Recent neuroimaging, neurophysiological, and neuropathological advances for the understanding of NPC

Niemann–Pick disease type C (NPC) is a rare autosomal recessive lysosomal storage disorder with extensive biological, molecular, and clinical heterogeneity. Recently, numerous studies have tried to shed light on the pathophysiology of the disease, highlighting possible disease pathways common to other neurodegenerative disorders, such as Alzheimer’s disease and frontotemporal dementia, and identifying possible candidate biomarkers for disease staging and response to treatment. Miglustat, which reversibly inhibits glycosphingolipid synthesis, has been licensed in the European Union and elsewhere for the treatment of NPC in both children and adults. A number of ongoing clinical trials might hold promise for the development of new treatments for NPC. The objective of the present work is to review and evaluate recent literature data in order to highlight the latest neuroimaging, neurophysiological, and neuropathological advances for the understanding of NPC pathophysiology. Furthermore, ongoing developments in disease-modifying treatments will be briefly discussed.

Niemann-Pick disease type C (NPC) (MIM 257220) is an autosomal recessive neurovisceral lysosomal lipid storage disorder due to mutations of either the NPC1 (95% of families) 1 or the NPC2 gene 2 . The incidence of NPC is estimated to be 1 in 100,000 live births 3-5 , although the late-onset phenotypes or variant forms with visceral-only or neurologically mild presentations might have a much higher frequency 6 .
Although the exact functions of the NPC1 and NPC2 genes are still to be fully elucidated, mutations in these genes cause a loss of function, resulting in the accumulation of unesterified cholesterol and glycosphingolipids within the late endosome/ lysosome of all cells, leading to downstream effects on cholesterol homeostasis 7-9 . Recently, using structural biology approaches, such as crystallography and cryo-electron microscopy, researchers have gained insights into how NPC1 and NPC2 proteins function in tandem to facilitate cholesterol efflux from late endosomes/lysosomes [10][11][12][13][14][15][16][17][18][19] .
The broad clinical spectrum ranges from a neonatal, rapidly fatal disease to an adult-onset chronic neurodegenerative disorder, and patients can be classified into four general categories based on age at neurological onset: the early infantile, late infantile, juvenile, and adolescent/adult onset form 3 . In most patients, disease severity is defined by the neurological involvement but usually is preceded by systemic signs such as neonatal cholestatic jaundice or isolated spleen or liver enlargement in infancy or childhood 4 . In the majority of patients with NPC, the liver disease frequently resolves with time 3,4 .
Clinical pictures of NPC are widely heterogeneous, and there are a number of neurological signs and symptoms with different ages at onset and different rates of progression: gait ataxia, clumsiness, cataplexy, epilepsy, dystonia, supranuclear gaze palsy, dysarthria, dysphagia, cerebellar ataxia, psychiatric illnesses, or cognitive decline 3,4,20 . Psychiatric disturbances may be undiagnosed for several years and are often characterized by psychosis such as paranoid delusions, visual or auditory hallucinations, or behavioral abnormalities 20 . The age of onset of neurological manifestations usually correlates with the patient's prognosis 4 .
Slightly different phenotypes have been observed in patients with NPC2 gene mutations, which are frequently associated with a severe phenotype, characterized by pulmonary infiltrates, respiratory failure, and death in early age 21-23 . Adult-onset disease with frontal lobe atrophy has also been described 24 , and in some cases prolonged survival into middle adult life has been reported 25 .
Plasma oxysterols have been shown to be highly sensitive for NPC; however, the originally reported specificity has recently been widened to other disorders of sterol metabolism, including acid sphingomyelinase deficiency (Niemann-Pick type A and B disease), cerebrotendinous xanthomatosis, and lysosomal acid lipase deficiency (Wolman disease) [36][37][38] . Furthermore, oxysterols can be increased in neonates with non-NPC cholestasis 39 .
Until recently, filipin staining was considered the gold standard assay for NPC diagnosis 4,9,33,40  A major limitation of the filipin test is that it requires cell cultures of living fibroblasts from a skin biopsy, has relatively long turnaround times, and is performed in only a few specialized laboratories worldwide 49 .
Genetic testing, which involves the sequencing of the NPC1 and NPC2 genes, is also available; however, it is still inconclusive in 12 to 15% cases because of the unknown pathogenicity of the mutation, the lack of a study of allele segregation, and the possible existence of one unidentified mutant allele 6 .
