oxidative stress alters the subunits of proteasome, so that even reactive oxygen species might be responsible for proteasome impairment in Parkinson's disease.

### **4.2 Autophagy dysfunction**

120 Etiology and Pathophysiology of Parkinson's Disease

suggested that a dysfunction in proteasome might contribute to the accumulation and aggregation of alpha-synuclein and other neurotoxic proteins. The first confirmation of this hypothesis has derived from the identification of hereditary forms of Parkinson's disease

Parkin (PARK2) is an ubiquitin E3 ligase, an enzyme that catalyzes the addition of ubiquitin chains to substrate proteins which must be degraded by the proteasome. Mutations in parkin gene are responsible for almost half of autosomal recessive cases of hereditary Parkinson's disease (Kitada et al 1998); most of the pathogenetic mutations are associated with a defect in the E3 ubiquitin ligase activity of the protein. Furthermore, parkin is responsible for K63 ubiquitination of alpha-synuclein; this function is not involved in the degradation of substrate proteins through proteasome but favors protein aggregation and generation of insoluble inclusions. Mutations in the parkin gene prevent the formation of Lewy bodies in the substantia nigra, thus suggesting that the K63 ubiquitin ligase function of parkin is essential for the fibrillization of alpha-synuclein and formation of insoluble inclusions; the degeneration of dopaminergic neurons deriving from parkin loss of function supports the hypothesis that

Ubiquitin C-terminal hydrolase L1 (UCHL1/PARK5) is an enzyme that cleaves peptideubiquitin bonds and recycles ubiquitin monomers. Variants of UCHL1 gene have been associated to familial forms of Parkinson's disease. The I93M mutation was identified in a family with probable autosomal dominant Parkinson's disease; in vitro studies have demonstrated that this mutation results in partial loss of UCHL1 hydrolytic activity. In addition to the hydrolase activity, UCHL1 exerts a dimerization-dependent ubiquitin ligase activity that promotes alpha-synuclein aggregation. The single nucleotide polymorphism S18Y, which has been found to decrease the susceptibility to sporadic disease, seems to be

A direct proof of proteasome dysfunction in Parkinson's disease has been the finding of structural and functional alterations in the 20S proteasome subunit in the substantia nigra of patients with sporadic disease. The pathogenetic relevance of proteasome impairment has been reinforced by the observation that administration of proteasomal inhibitors to animals can produce the neuropathological and motor manifestations of Parkinson's disease, including selective nigral cell loss, Lewy-bodies-like inclusions and typical clinical signs. Two mechanisms are known to be responsible for a dysfunction in the ubiquitin-proteasome system: aging is associated with a physiologic decrease of proteasome efficiency and ubiquitination activity; oxidative species and alpha-synuclein protofibrils exert a deleterious

Alpha-synuclein oligomers have demonstrated to inhibit proteasome function through direct interaction with 20S subunit; this effect might favor further accumulation of alpha-

The mechanisms behind proteasome inhibition by oligomeric alpha-synuclein require further investigation in order to be clarified. Synphilin-1 has been associated to the pathogenesis of Parkinson's disease since its identification as an alpha-synuclein-interacting protein, a component of Lewy bodies and a substrate of the E3 ligase parkin. This protein has been found to interact with the protein S6 ATPase, which exerts a regulatory function on proteasome. Furthermore, recent studies indicate that synphilin-1 exerts an inhibitory effect on proteasome activity partially due to the interaction with alpha-synuclein (Alvarez-Castelao et al 2010); mutations in synphilin-1 gene have been detected in patients with

synuclein, which in turn may worsen proteasome impairment.

linked to two genes within the ubiquitin-proteasome system: parkin and UCHL1.

Lewy bodies might contrast the toxic effects of the protofibrillar protein.

associated with reduced ligase activity.

effects on proteasome subunits.

sporadic Parkinson's disease.

