S.I. : Genetic pathways to Neurodegeneration

Neurodegenerative diseases: model organisms, pathology and autophagy

SN Suresh1, Vijaya Verma2, Shruthi Sateesh2, James P Clement2,

Ravi Manjithaya1,2*

Molecular Biology and Genetics Unit1, Neuroscience Unit2, Jawaharlal Nehru

Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore 560 064

*Corresponding author: Ravi Manjithaya (ravim@jncasr.ac.in)

Abstract

A proteostasis view of neurodegeneration (ND) identifies protein aggregation as a

leading causative reason for damage seen at the cellular and organ level. While

investigative therapies that aim at dissolving aggregates have failed, and the promises

of silencing expression of ND associated pathogenic proteins or the deployment of

engineered iPSCs are still in the horizon, emerging literature suggests degrading

aggregates via autophagy related mechanisms hold the current potential for a possible

cure. Macroautophagy (hereafter autophagy) is an intracellular degradative pathway

where superfluous or unwanted cellular cargoes (such as peroxisomes, mitochondria,

ribosomes, intracellular bacteria and misfolded protein aggregates) are wrapped in

double membrane vesicles called autophagosomes that eventually fuses with lysosomes

for their degradation. The selective branch of autophagy that deals with identification,

capture and degradation of protein aggregates is called aggrephagy. Here, we cover the

workings of aggrephagy detailing its selectivity towards aggregates. The diverse

cellular adaptors that bridge the aggregates with the core autophagy machinery in terms

of autophagosome formation are discussed. In ND, essential protein quality control

mechanisms fail as the constituent components also find themselves trapped in the

aggregates. Thus, although cellular aggrephagy has the potential to be upregulated, its

dysfunction further aggravates the pathogenesis. This phenomenon when combined

with the fact that neurons can neither dilute out the aggregates by cell division nor the

dead neurons can be replaced due to low neurogenesis, makes a compelling case for

aggrephagy pathway as a potential therapeutic option.

Introduction

Cellular homeostasis is achieved through a balance of anabolic and catabolic states.

Cells possess several quality control mechanisms to identify, correct or remove

dysfunctional or potential toxic cellular components such as proteins and organelles.

For example, inside cells, at steady state, misfolded proteins are formed continuously

and are fixed by chaperones or cleared via the ubiquitin proteasome system and

autophagy related pathways. This maintains the proteostatic equilibrium in cells.

Altered cellular states resulting from expression of pathogenic levels or mutant forms

of aggregate prone proteins overwhelm these quality control systems and their build-

up can eventually result in cell death. This is the fate of brain cells in neurodegenerative

diseases (NDD). In this review, we unravel this proteostatic central view as a cause for

ND, discuss the potential reasons behind the failure of quality control systems with an

emphasis on an aggrephagy, autophagy related pathway.

Proteostasis

Protein quality control machineries ensure the proper folding of newly synthesized

proteins for their distinct function. This process is critical as 30% of these new proteins

are prone to misfolding (Mymrikov et al., 2017). The cellular quality control measures

include unfolding, refolding and/or degradation of the misfolded proteins (Sontag et al.,

2017). Not surprisingly, failure of protein quality control poses a threat to the cellular

vitality (Balch et al., 2008). This qualitative process of maintaining the homeostasis of

intracellular pool of functional and “healthy” proteins is called proteostasis.

Proteostasis as a function for cell survival becomes even more critical for those cells

such as neurons that cannot divide and thus dilute out the toxic protein aggregates

(Balch et al., 2008). In addition, with age, the neuronal proteostatic machineries

become incompetent and prone for accumulation of damaged organelles and misfolded

proteins (Labbadia & Morimoto, 2015; Walther et al., 2017).

Protein misfolding is not an uncommon phenomenon inside cells. The presence of

misfolded protein activates chaperones to unfold and refold them in an ATP-dependent

manner. Misfolded proteins induce heat shock response (HSR) and heat shock (HS)

transcription factor HSF-1 that include the upregulation of heat shock cognate protein

(Hsc70) (Kampinga & Craig, 2010). The aim of enhancing chaperone expression and

its activity is to prevent protein aggregation. Once the proteins remain misfolded

despite the efforts by chaperones, they will form the higher order structures such as

oligomers and aggregates that will eventually accumulate inside cells. In certain

scenarios, when protein aggregates overwhelm the chaperone capacity, the available

chaperones themselves get trapped in protein aggregates (Anckar & Sistonen, 2011;

Raychaudhuri et al., 2014). Subsequently, these events also aggravate disease

pathogenesis. An additional mechanism that has been recently described identifies

misfolded proteins as early as they are translating and subsequently marks them for

degradation. Such nascent misfolded polypeptides are ubiquitinated at K-48 residue as

a degradation mark in a process termed as cotranslational ubiquitination (CTU) (Wang

et al., 2013). The polypeptide with this mark will be acted upon by proteasome for

their effective degradation. CTU happens within the active translation complexes

(Wang et al., 2013).

At the organellar level, accumulation of misfolded proteins inside endoplasmic

reticulum (ER) lumen induces ER stress, which triggers one of the vital cellular

adaptive mechanisms known as unfolded protein response (UPR) (Powers et al., 2009).

UPR leads to suppression of active protein translation, increase ER chaperone

accumulation to unfold and/or degrade these proteins to ameliorate the proteotoxicity

(Hetz, 2012). Post-mortem brain analyses of Alzheimer’s disease (AD), Parkinson’s

disease (PD) and Huntington’s disease (HD) have revealed the correlation of UPR

markers with protein aggregation and onset of disease pathogenesis (Hetz & Mollereau,

2014).

The fate of misfolded proteins, not refolded by chaperones, are marked for degradation

through ubiquitin-proteasome system (UPS) and/or autophagy. UPS degrades

ubiquitinated, soluble and short-lived proteins. The target protein is tagged by ubiquitin

through three enzymatic ubiquitin- activating (E1), - conjugating (E2) and –ligating

(E3) reactions (Goldberg, 2003). Ubiquitin is attached through its carboxyl residue to

specific lysine residue through isopeptide linkage. One ubiquitin molecule is a target

for another ubiquitin molecule and forms a polyubiquitin chain at its lysine residues at

48, 63 or at N-terminal methionine residue (Kirkin et al., 2009). The polyubiquitin

signal at K48 serves as a proteasome degradation signal whereas K63 and N-term

methionine signals serve other functions (Kirkin et al., 2009). It is also proposed that

K63 polyubiquitination may target a protein to autophagy, but the exact mechanism is

unclear.

UPS has been shown to degrade several neurodegenerative disease related proteins such

as tau, SOD1 and α-synuclein (Goldberg, 2003). Inhibiting UPS accumulate disease

related proteins to aggregate and form inclusion bodies inside cells. Thus, UPS is

essential for cells to prevent toxic protein aggregate formation (Lim & Yue, 2015). It

is believed that larger aggregates that cannot be resolved by UPS are the substrates of

the autophagy machinery.

Aggrephagy: definition and modes

In 1960s, the Nobel laureate Christian De Duve observed double membrane vesicles

entrapping intracellular organelles and proteins and coined the term “self-eating” or

autophagy (De Duve & Wattiaux, 1966). Nobel laureate Yoshinori Ohsumi tapped the

power of yeast genetics to elucidate the molecular mechanism governing autophagy

and contributed to the discovery of its conserved nature from yeast to mammals

(Harnett et al., 2017).

Depending on the distinct molecular mechanisms, autophagy is broadly classified into

three types: Macroautophagy, Microautophagy and Chaperone mediated autophagy

(CMA). Macroautophagy is the most extensively studied process that has an

indispensable role in maintaining cellular and organismal homeostasis. During

macroautophagy, a phagophore or isolation membrane forms and expands to form

double membrane autophagosomes that engulfs cellular cargoes. These cargoes are

parts of cytosol constituting superfluous organelles, pathogenic organisms, misfolded

protein aggregates and/or damaged mitochondria (Zaffagnini & Martens, 2016). These

autophagosomes fuse with lysosomes to form autolysosomes and eventually degrade

its intraluminal cargoes. The degraded entities result in building blocks that are recycled

back to the cytosol for fuelling other cellular pathways. Apart from randomly

sequestering portions of cytosol for degradation (general autophagy), macroautophagy

can be highly selective in the cargoes it captures. The selective autophagy pathway that

is involved in clearance of misfolded protein aggregates is called aggrephagy (Hyttinen

et al., 2014). Here, misfolded proteins that are tagged by ubiquitin are recognized by

adaptor proteins such as p62, NBR1 and NDP52 which in turn recruit autophagy

proteins such as LC3 to facilitate selective capture (Figure 1).

Thus, aggrephagy is a prominent defense mechanism against misfolded protein

aggregate mediated cellular toxicity. Microautophagy is the direct uptake of cargo by

lysosomes through membrane invagination. Chaperone Mediated Autophagy (CMA) is

the selective degradative process of proteins that involves Hsc70 but is a vesicle

independent process. During CMA, the target protein is recognized by Hsc70 through

a specific amino acid motif, KFERQ and delivered to lysosome by interacting with

LAMP2A in an ATP-dependent manner.

