**4.1 Clinical aspects**

Clinically, they are progressive neurological diseases characterized almost in all cases by a combination of retinopathy, dementia and epilepsy. They have been originally clinically classified according to the age at onset in four main forms: infantile (INCL), late infantile (LINCL), juvenile (JNCL) and adult (ANCL). However, they are currently classified on the bases of the genetic defect (Wisniewski et al., 2001; Haltia, 2003, Mole et al., 2005, Jalanko et al., 2009, Kohlschütter & Schulz, 2009) (Table 1).

The clinical spectrum of NCL1 includes all four forms. Patients with NCL2 can present the late infantile or juvenile phenotype. The late infantile presentation has been reported in NCL5, NCL6, NCL7, NCL 8; the juvenile presentation has been reported in NCL3 and NCL9 and the adult phenotype has been reported in CLN4 (Table 1).

The ultrastructural pattern of accumulated lipopigment is different in different types of NCL: NCL1 and NCL10 present a pattern referred as granular osmiophilic deposits (GROD), while NCL2 and NCL3 are characterized by the presence of curvilinear (CLP) and fingerprint (FPP) profiles, respectively. The other forms, NCL4, NCL 5, NCL 6, NCL 7, and NCL8, show a mixed combination of CLP, FPP and rectilinear profiles (RLP) (Table 1).

Despite the wide molecular heterogeneity, the clinical findings are quite monomorfic. In fact, neuromotor impairment (tremor, ataxia, myoclonus, dysarthria, speech loss), ocular involvement (pigmentary retinal degeneration, optic atrophy, blindness), myoclonic epilepsy, progressive mental deterioration and behavior modifications are common clinical signs shared by all forms of NCLs. The main clinical signs and symptoms are summarized in table 1.


Clinically, they are progressive neurological diseases characterized almost in all cases by a combination of retinopathy, dementia and epilepsy. They have been originally clinically classified according to the age at onset in four main forms: infantile (INCL), late infantile (LINCL), juvenile (JNCL) and adult (ANCL). However, they are currently classified on the bases of the genetic defect (Wisniewski et al., 2001; Haltia, 2003, Mole et al., 2005, Jalanko et

The clinical spectrum of NCL1 includes all four forms. Patients with NCL2 can present the late infantile or juvenile phenotype. The late infantile presentation has been reported in NCL5, NCL6, NCL7, NCL 8; the juvenile presentation has been reported in NCL3 and NCL9

The ultrastructural pattern of accumulated lipopigment is different in different types of NCL: NCL1 and NCL10 present a pattern referred as granular osmiophilic deposits (GROD), while NCL2 and NCL3 are characterized by the presence of curvilinear (CLP) and fingerprint (FPP) profiles, respectively. The other forms, NCL4, NCL 5, NCL 6, NCL 7, and NCL8, show a mixed combination of CLP, FPP and rectilinear profiles (RLP) (Table 1). Despite the wide molecular heterogeneity, the clinical findings are quite monomorfic. In fact, neuromotor impairment (tremor, ataxia, myoclonus, dysarthria, speech loss), ocular involvement (pigmentary retinal degeneration, optic atrophy, blindness), myoclonic epilepsy, progressive mental deterioration and behavior modifications are common clinical signs shared by all forms of NCLs. The main clinical signs and symptoms are summarized

**Clinical signs** 

muscular hypotonia, growth impairment,

seizures, retinal blindness, microcephaly. ataxia, myoclonic jerks, seizures, vision deterioration, mental deterioration. ataxia, parkinsonism, verbal impairment,

hallucinations, mental deterioration

vaculated lymphocytes

deterioration

psychomotor deterioration, ataxia, myoclonic jerks,

pigmentary retinopathy, tunnel vision, depression,

spasticity, ataxia, myoclonus, seizures, optic atrophy, rapid mental deterioration, dementia; no

motor deterioration, dysarthria, parkinsonism, myoclonus, seizures, pigmentary retinopathy, optic

atrophy with rapid visual loss, early mental

motor deterioration, athetoid movements, myoclonic epilepsy (in type A), tonic-clonic

dementia, psychosis, stupors. No visual impairment (generally).

seizures, hearing impairment, mental deterioration,

**4.1 Clinical aspects** 

in table 1.

