Written by: Gabrielle Trépanier
Neuronal ceroid lipofuscinosis type 2 is a deadly pediatric neurodegenerative disease that manifests itself through psychomotor decline and the accumulation of autofluorescent storage material. Recent research has revealed the role of enzyme deficiency in the pathophysiology of the disease, though the precise mechanisms by which neurotoxicity occurs remain misunderstood. Despite the improvement of diagnostic accuracy due to the advent of DNA testing, diagnosis remains challenging primarily as a result of varying clinical presentation. Promising therapies such as cerliponase alfa are emerging, while other treatment options have yet to show clinical results. This review evaluates the current understanding of disease pathways, while highlighting challenges for novel discoveries such as diagnosis latency, clinical research design flaws, and pharmacokinetic barriers to treatment solutions.
Primarily affecting infants and youth, neuronal ceroid lipofuscinoses (NCLs) are a group of rare neurodegenerative autosomal recessive lysosomal storage disorders that are estimated to occur in 0.56 to 13.6 per 100 000 live births.1–5 These disorders are grouped together due to their broad hallmark characteristics: neuronal loss resulting in cerebral atrophy and excessive accumulation of ceroid lipofuscin in lysosomes.6 While NCLs are similar in their neuropathology, they differ notably in age of onset, rate of disease progression, as well as chronology and severity of clinical symptoms.2,7 NCL symptoms commonly include deterioration of motor abilities, epileptic seizures, language development delays, behavioural changes, and vision loss.2,4,7 Classified by their locus of mutation, 13 forms of NCL have been identified.2,8 The most common genetic mutations in NCL patients in the United States are variants in the CLN2 gene, which codes for the lysosomal enzyme tripeptidyl peptidase I (TPP1).1,9 In neuronal ceroid lipofuscinosis type 2 (CLN2), also known as Jansky-Bielschowsky disease, the deficient TPP1 enzyme is responsible for accumulation of ceroid lipofuscin, thereby provoking neuronal degeneration.7,9,10 Though NCLs are fatal and incurable, there are several therapies available for the treatment of CLN2 disease.7 The following review aims to discuss the underlying genetic and enzymatic mechanisms of CLN2, understand the shortcomings of current diagnostic methods, and provide insight on novel therapeutic interventions.
Tripeptidyl peptidase I (TPPI) is a serine carboxypeptidase enzyme present in various human organs and tissues.5,11 Encoded by mapping the CLN2 gene to chromosome 11p15, TPPI is normally synthesized as an inactive enzyme before being transported into lysosomes through the mannose 6-phosphate-dependant pathway, where it is proteolytically converted into its mature form.11–14 In this form, TPPI cleaves tripeptides from the N termini of oligopeptides undergoing degradation in the lysosome, and exhibits minor endopeptidase activity.5,14,15 The cleavage of these tripeptides is assumed to be crucial to the neuropeptide degradation process, as this process is inhibited in CLN2.16 To date, 140 disease-causing changes in the CLN2 gene have been discovered, which account for the three variations of CLN2: infantile, juvenile, and most commonly, classic-late infantile phenotypes.11 Among these mutations, c.509-1G>C, a splicing mutation, and c.622C>T, a nonsense mutation, occur in 89% of CLN2 cases worldwide.11,13,17 Most mutations are believed to affect the structural stability of TPPI by cleaving the precursor of its catalytic domain and its essential five N-glycosylation sites, resulting in undetectable enzymatic and proteolytic activity.6,11,18
Loss of TPPI activity leads to the lysosomal overaccumulation of ceroid lipofuscin, an autofluorescent lipopigment storage material similar to the lipofuscin that normally accrues as a result of the aging process.5,19 This lipopigment storage material accumulates in deposits that cannot be degraded nor expelled through exocytosis.8,19 While the mechanisms by which TPPI deficiency results in massive ceroid lipofuscin deposits remain misunderstood, multiple theories have been proposed. The first suggests that TPPI deficiency blocks specific biochemical pathways, resulting in the precipitation of enzyme substrates possessing autofluorescent properties.20,21 The second theory notes that the accumulated enzyme substrates may instead alter the cellular or lysosomal environment, causing the accumulation of autofluorescent lysosomal components.20 These enzyme substrates remain to be