Synaptic dysfunction in NDD

Synaptic plasticity refers to the ability of synapses to modify their structure and tonus

after persistent electrical activity and/or signalling. In fact, the number, morphology,

position, molecular phenotype, and strength of synapses continue to modify as a

function of neurons’ requirements. These events take place in the nervous system

during its development, which represents the basis for learning and memory (Bliss &

Collingridge, 1993; Citri & Malenka, 2008). In case of neurons, in addition to the

autophagy process in soma, autophagy occurring at the synapse, of late, has received

much attention as it involves immediate turnover of proteins and, thus, affects synaptic

transmission. Mutations in genes involved in several neurodegenerative diseases such

as Alzheimer, Huntington, ALS and Parkinson disease affect synaptic proteins levels

and functions (Lepeta et al., 2016). Synaptic dysfunction throughout the central and

peripheral nervous systems has shown to be an early hallmark of neurodegenerative

diseases preceding neuronal death and the subsequent onset of clinical symptoms

(Figure 2 and Table 1) (Brose et al., 2010). These events have been validated using

transgenic mouse models available for different neurodegenerative diseases

(Trancikova et al., 2011).

Neurodegeneration model systems as tools to study aggrephagy

Numerous model systems have been utilized to study the aggrephagy and modulate it

to clear the protein aggregates. It is important to note that the cellular proteostatic

machineries are conserved from simple yeast to higher model rodents. We will briefly

discuss the different model systems used to understand the neurodegeneration

pathogenesis with a special emphasis on autophagy.

Animal models to study NDD

Historically, mouse has been used as a model to study genetic mutation-causing

diseases in humans, especially brain related disorders including neurodegeneration

diseases, due to the similarity of mammalian neuronal physiology and anatomy to

human brain. The main rationale to model human disorders in non-human organisms is

the identification of fundamental pathogenic mechanisms that could lead to potential

novel therapeutic targets, and the elevation of efficacy and safety of potential drugs. A

prerequisite for clinical trials of a compound in humans is the successful alleviation of

the disease or symptoms in animal models.

The occurrence of neurodegeneration is majorly a sporadic event with poorly

understood etiological basis. However, in familial cases of AD, PD, HD and ALS, the

genetic mutations are the underlying causes of disease phenotypes and these can be

recapitulated in animal models. Due to a rapid increase in the availability of the type of

genetically modified mice (Branchi et al., 2003), it is critical to meticulously

characterise the biochemical, pathological and behavioural features of these mice and

compare them with human phenotypes (Crawley, 2008). Generally, laboratories

involved in testing the phenotypes of genetically modified mice subject these mice to a

battery of behavioural features to assess cognitive, motor and sensory functions. To

consider a genetically modified mouse as a disease model, it must fulfil three levels of

validity to judge its psychopharmacology (van der Staay et al., 2009). An animal model

should score high on the following validities: face validity, i.e., resemblances of

behavioural phenotypes of mouse model to that of human disorder; construct validity,

i.e., closely reconstructs and mimics the underlying cause of the disease or disorder;

and predictive validity, i.e., treatments alleviate symptoms in mouse and human. A

successful mouse model should fulfil face and construct validity before being tested for

therapeutics (Predictive validity). Due to these varied features and their behavioural

readouts, the mouse models of the respective human diseases are highly useful in NDD

research and in preclinical studies.

1. Parkinson disease (PD)

Parkinson disease (PD) is characterized by progressive degeneration of nigrostriatal

dopaminergic neurons, leading to loss of motor function, rigidity, postural instability,

tremor, and bradykinesia as described in the 1800’s by James Parkinson (Mhyre et al.,

2012; Rouse et al., 2000). Both familial (5–10% of all cases) and acquired (90–95% of

all cases) forms of Parkinsonism are usually caused by defects in dopamine (DA)

metabolism. Decrease in DA inputs from basal ganglia (BG) result in impaired control

of motor circuits that ultimately leads to clinical symptoms (Spillantini et al., 1997).

For the PD mouse model, the transgenics express pathogenic mutant version of PD

associated genes such as α-Synuclein, Parkin, Pink1 and Park7. α-Synuclein transgenic

mice exhibit Lewy bodies that are the histopathological hallmark of PD. These animals

exert age-related progressive movement deficits, which is associated with

dopaminergic neuronal loss. However, not all transgenic mutants exert significant

dopaminergic neuronal loss.

Activity-dependent modifications in synaptic efficacy, such as long-term depression

(LTD) and long-term potentiation (LTP) represent the key cellular substrates for

adaptive motor control and procedural memory (Bliss & Collingridge, 1993).

Dopamine (DA) acting on D1- and D2-like receptors play a critical role in driving the

above-mentioned forms of synaptic plasticity in striatum. D1 and D2 receptor signalling

pathways converge in opposite manners on a common target, DARPP-32. The

involvement of DA in these phenomena has been thoroughly established by the study

of synaptic plasticity in striatal neurons recorded from rodent models of PD (Calabresi

et al., 1992a; Calabresi et al., 1992b). The absence of synaptic plasticity and abnormal

synapse structure implied the cellular basis underlying the abnormalities in striatal

output within the Basal ganglia (BG), and consequently resulting in PD symptoms.

Abnormalities in the subcellular distribution of N-methyl-D-aspartate receptor

(NMDAR) subunit GRIN2B represent one of the major changes that take place at

corticostriatal glutamatergic synapses. In fact, studies using dyskinetic mouse models,

increased levels of GRIN2A and lower levels of GRIN2B were observed in extra

synaptic sites which altered binding of NMDAR subunits with their cargo proteins such

as Synapse-associated protein 97 (SAP97) and SAP102 (Gardoni et al., 2006; Sheng &

Sala, 2001). Moreover, activation of DARPP-32 (dopamine- and cAMP-regulated

phosphoprotein-32) results in increased opening of the L-type Ca2+ channels promoting

the transition of Medium Spiny Neurons (MSN) to a higher level of excitability, which

in turns phosphorylates AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid) and NMDARs providing a mechanism for the direct control of glutamatergic

transmission by DA signalling, that is altered in PD (Greengard et al., 1998; Vergara et

al., 2003). This is further evident from the studies that show the dopamine-denervation

augments neuronal excitability in the striatum leading to excitability of striatal neurons,

which is caused due to the increased glutamatergic cortical inputs to the striatum.

(Calabresi et al., 1993; Centonze et al., 2004). The increased glutamate level in the

synaptic cleft (Herrera-Marschitz et al., 1994), is consequently responsible for the

overactivity of NMDARs and AMPARs on MSNs which correlate with the motor

behaviour abnormalities observed in a rat model of PD. Thus, functional changes of the

striatal neurons may alter the output signals from the striatum to the other structures of

the basal ganglia that leads to pathophysiological changes observed in Parkinson's

disease.

Evidences also suggest that the genes linked to PD play a critical role in regulating

proper presynaptic and synaptic vesicular transport, modification of DA flow and

altered presynaptic plasticity (Dihanich & Manzoni, 2011). Once Parkinsonism is well

established, i.e. parkinsonian state, most BG mechanisms are insufficient and cortical

mechanisms become important (Blesa et al., 2017; Dihanich & Manzoni, 2011). At the

postsynaptic level, decreased activation of D2 receptors leads to a disinhibition of

voltage-gated ion channels and increased influx of Ca2+ that leads to degeneration of

the synapses observed in PD-affected animals and in PD patients (Arbuthnott et al.,

2000; Calautti et al., 2007; Nitsch & Riesenberg, 1995). In DA-denervated striatum,

NMDAR subunit GRIN1, and its interacting protein at synapse, PSD-95, levels are

selectively reduced in the post-synapse (Lundblad et al., 2004).

2. Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease

characterized by progressive loss of upper motor neurons in the motor cortex (cortical

layer of pyramidal cells of cortical layer V), and of lower motor neurons in the

brainstem and spinal cord as first described by Jean-Martin Charcot in 1869 (Kumar et

al., 2011). ALS typically affects adults in mid-life, with an incidence of 1–

2/100,000/year (Rosen, 1993). Most ALS patients have no affected family members

and are considered to have sporadic ALS. Familial ALS occurs in 5–10% of cases and

has an autosomal dominant inheritance. Mutations in genes such as TARDP (TDP-43),

FUS and SOD1 drive the familial forms of ALS. In 20% of familial cases, mutations in

the superoxide dismutase-1 (SOD1) gene on chromosome 21q were identified (ALS1)

(Rosen, 1993). The recessive, juvenile ALS2, is caused by mutations in ALSIN, which

codes for a protein containing guanine exchange factor domains (Hadano et al., 2001;

Yang et al., 2001). Mice transgenics overexpressing wild type or mutant TDP-43

proteins cause TDP-43 inclusion bodies and loss of motor neurons with behavioural

impairments. In addition, mice overexpressing mutant SOD-1 develops inclusion

bodies, neuronal loss, gliosis, tremor, hindlimb paralysis with significant reduction in

lifespan.