**NCL1** 

**NCL 3 (Batten disease)** 

**NCL4** ANCL

**NCL Clinical** 

**phenotype** 

LINCL/JNCL

ICLN

ANCL

**NCL2** LINCL/JNCL CLP

JNCL FPP

al., 2009, Kohlschütter & Schulz, 2009) (Table 1).

and the adult phenotype has been reported in CLN4 (Table 1).

**Storage pattern** 

GROD


INCL: infantile, LINCL: late infantile, JNCL: juvenile, ANCL: adult, GROD: granular osmiophilic deposits; CLP: curvilinear profiles; FPP: fingerprint profiles; RLP: rectilinear profiles.

Table 1. NCLs classification, age at onset, storage pattern and clinical signs.

As other neurodegenerative disorders, which manifest during the first year of life, generalized hypotonia and psychomotor regression are the first clinical signs of classic INCL. They are generally accompanied by head growth impairment (leading to microcephaly), seizure and myoclonic jerks. Behavior and sleep disturbance are frequently reported. Disease progression leads to visual and language deterioration. Death usually occurs within the first decade of life (Williams et al., 2006).

The late-infantile forms present with a similar clinic phenotype, showing progressive neurological deterioration during pre-school age. The classical late-infantile form of NCL2, generally begins during the second year of life, with slow cognitive regression and language deterioration. Epilepsy appears later, becoming rapidly intractable and accompanied with cognitive loss, myoclonic jerks and retinopathy. Patient autonomy is completely lost within the age of 6-8 and death occurs within adolescence period (Zhong et al., 2000; Steinfeld et al., 2002; Kohan et al., 2009).

Two major distinct phenotypes have been described for classical juvenile phenotype of NCL3 (Batten disease), according to the patient's genotype: a. patients carrying the 1-kb deletion in homozygous, (firstly described in Finland and Northern Europe), and b. patients carrying a compound of 1-kb deletion with other mutations (Munroe et. al., 1997).

Myoclonic Epilepsy in Lysosomal Storage Disorders 229

Most NCL1 patients accumulate autofluorescent lysosomal deposits, consisting mainly in

In neurons palmitoylation targets proteins for transport to nerve terminals and regulates trafficking at synapses (Huang et al., 2005). It is worth of note that PPT1 has been detected in non lysosomal compartments such as cells soma, varicosities and presynaptic terminals

> thioesterase (PPT1), lysosomal

peptidase 1 (TPP1), lysosomal

transmembrane

soluble protein

transmembrane ER protein

transmembrane

transmembrane ER protein

D, lysosomal enzyme

To date, 48 disease causing mutations distributed throughout the entire *CLN1* gene have been described (http://www.ucl.ac.uk/ncl) , most of them have been found in individual families. The only exception is represented by the missense mutation (c.364A>T, R122W), which has been found in most Finnish families. Most mutations cause the severe infantile form of NCL (MIM256730). However, mutations causing late infantile, juvenile and adult form have also been reported. No clear correlation between the phenotype and the genotype has been demonstrated (Das et al., 1998; Mitchison et al., 1998; van Diggelen et al., 2001;

Table 2. NCL genes, localization, encoded proteins and storage materials (Jalanko &

protein

Saposins A and D

Subunit c of ATP synthase

Subunit c of ATP synthase

Subunit c of ATP synthase

Subunit c of ATP synthase

Subunit c of ATP synthase

Subunit c of ATP synthase

Saposins A and D

enzyme

enzyme

protein

**Gene Chromosome Protein Main storage material** 

sphingolipids activation proteins A and D.

(Lehtovirta et al., 2001; Ahtiainen et al., 2003).

*CNL1* 1p32 palmitoyl protein

*CNL3* 16p12 CNL3, lysosomal

*CNL5* 13q21-q32 CNL5, lysosomal

*CNL7* 4q28.1-q28.2 CNL7, lysosomal

*CNL10* 11p15.5 CTSD, cathepsin

*CNL2* 11p12 ripeptidil

*CNL6* 15q23 CNL6,

*CNL8* 8p23 CNL8,

Braulke, 2009)

Williams et al., 2006).

In homozygous patients, visual impairment represent the onset sign, appearing during the first school years, with a pigmentary retinopathy; frequently a first diagnosis of retinitis pigmentosa or cone dystrophy is made. Often the cognitive skills are normal until teenage period, with subsequent deterioration and development of generalized or partial epilepsy, responsive to therapy. Behavior becomes aggressive; mood disturbance and psychotic symptoms are present. With disease progression, motor skills regress as well as speech articulation and parkinsonism and myoclonus become prominent.