identified.22 In CLN2, ceroid lipofuscin is primarily composed of subunit C of mitochondrial synthase, in addition to protein, lipid, and trace amounts of carbohydrates and metals.20,23,24 As such, it was previously hypothesized that subunit C of mitochondrial synthase was the foremost substrate involved in CLN2.23,24 However, subunit C accumulation occurs in other NCL forms in addition to CLN2, suggesting that the primary metabolic error in TPPI deficiency lies elsewhere.11,14 Other substrates for TPPI have been proposed, including β-amyloid peptides, angiotensin I and II, glucagon, cholecystokinin, and neuromedin.22 However, these substrates require further study, as most have only been observed in vitro.25
Though lipopigment storage material deposits occur in various tissues and organs, they only present major implications for the central nervous system.5 In CLN2 patients, pronounced neuronal degeneration occurs in the cerebral and cerebellar cortices as well as the retina, while depletion is suggested to occur to a smaller extent in subcortical grey matter regions and nuclei.3,5 Though the mechanism of ceroid lipofuscin neurotoxicity remains poorly understood, several theories have been suggested to explain the connection between lipopigment accumulation and neurodegeneration. It has been proposed that the formation of ceroid lipofuscin induces the excessive generation of reactive oxygen species through Fenton reactions; this oxidative stress may result in cell death.24,26–29 Ceroid lipofuscin may also exhibit neurotoxic effects by reducing proteasome activity.26,30 Unsuccessful binding attempts of proteasomes to ceroid lipofuscin result in decreased proteolytic activity, causing inhibition of normal protein turnover and failure to degrade proapoptotic proteins.30–32 This disruption of proteasome-regulated pathways by ceroid lipofuscin may results in cell death, primarily by apoptosis.30 Additionally, ceroid lipofuscin may cause neurotoxic effects by gradually filling cytoplasmic space, thereby inhibiting normal cellular function and eventually causing neuronal death.30,33,34 Conclusively, the proposed mechanisms of cell death subsequently lead to loss of neurons, cerebral atrophy, and cognitive decline.3,4
Symptoms & Diagnosis
CLN2 presents itself in three age-related phenotypes: infantile, classic late-infantile, and juvenile.10,17 Symptomatic progression remains similar for the two former phenotypes.11 Language acquisition delay is frequently the first presentation of infantile and classic late-infantile CLN2, though it is often overlooked.10,17 Rather, the first symptom noticed by parents and pediatricians is commonly the onset of myoclonic seizures (epilepsy).10,17,35 Epilepsy commonly coexists with motor decline, truncal and peripheral ataxia, and behavioural changes.2,17 In the subsequent years following epileptic onset, rapid deterioration of motor, visual and cognitive function occurs, eventually resulting in loss of movement, vision and speech, as well as the development of motor disorders.4,17 While the juvenile CLN2 phenotype presents many of the symptoms noted above, its chronology varies: visual deterioration and behavioural changes occur first, followed by motor decline and epilepsy onset.2 Dependent on the phenotype, death may occur anytime between late childhood and early adulthood.2,4,17
The most definitive method of CLN2 diagnosis is enzymatic and molecular testing, which includes the detection of deficient TPPI activity and identification of specific mutations in the CLN2 gene.7,36 Electroencephalography (EEG) was previously suggested as a method of CLN2 diagnosis, but cast aside in favour of more accurate testing.37 However, recent studies have identified EEG benefits in areas where molecular testing remains inaccessible, as CLN2 patients may exhibit photoparoxysmal responses to intermittent photic stimulation on account of metabolic changes caused by deficient enzymatic activity.37,38 However, this response is characteristic of multiple neurodegenerative disorders, and it remains unclear whether EEG findings are consistent in all CLN2 patients.37–41 Magnetic resonance imaging (MRI) may also be useful in identifying and monitoring progressive brain atrophy as well as alterations in periventricular white matter tracts.7,17,36 Similar to EEG results, MRI findings are nonspecific to CLN2 and may appear normal in the early stages of the disease.7 Nonetheless, both EEG and MRI, paired with neurophysiological examination, remain viable methods of CLN2 biomarker detection, which may be followed up with more accurate testing.