Autophagy is a finely regulated intracellular process that mediates lysosomal degradation of proteins and organelles. Its function allows the clearance of substrates characterized by alterations limiting their physiologic function or responsible for a cytotoxic effect. This degradative process exerts a cytoprotective role that is probably dependent on the clearance of toxic intracellular structures and the catabolism of substrates in order to obtain energy during starvation. Anyway, in particular situations autophagy seems to mediate a specific pathway of programmed cell death; this function requires a strong activation of autophagy and until now, in vivo, has been identified only during involutional physiologic processes in embryonic tissues (Nixon et al 2006).

In mammalian cells autophagy encompasses three main processes: microautophagy, macroautophagy and chaperone-mediated autophagy. Microautophagy is a constitutive, non selective process consisting on endocytosis of small amounts of cytoplasm into lysosomes through invagination of lysosomal membrane. Macroautophagy and chaperonemediated autophagy are inducible processes: the first one allows lysosomal degradation of organelles and proteins after their sequestration within a double-membrane-limited vacuole called autophagosome; the second one is a selective device for degradation of aberrant proteins, which are directly transported into the lysosomal lumen by a translocation system constituted by specific carrier proteins.

Recent research has revealed the existence of a dysfunction in autophagy pathways in the cerebral regions involved in neurodegenerative processes. A decrease in the activity of both autophagic-lysosomal pathway and the ubiquitin-proteasome system has been reported during aging in every tissue, included neurons (Martinez-Vicente et al 2005). This deficit might be a major responsible for the intracellular accumulation of misfolding proteins and aberrant mitochondria, representing factors leading to oxidative stress and neuronal damage. Therefore, autophagy impairment might be a primum movens of the neurodegenerative processes and, at the same time, a limiting factor that is necessary for preventing early onset of massive neuronal loss.

In the last years the pathogenetic role of altered clearance machinery has become a central subject of research in Parkinson's disease: indeed, the impairment of chaperone-mediated autophagy and macroautophagy, together with the dysfunction of ubiquitin-proteasome system, seems to give a major contribute to the development and progression of nigral degeneration. The loss of function of these degradative pathways might produce a deleterious effect through induction of the pathogenetic processes more directly responsible for neuronal death, such as the accumulation and oligomerization of alpha-synuclein, the persistence of damaged mitochondria and the consequent production of reactive oxygen species. In turn, these key mediators of dopaminergic neurodegeneration can further worsen the impairment of clearance machinery and, in particular, of autophagic pathways (Cuervo et al 2010).

### **4.2.1 Impairment of chaperone-mediated autophagy**

Chaperone mediated autophagy mediates the translocation of proteins containing a Lys-Phe-Glu-Arg-Gln (KFERQ) motif into lysosomes. This process requires the presence of three

Alpha-Synuclein, Oxidative Stress and

**4.2.2 Macroautophagy failure** 

growth factors stimulate the cell.

1. Nucleation of the autophagosome.

3. Maturation of the autophagosome.

activation of macroautophagy.

(Xilouri et al 2009).

and lysosomal clearance of autophagosomes.

pathways.

Autophagy Failure: Dangerous Liaisons in Dopaminergic Neurodegeneration 123

Macroautophagy starts with sequestration of a region of cytoplasm containing proteins and organelles designed for degradation within a double-membrane vacuole called autophagosome. Once formed, the autophagic vacuole undergoes a process of maturation, which is essential for the subsequent fusion with lysosome and the degradation of substrates. The proper function of macroautophagy allows the removal of misfolded proteins and aberrant organelles, which are unsuitable for degradation through other

Macroautophagy is regulated by the mammalian target of rapamycin (mTOR), a

Macroautophagy is suppressed by mTOR in presence of high levels of nutrients or when

Several exogenous molecules are known to activate macroautophagy independently of mTOR activity. These include drugs actually used in the treatment of neurologic and non

Macroautophagy proceeds through various steps, each requiring the presence of specific

Therefore, regulation and biological function of macroautophagy appear to be characterized by a remarkable complexity. Since different variants of this process exist, each one

Although macroautophagy, chaperone mediated autophagy and ubiquitin-proteasome system are responsible for the degradation of different preferential substrates, a functional correlation among them have been demonstrated. In particular, macroautophagy is able to modulate its own activity depending on the efficiency of the other two pathways. Both proteasome and chaperone mediated autophagy inhibition determine a cytoprotective