The pathological hallmark of ALS is the degeneration of lower motor neurons in the

brainstem and spinal cord, upper motor neurons in the motor cortex, and of the

corticospinal tracts, accompanied by reactive gliosis (Pehar et al., 2005). The exact

pathogenic mechanism underlying the selective motor neuron death in ALS is yet to be

elucidated, although many possible mechanisms in sporadic ALS, and SOD1-linked

ALS have been proposed (Brown & Robberecht, 2001; Cleveland & Rothstein, 2001;

Heath & Shaw, 2002; Julien, 2001). One of the common reasons for neuronal

degeneration is because of the overstimulation of glutamate receptors induced

excitotoxicity (Lipton & Rosenberg, 1994).

Evidences from the past studies have strengthened a link between glutamate-mediated

toxicity and sporadic ALS. Motor neurons express Ca2+-permeable AMPARs, GRIA2

subunit, and are, therefore, particularly vulnerable to AMPAR-mediated excitotoxicity

(von Lewinski & Keller, 2005). Transgenic mice expressing GRIA2 subunits with an

asparagine at the Q/R site (conferred a two-fold increase in the Ca2+ permeability of

AMPARs) (Feldmeyer et al., 1999), and showed late-onset of degeneration of spinal

neurons and a decline in motor function (Kuner et al., 2005). Crossing these mice with

those carrying a mutation linked to familial ALS accelerated disease progression and

motor decline (Kuner et al., 2005), and the phenotype is exaggerated when GRIA2 is

deleted (Van Damme et al., 2005).

The NMDAR-mediated neurotoxicity and subsequent overload of mitochondrial Ca2+

and ROS production has been shown to take place in cultured motor neurons (Carriedo

et al., 2000). Furthermore, the NMDAR/Ca2+ mediated excitotoxicity has been

demonstrated in the neuronal loss observed in spinal neurons obtained from human low

molecular weight Neuro filament (NF) protein (hNfl+/+) mice, an ALS mouse model

(Kambe et al., 2011; Nicholls et al., 2007; Sanelli et al., 2007). Compelling evidence

supports the fact that excessive Ca2+ influx through NMDARs targets mitochondria

which results in excitotoxicity in ALS-related MN death (Peng et al., 1998). It is

intriguing to hypothesize that regain control of the NMDAR functionality will in turn

affect AMPAR function, due to the close interaction between these receptors, thereby

further hampering the ALS-related excitotoxic drive.

Apart from the extensive loss of motor neurons, there is degeneration of midbrain

dopaminergic cells and reduced tyrosine hydroxylase positive neurons which has been

described in both familial and sporadic forms of ALS, (Andreassen et al., 2000; Kostic

et al., 1997), and functional alteration of the voltage-dependent Na2+ channel (Zona et

al., 1998). The dysfunction in the DA neurons is shown by investigating the

corticostriatal synaptic plasticity in mice overexpressing the human SOD1 and mutating

the (Gly933Ala) form (G93A) of the same enzyme(Calabresi et al., 2000; Lovinger et

al., 1993).

The pivotal role of DA in corticostriatal LTD is well established. For example, injection

of 6-hydroxydopamine in rodent model leads to DA denervation that modifies the

corticostriatal plasticity (Calabresi et al., 1992a; Calabresi et al., 1992b) . Moreover, a

decreased striatal D2 receptor binding in sporadic ALS has been described and is likely

to occur because of an excessive glutamatergic corticostriatal neurotransmission

(Vogels et al., 1999; Vogels et al., 2000). This pattern of degeneration is consistent

with the observation that elevated levels of SOD1 are expressed in basal ganglia cells,

including the midbrain dopaminergic neurons (Pardo et al., 1995). In conclusion,

perturbations in these systems may cause the cell to become more susceptible to

excitotoxic damage.

There is spectrum of synaptic changes that occur in ALS during the process of anterior

horn neuron degeneration. One such probable cause is due to the decreases in cell body

area, number of synapses, and synaptic contact length which is evident even during the

early stage of the disease. It is noteworthy that despite decreases in cell body area,

synaptic numbers and synaptic contact length, the length of the active zone was not

reduced in the normally appearing neurons of the ALS patients (Sasaki & Maruyama,

1994). The continuous loss of synapses that is observed from myriad studies implies a

decrease in the global connectivity of the motor system and a decreased potential for

motoneuronal interaction.

The neuromuscular junction (NMJ) is the synapse where the axon terminal of a motor

neuron (MN) meets the motor endplate. Recent studies suggest that distal degeneration

in the skeletal muscle plays a key role in the progression of ALS. Several studies using

SOD1G93A mice have shown that NMJ degeneration occurs in the initial stages of

disease progression, long before MN loss (Brooks et al., 2004; Fischer & Glass, 2010;

Kanning et al., 2010)

3. Huntington’s Disease (HD)

Huntington`s Disease (HD) is a progressive neurological disorder characterised by

chorea (uncontrolled dance like movement), cognitive disturbances, depression and

other psychiatric symptoms (Harper, 1996). It is inherited in an autosomal dominant

fashion, and severity of the disease depends on number of CAG repeats (Jones et al.,

1997; Walker, 2007). Both HTT and mutant HTT (mHTT) proteins are ubiquitously

expressed, predominantly in the striatum, although it is the least affected in HD, and in

moderate levels in other parts of the brain (Bhide et al., 1996). HTT is shown to be

expressed in the brain from midgestational period, and the expression of mHTT was

observed as early as 10-week old infant’s brain suggesting an implication for proper

neuronal development and function, especially when the brain is vulnerable to

excitotoxic injury (DiFiglia, 1997).

To further understand the function of HTT and mHTT in normal neuronal function and

in diseased states respectively, several mouse models of HD has been created (Cepeda

et al., 2010; Levine et al., 2004; Menalled & Brunner, 2014; Menalled & Chesselet,

2002). There are two types of mouse models of HD: ‘transgenics’, in which the mutant

gene or part of it, is inserted randomly into the mouse genome, leading to the expression

of mutant protein along with the endogenous normal HUNTINGTIN; and ‘knock-ins’,

in which the mutant gene is inserted into the mouse Hdh (mouse Huntington) gene,

leading to either homozygotes or heterozygotes for the mutation [for further

information please refer (Menalled & Brunner, 2014; Menalled & Chesselet, 2002)].

Myriad of transgenic mice with expanded polyQ repeats like R6/1, R6/2 and N171-Q82

have been generated that recapitulate the symptoms of HD. All these models develop

aggregate inclusions, neuronal loss and motor-coordination deficits. The first and most

widely used transgenic mouse model of HD was generated by overexpression of exon

1 of the human gene encoding HUNTINGTIN (IT15) with 141-157 CAG-repeat

expansions, which was termed as R6 (Mangiarini et al., 1996). Unlike Htt homozygous

knock-out (embryonically lethal but not Htt conditional Knock-out) (Nasir et al., 1995;

Wang et al., 2016), R6 mice survive for 13 weeks. Apart from the motor deficits, these

different mouse models exhibit altered synaptic function and cognitive deficits.

Research over many years suggests that cognitive defects appear long before the onset

of overt motor dysfunction in HD (Paulsen et al., 2008). These studies have shown that

synaptic dysfunction precedes cell death by many years in humans (Murer et al., 2002).

It is observed that loss of medium-shaped spiny (MSN) projection neurons in striatum,

and pyramidal neurons from cortex is considered as prominent pathological

characteristics of HD (Milnerwood et al., 2010; Milnerwood & Raymond, 2010;

Vonsattel et al., 1985).

Impaired dopamine homeostasis is one of the major consequences of HTT (including

Htt) mutation that contributes to the impaired information processing from the cortical

inputs to striatum (Andre et al., 2010). Dopaminergic neurons from substantia nigra

and ventral tegmental area (VTA) project to the dorsal striatum to regulate

glutamatergic neurons (direct pathway; rich in D1 receptors; facilitates movement),

whereas, indirect pathway projects to external globus pallidus (rich in D2 receptors;

suppresses movement) (Andre et al., 2010; Sepers & Raymond, 2014), which are found

to be degenerated in HD. On the contrary, Andre et al, have shown that hyperactivation

of the nigrostriatal pathway may elicit the characteristics of chorea observed in HD.

Therefore, agents affecting dopamine transmission are used to modulate HD (Murer et

al., 2002).

Proper glutamatergic function of these neurons is driven majorly by glutamate

(excitatory neurotransmitter), and, thus have an increased sensitivity towards NMDAR

activation leading to neuronal death, especially in HD (Fonnum et al., 1981; McGeer

et al., 1977; Schwarcz et al., 1983). By the time motor symptoms were observed,

majority of striatal glutamatergic neurons and spine densities were lost (Cepeda et al.,

2010; Cepeda et al., 2007; Li, 1999). Thus, reduced glutamatergic signalling in the

striatum could lead to loss of spines and, eventually, contribute to motor deficits.