In compound heterozygous patients, visual impairment is also the first accused symptom, but cognitive and motor deterioration are less pronounced and slower. Some patient have been reported as completely free from motor and cognitive signs (Lauronen et al. 1999, Jarvela et al. 1999).

Adult phenotypes are described in NCL1 and in the very rare form of NCL4 (Kukfs disease) (Martin, 1991; Ruchoux & Goebel, 1996). In the NCL1 patients, neurological and mental degeneration, depression, retinal and optic atrophy have been described, while the ocular involvement is not present in NCL4.

Absence of visual impairment, has also been reported in NCL8. This form comprises a subgroup of patients (described as Northern Epilepsy Variant) who develop generalized tonic-clonic epilepsy during early school age, followed by progressive mental retardation. With ageing epilepsy severity decreases but cognitive deterioration is maintained. Survival may last to fifth-sixth decade (Herva et al., 2000) .

Finally, a rare congenital form of NCL has been described in NCL10 (Siintola et al., 2006). Clinical course is characterized by microcephaly and severe neurological involvement (rigidity, tremor, status epilepticus) in the first hours of life. Respiratory insufficiency and apnea crisis follow with precocious death (generally within the first weeks of life).

Electrophysiological exams (EEG, ERG,VEP, ABR, SSP) show a wide spectrum of abnormalities in the different phenotypes (Topçu et al., 2004; Weleber et al., 2004; Caraballo et al., 2005; Collins et al., 2006). While brain imaging studies show a variable degree of cerebral and cerebellum atrophy accompanied with abnormalities in the signal pattern of the periventricular white matter and other brain areas (thalami, basal ganglia and putamen) (D'Incerti , 2000; Santavuori et al., 2001; Vanhanen et al., 2004).

#### **4.2 Molecular aspects**

NCLs are caused by mutations in at least 10 different recessively inherited human genes. Eight of them have been identified. These genes encode soluble or transmembrane proteins localized to the endoplasmic reticulum (ER) or the endosomal/lysosomal organelles.

The genes involved in the NCLs, their chromosomal localization, the encoded proteins and the storage materials are summarized in table 2.

The human *CLN1* gene has been located to chromosome 1p32 and encodes a palmitoyl protein thioesterase (PPT1), an enzyme that removes palmitate residues from proteins (Vesa et al., 1995). The enzyme consists in a 306 aminoacid polypeptide including a N-terminal signal sequence which is cleaved cotraslationally. Overexpressed PPT1 is directed to lateendosomes/lysosomes via mannose-6-pèhosphate receptor (M6PR) mediated pathway in non neuron cells (Verkruyse & Hofmann, 1996; Hellsten et al., 1996). It has not been demonstrated that this pathway is utilize to target the PPT1 in neurons. However, PPT1 has been found as part of the human brain mannose 6-phosphoproteasome ( Sleat et al., 2005).

In homozygous patients, visual impairment represent the onset sign, appearing during the first school years, with a pigmentary retinopathy; frequently a first diagnosis of retinitis pigmentosa or cone dystrophy is made. Often the cognitive skills are normal until teenage period, with subsequent deterioration and development of generalized or partial epilepsy, responsive to therapy. Behavior becomes aggressive; mood disturbance and psychotic symptoms are present. With disease progression, motor skills regress as well as speech

In compound heterozygous patients, visual impairment is also the first accused symptom, but cognitive and motor deterioration are less pronounced and slower. Some patient have been reported as completely free from motor and cognitive signs (Lauronen et al. 1999,

Adult phenotypes are described in NCL1 and in the very rare form of NCL4 (Kukfs disease) (Martin, 1991; Ruchoux & Goebel, 1996). In the NCL1 patients, neurological and mental degeneration, depression, retinal and optic atrophy have been described, while the ocular

Absence of visual impairment, has also been reported in NCL8. This form comprises a subgroup of patients (described as Northern Epilepsy Variant) who develop generalized tonic-clonic epilepsy during early school age, followed by progressive mental retardation. With ageing epilepsy severity decreases but cognitive deterioration is maintained. Survival

Finally, a rare congenital form of NCL has been described in NCL10 (Siintola et al., 2006). Clinical course is characterized by microcephaly and severe neurological involvement (rigidity, tremor, status epilepticus) in the first hours of life. Respiratory insufficiency and