Macroautophagy has been implied in the pathogenesis of several neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, Huntington's disease and frontotemporal dementia, as well as acute injuries. A role of macroautophagy in the development of these pathologies was proposed when intraneuronal accumulation of autophagic vacuoles was detected in postmortem brains of diseased patients; increasing experimental data indicate that this feature is likely to mirror a dysfunction in maturation

Macroautophagy seems to play a relevant role in the clearance of alpha-synuclein. Both pathogenetic mutations and over-expression of alpha-synuclein determine an induction of macroautophagy, which is dependent on the inhibition of chaperone mediated autophagy

Anyway, the impairment of alpha synuclein degradation seems not to have a major role in mediating the deleterious effect of macroautophagy impairment: the accumulation of other substrates might be more decisive. In this regard, it is important to remind that

serine/threonine kinase on which two main signal transduction pathways converge:

• The AMPK pathway, modulated by intracellular levels of aminoacids and ATP.

• The AKT pathway, modulated by neurotrophic growth factors.

neurologic diseases, such as calcium antagonists, lithium and valproate.

autophagy related genes (Atg). Four main phases can be distinguished:

4. Fusion with lysosome and degradation of substrates.

2. Sequestration of a region of cytoplasm containing substrates to be degraded.

characterized by specific activators, regulatory pathways and biological effects.

main proteins: cytosolic heat shock protein 70 (hsc70), lysosomal hsc70, lysosomalassociated membrane protein 2A (lamp2A). Cytosolic hsc70 binds the KFERQ sequence of substrate proteins and carries them to the lysosomal membrane, where lamp2A, after interaction with cytosolic hsc70, multimerizes and forms a translocation complex with lysosomal hsc70, thus mediating the transport of the substrate protein into the lysosomal lumen. Several proteins with neuropathologic relevance, such as alpha-synuclein, amyloid precursor protein β and huntingtin, contain a KFERQ motif.

The binding of the substrate protein to lamp2A represents the limiting step of chaperone mediated autophagy. Oxidative stress, accumulation of substrates and the lack of nutrients and growth factors are all conditions determining a compensatory and cytoprotective activation of chaperone mediated autophagy through an increase of lamp2A levels on lysosomal membrane.

The hypothesis that a dysfunction of chaperone mediated autophagy might be involved in the pathogenesis of Parkinson's disease was proposed when this process was demonstrated to be the main degradative pathway for alpha-synuclein; indeed, this protein accumulates when chaperone-mediated autophagy is suppressed by downregulation of lamp2A expression. Therefore, the efficiency of this clearance pathway appears to be crucial in regulating the intraneuronal levels of alpha-synuclein. The evidence of a strong correlation between the functional state of chaperone mediated autophagy and the deleterious action of alpha-synuclein has been reinforced by demonstration that both pathogenetic mutations and overexpression of α-synuclein inhibit this process (Cuervo et al 2004, Xilouri et al 2009). Experiments performed on animal models over-expressing alpha-synuclein have revealed that down-regulation of chaperone mediated autophagy is responsible for part of the alpha-synuclein toxicity in dopaminergic neurons. A reduced turnover of proteins directly involved in the neuronal survival and in the apoptotic machinery may mediate the deleterious effect of alphasynuclein-mediated inhibition of this clearance pathway.

Therefore, it is conceivable to assume that other substrates of chaperone mediated autophagy contribute to neuronal death through their accumulation. For example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Tatton 2000) and myocyte enhancer factor 2D (MEF2D) (Yang et al 2009) are substrates of this degradative pathway and, at the same time, are specifically involved in the molecular physiopathology of Parkinson's disease; these factors could represent key mediators of the neuronal death when chaperone mediated autophagy is impaired.

Besides mutations and multiplications of alpha-synuclein gene, other mutations linked to hereditary Parkinson's disease are responsible for the impairment of chaperone mediated autophagy. Pathogenetic mutation I93M of PARK5 gene has been demonstrated to determine the inhibition of chaperone mediated autophagy and the accumulation of alphasynuclein (Kabuta et al 2008).