A series of therapies for NPC are actively being developed.
Miglustat, an iminosugar that is a competitive inhibitor of glucosylceramide synthase and specifically targets the metabolic pathway that leads to the synthesis of glycosphingolipids in neurons and other cells, has been approved for the treatment of NPC in Europe and elsewhere 51 .
Very recently, the most up-to-date clinical guidelines on NPC were published by an expert group 52 .
In recent years, numerous studies have increased the understanding of NPC, highlighting the very complex and multifaceted nature of the disease, and reported possible links to other neurodegenerative disorders, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD).
The objective of the present work is to review and evaluate recent literature data in order to highlight the latest neuroimaging, neurophysiological, and neuropathological advances for the understanding of NPC pathophysiology. Furthermore, recent developments in disease-modifying treatments will be briefly discussed.

Neuroimaging
Historically, brain magnetic resonance imaging (MRI) and computed tomography scans were not usually considered very useful for the diagnosis of NPC, as most of the findings are unspecific, sometimes showing cerebellar or cortical atrophy or, in the severe infantile form, white matter changes 3 . However, in recent years, numerous studies have shown the involvement of several cerebral structures in patients with NPC, even highlighting the possible modifications induced by therapeutic interventions (Table 1).
Voxel-based morphometry analyses have shown a significant involvement of the hippocampus, thalamus, striatum, and cerebellum in NPC 53-57 . In particular, cerebellar gray matter and left thalamus volume loss were significantly correlated with Iturriaga disability scale changes and ataxia measures 53,58 . Furthermore, untreated patients exhibited what may appear to be greater thalamic and cerebellar gray and white matter reductions over time compared with both controls and patients treated with miglustat 53 .
As in progressive supranuclear palsy, the pontine-to-midbrain ratio is increased in adult patients with NPC compared with controls, and the strong correlation with illness and oculomotor variables suggests that it may be a useful marker for illness progression in NPC 59 .
The atrophy pattern in the thalamus, hippocampus, and caudate nucleus, observed with cortical thickness analyses, showed a significant correlation with memory, executive functions, and motor control dysfunction 58 .
The involvement of deep gray nuclei has also been confirmed by 123 I-FP-CIT (ioflupane I 123 DaTSCAN) single-photon emission computed tomography (SPECT) imaging in a case of NPC, showing a marked, symmetrical loss of dopamine transporter binding, especially in the putamen 57 . This pattern has also been observed in a heterozygote patient with a "variant phenotype" in filipin staining and with high levels of plasma oxysterols 60 .
Diffusion tensor imaging (DTI) analyses showed decreased fractional anisotropy in NPC patients compared with controls, especially in the corpus callosum, internal capsule, corona radiate, and the cingulate gyrus, with an early but transient improvement of DTI metrics after miglustat treatment 61-63 . Global callosal measures correlated significantly with duration of illness and symptom score and at trend level with degree of filipin staining 64 .
In agreement with these studies, myelin water imaging, a technique that measures the amount of water present within the myelin of white matter tracts 65,35 , has shown large reductions of myelin water fraction in large association tracts and the corpus Functional imaging with 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) and SPECT has highlighted the involvement of frontal and temporal structures, even in the very initial phases of the disease 28,68-72 , possibly reflecting disease severity 68 .
Proton magnetic resonance spectroscopic imaging (H-MRSI) studies have shown a decreased N-acetyl aspartate/creatine ratio in the frontal and parietal cortices, centrum semiovale, and caudate nucleus, and there were significant correlations between clinical staging scale scores and H-MRSI abnormalities 59,73,74 .
As it clearly emerges, MRI is the modality of choice for identifying reported abnormalities in the clinical setting, such as frontal lobe and cerebellar atrophy 4,75 , white matter hyperintensities in parieto-occipital periventricular regions 75 , deep gray matter and hippocampal atrophy particularly in adult-onset patients 54 , and reduced midbrain-to-pons ratio 59 . However, specific neuroimaging findings are lacking in NPC, providing little aid in the clinical setting and diagnostic workup, highlighted by the absence of imaging tests in diagnostic criteria or from the NPC suspicion index 76 . Furthermore, these biomarkers have not been validated regarding accuracy in the differential diagnosis with other neurodegenerative diseases or compared with healthy controls; thus, specificity and sensitivity measures are currently unavailable. However, new imaging modalities have provided a solid basis for the development of biomarkers to understand disease pathophysiology and to monitor disease progression and response to treatments in the research setting.