Electrophysiological exams (EEG, ERG,VEP, ABR, SSP) show a wide spectrum of abnormalities in the different phenotypes (Topçu et al., 2004; Weleber et al., 2004; Caraballo et al., 2005; Collins et al., 2006). While brain imaging studies show a variable degree of cerebral and cerebellum atrophy accompanied with abnormalities in the signal pattern of the periventricular white matter and other brain areas (thalami, basal ganglia and putamen)

NCLs are caused by mutations in at least 10 different recessively inherited human genes. Eight of them have been identified. These genes encode soluble or transmembrane proteins

The genes involved in the NCLs, their chromosomal localization, the encoded proteins and

The human *CLN1* gene has been located to chromosome 1p32 and encodes a palmitoyl protein thioesterase (PPT1), an enzyme that removes palmitate residues from proteins (Vesa et al., 1995). The enzyme consists in a 306 aminoacid polypeptide including a N-terminal signal sequence which is cleaved cotraslationally. Overexpressed PPT1 is directed to lateendosomes/lysosomes via mannose-6-pèhosphate receptor (M6PR) mediated pathway in non neuron cells (Verkruyse & Hofmann, 1996; Hellsten et al., 1996). It has not been demonstrated that this pathway is utilize to target the PPT1 in neurons. However, PPT1 has been found as part of the human brain mannose 6-phosphoproteasome ( Sleat et al., 2005).

localized to the endoplasmic reticulum (ER) or the endosomal/lysosomal organelles.

apnea crisis follow with precocious death (generally within the first weeks of life).

articulation and parkinsonism and myoclonus become prominent.

Jarvela et al. 1999).

**4.2 Molecular aspects** 

involvement is not present in NCL4.

may last to fifth-sixth decade (Herva et al., 2000) .

the storage materials are summarized in table 2.

(D'Incerti , 2000; Santavuori et al., 2001; Vanhanen et al., 2004).

Most NCL1 patients accumulate autofluorescent lysosomal deposits, consisting mainly in sphingolipids activation proteins A and D.

In neurons palmitoylation targets proteins for transport to nerve terminals and regulates trafficking at synapses (Huang et al., 2005). It is worth of note that PPT1 has been detected in non lysosomal compartments such as cells soma, varicosities and presynaptic terminals (Lehtovirta et al., 2001; Ahtiainen et al., 2003).


Table 2. NCL genes, localization, encoded proteins and storage materials (Jalanko & Braulke, 2009)

To date, 48 disease causing mutations distributed throughout the entire *CLN1* gene have been described (http://www.ucl.ac.uk/ncl) , most of them have been found in individual families. The only exception is represented by the missense mutation (c.364A>T, R122W), which has been found in most Finnish families. Most mutations cause the severe infantile form of NCL (MIM256730). However, mutations causing late infantile, juvenile and adult form have also been reported. No clear correlation between the phenotype and the genotype has been demonstrated (Das et al., 1998; Mitchison et al., 1998; van Diggelen et al., 2001; Williams et al., 2006).

Myoclonic Epilepsy in Lysosomal Storage Disorders 231

The human *CLN6* gene has been located to chromosome 15q23 and encodes a 311 amoniacid non glycosilated membrane protein. It is localized in the ER and in neuronal cells it is additionally found along neural extension in subdomains of a tubular ER network. It contains a N-terminal cytoplasmic domain, seven putative transmembrane domains and a

The main storage component in NCL6 cells is the subunit c of the mitochondrial ATP

Forty six disease mutations have been described to cause a late infantile variant of NCL (MIM601780) (http://www.ucl.ac.uk/ncl). The nonsense mutation c.214G>T (p.E72X) has been demonstrated to be highly frequent in patients from Costa Rica probably due to

The human *CLN7* gene has been recently located to chromosome 4q28.1-q28.2 and encodes a transmembrane protein of 518 aminoacids. The CLN7 protein belongs to the major facilitator superfamily (MFS), which transport specific substrates. However, its specific substrate has not been identified yet (Kasho et al., 2006). Overexpressed CLN7 is located in lysosomes

Mutations in the CLN7 gene cause a variant late infantile NCL (MIM610951). Twenty-three disease-causing mutations have been described to date (http://www.ucl.ac.uk/ncl). Mutations in CLN7 gene have been initially described in Turkish patients (Siintola et al, 2007) and therefore it has been considered the Turkish variant late infantile NCL. However, it has been recently shown that CLN7 defects are geographically widespread (Aiello et al., 2009; Aldahmesh et al., 2009; Stogmann et al., 2009; Kousi et al., 2009). The missense mutation c.881C>A (p.T294K) was found in most patients of Romany origin previously studied by Elleder et al. (Elleder et al., 1997). Haplotype analysis of these patients was