The finding of low levels of lamp2A and total hsc70 in post-mortem substantia nigra of patients with sporadic disease indicates that a reduced activity of chaperone mediated autophagy is likely to be a pathogenetic mechanism even in idiopathic Parkinson's disease (Alvarez-Erviti 2010).

In conclusion, the failure of chaperone mediated autophagy seems to be a pathogenetic mechanism favoring the death of dopaminergic neurons and, as a consequence, contributing to the development and progression of Parkinson's disease.

### **4.2.2 Macroautophagy failure**

122 Etiology and Pathophysiology of Parkinson's Disease

main proteins: cytosolic heat shock protein 70 (hsc70), lysosomal hsc70, lysosomalassociated membrane protein 2A (lamp2A). Cytosolic hsc70 binds the KFERQ sequence of substrate proteins and carries them to the lysosomal membrane, where lamp2A, after interaction with cytosolic hsc70, multimerizes and forms a translocation complex with lysosomal hsc70, thus mediating the transport of the substrate protein into the lysosomal lumen. Several proteins with neuropathologic relevance, such as alpha-synuclein, amyloid

The binding of the substrate protein to lamp2A represents the limiting step of chaperone mediated autophagy. Oxidative stress, accumulation of substrates and the lack of nutrients and growth factors are all conditions determining a compensatory and cytoprotective activation of chaperone mediated autophagy through an increase of lamp2A levels on

The hypothesis that a dysfunction of chaperone mediated autophagy might be involved in the pathogenesis of Parkinson's disease was proposed when this process was demonstrated to be the main degradative pathway for alpha-synuclein; indeed, this protein accumulates when chaperone-mediated autophagy is suppressed by downregulation of lamp2A expression. Therefore, the efficiency of this clearance pathway appears to be crucial in regulating the intraneuronal levels of alpha-synuclein. The evidence of a strong correlation between the functional state of chaperone mediated autophagy and the deleterious action of alpha-synuclein has been reinforced by demonstration that both pathogenetic mutations and overexpression of α-synuclein inhibit this process (Cuervo et al 2004, Xilouri et al 2009). Experiments performed on animal models over-expressing alpha-synuclein have revealed that down-regulation of chaperone mediated autophagy is responsible for part of the alpha-synuclein toxicity in dopaminergic neurons. A reduced turnover of proteins directly involved in the neuronal survival and in the apoptotic machinery may mediate the deleterious effect of alpha-

Therefore, it is conceivable to assume that other substrates of chaperone mediated autophagy contribute to neuronal death through their accumulation. For example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Tatton 2000) and myocyte enhancer factor 2D (MEF2D) (Yang et al 2009) are substrates of this degradative pathway and, at the same time, are specifically involved in the molecular physiopathology of Parkinson's disease; these factors could represent key mediators of the neuronal death when chaperone

Besides mutations and multiplications of alpha-synuclein gene, other mutations linked to hereditary Parkinson's disease are responsible for the impairment of chaperone mediated autophagy. Pathogenetic mutation I93M of PARK5 gene has been demonstrated to determine the inhibition of chaperone mediated autophagy and the accumulation of alpha-

The finding of low levels of lamp2A and total hsc70 in post-mortem substantia nigra of patients with sporadic disease indicates that a reduced activity of chaperone mediated autophagy is likely to be a pathogenetic mechanism even in idiopathic Parkinson's disease

In conclusion, the failure of chaperone mediated autophagy seems to be a pathogenetic mechanism favoring the death of dopaminergic neurons and, as a consequence, contributing

precursor protein β and huntingtin, contain a KFERQ motif.

synuclein-mediated inhibition of this clearance pathway.

to the development and progression of Parkinson's disease.

mediated autophagy is impaired.

synuclein (Kabuta et al 2008).

(Alvarez-Erviti 2010).

lysosomal membrane.

Macroautophagy starts with sequestration of a region of cytoplasm containing proteins and organelles designed for degradation within a double-membrane vacuole called autophagosome. Once formed, the autophagic vacuole undergoes a process of maturation, which is essential for the subsequent fusion with lysosome and the degradation of substrates. The proper function of macroautophagy allows the removal of misfolded proteins and aberrant organelles, which are unsuitable for degradation through other pathways.