To study the functional insights of HD pathology, electrophysiological studies have

dissected out intrinsic and synaptic neuronal properties (Sepers & Raymond, 2014).

Studies have shown that, in R6/2 mice, basal synaptic transmission and presynaptic

release were progressively altered (Cepeda et al., 2001; Khedraki et al., 2017; Klapstein

et al., 2001; Milnerwood et al., 2006). Presymptomatic R6/2 mice showed increased

neuronal input resistance and lower stimulus intensity to evoke action potentials

(rheobase), whereas, symptomatic R6/2 mice exhibited increased resting membrane

potentials, input resistance and decreased membrane time constants and synaptic

plasticity. Taken together, these findings indicate that passive and active membrane and

synaptic properties of medium-sized spiny neurons are altered in the R6/2 transgenic.

Many studies have observed an early augmentation of NMDAR activity in MSNs of

HD mouse (Cepeda et al., 2010; Cummings et al., 2010; Milnerwood & Raymond,

2007). Further, overexpression of GluN2B subunit in HD has been shown to exacerbate

the phenotype and pathology. Studies have shown that brief stimulation of synaptic

NMDAR (containing GluN1/GluN2A) leads to survival signalling via BNDF activation

(Martire et al., 2013) but activation of extrasynaptic NMDAR (containing

GluN1/GluN2B) is neurotoxic and leads to cell death (Hardingham et al., 2002). These

studies suggest that hyperactivation of extrasynaptic NMDAR leads to neuronal loss in

HD.

In another mouse model of HD, Yeast Artificial Chromosome (YAC)-Htt (YAC128),

containing entire human HTT to have 128 CAG-repeat expansions (Slow et al., 2003),

it was found that the probability of release at D1-presynapse was increased in 1.5

months, but decreased at 12 months. However, D2-presynapse release probability was

not altered in these mice (Cummings et al., 2010). Further studies have shown that the

synaptic transmission and function are altered in YAC128 mouse model of HD (Joshi

et al., 2009; Miller et al., 2008). Similar observations were found in other mouse

models of HD (Akopian et al., 2016; Cummings et al., 2010; Graham et al., 2009;

Klapstein et al., 2001; Kolodziejczyk & Raymond, 2016; Levine et al., 2004; Levine

et al., 1999; Pouladi et al., 2009).

4. Alzheimer`s Disease (AD)

Alzheimer`s Disease (AD) is the most common cause of senile dementia and a

devastating neurodegenerative disease. Formation of senile plaques, neurofibrillary

tangles (NFT), massive loss of synapses, and eventual neuronal cell death are the

pathological hallmarks of AD. The pathogenesis is associated with increased β-amyloid

(Aβ) levels in the brain generated by proteolysis of amyloid precursor protein (APP)

with the help of Presenilin1 (PSEN1), and part of γ- secretase complex (De Strooper

et al., 1998; Selkoe, 2002). Mutations in APP and Presenilin1 or 2 genes result in an

autosomal dominant form of AD. To understand the mechanism of AD, temporal

evolution, and to translate it for therapeutic purposes, several transgenic mouse models

of AD has been used till date. There are currently 127 animal models of AD in use

having wide variety of mutations (single, double and triple mutations) that are

implicated in AD [(Higgins & Jacobsen, 2003; LaFerla & Green, 2012)

www.alzforum.org]. Recently, very robust mouse model, 5xFAD, having five familial

AD mutations, causes relatively early and aggressive presentation of AD, has been

widely used as compared to other models. It recapitulates the following disease

phenotypes: β-amyloidosis, plaques, neurite dystrophy, dendritic spine loss,

neuroinflammation, neuronal loss and age-dependent cognition decline. Limitations of

these models include the following: a) evidences from positron-emission tomography

(PET) suggest that mouse plaques are significantly differ from human biochemically,

and b) another caveat is the neuronal loss is not profound as that of humans (Sasaguri

et al., 2017). A detailed review on various animal models of AD can reviewed here

(LaFerla & Green, 2012; Pozueta et al., 2013; Sasaguri et al., 2017). These transgenic

models show synaptic dysfunction, and, thus, provide an opportunity to understand the

mechanisms of neurophysiological deficits observed in AD, mainly the synaptic and

cognitive decline (Rowan et al., 2003; Spires-Jones & Knafo, 2012). Abnormalities in

synaptic function in AD was observed more than four decades ago by Gonatas et al

(Gonatas et al., 1967). Since then, there are many studies that focused on reinforcing

the idea that loss of synaptic function is the main characteristics of AD. Indeed, this

was further confirmed by quantitative ultrastructural and immunohistochemical post-

mortem studies from human AD patients with symptoms ranging from mild-cognitive

impairments to early-mild AD (Masliah et al., 1989; Scheff & Price, 1993; Terry et al.,

1991). Most of the electrophysiological studies of amyloid depositing mouse models

have investigated alterations in synaptic strength between hippocampal pyramidal

neurons by measuring basal synaptic strength, synaptic transmission, and neuronal

intrinsic properties (Nistico et al., 2012). A study from a mouse model of AD

expressing single or double transgenic mutations showed normal basal synaptic

transmission but impaired LTP in early stage of AD (Liu, 2008), while in another study

using App695Swe mutation, normal LTP with deficits in basal synaptic transmission was

observed for both 12- as well as 18-month old mice (Fitzjohn et al., 2001). These initial

studies have shown that disruption in synaptic function is the major cause of cognitive

decline in AD. Studies have shown that AD pathology increases in an age dependant

manner (Hsia et al., 1999; Sun et al., 2017). By using Apoe4 mouse (4 months and

older), alteration in short as well as long-term plasticity has been observed (Sun et al.,

2017). A similar age dependent decline in basal synaptic function was observed in other

models (Hsia et al., 1999; Puzzo et al., 2017). These studies clearly show that the

decline in basal synaptic function is an early indicator cognitive decline observed in

later stages of AD. To further understand whether amyloidogenic processing of APP is

responsible for Amyloid Beta and Tau toxicity, toxic proteins are administered in acute

brain slices prepared from WT as well as BACE1 KO mice. No affect in basal synaptic

transmission was observed in WT or BACE1 (β Secretase) KO mice but LTP was

impaired in both genotypes, suggesting toxicity did not depend on APP processing

alone (Puzzo et al., 2017). Apart from the alterations in synaptic function, abnormal

dendritic spine structures were also observed in AD. Dendritic spines are specialised

anatomical structures on neuronal cells that serve as the postsynaptic component for the

excitatory dendritic spines. Several studies using different mouse models of AD have

identified severe dendritic spine loss and plaque-associated structural plasticity deficits

in AD. This is extensively reviewed here (Pozueta et al., 2013; Rowan et al., 2003;

Spires-Jones & Knafo, 2012). Based on these studies, basal synaptic function can be

used as a biomarker to detect AD in an early stage of development.

Drosophila melanogaster

It is a multicellular eukaryote that exhibits both histological and behavioural phenotype

associated with neurodegeneration. Flies expressing the proteins such as α-synuclein,

β-amyloid, polyglutamine repeat, tau recapitulate many of the histological and

behavioural disease phenotypes associated with neurodegeneration. In this model, the

toxic proteins are overexpressed in retina leading to rough-eye phenotype that forms a

platform, which is used extensively for genetic and chemical screens (Luheshi et al.,

2007). The advantages working with flies are short doubling time, multicellularity,

availability of genetic resources and ability to express proteins in a tissue-specific

manner. Also, the fly model can unravel the basic pathophysiological mechanisms

underlying neurodegeneration. It is often used synergistically with another disease

model to validate the basic mechanisms. For e.g. a small molecule screen performed

on fly model identified mGLUR5 (GPCR) as a druggable target that is abundantly

expressed in brain (Chang et al., 2008). Recently, regulation of protein QPCT

(glutaminyl cyclase) has been shown to curb neurodegeneration symptoms in fly model

and mammalian cells (Jimenez-Sanchez et al., 2015).

Caenorhabditis elegans

It is one of the widely used models to study neurodegeneration for more than a decade.

Pros are the availability of genetic resources of this tiny transparent animal where all

302 neurons are mapped for their interactions and their short generation time

(Nussbaum-Krammer & Morimoto, 2014). Genetic strains were constructed that

express toxic protein aggregates such as Aβ, α-synuclein, TDP43 and polyglutamine

repeats in either whole body or tissue specific manner. They not only form visible

aggregates but also show the behaviour phenotype such as paralysis. Number of genetic

and chemical screens was conducted to identify modulators that rescue the behavioural

phenotype. This led to identification of small molecules that cleared toxic protein

aggregates and were further validated in higher model systems (Narayan et al., 2014).

Successful genetic screens too have identified many genes such as LRRK2, the

reduction of its kinase activity rescued the dopaminergic deficit motor phenotype (Yao

et al., 2013). Various regulators of HSR have been tested in the worm model to treat

proteotoxicity. Its transparent tissue enables for high content imaging. More

importantly, they also show a typical ageing symptoms making them an ideal tool to

investigate the interplay of metabolism, ageing and neurodegeneration

comprehensively (Chung et al., 2008).