Neurophysiology
As patients with NPC can experience any type of seizure (partial/focal, generalized, absence, myoclonic, or tonic-clonic), which vary in intensity and frequency, electroencephalography (EEG) should be used for confirmation and for differentiating epilepsy from cataplexy 3,77-83 . Patients who develop severe epilepsy generally have a worse prognosis and reduced life span compared with patients who are seizure-free 3 .
Neurophysiological evaluation with evoked potentials has shown the involvement of central tracts in most patients with NPC. Indeed, somatosensory evoked potentials have been shown to be impaired in patients with NPC, particularly in the lower limbs 84,85 , whereas brain auditory evoked potentials showed a bilateral absence of most waves, highlighting the involvement of auditory pathways ranging from the auditory nerve to the midbrain 84-87 .
Pyramidal involvement 85,88 and abnormalities of visual evoked potentials 85 have been reported in patients with infantileonset NPC but appear only at advanced stages of disease 84 . Peripheral neuropathy has also been reported as a rare manifestation in patients with NPC 3,68,89,90 .
Recently, transcranial magnetic stimulation (TMS) pairedpulse paradigms have been used to evaluate cortical excitability and intracortical connectivity measures. In this context, an impairment in short-latency afferent inhibition (SAI), a measurement of sensorimotor integration, has been observed in NPC. SAI is thought to be mediated largely by central cholinergic transmission and has been shown to be impaired in patients with AD 91,92 , further supporting the link between NPC and AD 60,93,94 . Moreover, long-term potentiation (LTP)-like cortical plasticity, evaluated with a paired associative stimulation protocol, has been shown to be impaired in patients with NPC and in a symptomatic heterozygous carrier, confirming previous reports of impaired hippocampal synaptic plasticity in Npc1-mutant mice 95 . The impairment in LTP-like cortical plasticity has also been observed in patients with AD and FTD, further highlighting the possible parallelism between these disorders 96,97 . Interestingly, after 12 months of treatment with miglustat, a considerable improvement in SAI and LTP-like plasticity was observed in patients with NPC 60 .
Only a limited decrease in short-interval intracortical inhibition, a marker of GABAAergic transmission, and intracortical facilitation, a marker of glutamatergic transmission, has been observed. Long-interval intracortical inhibition, reflecting GABABergic transmission, was reported to be within normal range 60 .
In conclusion, neurophysiological tests still fall short in providing invaluable information for the diagnostic workup of NPC diagnosis because findings are not specific for NPC and may not be found in all cases of NPC. EEG remains the exam of choice for confirming and differentiating epilepsy from cataplexy and for monitoring response to antiepileptic drug treatment.
However, in the research setting, a new series of non-invasive tests have shown a selective impairment of specific intracortical circuits that, if confirmed in a wider population of patients, might provide valuable information on disease pathophysiology and disease progression and possibly be used to monitor response to therapeutic interventions.

Neuropathology
Although cholesterol is able to flow freely through most cellular membranes, it cannot exit from lysosomes without the aid of NPC1 and NPC2 proteins. The so-called "hydrophobic handoff" model has been proposed 98 , and recent studies with structural biology approaches, such as crystallography and cryo-electron microscopy, have corroborated this model hypothesis, highlighting how the luminal NPC2 protein picks up cholesterol from endocytosed cholesterol as well as from the significant lipid content present in the lumen of degradative lysosomes, eventually interacting with the membrane-bound NPC1 protein 10-19 . However, how cholesterol is transferred across the membrane by the NPC1 protein is still a matter of debate 99 .
Both NPC1 and NPC2 proteins are involved in the trafficking of low-density lipoprotein (LDL)-derived cholesterol from the lysosome to the cellular membranes of the endoplasmic reticulum, Golgi apparatus, and plasma membrane 17,100 . The decrease of cholesterol levels in the endoplasmic reticulum consequently enhances the synthesis and uptake of cholesterol by the sterol response element-binding protein pathway, leading to the accumulation of cholesterol and other lipids in many types of cells, including lipid-laden macrophages (called foam cells) and neuronal and glial cells 101-103 .
Compared with most other lipid storage disorders, NPC does not arise from defective substrate degradation but from the impairment of LDL-derived cholesterol export out of the lysosome, followed by the disruption of lipid homeostasis 103 , affecting multiple cellular functions such as lysosomal calcium homeostasis 104 , oxidative stress 105,106 , Rab-mediated vesicle trafficking 107,108 , or fusion of lysosomes 109 , leading to impaired autophagy 110,111 . The accumulation of lipids in the central nervous system causes neuronal distension, axonal swelling, and the formation of axonal spheroids 112-115 .