Macroautophagy is regulated by the mammalian target of rapamycin (mTOR), a serine/threonine kinase on which two main signal transduction pathways converge:


Macroautophagy is suppressed by mTOR in presence of high levels of nutrients or when growth factors stimulate the cell.

Several exogenous molecules are known to activate macroautophagy independently of mTOR activity. These include drugs actually used in the treatment of neurologic and non neurologic diseases, such as calcium antagonists, lithium and valproate.

Macroautophagy proceeds through various steps, each requiring the presence of specific autophagy related genes (Atg). Four main phases can be distinguished:


Therefore, regulation and biological function of macroautophagy appear to be characterized by a remarkable complexity. Since different variants of this process exist, each one characterized by specific activators, regulatory pathways and biological effects.

Although macroautophagy, chaperone mediated autophagy and ubiquitin-proteasome system are responsible for the degradation of different preferential substrates, a functional correlation among them have been demonstrated. In particular, macroautophagy is able to modulate its own activity depending on the efficiency of the other two pathways. Both proteasome and chaperone mediated autophagy inhibition determine a cytoprotective activation of macroautophagy.

Macroautophagy has been implied in the pathogenesis of several neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, Huntington's disease and frontotemporal dementia, as well as acute injuries. A role of macroautophagy in the development of these pathologies was proposed when intraneuronal accumulation of autophagic vacuoles was detected in postmortem brains of diseased patients; increasing experimental data indicate that this feature is likely to mirror a dysfunction in maturation and lysosomal clearance of autophagosomes.

Macroautophagy seems to play a relevant role in the clearance of alpha-synuclein. Both pathogenetic mutations and over-expression of alpha-synuclein determine an induction of macroautophagy, which is dependent on the inhibition of chaperone mediated autophagy (Xilouri et al 2009).

Anyway, the impairment of alpha synuclein degradation seems not to have a major role in mediating the deleterious effect of macroautophagy impairment: the accumulation of other substrates might be more decisive. In this regard, it is important to remind that

Alpha-Synuclein, Oxidative Stress and

vulnerability to systemic biochemical alterations.

clinical research aimed at identifying biomarkers of disease.

Different rationales justify the use of biomarkers for Parkinson's disease:

**5.1 Peripheral biomarkers** 

neurons.

**degeneration** 

Autophagy Failure: Dangerous Liaisons in Dopaminergic Neurodegeneration 125

Glucocerebrosidase (GBA) is a lysosomal enzyme that catalyzes the hydrolysis of the lipid glucosylceramide into glucose and ceramide. Homozygous mutations of GBA gene are responsible for Gaucher syndrome, a multisystem lysosomal storage disease characterized by neurodegenerative manifestations. Recently, carriers of GBA pathogenetic mutations have been identified at higher risk of sporadic Parkinson's disease (Sidransky et al 2009). This confirms the great importance of lysosomal function for the survival of dopaminergic

**5. Neuropathologic outcome of molecular physiopathology: More than nigral** 

In the previous paragraphs we have reported evidences regarding the occurrence of specific deleterious processes in degeneration of dopaminergic neurons; furthermore, we have highlighted the functional correlations that have been demonstrated among these processes. A wide body of data confirms that each of these mechanisms can be responsible for degeneration of dopaminergic neurons and involutional modifications of the substantia nigra. These modifications are responsible for the motor symptoms that represent the major criteria for diagnosis of Parkinson's disease. The reasons of the selective damage of nigral neurons is not currently known; particular biological conditions might favor the onset of pathogenetic mechanisms in these neurons or could be responsible for an extreme