Cellular Models

1. Yeast

The budding yeast (Saccharomyces cerevisiae) is a simple yet powerful tool to study

the cellular pathophysiological mechanisms underlying the protein toxicity(Smith &

Snyder, 2006). It recapitulates the toxicity of protein aggregates as that of neurons. This

is because the fundamental cellular features and vital cellular pathways are

conserved(Khurana & Lindquist, 2010). For instance, the membrane-bound organelles

such as mitochondria, Golgi, lysosome, endoplasmic reticulum and so on exist in yeast.

More importantly, the unfolded protein response is conserved as that of the mammalian

cells. The major advantages of yeast are short doubling time, existence of genetic

sources, amenable to genetic manipulation and scalable to high throughput genetic and

chemical screens. Also, around 3500 genes are found to be homologues as that of

human cells (Botstein et al., 1997; Kachroo et al., 2015). Pioneering work by Susan

Lindquist and her colleagues have shown that yeast cells expressing aggregate prone

genes and their variants that have been identified in ND have been used to understand

their pathobiology (Outeiro & Lindquist, 2003). The ER-Golgi trafficking defect due

to α-synuclein overexpression leads to cellular toxicity was first noted in yeast and

further validated in mammalian cells (Cooper et al., 2006; Winslow et al., 2010).

Several studies including ours have identified novel peptidomimetics and small

molecule autophagy modulators that ameliorate ND related aggregate proteotoxicity

(Khurana & Lindquist, 2010; Rajasekhar et al., 2015; Sarkar et al., 2007b). Some of

the obvious limitation of the yeast model is that it cannot recapitulate the complexities

of neuronal network and multicellularity that is present at the organ level.

2. iPSC

iPSC technology enables researchers to generate human specific, disease relevant cell

type such as neurons directly from patient fibroblasts. The advancement of gene editing

techniques such as CRISPR-Cas9 has simplified genetic knock out/in studies in iPSC-

derived neurons to understand the pathogenesis of neurodegeneration. The potential of

this model system to shed light on basic disease pathology mechanisms is massive

(Narayan et al., 2014). iPSC lines for AD, PD, Niemann-Pick disease type C and ALS

disease have been generated for their biological investigations (Chung et al., 2013;

Kiskinis et al., 2014; Ryan et al., 2013). Consequently, genetic or small molecule

screens can be performed using iPSC. The current limitations are 1) hurdle in scaling

up for high throughput screening due to their relative slow propagation rates, 2)

extensive and time-consuming neuronal differentiation protocols. However, they can

be used to validate the data generated from nonhuman disease models. For example,

yeast screen yielded the NAB2 compound where it rescued the disease phenotype and

validated its disease modifying mechanisms in iPSC(Chung et al., 2013).

Current understanding on the mechanistic insights of aggrephagy

These and other model systems have contributed immensely to unravel molecular

aspects of the process of aggrephagy. And how aggrephagy is perturbed by various

protein aggregates is detailed below.

Substrate recognition

Protein aggregate (substrate) recognition by receptors/adaptors such as Alfy,

p62/SQSTM1, Optineurin and Cue5/Tollip, is one of the first steps of autophagy for its

clearance (Stolz et al., 2014). The selectivity of autophagic cargo depends on specific

receptors/adaptors that recognize it by degradation marks. Autophagic receptors bind

to the ubiquitinated substrates through their respective domains to capture the

aggregates. They also tether them to autophagic machinery like LC3 to enable

autophagosome formation around the aggregates. The brief description pertaining to

autophagic receptors are as follows:

Alfy: This multidomain scaffolding protein recognizes ubiquitinated protein aggregates

for their clearance. It contains following domains: 1) Pleckstrin homology (PH)-

BEACH domain that binds to p62, 2) FYVE domain for PI3P binding, and 3) WD40

repeats that interacts with Atg5. It colocalizes with ubiquitin positive p62 aggregates

(Isakson et al., 2013). It stabilizes the LC3 and p62 interaction and also recruits Atg5

for the formation of autophagosomes that facilitated huntingtin polyQ aggregate

clearance (Filimonenko et al., 2010).

p62/SQSTM1: p62 is the adaptor for aggrephagy (Pankiv et al., 2007), mitophagy

(Geisler et al., 2010), pexophagy (Kim et al., 2008b) and xenophagy (Bah & Vergne,

2017). It possesses several domains namely: 1) UBA domain that binds ubiquitin, 2)

PB1 domain for aggregation of cargoes, and 3) LC3 Interacting Region (LIR) motif for

LC3 binding (Lim & Yue, 2015). It also has nuclear localization signal (NLS) and

nuclear export signal (NES) for nucleocytoplasmic shuttling (Pankiv et al., 2007). p62

mutations have been linked to sporadic and familial ALS through impaired clearance

of TDP-43 and SOD1 (Fecto et al., 2011). Paget’s disease of bone patients with

mutations in p62 are predisposed to ALS, a disease that is characterized by p62 positive

inclusions in neurons. Mutant p62 contributes to pathogenesis through multitude of

mechanisms and one of them is via over activation of nuclear factor-κB (NF-κB)

signaling (Chamoux et al., 2013).

Optineurin: It binds to ubiquitinated proteins through its UBA and NF-kB Essential

Modulator (NEMO) domains (Kachaner et al., 2012). On its other end, it binds to LC3

through LIR motif. Genetic mutations of optineurin are also associated with ALS.

Optineurin mutation is associated with rare ALS mutations leading to TAR DNA-

binding protein 43 (TDP-43) inclusions (Ito et al., 2011).

Cue5: Cue5 is the recently identified yeast autophagy receptor that binds to ubiquitin

through its CUE domain and Atg8 via its AIM1 and AIM2 motifs (Lu et al., 2014). Its

mammalian homologue is Tollip which is essential for the huntingtin polyQ clearance.

Valosin-Containing Protein (VCP): It is a member of AAA+ family of ATPase and

is involved in the sorting of ubiquitinated cargoes through endolysosomal pathway.

VCP mutation is associated with Paget’s disease of bone and Fronto Temporal dementia

(FTD). Overexpression or knockdown of VCP in cells leads to appearance of

autophagosomes that are immature with accumulation of ubiquitin positive cargoes

(Ritz et al., 2011).

Autophagosome formation

mTOR complex 1 (TORC1) negatively regulates autophagy. Various stimuli in neurons

such as ATP, ER stress, and specific amino acids repress TORC1 to activate ULK

complex (ULK1, FIP200, ULK2, Atg101 and Atg13) (Mizushima, 2010).

Phosphorylation of ULK1 at its active site leads to subsequent phosphorylation of other

components of complex for initiating autophagy (Chan et al., 2009). Although ULK1

phosphorylation is primarily regulated by TORC1, it can be also independently

modulated by AMP-activated protein kinase (AMPK) (Khan & Kumar, 2012). Also,

ULK1 phosphorylation leads to the translocation of class III PI3 Kinase complex

(beclin1, Atg14, Vps15 and Vps34) to isolation membranes (also known as omegasome

formation at the ER) by phosphorylating one of its components, AMBRA (Di

Bartolomeo et al., 2010). Vps34 activity generates phosphatidylinositol-3-phosphate

(PI3P) to bind to its effectors such as WIPI1 (WD repeat domain phosphoinositide

interacting 1) and WIPI2 (WD repeat domain phosphoinositide interacting 2) (Jaber &

Zong, 2013). This is to catalyse the ubiquitination like reactions at the isolation

membrane that aid autophagosome formation. Atg5-Atg12-Atg16L complex is formed

on the isolation membrane in presence of Atg7 and Atg10. This Atg5-Atg12-Atg16L

catalyses the covalent attachment of phosphatidylethanolamine to LC3 that facilitates

the closure of autophagosome membranes (Ichimura et al., 2000). Atg9 aims to supply

membranes for the growing autophagosomes by shuttling from various sources such as

endoplasmic reticulum-mitochondria contact sites (Hamasaki et al., 2013), Golgi

(Geng & Klionsky, 2010) and plasma membrane (Ravikumar et al., 2010) to

autophagosome formation site. ER has been suggested as a membrane source for

autophagosomes formed at synapse but the main source is not yet clear (Maday &

Holzbaur, 2014).

In neurons, how autophagy induced is still an enigma. Conventionally, the key trigger

for autophagy is starvation that induces autophagy in numerous cell types(Kim et al.,

2008a). However, even 48 hours of starvation did not induce GFP-LC3 puncta in brain

but profoundly induced autophagy in the liver (Mizushima et al., 2004). On the other

hand, while caloric restriction induced autophagy in cortical, Purkinje and motor

neurons, nutrient starvation failed to induce autophagy in cultured hippocampal

neurons (Kaushik et al., 2011). These contradicting results may mirror the neuronal

specificity in inducing and regulating autophagy. Several studies have shed light about

“other” ways of modulating autophagy in the brain.