The human *CLN8* gene has been located to chromosome 8p23 (Ranta et al., 1999). It encodes a non glycosylated membrane protein of 286 aminoacids. The CLN8 protein belongs to the TRAM-Lag1p-CLN8 (TLC) family. Members of this family are involved in the biosynthesis, metabolisms, transport and sensing of lipids (Winter & Ponting, 2002). However, the

The overexpressed protein has been localized in the ER but it seems to shuttle between ER and the ER-Golgi intermediate complex (ERGIC) (Lonka et al., 2000). The storage material in

Sixteen mutations in the *CLN8* gene have been reported to date (http://www.ucl.ac.uk/ncl). They have been identified in Finnish families with Northern Epilepsy (Ranta et al., 1999) and in patients of other ethnic origins affected with a more severe variant of NCL (Ranta et al., 2004; Cannelli et al., 2006; Vantaggiato et al., 2009; Kousi et al., 2009; Reinardt et al., 2010; Zelnik et al., 2007; Mole et al., 2005) . All but one Finnish patient present the missense mutation c.70C>G (p.R24G)in homozygous, suggesting that this mutation would be associated to a protracted and atypical NCL (Ranta et al., 1999). The human *CLN10* gene has been located to chromosome 11p15.5 and encodes the major lysosomal aspartic protease cathepsine D (CTSD). The CLN10 protein consists in 412 aminoacids and it is synthesized as a preproenzyme, which becomes posttranslationally modified by glycosilation and proteolysis leading to intermediates and mature forms (Gieselmann et al., 1985). Depending on the cell type it is trafficking to the lysosomes as a M6PR dependent or independent manner (Dittmer et al., 1999). CTSD is involved in limited proteolysis in the lysosomes and several proteins function as CTSD substrates, including

NCL8 patients consists mainly in the subunit c of the mitochondrial ATP Synthase.

consistent with the existence of a common founder effect (Kousi et al., 2009).

C-terminal luminal domain (Heine et al., 2004; Mole et al., 2004).

founder effect (Gao et al, 2002; Wheeler et al., 2002).

Synthase (Elleder et al.., 2006).

(Siintola et al., 2007).

function of the CLN8 is not known.

The human *CLN2* gene has been located to chromosome 11p12 and encodes the CLN2 protein tripeptidil peptidase 1 (TPP1) (Sleat et al., 1997), a lysosomal hydrolase that removes tripeptides from the N-terminus of small polypeptides (Golabek et al., 2006) such as the subunit c of mitochondrial ATP synthase. TPP1 consists in a 563 aminoacids, which includes a 19 aminoacid signal peptide and a 176 aminoacid prosegment that is autocatalytically cleaved within the lysosomes (Golabek et al., 2003). It is transported to the lysosomes in a M6PR-dependent manner (Chang et al., 2008).

The storage bodies contain mainly the subunit c of mitochondrial ATP synthase and to a less extent saposin A and D.

To date, 72 disease-causing mutations have been reported (http://www.ucl.ac.uk/ncl) leading to the classic late infantile NCL or Jansky-Bielschowsky disease (MIM 204500). Among them, the splice site mutation c.509-1G>C and the nonsense mutation c.622C>T (R208X) are quite frequent (Mole et al., 2005) and they result in very similar phenotypes.

The human **CLN3** has been located to chromosome 16p12 and encodes an integral membrane glycoprotein of 438 aminoacids (International Batten Disease Consortium, 1995). It possesses six transmembrane domains and the glycosilation varies in different tissues (Ezaki et al., 2003; Storch et al., 2007). Overexpressed CLN3 protein is localized in the lysosomes in non neuronal cells while it is detected in the endosomal/lysosomal structures and in the synaptosome in neurons (Kyttala et al., 2004; Luiro et al., 2001). In addition, CLN3 protein has also been detected in the plasma membrane and in lipid rafts (Rakheja et al., 2004; Rusyn et al., 2008). Many different functions have been attributed to CLN3 protein, including lysosomal acidification (Holopainen et al., 2001), lysosomal import of basic aminoacids (Kim et al., 2003), autophagy (Cao et al., 2006), membrane fusion, vesicular transport, , cytoskeletal organization (Brooks et al., 2003; Luiro et al., 2006) and apoptosis (Persaud-Sawin & Boustany, 2005; Wang et al., 2011).