NPC is also characterized by the accumulation of β-amyloid 116,117 and neurofibrillary tangles 24,118-122 , which are immunologically and ultrastructurally similar to those seen in AD 123 . Indeed, in one of the first descriptions of NPC neuropathology, a widespread neurofibrillary degeneration with a distribution similar to that of advanced AD was reported 124 . However, subsequent studies highlighted a somewhat different distribution of neurofibrillary tangles in NPC compared with AD 71 ; there was a primary involvement of subcortical structures, including hippocampus, thalamus, and striatum in NPC 24,118-120 , and a more cortical distribution in AD 125-127 . Neurofibrillary tangles in NPC tend to be associated with lipid accumulations in swelling neurons, possibly suggesting a triggering effect of intracellular accumulations on tau aggregation 71,118 .
Beyond cholesterol pathway NPC and AD have other similarities, which involve AD pathogenesis 128 . This parallelism between NPC and AD is further strengthened by the observation that cholesterol levels may modulate the processing of amyloid precursor protein 129 and accumulation of β-amyloid 130 is supported by the disease-modifying effect of the ε4 isoform of apolipoprotein E on disease progression in both NPC and AD 131-133 . Moreover, a possible effect of mutations in the NPC genes as AD risk factors has been speculated 133,134 .
Just recently, a novel link with another intracellular proteinopathy has been established. Indeed, both in NPC mouse and in a human neuronal model of the disease, an altered expression or mislocalization of the TAR-DNA binding protein 43 (TDP-43) or both were reported 135 . From a functional point of view, the TDP-43 mislocalization observed in human experimental neuronal models of NPC was associated with specific alterations in TDP-43 controlled genes. Most interestingly, N-acetyl-cysteine or 2-hydroxypropyl-β-cyclodextrin may partially restore TDP-43 metabolism 135 . TDP-43 inclusions have been reported as the main pathological signature of FTD due to C9orf72 and GRN mutations 136 and are also described in AD cases 137 .
Whereas β-amyloid and α-synuclein may accumulate biochemically in the NPC brain, senile plaques or Lewy bodies are not characteristic of the disease, and further studies are needed to assess the possible overlap between these neurodegenerative disorders. As highlighted above, these speculations have not been validated and replicated in larger studies and thus a cautious interpretation is warranted.

Treatments
Miglustat, a small iminosugar molecule that reversibly inhibits glycosphingolipid synthesis, has been licensed in the European Union and elsewhere for the treatment of progressive neurological manifestations of NPC in both children and adults 3,13,83,106 . It has been shown to stabilize or improve certain neurological manifestations in six clinical trials 28,88,[138][139][140][141][142] , none of which is randomized or placebo-controlled, and to partially restore neurophysiological markers of cholinergic impairment (such as SAI) and LTP-like cortical plasticity 60,95 .
The effect of cholesterol-lowering agents on hepatic and plasma cholesterol in NPC has been assessed with dimethyl sulfoxide, nicotinic acid, lovastatin, cholestyramine, and combinations of the above drugs. The treatment effects on total cholesterol varied depending on the drug combinations and overall improved with the number of drugs. However, efficacy for neurological outcomes was not reported, and safety findings discouraged widespread application of cholesterol-lowering agents to patients with NPC 143,144 . Many other therapies, including 2-hydroxypropyl-β-cyclodextrin (NCT02912793, NCT02939547, NCT01747135, and NCT02534844), arimoclomol (NCT02612129), vorinostat (NCT02124083), lithium carbonate (NCT03201627), and δ-tocopherol, are currently under clinical investigation for NPC 145 .
Just recently, a phase 1-2 clinical trial with monthly 2-hydroxypropyl-β-cyclodextrin was performed on 14 patients with NPC and showed slowed disease progression, in particular in ambulation, cognition, and speech, with an acceptable safety profile 155 . Since 2-hydroxypropyl-β-cyclodextrin does not efficiently cross the blood-brain barrier 156 and high-dose systemic delivery can be associated with pulmonary toxicity 149,157 , lumbar intrathecal administration is the route of choice but has common adverse events, such as post-lumbar puncture headache, reported in 64% of cases 155 . At doses above 600 mg, unexpected adverse events included post-administration unsteadiness and fatigue, which were transient and typically occurred 24 to 72 hours after dosing 155 . Ototoxicity, with mid-to high-frequency hearing loss, an expected adverse event, was documented in all participants and was probably due to outer hair cell loss 155,158 .