Recently, the neuronal activity such as acute stimuli can trigger autophagy. Upon high

frequency stimulation, the Atg8 puncta is increased at neuromuscular junctions

(presynapse) in D. melanogaster (Vanhauwaert et al., 2017). Although the molecular

players and its mechanism(s) are not yet elucidated, calcium signalling can be a key

player. During exocytosis driven by action potential, there is remarkable increase in

calcium levels at presynapse (Rizzoli, 2014). Calcium can either enhance or inhibit

autophagy depending on the context and calcium responsive proteins are abundant at

presynapse. It is noteworthy to mention that investigating calcium-mediated autophagy

at presynapse can shed light on the novel mechanistic aspect of neuronal autophagy.

Interestingly, the presynaptic resident proteins such as Endophilin A, Basoon,

Synaptojanin 1 regulate autophagy by interacting with autophagy related proteins

(Vijayan & Verstreken, 2017).

Regulation of protein synthesis by TORC1 is implicated in synaptic plasticity (Kovacs

et al., 2007). However, TORC1 mediated autophagy induction at presynapse has not

been studied extensively. Autophagy mediated degradation of GABA receptors and

AMPA receptors (that are involved in synaptic plasticity) at post-synapse induce long

term depression (Shehata et al., 2012).

In PD model, α-synuclein perturbs Rab1a mediated Atg9 translocation to isolation

membrane leading to impairment of omegasome formation (Cooper et al., 2006). Atg9

trafficking is also abnormal in vacuolar protein sorting-associated protein 35 (VPS35)

D620N mutation that results in autosomal dominant case of PD (Zhou et al., 2017).

VPS35 (component of retromer complex) recruits actin nucleation-promoting WASP

and Scar homolog (WASH) complex to endosomes. Mutation in VPS35 perturbs this

recruitment to cause the abnormal Atg9 trafficking to impair autophagy(Zavodszky et

al., 2014).The E122D Atg5 mutation has been identified in childhood ataxia(Kim et al.,

2016). This disease is characterized by loss of motor coordination and cerebellar

hypoplasia. This mutation decreases the affinity of Atg5 to Atg12 and reduces the

autophagosome biogenesis and its flux (Kim et al., 2016).

β-propeller protein-associated neurodegeneration (BPAN), the static encephalopathy of

childhood with neurodegeneration in adulthood (SENDA) is caused by de novo

mutations in WDR45(Haack et al., 2013). WDR45 gene encodes WIPI4, the key

protein that bridges PI3P production and LC3 lipidation for autophagosome maturation

(Zhao et al., 2015). Neuronal specific knockout of WDR45 recapitulates some BPAN

disease phenotype such as impaired motor coordination, cognitive deficit, axonal

swelling with accumulation of ubiquitin positive aggregates (Saitsu et al., 2013).

Hexanucleotide repeats in C9ORF72 gene is one of the most common causes of ALS.

Different mechanisms have been proposed for this disease pathogenesis and

perturbation of autophagy is one of them. C9ORF72 interacts with ULK1 and affects

the phagophore initiation complex(Webster et al., 2016). In addition, C9ORF72 is a

part of WD repeat domain 41 (WDR41) and Smith-Magenis syndrome chromosome

region, candidate 8 (SMCR8) complex that is involved in both vesicular trafficking and

autophagy. This complex is a guanosine diphosphate (GDP)/guanosine triphosphate

(GTP) exchange factor (GEF) that activates the small GTPases Rab39 and Rab8 for

autophagosome formation (Amick & Ferguson, 2017).

Wild type huntingtin protein acts as a scaffold for autophagosome formation whereas

its mutant fails to recognize the cargo leading to “empty” autophagosomes(Martinez-

Vicente et al., 2010). It also impairs autophagosome transport and further accumulation

of autophagic substrates(Wong & Holzbaur, 2014). Mutant huntingtin interacts with

Rhes that are selectively expressed in striatum to inactivate it. Rhes interacts with beclin

1 to induce autophagy by reducing the bcl2-beclin 1 interaction (Subramaniam et al.,

2009). Furthermore, mTOR, is also sequestered in HD and spino-cerebellar ataxia 7

(SCA7) aggregates (Jimenez-Sanchez et al., 2012).

Autophagosome-lysosome fusion

Autophagosomes fuse with lysosomes to generate autolysosomes that digest the toxic

protein aggregates. In neurons, autophagosomes generated in axons are transported to

the lysosomes that are abundant near perinuclear MicroTubule-Organizing Centre

(MTOC). Microtubules and motor proteins such as dynein-dynactin complex are

involved in retrograde transport of autophagosomes towards neuronal cell body.

Mutations in dynein (axonal Charcot-Marie-Tooth hereditary neuropathy type 2) and

dynactin (motor neuron disease) motor protein complexes are implicated in

neurodegeneration pathogenesis indicating the importance of retrograde transport of

autophagosomes to fuse with lysosomes (Ferrucci et al., 2011). Interestingly, decreased

dynein activity resulted in the accumulation of autophagosomes, autolysosomes and

p62 positive inclusions in motor neurons before the onset of symptoms where they

degenerate upon clinical manifestations (Sasaki, 2011).

Amphisome formation (a fusion product of endosomes with autophagosome) is

perturbed in familial ALS and FTD and is associated with loss-of-function mutations

in CHMP2B (charged multivesicular body protein 2B). Mutant CHMP2B neurons

accumulate ubiquitin positive inclusion by disrupting ESCRT machinery

activity(Skibinski et al., 2005). Autophagy and endolysosomal pathways have

significant crosstalk pertaining to usage of cellular protein machineries. Mutations in

an endolysosomal pathway affects autophagy and vice versa (Otomo et al., 2011).

Endosomal maturation protein Rab7 is essential for autophagic flux and its mutation is

associated with bone related osteopetrosis (lysosomal perturbation) and

neurodegenerative CMT2 (autophagy flux perturbation) diseases (Tabata et al., 2010).

Furthermore, Rab7 significantly influences lysosome positioning. ALS2 protein

mutation, an activator of Rab5 perturbs ALS2 mediated Rab5 activation that hampers

amphisome formation as observed in familial cases of ALS (Otomo et al., 2011). The

coordinated crosstalk of endolysosomal and autophagy pathways to clear toxic protein

aggregates in healthy cells goes awry in neurodegeneration.

In neuronal specific interferon-β knockout of transgenic mouse model, the

autophagosome-lysosome fusion is affected with concomitant accumulation of

ubiquitin positive inclusion in neurons that leads to motor coordination and behavioural

impairments (Ejlerskov et al., 2015).

Regulation of lysosomal activity

Autophagosomes fuse with lysosomes for the clearance of its contents. For this process

to be efficient, the uncompromised lysosomal activity is essential. The lysosomal pH is

optimally (~4.5 to 5) maintained by proton pump vacuolar ATPase for the activity of

hydrolytic enzymes (proteases, peptidases, lipases and nucleases). Ionic balances of

lysosomes are regulated by presence of numerous ion channels (calcium, chloride and

potassium). Lysosomal associated membrane protein-1 (LAMP1) and LAMP2 prevents

the self-digestion of lysosomes (Nixon, 2013). The functional integrity of lysosomes is

compromised in most neurodegenerative disorders (Kroemer & Jaattela, 2005).

Lysosomes serve as key players in the initial stage of autophagy induction. Upon

perceiving the autophagy inducing stimuli, the TORC1 localized on the lysosomal

membrane is repressed and that further allows activation of transcription factor EB

(TFEB) to translocate to the nucleus. TFEB activates genes involved in lysosomal

biogenesis and autophagy process. TORC1 regulation of TFEB is achieved through

Rag-GTPase pathway (Kim et al., 2008a). TORC1 and TFEB activities are tightly

controlled by the presence of intracellular and lysosomal amino acid contents. If amino

acids are limiting inside the cells, for instance as in starvation, the TORC1 activity is

repressed and is followed by translocation of dephosphorylated TFEB to nucleus

resulting in upregulation of autophagy. On the other hand, the surplus amino acids lead

to reactivation of TORC1 to terminate the autophagy induction (Avruch et al., 2009).

The androgen receptor polyQ repeat mutant (implicated in SBMA) interacts with TFEB

and suppresses autophagy at transcription level (Cortes et al., 2014). SCA3 transgenic

animals show low levels of sirtuin 1 (deacetylates numerous autophagy and its related

proteins), parkin and beclin 1(Cunha-Santos et al., 2016).

Zinc-finger protein with KRAB and SCAN domains 3 (ZKSCAN3) is repressor of

autophagy and has opposite function that of TFEB. Inhibition of mTOR enhances its

accumulation in the cytosol. Down regulating ZKSCAN3 induces autophagosome and

lysosomal biogenesis (Chauhan et al., 2013). Apart from this, around 20 different

transcription factors are reported to regulate the autophagy at gene transcription level

depending on the context of stimuli. For instance, p53, forkhead box O3 (FOXO3) and

microphthalmia-associated transcription factor (MITF), all profoundly affect

autophagy gene expression (Li et al., 2017).