The storage deposits contain mainly subunit c of the mitochondrial ATP Synthase (Lake & Hall, 1993). NCL3 is the only NCL typified by vacuolated lymphocytes (Mole et al., 2005)

So far, 49 disease causing mutations have been described in the *CLN3* gene (http://www.ucl.ac.uk/ncl), causing the juvenile NCL or Batten disease (MIM 204200). Many patients present the ancestral 1 kb deletion mutation, which results in the deletion of 2 exons. This mutation is predicted to produce an inactive truncated protein. However, it has been recently proposed that this mutated protein may retain some degree of residual function (Kitzmuller at al, 2008).

The human *CLN5* gene has been located to chromosome 13q21-q32 and encodes a 407 aminoacid polypeptide. Sequence analysis shows the presence of four initiation methionines and the production of four different polypeptides with a molecular weight ranging from 39 to 47 kDa has been described (Vesa et al., 2002). The human CLN5 contains mannose-6 phosphate residues on high-mannose type oligosaccharides, suggesting that at least some variants would be soluble. (Sleat et al., 2006). Overexpressed protein is localized to lysosomes, however it has also been detected in axons in neuronal cells (Holmberg et al., 2004). It has been demonstrated that CLN5 interacts with both NCL2 and NCL3 (Vesa et al., 2002).

The main storage component in NCL5 patients is the subunit c of the mitochondrial ATP Synthase (Tyynela et al., 1997).

Mutations in the *CLN*5 gene cause the Finnish variant form of late infantile NCL (MIM 256731). Twenty seven mutations have been reported to date (http://www.ucl.ac.uk/ncl). A frequent mutation consists in a 2bp deletion in exon 4 (c.1175delAT) and has been found in 94% of Finnish NCL5 alleles.

The human *CLN2* gene has been located to chromosome 11p12 and encodes the CLN2 protein tripeptidil peptidase 1 (TPP1) (Sleat et al., 1997), a lysosomal hydrolase that removes tripeptides from the N-terminus of small polypeptides (Golabek et al., 2006) such as the subunit c of mitochondrial ATP synthase. TPP1 consists in a 563 aminoacids, which includes a 19 aminoacid signal peptide and a 176 aminoacid prosegment that is autocatalytically cleaved within the lysosomes (Golabek et al., 2003). It is transported to the

The storage bodies contain mainly the subunit c of mitochondrial ATP synthase and to a less

To date, 72 disease-causing mutations have been reported (http://www.ucl.ac.uk/ncl) leading to the classic late infantile NCL or Jansky-Bielschowsky disease (MIM 204500). Among them, the splice site mutation c.509-1G>C and the nonsense mutation c.622C>T (R208X) are quite frequent (Mole et al., 2005) and they result in very similar phenotypes. The human **CLN3** has been located to chromosome 16p12 and encodes an integral membrane glycoprotein of 438 aminoacids (International Batten Disease Consortium, 1995). It possesses six transmembrane domains and the glycosilation varies in different tissues (Ezaki et al., 2003; Storch et al., 2007). Overexpressed CLN3 protein is localized in the lysosomes in non neuronal cells while it is detected in the endosomal/lysosomal structures and in the synaptosome in neurons (Kyttala et al., 2004; Luiro et al., 2001). In addition, CLN3 protein has also been detected in the plasma membrane and in lipid rafts (Rakheja et al., 2004; Rusyn et al., 2008). Many different functions have been attributed to CLN3 protein, including lysosomal acidification (Holopainen et al., 2001), lysosomal import of basic aminoacids (Kim et al., 2003), autophagy (Cao et al., 2006), membrane fusion, vesicular transport, , cytoskeletal organization (Brooks et al., 2003; Luiro et al., 2006) and apoptosis

The storage deposits contain mainly subunit c of the mitochondrial ATP Synthase (Lake & Hall, 1993). NCL3 is the only NCL typified by vacuolated lymphocytes (Mole et al., 2005) So far, 49 disease causing mutations have been described in the *CLN3* gene (http://www.ucl.ac.uk/ncl), causing the juvenile NCL or Batten disease (MIM 204200). Many patients present the ancestral 1 kb deletion mutation, which results in the deletion of 2 exons. This mutation is predicted to produce an inactive truncated protein. However, it has been recently proposed that this mutated protein may retain some degree of residual