Early diagnosis of CLN2 is crucial to the provision of adequate therapies. However, the mean age of diagnosis for CLN2 is 22.7 months following the initial onset of symptoms.35 Diagnosis latency may be explained by multiple factors. One such factor is poor awareness of the disease: speech delays and vision loss, common first CLN2 symptoms, easily go unnoticed by parents and healthcare providers; epilepsy treatment may be considered more pressing than its etiology; motor deterioration can mistakenly be attributed to antiepileptic medication.7,10,17 A second factor is the non-uniform symptomatic and clinical presentation of CLN2, thus complicating diagnosis.37,41 Diagnosis may also be hindered by a lack of access to enzymatic and molecular testing; healthcare providers may have only have access to non-specific neurodegeneration findings.36 Ultimately, a more widespread understanding of CLN2 clinical presentation and testing will lead to earlier and more effective diagnosis.
Many CLN2 therapies aiming to reduce disease progression are emerging. In 2017, the U.S. Food and Drug Administration was the first to approve cerliponase alfa (Brineura™), a recombinant human TPPI (rhTPPI) enzyme replacement therapy.42 Administered intracerebroventricularly through a Rickham or Ommaya reservoir every two weeks, the drug enters neurons by a mannose 6-phosphate-dependent pathway and is targeted to lysosomes.2,7 Upon entry, rhTPPI is activated to its proteolytic form and completes normal TPPI function.2,9,43 To date, cerliponase alfa clinical trials have shown significant reduction in the progression of motor and language decline; most adverse effects were mild, short-term, and clinically manageable.2,9,44 The development and approval of cerliponase alfa has paved the route for similar novel therapies targeting other forms of NCL, utilizing a similar enzymatic delivery method.
Many studies note that cerliponase alfa may be used solely and regularly, or in alternation with other therapies such as stem-cell therapy.7,45 This therapy aims to preserve remaining motor and cognitive function by transplanting healthy human neural stem cells into the CLN2 patient’s brain, in order to increase TPPI expression.2 A phase I clinical trial was conducted on 4 CLN2 patients; however, there was no slowing of disease progression following transplant.46 Cerliponase alfa has also been suggested in conjunction with gene therapy, which utilizes a gene therapy vector to introduce a healthy CLN2 gene through intracranial injection. Similar to the stem-cell therapy trial, the phase 1 CLN2 gene therapy trial noted no clinical benefit.47 Nonetheless, these trials demonstrate the feasibility of both stem-cell and gene therapy; ongoing trials are further studying the effectiveness of such therapies in CLN2 patients.7
Other novel pharmacological treatments (NMDA receptor antagonists, Bcl-2 and TPPI mRNA upregulators) have not been able to replicate their animal model successes in clinical trials.46,48,49 The development of a CLN2 drug is particularly complex for many reasons. Lack of understanding of TPPI deficiency and ceroid lipofuscin toxicity mechanisms renders the identification of viable drug pathways difficult.7 Furthermore, any CLN2 therapeutic drug must cross the blood-brain barrier, which is particularly difficult due to the semipermeability of the membrane.22,50 Moreover, designing generalizable clinical trials for CLN2 proves difficult due to the low prevalence of the disease, producing small cohort sizes and single-center studies.35 In the meanwhile, uttermost importance is being placed on multidisciplinary palliative care: physical and speech therapy, family support, seizure and movement disorder management, as well as behaviour management.2,17
The pathways by which TPPI causes ceroid lipofuscin accumulation and subsequent neurotoxicity are crucial to understanding CLN2, a rare pediatric neurodegenerative lysosomal storage disorder characterised by the accumulation of ceroid lipofuscin and severe cerebral atrophy. Though existing therapeutic treatments are promising, novel discoveries will be required to overcome challenges in clinical presentation, diagnosis latency, drug delivery, and most notably, the complexity of CLN2 mechanisms.
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