Lysosomal storage disorders (LSDs) illustrates the nexus between lysosomal

dysfunction and neurodegeneration. In LSDs, the unavailability of functional lysosomal

hydrolytic enzymes leads to defective autophagy that manifests disease pathology in

brain. In Niemann Pick type C (NPC) disease, the mutations of NPC1 and/or NPC2

genes lead to defective cholesterol trafficking where profound autophagic vacuole are

observed in neurons (Elrick et al., 2012). In neuronal ceroid lipofuscinosis, the mutation

in CLN3 gene reduces autolysosome formation, whereas in Cathepsin D mutation that

causes the same disease where declined lysosomal proteolysis is observed (Nixon,

2004).

Therapeutic strategies for ND

Current therapeutic interventions for neurodegeneration include three different

strategies. First, preventing the toxic protein aggregate build-up. For instance, the

antisense oligonucleotides ameliorated the amyotrophic lateral sclerosis disease

phenotype under preclinical settings and showed significant safety in phase I clinical

trial (Nizzardo et al., 2016). Second, dissolving the already culminated toxic protein

aggregates inside the neurons. Various groups identified small molecules and peptides

that can bring about disintegration of the aggregate. This has been a widely used

strategy for many years as a promising anti-neurodegeneration therapeutic. For AD,

few small molecule candidates that dissolve the amyloid plaques are currently in the

clinical trials in USA and Europe. The third and recent approach is to promote cellular

pathways to capture and degrade the protein aggregates by either ubiquitin proteasome

(UPS) or autophagy systems. Numerous groups have demonstrated that

neurodegeneration could be ameliorated by upregulating the activities of cellular

clearance systems such as proteasome or autophagy through small molecules or

peptides. Many small molecule screens have been reported that have identified the

druggable candidate(s) (target proteins or pathways). For e.g., Rilmenidine, an

autophagy inducer is currently in clinical trial as a HD therapeutic in Europe.

Chemical modulation of autophagy

As mentioned earlier that upregulation of autophagy gene expression enhances the

longevity of several model organisms (Pyo et al., 2013). Convincing evidences

demonstrate that dysfunctional autophagy contribute to the pathogenesis of

neurodegeneration (Nixon, 2013). Importantly, autophagy can clear the toxic misfolded

protein aggregates. Mechanisms that govern degradation of toxic misfolded protein

aggregates is thus a therapeutically attractive target (Sarkar et al., 2007b). Numerous

studies demonstrated that pharmacological upregulation of autophagy to clear protein

aggregates is beneficial in ameliorating neurodegeneration.

mTOR dependent autophagy modulators

mTOR dependent modulators induce autophagy by repressing mTOR and it can be of

ATP competitive (e.g., Torin1) or non-ATP competitive (e.g., rapamycin). mTOR can

be found in two complexes such as TORC1 (negative regulator of autophagy) and

TORC2 (positive regulator of autophagy). Rapamycin and their analogues are relatively

safer owing to its non-ATP competitive mode of inhibition. The allosteric TORC1

inhibitor, rapamycin was the first identified small molecule autophagy inducer.

Rapamycin interacts with FK506-binding protein 12 (FKBP12) to inhibit the TORC1

activity(Lorenz & Heitman, 1995). Rapamycin has been demonstrated to be

neuroprotective in various diseases such as PD, HD, AD and FTD in an autophagy

dependent manner(Jahrling & Laberge, 2015). Limited absorption of rapamycin led to

the development of its analogues (rapalogs) such as everolimus, temsirolimus, and

ridaforolimus. Rapalogs are under rigorous investigation for their therapeutic potential

in neurodegeneration. For instance, Food and Drug Administration (FDA) approved

everolimus as a tuberous sclerosis therapeutic(Bissler et al., 2017). Torin1 inhibits both

TORC1 and TORC2 in concentration dependent manner (Ma et al., 2013). There are

classes of modulators that indirectly inhibit mTOR activity. For instance, metformin

(Type II diabetes therapeutic) inhibits mTOR through regulating AMPK pathway.

Another AMPK activator, Nilotinib also exerts neuroprotection by inhibiting mTOR

activity and is currently under phase II clinical trial for PD therapy (Karuppagounder

et al., 2014). PI103 belong to dual mTOR/class III PI3Kinase modulators that inhibit

both mTOR and AKT pathway to induce autophagy. However, due to its rapid in vivo

metabolism, PI103 is currently unsuitable for neurodegeneration therapy (Hassan et al.,

2013).

mTOR independent autophagy modulators

mTOR independent autophagy modulators are those that induce autophagy without

repressing the mTOR activity. mTOR being the epicentre for cellular growth signalling

whose chronic inhibition exerts significant toxicity. These inhibitors are preferred

therapeutically as significant side effects such as impaired wound healing and

immunosuppression that are classically associated with mTOR inhibitors can be

avoided. Thus, these are also more suited for long-term drug administration. Small

molecule screening for FDA approved drugs revealed that several modulators

ameliorated neurodegeneration pathogenesis in various model systems in mTOR

independent manner. This study revealed the involvement of two cyclical pathways

namely Gsα/calpain/Ca2+ (Cardenas et al., 2010) and EPAC/cAMP/PLCε/Ins(1,4,5)P3

(Williams et al., 2008) that are important for the mTOR independent clearance of

aggregates.

Rilmenidine, the imidazoline receptor inhibitor, enhances autophagy by reducing

cAMP levels. It has been shown to ameliorate neurodegeneration in various model

systems such as primary neurons and transgenic mouse models of HD. It is important

to note that rilmenidine is currently undergoing safety trials for HD in Europe (Williams

et al., 2008).

Other drugs identified to regulate autophagy through the cyclical pathway are lithium,

valproic acid and carbamazepine. Carbamazepine and valproic acid repress inositol

synthesis, while lithium inhibits inositol monophosphatase to reduce Ins(1,4,5)P3

levels and thus induce autophagy (Williams et al., 2008).

Modulators of Gsα/calpain/Ca2+pathway such as verapamil and amiodarone along with

calpain inhibitors such as calpeptin and calpastatin have shown to exert neuroprotective

potential by degrading the protein aggregates through an autophagy-mediated

mechanism (Williams et al., 2008).

Trehalose clears α-synuclein and mutant huntingtin protein aggregates in autophagy

dependent manner in various model systems (Sarkar et al., 2007a). It has been shown

to be neuroprotective in tauopathy models as well (Schaeffer et al., 2012). It extends

life span, curbs motor and cognitive deficits in transgenic HD and ALS mouse models

(Tanaka et al., 2004).

Perspectives

The etiological components, as well as the molecular and cellular points of convergence

in ND, remain elusive and hence optimizing a defined therapeutic approach has been

challenging. Cellular mechanisms that directly impact synaptic function, including

autophagy, endosomal trafficking, and mitochondrial homeostasis, are likely to

constitute effective interventional targets in therapy in future. It is becoming

increasingly evident from recent literature that clearing protein aggregates via protein

quality control systems such as aggrephagy hold therapeutic promise for curbing

neurodegenerative diseases.

Acknowledgments

We thank members of the autophagy lab (JNCASR), Mridhula Giridharan and Aparna

Hebbar for critical reading of the manuscript. We apologize to researchers whose work

could not be included due to constraint in space. We acknowledge Wellcome

Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant

(EMR/2015/001946) and LSRB-DRDO grant (LSRB-31012017-2018) to RM, DST-

SERB grant (EMR/2015/001946) to JC, and JNCASR intramural funds.

Competing financial interests

All the authors declare no competing interests.

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Received 21 December 2017; revised 9 March 2018; accepted 20 March 2018

Table 1: Summary of synaptic dysfunction observed in different

neurodegenerative diseases.

Properties Amyotrophic

Lateral Sclerosis

Parkinson`s

Disease

Alzheimer`

s Disease

Huntington

`s Disease

Basal Synaptic

Transmission

(BST)

Impaired

transmission (()

presynaptic relase)

(Maselli, Wollman

et al. 1993)

Unaltered in

Medium spiny

neurons

(MSN’s) of

LRRK2 over

expressing

mice

(Beccano-

Kelly, Volta et

al. 2015)

Altered

BST was

observed in

App695Swe

mutant,

Apoe4 mice

(), AppInd

mice ()

(Hsia,

Masliah et

al. 1999,

Fitzjohn,

Morton et

al. 2001,

Puzzo,

Piacentini

et al. 2017,

Sun, He et

al. 2017)

In R6/2,

BST is

altered ()

(Cepeda,

Ariano et al.

2001,

Klapstein,

Fisher et al.

2001,

Milnerwood

, Cummings

et al. 2006,

Khedraki,

Reed et al.

2017).

Neurotransmitt

er Release

Increase in

Glutamate-induced

excitotoxicity;

Presynaptic

transmission

deficit

(Coyle and

Schwarcz 1976,

Lipton and

Rosenberg 1994,

Doble 1999,

Kiernan, Vucic et

al. 2011)

Deficiency of

dopamine in

the

nigrostriatal

system, and in

levels of

somatostatin.