The human *CLN5* gene has been located to chromosome 13q21-q32 and encodes a 407 aminoacid polypeptide. Sequence analysis shows the presence of four initiation methionines and the production of four different polypeptides with a molecular weight ranging from 39 to 47 kDa has been described (Vesa et al., 2002). The human CLN5 contains mannose-6 phosphate residues on high-mannose type oligosaccharides, suggesting that at least some variants would be soluble. (Sleat et al., 2006). Overexpressed protein is localized to lysosomes, however it has also been detected in axons in neuronal cells (Holmberg et al., 2004). It has been

The main storage component in NCL5 patients is the subunit c of the mitochondrial ATP

Mutations in the *CLN*5 gene cause the Finnish variant form of late infantile NCL (MIM 256731). Twenty seven mutations have been reported to date (http://www.ucl.ac.uk/ncl). A frequent mutation consists in a 2bp deletion in exon 4 (c.1175delAT) and has been found in

demonstrated that CLN5 interacts with both NCL2 and NCL3 (Vesa et al., 2002).

lysosomes in a M6PR-dependent manner (Chang et al., 2008).

(Persaud-Sawin & Boustany, 2005; Wang et al., 2011).

function (Kitzmuller at al, 2008).

Synthase (Tyynela et al., 1997).

94% of Finnish NCL5 alleles.

extent saposin A and D.

The human *CLN6* gene has been located to chromosome 15q23 and encodes a 311 amoniacid non glycosilated membrane protein. It is localized in the ER and in neuronal cells it is additionally found along neural extension in subdomains of a tubular ER network. It contains a N-terminal cytoplasmic domain, seven putative transmembrane domains and a C-terminal luminal domain (Heine et al., 2004; Mole et al., 2004).

The main storage component in NCL6 cells is the subunit c of the mitochondrial ATP Synthase (Elleder et al.., 2006).

Forty six disease mutations have been described to cause a late infantile variant of NCL (MIM601780) (http://www.ucl.ac.uk/ncl). The nonsense mutation c.214G>T (p.E72X) has been demonstrated to be highly frequent in patients from Costa Rica probably due to founder effect (Gao et al, 2002; Wheeler et al., 2002).

The human *CLN7* gene has been recently located to chromosome 4q28.1-q28.2 and encodes a transmembrane protein of 518 aminoacids. The CLN7 protein belongs to the major facilitator superfamily (MFS), which transport specific substrates. However, its specific substrate has not been identified yet (Kasho et al., 2006). Overexpressed CLN7 is located in lysosomes (Siintola et al., 2007).

Mutations in the CLN7 gene cause a variant late infantile NCL (MIM610951). Twenty-three disease-causing mutations have been described to date (http://www.ucl.ac.uk/ncl). Mutations in CLN7 gene have been initially described in Turkish patients (Siintola et al, 2007) and therefore it has been considered the Turkish variant late infantile NCL. However, it has been recently shown that CLN7 defects are geographically widespread (Aiello et al., 2009; Aldahmesh et al., 2009; Stogmann et al., 2009; Kousi et al., 2009). The missense mutation c.881C>A (p.T294K) was found in most patients of Romany origin previously studied by Elleder et al. (Elleder et al., 1997). Haplotype analysis of these patients was consistent with the existence of a common founder effect (Kousi et al., 2009).

The human *CLN8* gene has been located to chromosome 8p23 (Ranta et al., 1999). It encodes a non glycosylated membrane protein of 286 aminoacids. The CLN8 protein belongs to the TRAM-Lag1p-CLN8 (TLC) family. Members of this family are involved in the biosynthesis, metabolisms, transport and sensing of lipids (Winter & Ponting, 2002). However, the function of the CLN8 is not known.

The overexpressed protein has been localized in the ER but it seems to shuttle between ER and the ER-Golgi intermediate complex (ERGIC) (Lonka et al., 2000). The storage material in NCL8 patients consists mainly in the subunit c of the mitochondrial ATP Synthase.

Sixteen mutations in the *CLN8* gene have been reported to date (http://www.ucl.ac.uk/ncl). They have been identified in Finnish families with Northern Epilepsy (Ranta et al., 1999) and in patients of other ethnic origins affected with a more severe variant of NCL (Ranta et al., 2004; Cannelli et al., 2006; Vantaggiato et al., 2009; Kousi et al., 2009; Reinardt et al., 2010; Zelnik et al., 2007; Mole et al., 2005) . All but one Finnish patient present the missense mutation c.70C>G (p.R24G)in homozygous, suggesting that this mutation would be associated to a protracted and atypical NCL (Ranta et al., 1999).