The main drawback of this approach is due to the route of administration; owing to the ability of molecular chaperones of the heat shock protein 70 (HSP70) family to protect pathologically challenged cells, HSP70-based therapies are emerging as attractive treatment options for many degenerative diseases [159][160][161][162][163] , including lipid storage disorders due to their direct interaction with lysosomes 162,164 , and for the proper folding and activity of the NPC1 protein 165,166 . In this view, arimoclomol and small-molecule HSP70 co-inducer have been tested in a number of clinical trials 167,168 and are currently under investigation for the treatment of NPC.
Vorinostat, currently used for cutaneous T-cell lymphoma, is a histone deacetylase inhibitor that was able to increase NPC1 protein and decrease unesterified cholesterol deposits [169][170][171][172][173] . Indeed, in selected genetic disorders, histone deacetylase inhibitors have been shown to induce histone modifications that not only can result in increased or decreased transcriptional expression of mutated genes 174 but also confer indirect benefits through acetylation of non-histone proteins, such as transcription factors and heat shock proteins, that modulate chaperones and proteostatic networks 169,174-176 . Interestingly, however, treatment of NPC2-deficient human fibroblasts with a histone deacetylase inhibitor did not reduce cholesterol storage in lysosomes and late endosomes 171 . FTY720/fingolimod, an inhibitor of class I histone deacetylases used for the treatment of multiple sclerosis, has been shown to increase the expression of NPC1 and NPC2 in human NPC1mutant fibroblasts and to significantly reduce the accumulation of cholesterol and glycosphingolipids 177 .
Other therapeutic approaches-including several that use human stem cells, such as hematopoietic stem cell transplantation (NCT00668564, NCT00730314, and NCT01372228), human placental-derived stem cell transplantation (NCT01586455), and intrathecal umbilical cord blood-derived oligodendrocyte-like cells (NCT02254863)-are currently under development for the treatment of NPC.
In regard to NPC2 mutations, given that the NPC2 protein is soluble, secreted, and recaptured, there is a rationale supporting early hematopoietic stem cell transplantation 178,179 .
Other attractive approaches, evaluated in preclinical models of disease, include the systemic delivery of adenoassociated virus vectors to NPC1 -/mice to increase the expression of a therapeutic NPC1 transgene, which has resulted in an improved clinical appearance, delayed weight loss, significantly increased life span, reduced cholesterol storage, and decreased cerebellar Purkinje cell degeneration compared with untreated NPC1 -/mice 180,181 .

Conclusions
NPC represents an autosomal recessive disorder with extensive biochemical, molecular, and clinical variability, which probably results in an underestimation of the burden of NPC cases worldwide. The relatively low incidence of the disease increases the difficulties in developing high-quality observational studies or randomized clinical trials.
As outlined above, recent studies have tried to shed light on the pathophysiology of this disease, further underlying its complex nature. Nevertheless, numerous biomarkers reflecting disease pathogenesis have emerged, thus representing a useful aid to diagnose disease or to evaluate disease progression and response to therapeutic interventions. Indeed, in this view, imaging and neurophysiological markers have been shown to reflect disease severity and to respond to disease-modifying treatments.
Intriguingly, a close parallelism has been observed between NPC and other neurodegenerative disorders, highlighting the possible involvement of multiple, but intertwined, disease pathways. Thus, unravelling the connection between neurofibrillary tangles, TDP-43 pathology, and neurodegeneration could result in important advances not only for NPC but also for AD and FTD/amyotrophic lateral sclerosis.
Diagnosing NPC represents a challenge for physicians, and delays in diagnosis and ensuing miglustat treatment and eventually future disease-specific interventions may affect disease outcomes because of irreversible anatomical damage and progressive neurodegeneration 182 . In this view, a prompt diagnosis is essential, and development of clinical tools, such as suspicion index to provide a risk prediction score 76 , along with instrumental diagnostic and prognostic markers, is mandatory. Efforts to increase awareness of NPC among clinicians are still needed, but the recent development of rapid and relatively simple instrumental and laboratory tests should improve the diagnostic and prognostic approach to NPC 49 . 135.