However, the selectiveness of nigral involvement in Parkinson's disease is not absolute. In fact, the cardinal symptoms of Parkinson's disease are often preceded by a series of early manifestations, such as depression, sleep disturbances, eye movement disorders, hyposmia, constipation and loss of cardiac and vasomotor reflexes, which can start several years before the motor disturbances. The neuropathologic substrate of these aspecific manifestations does not seem to be ascribable to the degeneration of substantia nigra, rather they would derive from the involvement of other monoaminergic neurons in central and peripheral nervous system. Postmortem studies in patients with Parkinson's disease have provided evidence of a typical temporal pattern of pathology progression from peripheral neurons, spinal cord and brainstem to basal ganglia and cortical regions. These studies have revealed the precocious degeneration of autonomic ganglia of mesenteric plexus, dorsal motor nucleus of vagus, rafe nuclei, locus ceruleus and ventral tegmental area; the involvement of these centers is consistent with the clinical symptoms preceding parkinsonian signs. Based on these premises, the degeneration of substantia nigra appears as a late manifestation of the neuropathologic process; moreover, the onset of the characteristic motor manifestations is usually insidious and can be appreciated only when about 50% to 60% of dopaminergic neurons have been lost. These remarks indicate that the diagnosis of Parkinson's disease is always made when the underlying neuropathologic process has already reached an advanced phase; this limits the benefits that can be expected from putative neuroprotective therapies. Hence, it would be desirable to be endowed of diagnostic tools useful to make diagnosis of Parkinson's disease during early or even pre-clinical stages. This assumption has promoted the pre-clinical and

macroautophagy represents the only mechanism able to mediate the clearance of damaged mitochondria through a process named mitophagy. The intraneuronal accumulation of aberrant mitochondria determines neurotoxic effects linked to the generation of reactive oxygen species and the release of pro-apoptotic mediators. Increasing evidence from transgenic models of disease suggests that a defect in the mitophagy pathway might exert an key pathogenetic role in Parkinson's disease. In fact, genes responsible for hereditary disease are essential components of mitophagy machinery: PINK1 and parkin, two genes linked to recessive forms of Parkinson's disease, encode proteins that work synergistically to ensure the sequestration of aberrant mitochondria within the autophagic vacuole Narendra et al 2010).

Even DJ1, a gene linked to autosomal recessive Parkinson's disease, encodes for a protein that activates macroautophagy and favors mitochondrial turnover (Krebiehl et al 2010).

Loss of function of PINK1, parkin or DJ1 causes hereditary Parkinson's disease and the death of dopaminergic neurons in cell and animal models: the decreased efficiency of macroautophagy and mitophagy might be responsible for part of this neurotoxic effect.

Based on these assumptions, the efficacy of mitochondria turnover should be evaluated as a putative defective step linked to macroautophagy dysfunction even in idiopathic Parkinson's disease.

#### **4.2.3 Loss of lysosomal function**

The hypothesis that macroautophagy might be impaired in Parkinson's disease only apparently contrasts with the accumulation of autophagosomes and the increase of LC3II levels that have been demonstrated in postmortem substantia nigra of diseased patients. As previously reported, recent data suggest that a defect in maturation of autophagosome and in its fusion with lysosome could be the major responsible for the accumulation of autophagic vacuoles. Indeed, a depletion of the intraneuronal lysosomal pool and decreased levels of lysosome associated proteins, such as LAMP1, cathepsin D and heat shock protein 73, have been detected in nigral neurons of patients with sporadic Parkinson's disease. Furthermore, lysosomal depletion has been found in dopaminergic neurons of a MPTP mouse model (Dehay et al 2010); in this study the loss of lysosomes has been identified as an early alteration preceding the accumulation of autophagosomes and the neuronal degeneration. Oxidative injury has been found to favor lipid peroxidation of lysosomal membrane and, at last, to reduce the pool of lysosomes. Therefore, a strong and synergistic correlation seems to exist between oxidative stress and the dysfunction of autophagiclysosomal pathway.