The human *CLN10* gene has been located to chromosome 11p15.5 and encodes the major lysosomal aspartic protease cathepsine D (CTSD). The CLN10 protein consists in 412 aminoacids and it is synthesized as a preproenzyme, which becomes posttranslationally modified by glycosilation and proteolysis leading to intermediates and mature forms (Gieselmann et al., 1985). Depending on the cell type it is trafficking to the lysosomes as a M6PR dependent or independent manner (Dittmer et al., 1999). CTSD is involved in limited proteolysis in the lysosomes and several proteins function as CTSD substrates, including

Myoclonic Epilepsy in Lysosomal Storage Disorders 233

The human *NEU1* gene (Gen Bank AF040958) has been located to chromosome 6p21.3 within the region of the major histocompatibility complex (Bonten et al., 1996; Pshezhetsky et al., 1997). It contains 6 exons and spans approximately 3.5 kb of genomic DNA (Milner et

The *NEU1* gene encodes a protein of 415 aminoacids including a signal sequence, a central hydrophobic core and a more polar c-terminal domain (Bonten et al., 1996). After the removal of the signal peptide and glycosilation the protein would have a molecular mass of 45 kD. In fact, western blot studies have demonstrated the presence of two major bands of 44 -45 kD which yielded a 40 kD protein after de-glycoslation (Bonten et al., 1996). NEU1 exists as a multienzyme complex with at least two other proteins,-galactosidase and the protective protein/cathepsin A (PPCA) (d'Azzo et al., 2001). The association with PPCA is necessary for its enzymatic activity. The association with PPCA stabilizes the active conformation of NEU1 in lysosomes. Moreover, since NEU1 is poorly mannose 6-phosphorylated, it depends on PPCA for its correct compartmentalization and catalytic activation in lysosomes (van der Spoel

About 45 different mutations in *NEU1* gene have been reported to date (http://www.hgmd.org/). Almost all of them have been found in single families and most of them are missense mutations. Bonten et al. have studied the impact of some missense mutations on NEU1 protein distribution and catalytic activity and they classified these mutant proteins in 3 groups: 1-catalytically inactive and not lysosomal; 2-catalytically inactive and lysosomal and 3-catalytically active and lysosomal. A good correlation between the residual activity of mutant proteins and the severity of the disease has been found. In fact, patients with the severe type II infantile form presented mutations from group 1 while those with a mild form of type I disease had at least one mutation from group 3. Mutations from group 2 were found mainly in patients with the juvenile form of type II sialidosis with

Niemann Pick type C (NPC) disease (NPC1, MIM 257220; NPC2, MIM 607625) is an autosomal recessive neurodegenerative lysosomal storage disorder, caused by the abnormal function of NPC1 or NPC2 protein. Both proteins are involved in the intracellular trafficking of cholesterol and other lipids. The deficiency of either of them leads to the accumulation of

Endocyted low density lipoproteins are delivered to the late endosomes/lysosomes where they are hydrolized. In normal cells, free cholesterol is transported to the plasma membrane or to the endoplasmic reticulum through the action of NPC1 and NPC2 proteins. In NPC cells cholesterol accumulate within the lysosomes and the subsequent induction of all lowdensity lipoprotein cholesterol-mediated homeostatic responses, including cholesterol

In addition NPC-deficient cells also accumulate gangliosides and other GSLs. These findings show that the defect in NPC cells encompasses a global transport error. In fact, while unesterified cholesterol is the main lipid accumulated in peripheral tissues, GM3, GM2 and glucosylceramide are the mayor lipids accumulated in brain of NPC patients (Zervas et al.,

the endocytosed unesterified cholesterol within the lysosomes (Patterson et al., 2001).

et al., 1998; van der Spoel et al., 2000; Yamamoto et al., 1987).

an intermediate phenotype (Bonten et al., 2000).

**6. Niemann pick type C (NPC) disease** 

esterification, is compromised.

2001a).

**5.2 Molecular aspects** 

al., 1997).

prosaposin that can be cleaved to saposins A, B, C and D (Gopalakrishnan et al., 2004). Most patients accumulate autofluorescent lysosomal deposits with GRODs.

Only four disease-causing mutations have been described to date (http://www.ucl.ac.uk/ncl).