(Rinne, Rinne

et al. 1984)

SV trafficking

defects (Esposi

to, Ana Clara

et al. 2012)

Increased

activity of

GABA and 5-

HTT (Anden

1974,

Grabowska

and Michaluk

Increased

presynaptic

function

(Sun, He et

al. 2017)

In R6/2,

altered

presynaptic

release (),

Post Tetanic

Potentiation

(), and in

YAC128 in

12 months

()

(Murphy,

Carter et al.

2000,

Cepeda,

Ariano et al.

2001,

Klapstein,

Fisher et al.

2001,

Milnerwood,

Cummings

1974, Maj,

Pawlowski et

al. 1974,

Hollister,

Breese et al.

1976)

et al. 2006,

Andre,

Cepeda et al.

2011,

Khedraki,

Reed et al.

2017).

Synaptic

Plasticity

Impaired LTP in

Ubqln2P497H and

Sod1

mice (Geracitano,

Paolucci et al.

2003, Gorrie,

Fecto et al. 2014)

LTP observed

in original

LTD-inducing

stimulus.

(Kreitzer and

Malenka 2005,

Bagetta,

Ghiglieri et al.

2010)

LTP in

AAV-

App/Ps1

(), Apoe4

()

(Audrain,

Fol et al.

2016,

Puzzo,

Piacentini

et al. 2017,

Sun, He et

al. 2017).

Impaired

LTP and

LTD in

R6/2

(Murphy,

Carter et al.

2000).

Cell Intrinsic

Properties

Altered

excitability and

altered fast

afterhyperpolarizat

ion (fAHP) and

slow

afterhyperpolarizat

ion (sAHP) in

Sod1 (), Tardbp

(),

Ubqln2P497H(),

Sod1A4V () mice

(Fischer, Culver et

al. 2004, Murray,

Talbot et al. 2010,

Zhou, Huang et al.

2010, Wainger,

Kiskinis et al.

2014, Radzicki,

Liu et al. 2016,

Kim, Hughes et al.

2017)

Increased

postsynaptic

potentials,

Intrinsic

membrane

properties,

postsynaptic

responses to

different

agonists of

excitatory

amino acid

receptors not

altered in

neurons

recorded from

dopamine-

depleted slices

(Calabresi,

Mercuri et al.

1993)

Highly

disorganise

d synapses,

increased

and

dispersed

tonic

glutamaterg

ic current

amplitude

in AAV-

App/Ps1

(Audrain,

Fol et al.

2016).

Increased

NMDA /

AMPA

ratio (Hsia,

Masliah et

al. 1999).

Decreased

in the

frequency

of mEPSC

and no

change in

amplitude

Altered

input,

resting

membrane

potentials,

time

constants

and action

potentials in

CAG40 KI

(), R6/2

()and

YAC128

()

(Levine,

Klapstein et

al. 1999,

Klapstein,

Fisher et al.

2001,

Graham,

Pouladi et

al. 2009,

Cummings,

Cepeda et

al. 2010).

on

application

of

Amyloid-β

in culture

(Yu,

Polepalli et

al. 2012).

Regions

involved

Motor cortex upper and lower

motor neurons,

Brain stem and

Spinal cord

(Agosta, Chio et

al. 2010, Kumar,

Aslinia et al. 2011)

Substantia

nigra, Putamen

Caudate

nucleus,

Globus

pallidus and

Cerebral

cortex (Sian,

Dexter et al.

1994)

Initially

Entorhinal

Cortex,

Hippocamp

us and later

cerebral

cortex but

eventually

all regions

are

affected.

Prefrontal,

motor and

Striatum

(globus

pallidus and

the

substantia

nigra pars

reticulate).

HTT and

mutant HTT

(mHTT)

proteins

expressed

ubiquitously

,

predominant

ly in the

striatum

Receptors and

Neurotransmitt

ers involved

Acetylcholine

receptors

(AChRs) (Palma,

Reyes-Ruiz et al.

2016)

Glutamate

receptor (Paul and

de Belleroche

2014)

Loss of

metabotropic

glutamate

receptor-mediated

regulation

(Vermeiren,

Hemptinne et al.

2006)

Cholinergic-

muscarinic

receptors

GABA

receptor

(Rinne, Rinne

et al. 1984)

Overactivation

of glutamate

receptors

(Bergman,

Wichmann et

al. 1994,

Hassani,

Mouroux et al.

1996)

Nicotinic

and

muscarinic

acetylcholi

ne

receptors

(Kihara and

Shimohama

2004).

Cytokine

receptors

(Nagae,

Araki et al.

2016).

NMDA

receptor

and

Glutamate

(Danysz

and Parsons

2012).

Dopamine

(Martorana

NMDA

receptor

(Levine,

Klapstein et

al. 1999,

Paoletti,

Vila et al.

2008).

mGluR`s

( group

2,3),

AMPA,

Kainate,

Dopamine,

Muscarinic

and GABA

Receptors

(Cha,

Kosinski et

al. 1998,

Sepers and

Raymond

2014)

and Koch

2014).

Dopaminer

gic D1-like

receptors,

substance P

(SP) and

dynorphin

and D2-like

receptors

and met-

enkephalin

are

implicated

(Haber and

Nauta 1983,

Andre,

Cepeda et

al. 2010).

Impaired

Dopamine

and

Glutamate

Homeostasi

s (Andre,

Cepeda et

al. 2010).

Chromosome

/Genes

involved

21q22.1 SOD1

2q33-2q35 Alsin

9q34 SETX

15q15-21 SPG 11

16p11.2 FUS

20q13.3 VAPB

14q11.2 ANG

6q21 FIG 4

1p36.2 TARDBP

10p13 OPTN

9p13.3 VCP

Xp11 UBQLN2

9p21 C9ORF72

(Chen, Sayana et

al. 2013)

4q21 SNCA

12q12 LRRK2

6q25–q27

Parkin

1p35–p36

PINK1

1p36 DJ-1

1p36

ATP13A2

(Lesage and

Brice 2009)

APP

(21q21.3) (Yoshikai, Sasaki et al. 1990, Thinakaran and Koo 2008). PSEN1

(14q24.3)

(Esler and

Wolfe

2001).

PSEN2

(1q42.13)

(Steiner

2004).

4p16.3

chromosom

al fragment

containing

IT15 gene.

Summary of different synaptic function properties that were altered in

neurodegenerative diseases, mainly Alzherimer’s, Parkinson’s, Huntington’s and

Amyotrophic Lateral Sclerosis. () or () indicates decrease or increase in that

specific property in the said mouse model.

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Figure 1. Formation of aggrephagosome. Aggregate proteins are ubiquitinated (Ub)

which are recognized by adaptor proteins. p62, NDP-52, NBR1 and optineurin are some

of the examples of adaptor proteins that have a ubiquitin binding domain and

additionally have LC3 Interacting Region (LIR) that can recruit LC3 and thereby

facilitate cargo recognition and capture.

Figure 2. Signalling pathways affected in neurodegenerative disorders. Schematic

to show impaired synaptic function due to altered function of different signalling

pathways in different neurodegenerative disorders. Presence of aggregates in these

diseases leads to an increase in presynaptic release of glutamate or dopamine upon

stimulation that activates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

receptor (AMPAR), dopamine receptor1 (D1R) and dopamine receptor2 (D2R)

respectively.

a) Activation of AMPARs leads to N-methyl-D-aspartate receptors (NMDAR)

activation which is followed by Ca2+ influx. Additionally, Voltage gated Ca2+ channel

(VGCC) further facilitates the Ca2+ influx, leading to an increased intracellular Ca2+

levels at the postsynapse. This increased levels of Ca2+ results in: 1) Excitotoxicity, 2)

Mitochondrial Dysfunction, 3) Reactive Oxygen Species (ROS) activation, 4)

CASPASE activation, 5) Apoptosis, and 6) mTOR activation (autophagy inhibition).

These events eventually result in neuronal dysfunction, neurodegeneration, and

cognitive impairments.

b) Stimulation of metabotropic glutamate receptor5 (mGluR5) by glutamate leads to

activation of inositol 1,4,5-trisphosphate (IP3). In neurodegeneration, the aggregates

strongly bind to the IP3 receptor1 (IP3R1), which results in the release of the Ca2+

through IP3R1, in turn, further contributing to already increased intracellular Ca2+

levels, resulting in synaptic dysfunction.

c) Dopamine acting on DA1 receptor activates adenosine 3',5'-cyclic monophosphate

(cAMP), which further activates protein kinase A (PKA). Upon PKA activation, the

intracellular Ca2+ levels increase through IP3R1. Additionally, PKA activates

dopamine- and cAMP-regulated neuronal phosphoprotein (DARRP-32) and

extracellular signal-regulated kinases (ERK), which alters cAMP response element-

binding protein (CREB) activity resulting in altered transcription and cognitive

impairment.

d) Dopamine acting on DA2 receptor further contributes to the intracellular Ca2+ levels

through the activation of IP3R1. This Ca2+ influx simultaneously activates

CALCINEURIN followed by glycogen synthase kinase 3β (GSK-3β) activation,

leading to the phosphorylation of TAU. This event results in impaired cognition and

cell death.