An additional proof of the protective role of lysosomal function in dopaminergic neurons has derived from the finding that mutations of genes encoding lysosomal proteins can modify the risk of developing Parkinson's disease. ATP13A2 (PARK9) is a lysosomal transmembrane cation transporting ATPase that is necessary to maintain acidity in the lysosomal lumen; low pH is indispensable for the function of lysosomal hydrolases and, as a consequence, for degradation of autophagy substrates. Homozygous mutations of ATP13A2 gene causes Kufor-Rakeb syndrome, characterized by early-onset parkinsonism with pyramidal degeneration and dementia. In vitro experiments have demonstrated that loss of ATP13A2 impairs lysosomal degradation of alpha-synuclein and cause intraneuronal accumulation of this protein (Gitler et al 2009).

macroautophagy represents the only mechanism able to mediate the clearance of damaged mitochondria through a process named mitophagy. The intraneuronal accumulation of aberrant mitochondria determines neurotoxic effects linked to the generation of reactive oxygen species and the release of pro-apoptotic mediators. Increasing evidence from transgenic models of disease suggests that a defect in the mitophagy pathway might exert an key pathogenetic role in Parkinson's disease. In fact, genes responsible for hereditary disease are essential components of mitophagy machinery: PINK1 and parkin, two genes linked to recessive forms of Parkinson's disease, encode proteins that work synergistically to ensure the sequestration of aberrant mitochondria within the autophagic vacuole Narendra

Even DJ1, a gene linked to autosomal recessive Parkinson's disease, encodes for a protein that activates macroautophagy and favors mitochondrial turnover (Krebiehl et al 2010). Loss of function of PINK1, parkin or DJ1 causes hereditary Parkinson's disease and the death of dopaminergic neurons in cell and animal models: the decreased efficiency of macroautophagy and mitophagy might be responsible for part of this neurotoxic effect. Based on these assumptions, the efficacy of mitochondria turnover should be evaluated as a putative defective step linked to macroautophagy dysfunction even in idiopathic

The hypothesis that macroautophagy might be impaired in Parkinson's disease only apparently contrasts with the accumulation of autophagosomes and the increase of LC3II levels that have been demonstrated in postmortem substantia nigra of diseased patients. As previously reported, recent data suggest that a defect in maturation of autophagosome and in its fusion with lysosome could be the major responsible for the accumulation of autophagic vacuoles. Indeed, a depletion of the intraneuronal lysosomal pool and decreased levels of lysosome associated proteins, such as LAMP1, cathepsin D and heat shock protein 73, have been detected in nigral neurons of patients with sporadic Parkinson's disease. Furthermore, lysosomal depletion has been found in dopaminergic neurons of a MPTP mouse model (Dehay et al 2010); in this study the loss of lysosomes has been identified as an early alteration preceding the accumulation of autophagosomes and the neuronal degeneration. Oxidative injury has been found to favor lipid peroxidation of lysosomal membrane and, at last, to reduce the pool of lysosomes. Therefore, a strong and synergistic correlation seems to exist between oxidative stress and the dysfunction of autophagic-

An additional proof of the protective role of lysosomal function in dopaminergic neurons has derived from the finding that mutations of genes encoding lysosomal proteins can modify the risk of developing Parkinson's disease. ATP13A2 (PARK9) is a lysosomal transmembrane cation transporting ATPase that is necessary to maintain acidity in the lysosomal lumen; low pH is indispensable for the function of lysosomal hydrolases and, as a consequence, for degradation of autophagy substrates. Homozygous mutations of ATP13A2 gene causes Kufor-Rakeb syndrome, characterized by early-onset parkinsonism with pyramidal degeneration and dementia. In vitro experiments have demonstrated that loss of ATP13A2 impairs lysosomal degradation of alpha-synuclein and cause intraneuronal

et al 2010).

Parkinson's disease.

lysosomal pathway.

accumulation of this protein (Gitler et al 2009).

**4.2.3 Loss of lysosomal function** 

Glucocerebrosidase (GBA) is a lysosomal enzyme that catalyzes the hydrolysis of the lipid glucosylceramide into glucose and ceramide. Homozygous mutations of GBA gene are responsible for Gaucher syndrome, a multisystem lysosomal storage disease characterized by neurodegenerative manifestations. Recently, carriers of GBA pathogenetic mutations have been identified at higher risk of sporadic Parkinson's disease (Sidransky et al 2009). This confirms the great importance of lysosomal function for the survival of dopaminergic neurons.
